CN115844912A

CN115844912A - Specific active panaxadiol saponin composition for preventing and treating diabetes and its complication, and its preparation method and application

Info

Publication number: CN115844912A
Application number: CN202211638724.XA
Authority: CN
Inventors: 张治针; 连晓媛; 王延伟
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-12-20
Filing date: 2022-12-20
Publication date: 2023-03-28

Abstract

The invention provides a specific active panaxadiol saponin composition for preventing and treating diabetes and complications thereof, and a preparation method and application thereof, belonging to the technical field of medical health. The invention screens the types and specific proportions of the panaxadiol saponins influencing the prevention and treatment of diabetes and complications thereof through animal experiments, establishes a set of quality control standards of the specific active panaxadiol saponin composition for preventing and treating the diabetes and the complications thereof and the specific active total ginsenoside composition taking the panaxadiol saponin composition as a core active component, provides a new means for the medicine and health care application of the diseases related to the prevention and treatment of the diabetes and the complications thereof and metabolic disorders, improves the medical and health care value of the ginseng traditional Chinese medicinal materials, and effectively utilizes the resources of the ginseng stem and leaf medicinal materials.

Description

Specific active panaxadiol saponin composition for preventing and treating diabetes and its complication, and its preparation method and application

Technical Field

The invention belongs to the technical field of medical health, and particularly relates to a specific active panaxadiol saponin composition for preventing and treating diabetes and complications thereof, and a preparation method and application thereof.

Background

Pre-diabetes and diabetes are global epidemics, and diabetes and its complications have become the third leading killer threatening human life health after cardiovascular and cerebrovascular diseases, cancer. With the prevalence of global obesity and the aging world population, the threat of metabolic syndrome including diabetes to human health is increasing. Diabetes is a group of metabolic diseases characterized by a deficiency in insulin secretion or insulin action due to insulin resistance or both, leading to a syndrome of hyperglycemia and carbohydrate metabolism disorders accompanied by long-term damage and dysfunction or loss of different organs (in particular blood vessels, nerves, heart, muscles, eyes, kidneys) and associated diabetic complications. Diabetic complications involve a variety of diseases, and are the result of the combined actions of vasculopathy, nervous system disorders, metabolic disorders, and the like, and are commonly: diabetic skin disease, peripheral and central neuropathy, microvascular complications (including diabetic nephropathy and retinopathy), macrovascular complications (such as coronary heart disease, peripheral vascular disease and cerebrovascular disease), diabetic foot and wound of diabetic patients are difficult to heal, and the like.

The clinical manifestations of diabetic complications are complex and diverse, and diabetic autonomic neuropathy can appear: resting tachycardia, postural hypotension, gastroparesis, constipation, diarrhea, fecal incontinence, erectile dysfunction, neurogenic bladder dysfunction, gastrointestinal dysfunction, perspiration dysfunction, etc.; and sensory neuropathy can be manifested as: pain, paresthesia (e.g., burning discomfort, crawling worm feel, numbness, etc.), or loss of sensation without the ability to perceive external hazards. In the process of diabetes occurrence and development, the incidence rate of diabetic complications is up to 96 percent, 30 to 40 percent of patients suffer from at least one complication after the occurrence of the diabetes, the majority of the patients with the diabetes are accompanied with neuropathy and/or cardiac complication, and the complication is difficult to reverse once the medicine treatment is generated. Cardiovascular complications, including coronary heart disease, heart failure and diabetic cardiomyopathy, are the leading causes of death in diabetic patients, and the prognosis is also poor in diabetic patients who have suffered from myocardial infarction or heart failure. In addition, diabetes and its complications seriously reduce the working ability and the ability to withstand various physical and mental stresses of young and middle-aged patients. It can be seen that diabetic complications reduce the survival of patients at a low rate and threaten the lives of patients at a high rate.

Despite the long-term therapeutic drug development for diabetic complications on an international scale, there are no other methods and drugs that are effective in treating diabetic complications worldwide, in addition to improving lifestyle and controlling blood glucose of patients. Moreover, retrospective studies in clinical practice have shown that controlling blood glucose levels alone is not effective in preventing and treating diabetic complications. It can be seen that hyperglycemia is far from representing a metabolic disorder of diabetes, nor a cause of diabetic complications.

It must be pointed out here that the metabolic disorders of diabetes and its malignant events (including mitochondrial dysfunction and its induced tissue and organ energy insufficiency, oxidative stress injury and chronic inflammatory reaction) not only lead to a low and/or disordered cellular functional state, thus severely reducing the working capacity of young and middle-aged patients and the ability of patients of all ages to withstand various physical and mental stresses, but also are the beginners of various complications. In addition, diabetic metabolic disorders also increase the risk of osteoporosis, cancer and a variety of neurodegenerative diseases including alzheimer's disease (AD, also known as senile dementia), vascular dementia and parkinson's syndrome/disease in elderly diabetic patients. In particular, senile dementia metabolic disorders in the brain have a phenotype similar to the metabolic characteristics of type II diabetes, and therefore, senile dementia is referred to as developing intracerebral diabetes, also referred to as type III diabetes. In conclusion, correcting or relieving the metabolic disorder of diabetes is of great significance for improving the work and life quality of patients, preventing and treating diabetic complications and reducing the risk of senile diseases. However, how to correct or alleviate the metabolic disorders of diabetes is still an unsolved and serious scientific problem.

An increasing number of basic studies suggest that the circulation and metabolic blockade of glucose in the glycolysis link and tricarboxylic acid cycle or also with the mitochondrial respiratory chain leads to two types of cachexia: firstly, glucose is the most important way for various organs and tissues, particularly the heart to obtain energy substance Adenosine Triphosphate (ATP) through mitochondrial oxidative phosphorylation, so that ATP deficiency caused by glucose metabolism obstruction can directly influence the physiological functions of the organism; secondly, glucose flows to a polyol pathway in a large amount and glycolytic intermediate products flow to a glycosylation pathway continuously, so that the former can consume a large amount of antioxidant substance NADPH, the NADPH deficiency can directly limit the antioxidant capacity of Glutathione (GSH) and initiate oxidative stress injury and chronic inflammatory reaction, and the latter can generate a large amount of glycosylation end products (AGEs) to weaken and even destroy the physiological functions of macromolecules including metabolic enzymes. Since metabolic enzymes in the tricarboxylic acid cycle and mitochondrial respiratory chain are extremely vulnerable to oxidative stress, NADPH and GSH deficiencies will lead to secondary mitochondrial dysfunction and exacerbate ATP deficiency and oxidative stress damage. It is further pointed out that the correction or alleviation of metabolic disorders at the root of the disease is of great importance in the prevention and treatment of diabetes and its complications.

Disclosure of Invention

In view of the above, the present invention provides a specific active panaxadiol saponin composition, which defines a specific panaxadiol saponin composition that exerts drug effects in ginsenosides for diabetes and its complications, further determines a specific preparation ratio of the individual panaxadiol saponins in the composition, and provides a new idea for preventing and treating diabetes and its complications.

The invention also provides a preparation method of the specific active panaxadiol saponin composition, which has the characteristics of simple preparation process and production cost reduction.

The invention also provides application of the specific active panaxadiol saponin composition in preparing a medicament for preventing and treating diabetes or diabetic complications, and the specific active panaxadiol saponin composition generates breakthrough progress on the medicament effect of preventing and treating diabetes and systemic co-diseases and metabolic disorder related diseases thereof.

The invention provides a specific active panaxadiol saponin composition, which comprises the following panaxadiol saponins: GRb1, GRc, GRb3 and GRd; in the specific active panaxadiol saponin composition, the total mass percentage of GRc and GRb3 is more than that of GRb1 and GRd, and the difference between the total mass percentage of GRc and GRb3 and the total mass percentage of GRb1 and GRd is 4.55-21.30%;

The ratio of the total mass of GRc and GRb3 to the total mass of GRb1 and GRd is 1.13-1.64;

the mass ratio of GRb1 to GRd is 0.85-1.82;

the mass ratio of GRc to GRb3 is 0.60-0.86;

the mass ratio of GRb3 to GRb1 is 0.93-2.10;

the mass ratio of GRc to GRb1 is 0.72 to 1.42.

Preferably, the mass ratio of GRb1, GRc, GRb3 and GRd includes: 0.96-1.18: 0.90-1.10: 1.37 to 1.67: 1.02-1.24, 0.69-0.85: 0.90-1.10: 1.33 to 1.63: 0.66-0.80, 1.13-1.38: 0.90-1.10: 1.16 to 1.42: 0.70-0.86 or 0.99-1.21: 0.90-1.10: 1.17 to 1.43:0.59 to 0.73;

further preferably, the mass ratio of GRb1, GRc, GRb3, and GRd is 1.07:1.00:1.52:1.13, 0.77:1.00:1.48:0.73, 1.25:1.00:1.29:0.78 or 1.10:1.00:1.30:0.66;

preferably, the panaxadiol saponin composition further comprises GRb2, and the mass ratio of the GRb2 to the GRc is (0.32-1.11): 1.00.

the invention provides a preparation method of the specific active panaxadiol saponin composition, which comprises the following steps:

dissolving Ginseng radix total saponin in 30 vol% ethanol water solution, and subjecting to reversed phase C ₁₈ Separating by silica gel column chromatography, eluting with 27-33% ethanol water solution by volume percentage, eluting with 37-44% ethanol water solution by gradient volume percentage, collecting eluate, and stopping eluting when ginsenoside GRb1 is detected in the eluate; eluting with 50-60% ethanol water solution, collecting eluate, stopping eluting when ginsenoside GRd is not detected in the eluate, mixing the collected eluates, concentrating, and drying to obtain specific active panaxadiol saponin composition;

Or mixing the panaxadiol saponins GRb1, GRc, GRb3 and GRd in proportion to obtain specific active panaxadiol saponins composition;

the commercialized total ginsenoside comprises two or more of radix Panacis Quinquefolii root total saponin, radix Panacis Quinquefolii stem and leaf total saponin, radix Ginseng root total saponin, radix Ginseng stem and leaf total saponin and Notoginseng radix stem and leaf total saponin;

the Panax Chinese medicinal materials comprise two or more of radix Panacis Quinquefolii root, caulis Panacis Quinquefolii, radix Ginseng root, caulis Ginseng and folium Notoginseng; preferably, the commercial total ginsenosides comprise at least one of the following compositions: 0.8 to 1.2 parts by mass of American ginseng root total saponin and 1.6 to 2.4 parts by mass of American ginseng stem and leaf total saponin, 0.8 to 1.2 parts by mass of American ginseng root total saponin, 0.8 to 1.2 parts by mass of American ginseng stem and leaf total saponin and 1.4 to 2.1 parts by mass of pseudo-ginseng stem and leaf total saponin, 0.8 to 1.2 parts by mass of ginseng stem and leaf total saponin, 1.2 to 1.8 parts by mass of American ginseng root total saponin, 0.8 to 1.2 parts by mass of American ginseng stem and leaf total saponin and 2.4 to 3.6 parts by mass of pseudo-ginseng stem and leaf total saponin, 0.8 to 1.2 parts by mass of American ginseng root total saponin, 0.8 to 1.2 parts by mass of American ginseng stem and leaf total saponin and 0.8 to 1.2 parts by mass of pseudo-ginseng stem and leaf total saponin, and 0.8 to 1.2 parts by mass of pseudo-ginseng stem and leaf total saponin;

The ginseng traditional Chinese medicinal materials comprise at least one of the following raw materials: 0.8 to 1.2 parts by mass of American ginseng root, 1.6 to 2.4 parts by mass of American ginseng stem and leaf, 0.8 to 1.2 parts by mass of American ginseng root, 0.8 to 1.2 parts by mass of American ginseng stem and leaf, 1.4 to 2.1 parts by mass of pseudo-ginseng stem and leaf, 0.8 to 1.2 parts by mass of ginseng stem and leaf, 1.2 to 1.8 parts by mass of American ginseng root, 0.8 to 1.2 parts by mass of American ginseng stem and leaf, and 2.4 to 3.6 parts by mass of pseudo-ginseng stem and leaf, 0.8 to 1.2 parts by mass of American ginseng root, 0.8 to 1.2 parts by mass of American ginseng stem and leaf, and 0.8 to 1.2 parts by mass of pseudo-ginseng stem and leaf, and 0.8 to 1.8 parts by mass of pseudo-ginseng stem and leaf.

Preferably, the panaxadiol saponins GRb1, GRc, GRb3 and GR are obtained by the following preparation steps: dissolving the mixed total ginsenoside in 30 percent ethanol water solution by volume percentage, combining with on-line analysis, and performing reversed phase C ₁₈ Performing silica gel column chromatography separation, eluting with 30% ethanol water solution by volume percentage, eluting with 40% ethanol water solution, collecting the eluate until GRb1 or GRb1 is absent in the eluate, eluting with 43% ethanol water solution, collecting the eluate until GRc or GRc is absent in the eluate is extremely low, eluting with 47% ethanol water solution, collecting the eluate until GRb3 or GRb3 is absent in the eluate is extremely low, eluting with 55% ethanol water solution, and collecting the eluate until GRd or GRd is absent in the eluate is extremely low;

The total ginsenoside comprises commercial total ginsenoside or total ginsenoside extracted by taking ginseng as a raw material and adopting 30-80% ethanol water solution by volume percentage by using a conventional method.

The invention provides an active ginseng total saponin composition, which comprises more than two of American ginseng root total saponin, american ginseng stem leaf total saponin, ginseng root total saponin, ginseng stem leaf total saponin and panax notoginseng stem leaf total saponin, or is obtained by taking ginseng traditional Chinese medicinal materials as raw materials, extracting the raw materials by adopting 30-80% ethanol aqueous solution by volume percentage, and removing a solvent; the Panax Chinese medicinal material comprises two or more of radix Panacis Quinquefolii root, caulis Panacis Quinquefolii, radix Ginseng root, caulis Ginseng and folium Notoginseng;

the specific active panaxadiol saponin composition contains the specific active panaxadiol saponin composition with the mass percentage of more than 35%.

Preferably, at least one of the following compositions is included: 0.8 to 1.2 parts by mass of American ginseng root total saponin and 1.6 to 2.4 parts by mass of American ginseng stem and leaf total saponin, 0.8 to 1.2 parts by mass of American ginseng root total saponin, 0.8 to 1.2 parts by mass of American ginseng stem and leaf total saponin and 1.4 to 2.1 parts by mass of pseudo-ginseng stem and leaf total saponin, 0.8 to 1.2 parts by mass of ginseng stem and leaf total saponin, 1.2 to 1.8 parts by mass of American ginseng root total saponin, 0.8 to 1.2 parts by mass of American ginseng stem and leaf total saponin and 2.4 to 3.6 parts by mass of pseudo-ginseng stem and leaf total saponin, 0.8 to 1.2 parts by mass of American ginseng root total saponin, 0.8 to 1.2 parts by mass of American ginseng stem and leaf total saponin and 0.8 to 1.2 parts by mass of pseudo-ginseng stem and leaf total saponin, and 0.8 to 1.2 parts by mass of pseudo-ginseng stem and leaf total saponin;

The ginseng traditional Chinese medicinal materials comprise at least one of the following raw materials: 0.8 to 1.2 parts by mass of American ginseng root, 1.6 to 2.4 parts by mass of American ginseng stem and leaf, 0.8 to 1.2 parts by mass of American ginseng root, 0.8 to 1.2 parts by mass of American ginseng stem and leaf, and 1.4 to 2.1 parts by mass of pseudo-ginseng stem and leaf, 0.8 to 1.2 parts by mass of ginseng stem and leaf, 1.2 to 1.8 parts by mass of American ginseng root, 0.8 to 1.2 parts by mass of American ginseng stem and leaf, and 2.4 to 3.6 parts by mass of pseudo-ginseng stem and leaf, 0.8 to 1.2 parts by mass of American ginseng root, 0.8 to 1.2 parts by mass of American ginseng stem and leaf, and 0.8 to 1.2 parts by mass of pseudo-ginseng stem and leaf, and leaf.

The invention provides the application of the specific active panaxadiol saponin composition or the specific active panaxadiol saponin composition prepared by the preparation method alone or in combination with a hypoglycemic agent in preparing medicines for preventing and/or treating one or more of diabetes, metabolic disorder related diseases, diabetic complications, angiogenesis disorder related diseases and delaying aging.

Preferably, the diabetes comprises type I diabetes and type II diabetes;

preferably, the medicament has the efficacy of improving and relieving metabolic disorder states and insulin resistance states in type I and type II diabetes mellitus states;

preferably, the metabolic disorder states in type I and type II diabetes include, but are not limited to: disorders of carbohydrate metabolism, disorders of lipid metabolism, disorders of protein metabolism in negative balance, disorders of creatine metabolism and redox imbalances (chronic oxidative stress state).

Preferably, the disease associated with metabolic disorders comprises at least one of the following diseases: metabolic syndrome, therapeutic metabolic disorders, polycystic ovary, psychotic disorders associated with metabolic disorders, neurodegenerative disorders with metabolic syndrome as a risk factor, and tumor therapeutic toxic side effects characterized by metabolic disorders and oxidative stress injury;

preferably, the metabolic syndrome comprises at least one of the following diseases: pre-diabetes, atherosclerosis, hyperlipidemia, high blood viscosity, high creatinine, and hypertension;

preferably, the therapeutic metabolic disorder comprises hyperglycemia or lipid metabolism disorder caused by at least one drug treatment of glucocorticoid drugs, immunosuppressant drugs or antipsychotic drugs;

Preferably, the metabolic disorder-associated psychotic disorder comprises schizophrenia, autism, anxiety, bipolar disorder, major depression, attention deficit/hyperactivity disorder and post-traumatic stress disorder;

preferably, the neurodegenerative disease with metabolic syndrome as a risk factor comprises at least one of the following diseases: amnestic mild cognitive impairment, senile dementia, vascular dementia and parkinsonism/symptoms characterized by cerebrovascular disorders;

preferably, the diabetic complications include at least one of the following complications: diabetic skin disease, diabetic neuropathy, difficulty in healing of medical operation or accidental trauma for diabetic patients, diabetic cardiovascular complications, diabetic nephropathy, diabetic eye disease, diabetic foot, diabetic male sexual dysfunction, and diabetic female reproductive dysfunction;

further preferably, the diabetic skin disease includes, but is not limited to: itch, eczema and skin ulcers;

preferably, the diabetic neuropathy includes, but is not limited to, the following conditions: diabetic autonomic neuropathy, sensory neuropathy, and diabetic central complications;

preferably, the diabetic cardiovascular complications include, but are not limited to: coronary heart disease, heart failure and diabetic cardiomyopathy;

Preferably, the diabetic eye disease includes, but is not limited to: retinopathy-related vision decline, pupil narrowing, cataracts, diabetic fundus hemorrhage, and diabetic macular edema.

Preferably, the decrease in male sexual function in diabetes includes, but is not limited to: hyposexuality, erectile dysfunction, ejaculatory dysfunction, and premature ejaculation;

preferably, the diabetic female reproductive dysfunction includes, but is not limited to: diabetic menstrual disorder, diabetic polycystic ovary syndrome, pregnancy failure of diabetic pregnant women and abortion.

The invention provides application of the specific active ginsenoside composition in preparing health products for improving one or more of insulin resistance state, diabetes metabolic disorder state and diabetes complication.

Preferably, the application is that the specific active ginsenoside composition is combined with at least one of nutrients, metabolism regulating substances and medicinal and edible traditional Chinese medicines to prepare health care products;

preferably, the nutrients include at least one of: proteins, amino acids, vitamins and metabolic intermediates;

preferably, the metabolic regulation substances include NAD precursor substances, coenzyme Q10, vitamin B1, vitamin B2 and vitamin B6.

The invention provides a medicament for preventing and treating diabetes, metabolic disorders of diabetes and diabetic complications, which comprises the specific active panaxadiol saponin composition or the specific active panaxadiol saponin composition prepared by the preparation method as a sole active ingredient or the specific active panaxadiol saponin composition and a hypoglycemic drug which are combined as active ingredients; the hypoglycemic agent comprises insulin or metformin.

Preferably, the medicament further comprises a pharmaceutically acceptable excipient or carrier;

preferably, the dosage form of the medicament comprises at least one of: oral formulations, injectable formulations and microneedles;

further preferably, the oral formulation includes solid formulations and liquid formulations, and the solid formulation forms include but are not limited to: tablet, dispersible tablet, granule, pill, controlled release preparation, pellet and capsule.

The invention provides a health care product for improving diabetes, diabetic metabolic disorder and diabetic complication, which comprises the specific active ginsenoside composition as the only active component or the specific active ginsenoside composition combined with at least one of nutrient, metabolic regulation substance and medicine-food homologous traditional Chinese medicine as the active component;

The hypoglycemic agent comprises insulin or metformin.

Preferably, the health product further comprises acceptable excipient or carrier;

preferably, the dosage form of the health care product comprises a solid preparation and/or a liquid preparation;

further preferred, the solid dosage forms include, but are not limited to: granule, capsule, tablet, dispersible tablet and pill.

The invention provides a panaxadiol saponin binary composition, which comprises a GRb1+ GRd functional unit and a GRc + GRb3 functional unit;

the mass ratio of GRb1 to GRd in the functional unit of GRb1+ GRd is 1.48-1.82;

the mass ratio of the GRc and the GRb3 of the GRc + GRb3 functional unit is 0.69-0.85.

The invention provides application of the specific active panaxadiol saponin composition or the panaxadiol saponin binary composition in preparing a product for repairing skin injury or/and health care.

Preferably, the skin lesions include lesions of: skin damage in healthy people, skin damage in diabetics and skin damage caused by face-lifting, cosmetic and surgical procedures; preferably, the product comprises a pharmaceutical, cosmeceutical, cosmetic or cosmetology product.

The invention provides a specific active panaxadiol saponin composition (SAPDSC, also called as GRb effective or effective composition) for preventing and treating diabetes and complications thereof, which comprises the following panaxadiol saponins: GRb1, GRc, GRb3 and GRd; the total mass percentage of GRc and GRb3 is more than the total mass percentage of GRb1 and GRd, and the difference between the total mass percentage of GRc and GRb3 and the total mass percentage of GRb1 and GRd is 4.55-21.30%; the ratio of the total mass of GRc and GRb3 to the total mass of GRb1 and GRd is 1.13-1.64; the mass ratio of GRb1 to GRd is 0.85-1.82; the mass ratio of GRc to GRb3 is 0.60-0.86; the mass ratio of GRb3 to GRb1 is 0.93-2.10; the mass ratio of GRc to GRb1 is 0.72-1.42. The invention determines the quality standard of the specific active panaxadiol saponin composition influencing the treatment effect of the diabetes and the complications thereof through experiments, which comprises the difference value of 'GRc + GRb 3' and 'GRb 1+ GRd' and the mass ratio of (GRc + GRb 3)/(GRb 1+ GRd), GRb1/GRd, GRc/GRb3, GRb3/GRb1, GRc/GRb1 and GRb1/GRc/GRb3/GRd is seven important parameters, and further defines the parameter range in which the prevention and treatment effect can be realized by the important parameters, thereby producing breakthrough progress on the drug effect of preventing and treating the diabetes and the systemic co-disease and the metabolic disorder related diseases thereof, and providing a new means for the treatment of the diabetes and the complications thereof.

The invention provides a preparation method of the specific active panaxadiol saponin composition, which comprises the following steps: dissolving the total ginsenoside in 30 percent ethanol water solution by volume percentage, separating by reversed phase C18 silica gel chromatography column chromatography, eluting with 27 to 33 percent ethanol water solution by volume percentage, eluting with 37 to 44 percent ethanol water solution by volume percentage, collecting the eluent, stopping eluting when the ginsenoside GRb1 appears in the component 27 to 33, eluting with 50 to 60 percent ethanol water solution, starting collecting the eluent until the eluent does not appear in the panaxadiol saponin GRd any more, combining the collected eluents, and concentrating and drying the combined eluents by a conventional method to obtain the specific active panaxadiol saponin composition; or mixing high content of GRb1, GRc, GRb3 and GRd extracts at a certain proportion to obtain specific active panaxadiol saponin composition; the total ginsenoside comprises commercial total ginsenoside or total ginsenoside extracted by using ginseng traditional Chinese medicinal materials as raw materials and adopting 30-80% ethanol water solution by volume percentage by using a common method; the commercialized total ginsenoside comprises two or more of radix Panacis Quinquefolii root total saponin, radix Panacis Quinquefolii stem and leaf total saponin, radix Ginseng root total saponin, radix Ginseng stem and leaf total saponin and Notoginseng radix stem and leaf total saponin; the Panax Chinese medicinal materials comprise two or more of radix Panacis Quinquefolii root, caulis Panacis Quinquefolii, radix Ginseng root, caulis Ginseng and folium Notoginseng. Compared with the conventional method, the method comprehensively utilizes the ginseng medicinal materials or the ginseng total saponins to prepare the active panaxadiol (SAPDSC), thereby generating breakthrough effect. (1) Compared with the method for preparing the specific active panaxadiol saponin composition by only using the American ginseng root total saponin and the American ginseng stem leaf total saponin, the method for preparing the specific active panaxadiol saponin composition (SAPDSC, also called as GRb effective or effective composition) by comprehensively using the ginseng and the stem leaf total saponin thereof, the American ginseng and the stem leaf total saponin thereof and the panax notoginseng stem leaf total saponin has obvious advantages on effectively utilizing limited resources, reducing the preparation cost and obtaining the GRb effective or effective composition. The concrete points are as follows: the GRb optimal effect or effective composition product meeting the product quality standard is obtained, the GRb1 and the GRd are removed as the cost of the GRb optimal effect or effective composition product, the GRb optimal effect or effective composition product is obtained, the GRb1 and the GRd are removed as the cost of the GRb1, and the GRc and the GRb3 which are removed and used for removing the high proportion of the panax notoginseng stem and leaf total saponins and are low in cost are skillfully utilized to offset the high GRb1 content in the panax quinquefolium root total saponins and the GRd content in the panax quinquefolium stem total saponins; the use amount of the American ginseng root can be further reduced by jointly using the ginseng stem and leaf total saponin, the American ginseng stem and leaf total saponin and the panax notoginseng stem and leaf total saponin, so that the limited medicinal material resources are further saved, and the preparation cost of the product is greatly reduced; more importantly, the total saponins of all the medicinal materials of the ginseng are comprehensively utilized, so that the product of the high-quality GRb effective composition with the same content and quality as the GRb-PQN4 effective composition is easier to prepare. (2) The SAPDSC is prepared by taking the ginseng traditional Chinese medicinal material combination as the raw material, although the steps of the preparation process are increased, the preparation cost is not obviously increased, and the interconnection and intercommunication among different varieties of the ginseng traditional Chinese medicinal materials and between stems, leaves and roots are further increased, so that the flexibility of comprehensively utilizing the resources is improved, the economic and medicinal values of the medicinal materials are further improved, the raw material cost of the GRb effective composition is reduced, and the limitation of resource shortage on the preparation of the GRb effective composition is overcome. In particular, the content allocation between the core active panaxadiol saponins of the target specific active panaxadiol saponin composition (sapdpsc) can be precisely achieved, meeting the requirements of the seven-parameter quality standards.

The invention provides an application of the specific active panaxadiol saponin composition or the specific active panaxadiol saponin composition prepared by the preparation method in preparing a medicament for preventing and/or treating one or more of diabetes, metabolic disorder of diabetes and diabetic complication. The invention discovers the function of the specific active panaxadiol saponin composition (SAPDSC) in the comprehensive prevention and treatment of diabetes and the diseases related to the systemic co-morbid diseases and the metabolic disorders thereof and discloses a novel co-action mechanism thereof, so that the specific active panaxadiol saponin composition (SAPDSC) generates breakthrough progress on the drug effect of the prevention and treatment of the diabetes and the diseases related to the systemic co-morbid diseases and the metabolic disorders thereof. The specific active panaxadiol saponin composition (SAPDSC) can be used for safely and effectively preventing and treating diabetes and comprehensively preventing and treating diabetes systemic complications (diabetic complications) by being used alone or combined with insulin or metformin or other hypoglycemic drugs with coordinated action mechanisms.

Drawings

FIG. 1 shows the effect of the use of a panaxadiol saponin composition GRb-PQ1 or GRb-PQ2 in combination with insulin on the urinary output of type I diabetic mice.

FIG. 2 shows the effect of the combination of the panaxadiol saponin composition GRb-PQ1 or GRb-PQ2 and insulin on blood sugar level of type I diabetic mice.

FIG. 3 shows the effect of the panaxadiol saponin composition GRb-PQ1 or GRb-PQ2 in combination with insulin on the skin damage repair of type I diabetic mice. I, normal control group; II: model group: III: a group of insulins; IV: insulin + GRb-PQ1 (10) group; v: insulin + GRb-PQ1 (20) group; VI: insulin + GRb-PQ2 (10) group; VII: insulin + GRb-PQ2 (20) group. All data are expressed as mean ± SD; evaluating statistical significance difference by using t test and one-way analysis of variance (ANOVA), and performing significance analysis of difference between multiple groups of samples by Duncan multi-range test; compared with a normal control group: * p is a radical of<0.05; and (3) comparing with the model group: ^# p<0.05， ^## p<0.01；n＝5。

FIG. 4 shows the effect of the panaxadiol saponin compositions GRb-PQN1, GRb-PQN3, GRb-PQN4 and GRb-PQ2 in prolonging the hypoglycemic time of insulin. I, normal control group; II: a model group; III: a group of insulins; IV: insulin + GRb-PQN3 group; v: insulin + GRb-PQN4 group; VI: insulin + GRb-PQN4-1 group; VII: insulin + GRb-PQN1 group; VIII: insulin + GRb-PQ2 group. All data are expressed as mean ± SD; evaluating statistical significance difference by using t test and one-way analysis of variance (ANOVA), and performing difference significance analysis among multiple groups of samples by Duncan multi-range test; compared with a normal control group: * P <0.01; and (3) comparing with the model group: ^## p<0.01；n＝5。

FIG. 5 shows that the panaxadiol saponins GRb composition and insulin are combined to promote the effect of repairing the skin damage of the diabetic mouse. I, normal control group; II: a model group; III: a group of insulins; IV: insulin + GRb-PQN3 group (Whole treatment); v:insulin + GRb-PQN4 group (Whole treatment); VI: insulin + GRb-PQN4-1 group (3 weeks after treatment); VII: insulin + GRb-PQN1 group (3 weeks after treatment); VIII: insulin + GRb-PQ2 group (3 weeks after treatment). All data are expressed as mean ± SD; evaluating statistical significance difference by using t test and one-way analysis of variance (ANOVA), and performing significance analysis of difference between multiple groups of samples by Duncan multi-range test; compared with a normal control group: * p is a radical of<0.05; and (3) comparing with the model group: ^# p<0.05; in the insulin group ratio: ^& p<0.05；n＝5。

FIG. 6 shows the effect of the panaxadiol saponin composition GRb-PQN4 in reducing the urine output of diabetic mice in combination with insulin.

FIG. 7 shows that the panaxadiol saponin composition GRb-PQN4 and its combination with insulin can improve blood sugar fluctuation after insulin administration and avoid drug resistance caused by multiple times of insulin administration. All data are expressed as mean ± SD, n =5.

FIG. 8. Effect of Panaxadiol saponin composition GRb-PQN4 and its combination with insulin on plantar and caudal heat sensitivity in mice. All data are expressed as mean ± SD; evaluating statistical significance difference by using t test and one-way analysis of variance (ANOVA), and performing significance analysis of difference between multiple groups of samples by Duncan multi-range test; compared with a normal control group: * p is a radical of <0.05; and (3) comparing with the model group: ^# p<0.05; in the insulin group ratio: ^& p<0.05；n＝5。

FIG. 9 shows that the panaxadiol saponin composition GRb-PQN4 alone or in combination with insulin can significantly improve diabetic mouse skin rash. All data are expressed as mean ± SD; evaluating statistical significance difference by using t test and one-way analysis of variance (ANOVA), and performing difference significance analysis among multiple groups of samples by Duncan multi-range test; compared with a normal control group: * p is a radical of<0.05; and (3) comparing with the model group: ^# p<0.05; in the insulin group ratio: ^& p<0.05；n＝5。

FIG. 10 shows that the panaxadiol saponin composition GRb-PQN4 alone or in combination with insulin can significantly promote the repair of type I diabetic mouse skin damage. All data are expressed as mean ± SD; evaluating statistical significance difference by using t test and one-way analysis of variance (ANOVA), and performing significance analysis of difference between multiple groups of samples by Duncan multi-range test; compared with a normal control group: * p <0.05; and (3) comparing with the model group: # p <0.05; n =5.

Figure 11. Panaxadiol saponin composition GRb-PQN4 alone or in combination with insulin significantly protected the heart of type I diabetic mice. All data are expressed as mean ± SD; evaluating statistical significance difference by using t test and one-way analysis of variance (ANOVA), and performing significance analysis of difference between multiple groups of samples by Duncan multi-range test; compared with a normal control group: * P <0.01; and (3) comparing with the model group: ^# p<0.05; in the insulin group ratio: ^& p<0.05；n＝5。

figure 12 panaxadiol saponin composition GRb-PQN4 can significantly improve type II diabetic mouse skin rash and prevent dermatitis induced by metformin or dapagliflozin treatment. All data are expressed as mean ± SD; evaluating statistical significance difference by using t test and one-way analysis of variance (ANOVA), and performing difference significance analysis among multiple groups of samples by Duncan multi-range test; compared with a normal control group: * p is a radical of<0.05; and (3) comparing with the model group: ^# p<0.05；n＝5。

FIG. 13 shows that the panaxadiol saponin composition GRb-PQN4 alone or in combination with metformin can remarkably promote the repair of type II diabetic mouse skin damage. All data are expressed as mean ± SD; evaluating statistical significance difference by using t test and one-way analysis of variance (ANOVA), and performing difference significance analysis among multiple groups of samples by Duncan multi-range test; compared with a normal control group: * p is a radical of<0.05; and (3) comparing with the model group: ^# p<0.05；n＝5。

FIG. 14 shows that the combination of the panaxadiol saponin composition GRb-PQN4 and metformin or dapagliflozin can protect cardiac myocytes and cardiac vessels of diabetic mice [ hematoxylin-eosin (HE) staining ].

FIG. 15 combination of Panaxadiol saponin composition GRb-PQN4 with metformin or dapagliflozin protects cardiomyocytes and cardiovascular staining in diabetic mice [ Masson (MS) ] ]. All data are expressed as mean ± SD; statistical significance differences were assessed using t-test and one-way analysis of variance (ANOVA), by Duncan multiple range testAnalyzing the significance of the difference among a plurality of groups of samples; compared with a normal control group: * P<0.01; and (3) comparing with the model group: ^# p<0.05；n＝5。

Detailed Description

The invention provides a specific active panaxadiol saponin composition for preventing and treating diabetes and complications thereof, which comprises the following panaxadiol saponins: GRb1, GRc, GRb3 and GRd; the total mass percentage of GRc and GRb3 is more than the total mass percentage of GRb1 and GRd, and the difference between the total mass percentage of GRc and GRb3 and the total mass percentage of GRb1 and GRd is 5.06-19.36%; the ratio of the total mass of GRc and GRb3 to the total mass of GRb1 and GRd is preferably 1.13 to 1.64. The mass ratio of GRb1 to GRd is 0.94-1.65; the mass ratio of GRc to GRb3 is 0.66-0.78; the mass ratio of GRb3 to GRb1 is 1.03-1.91; the mass ratio of GRc to GRb1 is 0.80 to 1.29, and the upper and lower limits of the range of the difference are allowed to fluctuate by 10%.

In the present invention, the mass ratio of GRb1, GRc, GRb3 and GRd preferably includes, but is not limited to: 0.96-1.18: 0.9 to 1.1:1.37 to 1.67: 1.02-1.24, 0.69-0.85: 0.9 to 1.1:1.33 to 1.63: 0.66-0.80, 1.13-1.38: 0.9 to 1.1:1.16 to 1.42: 0.70-0.86: 0.99 to 1.21:0.9 to 1.1:1.17 to 1.43:0.59 to 0.73; the mass ratio of GRb1, GRc, GRb3 and GRd is more preferably 1.07:1.00:1.52:1.13, 0.77:1.00:1.48:0.73, 1.25:1.00:1.29:0.78 or 1.10:1.00:1.30:0.66, most preferably 1.10:1.00:1.30:0.66. in the embodiment of the invention, 4 compositions are obtained by screening, wherein the mass percentage content difference of (GRc + GRb 3) and (GRb 1+ GRd) of GRb-PQN1 is 19.36%, the mass ratio of (GRc + GRb 3) and (GRb 1+ GRd) is 1.64, the mass ratio of GRb1/GRd is 1.06, the mass ratio of GRc/GRb3 is 0.68, the mass ratio of GRb3/GRb1 is 1.91, the mass ratio of GRc/GRb1 is 1.29, the mass ratio of GRb1, GRc, GRb2, GRb3 and GRd is 0.77:1.00:0.79:1.48:0.73. the difference between the mass percentages of (GRc + GRb 3) and (GRb 1+ GRd) of GRb-PQN3 is 5.15%, the mass ratio of (GRc + GRb 3) to (GRb 1+ GRd) is 1.13, the mass ratio of GRb1/GRd (mass ratio) is 1.60, the mass ratio of GRc/GRb3 is 0.78, the mass ratio of GRb3/GRb1 is 1.03, the mass ratio of GRc/GRb1 is 0.80, the mass ratio of GRb1, GRc, GRb2, GRb3 and GRd is 1.25:1.00:0.34:1.29:0.78. the difference between (GRc + GRb 3) and (GRb 1+ GRd) in mass% of GR-PQN4 was 11.49%, (GRc + GRb 3) and (GRb 1+ GRd) in mass ratio of 1.31, GRb1/GRd (mass ratio) of 1.65, GRc/GRb3 of 0.77, GRb3/GRb1 of 1.19, GRc/GRb1 of 0.91, GRb1, GRc, GRb2, GRb3 and GRd in mass ratio of 1.10:1.00:0.32:1.30:0.66. the difference between the mass percentages of (GRc + GRb 3) and (GRb 1+ GRd) of GRb-PQ2 is 5.06%, (GRc + GRb 3) and (GRb 1+ GRd) is 1.14, GRb1/GRd (mass ratio) is 0.94, GRc/GRb3 is 0.66, GRb3/GRb1 is 1.42, GRc/GRb1 is 0.94, GRb1, GRc, GRb2, GRb3 and GRd is 1.07:1.00:1.11:1.52 and 1.13.

In the present invention, the specific active panaxadiol saponin composition preferably further comprises GRb2. The mass ratio of GRb2 to GRc is preferably (0.32 to 1.11): 1.00, the proportion comprises the natural existing proportion of the whole plants of ginseng and American ginseng and the stems and leaves of panax notoginseng, and the content of GRb2 and the relative proportion of the GRb2 and other panaxadiol saponins do not influence the drug effect of the composition, so the content of GRb2 in the composition can be present according to the natural content of raw materials.

In the invention, the structural formulas of GRb1, GRc, GRb2, GRb3 and GRd are shown in structural formula I;

wherein GRb1 represents ginsenoside Rb1 (R = β -D-glucopyranosyl), GRc represents ginsenoside Rc (R = α -L-arabinofuranosyl), GRb2 represents ginsenoside Rb2 (R = α -L-arabinopyranosyl), GRb3 represents ginsenoside Rb3 (R = β -D-xylopyranosyl), and GRd represents ginsenoside Rd (R = H).

dissolving the total ginsenoside in 30 volume percent ethanol water solution, separating by reversed phase C18 silica gel chromatography column chromatography, eluting with 27-33 volume percent ethanol water solution, eluting with 37-44 volume percent ethanol water solution, collecting eluent, eluting with 50-60 volume percent ethanol water solution, collecting eluent, stopping eluting when ginsenoside GRb1 is monitored in the eluent, merging the eluent components containing GRb1, GRc, GRb3 and GRd, concentrating under reduced pressure until complete drying to obtain the specific active panaxadiol saponin composition, wherein the total content of the panaxadiol saponins is over 75 percent, and the total content of the 4 core active panaxadiol saponins (GRb 1, GRc, GRb3 and GRd) is over 65 percent.

The preparation method of the specific active panaxadiol saponin composition is also preferably obtained by the following steps:

dissolving the ginseng total saponin in 30% ethanol aqueous solution by volume percentage, combining online analysis, separating by reversed phase C18 silica gel chromatography column chromatography, eluting with 30% ethanol aqueous solution by volume percentage, eluting with 40% ethanol aqueous solution and collecting eluent until the amount of GRb1 or GRb1 in the eluent is not extremely low, eluting with 43% ethanol aqueous solution and collecting eluent until GRc or GRc in the eluent is not extremely low, eluting with 47% ethanol aqueous solution and collecting eluent until GRb3 or GRb3 in the eluent is not extremely low, eluting with 55% ethanol aqueous solution and collecting eluent until GRd or GRd in the eluent is extremely low, finally respectively merging the eluents mainly containing GRb1, GRc, GRb3 and GRd, concentrating by a common method to be completely dried, and respectively obtaining four extracts of high-content GRb1, GRc, GRb3 and GRd; finally, the specific active panaxadiol saponins composition is obtained by quantitatively mixing the four extracts of GRb1, GRc, GRb3 and GRd according to the GRb1/GRc/GRb3/GRd ratio of claim 1, wherein the total content of panaxadiol saponins is over 75%, and the total content of the 4 core active panaxadiol saponins (GRb 1, GRc, GRb3 and GRd) is over 65%.

The total ginsenoside is obtained by mixing two or more of commercialized radix Panacis Quinquefolii root total saponin, radix Panacis Quinquefolii stem leaf total saponin, radix Ginseng root total saponin, radix Ginseng stem leaf total saponin, radix Notoginseng root total saponin, and radix Notoginseng stem leaf total saponin; or the self-made mixed ginseng total saponin is obtained by extracting two or more than two mixed medicinal materials of American ginseng root, american ginseng stem leaf, ginseng root, ginseng stem leaf and pseudo-ginseng stem leaf which are Chinese medicinal materials of ginseng by using an ethanol aqueous solution with the volume percentage of 30-80% by a conventional method, and removing a solvent.

In the present invention, the total ginsenosides preferably include at least one of the following compositions: the total saponin composition comprises, by mass, 0.8-1.2 parts of American ginseng root total saponin and 1.6-2.4 parts of American ginseng stem and leaf total saponin, 0.8-1.2 parts of American ginseng root total saponin, 0.8-1.2 parts of American ginseng stem and leaf total saponin and 1.4-2.1 parts of panax notoginseng stem and leaf total saponin, 0.8-1.2 parts of ginseng stem and leaf total saponin, 1.2-1.8 parts of American ginseng root total saponin, 0.8-1.2 parts of American ginseng stem and leaf total saponin and 2.4-3.6 parts of panax notoginseng leaf total saponin, 0.8-1.2 parts of American ginseng root total saponin, 0.8-1.2 parts of American ginseng stem and leaf total saponin, and 1.8-1.2 parts of panax notoginseng saponin, and 1.5 parts of panax notoginseng stem and leaf total saponin, and 1.5 parts of panax notoginseng root total saponin, 1.8-1.5 parts of panax notoginseng stem and 1.5 parts of panax notoginseng leaf total saponin, and 1.5 parts of panax notoginseng leaf total saponin. The sources of the American ginseng root total saponin, the American ginseng stem and leaf total saponin, the ginseng root total saponin, the ginseng stem and leaf total saponin and the panax notoginseng stem and leaf total saponin are not specially limited, and the panax ginseng root total saponin, the American ginseng stem and leaf total saponin and the panax notoginseng stem and leaf total saponin which are well known in the field can be used.

In the invention, according to the contents of GRb1, GRc, GRb3 and GRd in the ginseng total saponin raw materials from ginseng roots, ginseng stems and leaves, american ginseng roots, american ginseng stems and leaves and pseudo-ginseng stems and leaves and the complementary characteristics of the contents of the four types of saponins, the ginseng total saponin composition is prepared by taking mixed medicinal materials of more than two ginseng Chinese medicinal materials as raw materials and determining the feeding parts (proportion) of the raw materials through simple calculation and mixing and extracting. The mixed medicinal material of the ginseng traditional Chinese medicinal materials preferably comprises at least one of the following raw materials: the raw material comprises, by mass, 0.8-1.2 parts of American ginseng root, 1.6-2.4 parts of American ginseng stem and leaf, 0.8-1.2 parts of American ginseng root, 0.8-1.2 parts of American ginseng stem and leaf, and 1.4-2.1 parts of pseudo-ginseng stem and leaf, 0.8-1.2 parts of ginseng stem and leaf, 1.2-1.8 parts of American ginseng root, 0.8-1.2 parts of American ginseng stem and leaf, and 2.4-3.6 parts of pseudo-ginseng stem and leaf, 0.8-1.2 parts of American ginseng root, 0.8-1.2 parts of American ginseng stem and leaf, and 0.8-1.2 parts of pseudo-ginseng stem and leaf, and 0.8-1.2 parts of American ginseng stem and leaf, and 1.5 parts of pseudo-ginseng stem and leaf, more preferably, 1.8-1 part of American ginseng stem and leaf, 1.5 parts of pseudo-ginseng stem and leaf, 1.5 parts of American ginseng stem and leaf. When the mixed medicinal materials of the ginseng traditional Chinese medicinal materials are taken as raw materials and extracted by adopting an ethanol water solution with the volume percentage of 30-80%, the material-liquid ratio of the ginseng traditional Chinese medicinal materials to the ethanol water solution is preferably 1g:10 to 15mL, more preferably 1g:12mL. Preferably, the solvent is removed from the extracted Panax total saponins to obtain Panax total saponins powder derived from Panax mixed medicinal material. During sampling, the ginseng total saponin powder is preferably dissolved by adopting an ethanol water solution with the volume percentage of 30 percent, and the material-liquid ratio of the ginseng total saponin powder to the ethanol water solution with the volume percentage of 30 percent is 1g:8 to 10, more preferably 1g:9mL. Before the reversed-phase C18 silica gel chromatographic column chromatography separation, the reversed-phase C18 silica gel chromatographic column chromatography separation is preferably performed by adopting an ethanol water solution with the volume percentage of 30 percent for pre-equilibrium, wherein the volume of the ethanol water solution with the volume percentage of 30 percent is 8-10 times, and more preferably 9 times of that of the reversed-phase C18 silica gel. The mass of the C18 silica gel used for chromatographic column separation is 7-10 times, and more preferably 9 times of the mass of the total ginsenoside. The volume of the elution with the ethanol aqueous solution with the volume percentage content of 30 percent is 50 to 70 times, and more preferably 60 times of the volume of the reversed phase C18 silica gel. After collecting the eluate, the types of ginsenosides in the fractions are preferably analyzed by HPLC until the elution with 43% aqueous ethanol is stopped, preferably until ginsenoside GRb1 appears in the fractions, and then the elution is continued with 55% aqueous ethanol, and 55% ethanol eluate is collected to obtain the fractions, and the fractions are analyzed by HPLC until the elution with 55% aqueous ethanol is stopped when ginsenoside GRd does not appear in the fractions. Combining the collected components including GRb1, GRc, GRb2, GRb3 and GRd, and removing ethanol and water to obtain specific active panaxadiol saponin composition (SAPDSC). The respective contents of five panaxadiol saponins (GRb 1, GRc, GRb2, GRb3 and GRd) in the specific active panaxadiol saponin composition (SAPDSC) determined by HPLC method meet 7-parameter quality standard.

The invention provides a specific active ginseng total saponin composition, which is prepared by mixing two or more than two of commercialized American ginseng root total saponin, american ginseng stem and leaf total saponin, ginseng root total saponin, ginseng stem and leaf total saponin and panax notoginseng stem and leaf total saponin; or the mixed medicinal materials of more than two of panax quinquefolius roots, panax quinquefolius stems and leaves, ginseng roots, ginseng stems and leaves and panax notoginseng stems and leaves in the panax ginseng are taken as raw materials, ethanol water solution with the volume percentage content of 30-80% is adopted for extraction by a conventional method, and a solvent is removed to obtain the self-made specific active ginseng total saponin composition, wherein the total content of five types of panaxadiol saponins (GRb 1, GRc, GRb2, GRb3 and GRd) is 40%, and the total content of the 4 types of core active panaxadiol saponins (GRb 1, GRc, GRb3 and GRd) is more than 35%.

In the present invention, the specific active ginsenoside composition preferably comprises at least one of the following compositions compounded by commercial or homemade ginsenosides: the composition comprises 1 part by mass of American ginseng root total saponin and 2 parts by mass of American ginseng stem leaf total saponin, 1 part by mass of American ginseng root total saponin, 1 part by mass of American ginseng stem leaf total saponin and 1.75 parts by mass of panax notoginseng stem leaf total saponin, 1 part by mass of ginseng stem leaf total saponin, 1.5 parts by mass of American ginseng root total saponin, 1 part by mass of American ginseng stem leaf total saponin and 3 parts by mass of panax notoginseng stem leaf total saponin, 1 part by mass of American ginseng root total saponin, 1 part by mass of American ginseng stem leaf total saponin and 1 part by mass of panax notoginseng stem leaf total saponin, and a composition comprising 1 part by mass of American ginseng root total saponin, 1 part by mass of American ginseng stem leaf total saponin and 1.5 parts by mass of panax notoginseng stem leaf total saponin. The composition formed by adopting the mixture ratio contains five panaxadiol saponins (GRb 1, GRc, GRb2, GRb3 and GRd) which are determined by an HPLC method, and the content ratio of each panaxadiol saponin meets the quality standard of 7 parameters.

In the invention, the mixed medicinal materials of the ginseng traditional Chinese medicinal materials preferably comprise at least one of the following raw materials: the raw materials comprise, by mass, 1 part of American ginseng root and 2 parts of American ginseng stem leaf, 1 part of American ginseng root, 1 part of American ginseng stem leaf and 1.75 parts of pseudo-ginseng stem leaf, 1 part of ginseng stem leaf, 1.5 parts of American ginseng root, 1 part of American ginseng stem leaf and 3 parts of pseudo-ginseng stem leaf, and the raw materials comprise 1 part of American ginseng root, 1 part of American ginseng stem leaf and 1 part of pseudo-ginseng stem leaf, and the raw materials comprise 1 part of American ginseng root, 1 part of American ginseng stem leaf and 1.5 parts of pseudo-ginseng stem leaf. When the ginseng traditional Chinese medicinal materials are used as raw materials and extracted by ethanol aqueous solution with the volume percentage of 30-80%, the material-liquid ratio of the ginseng traditional Chinese medicinal materials to the ethanol aqueous solution is preferably 1g:10 to 15mL, more preferably 1g:12mL. Preferably, the solvent is removed from the extracted Panax total saponins to obtain Panax total saponins powder. The active ginsenoside composition prepared by extracting the raw materials formed by the proportion of the ginseng traditional Chinese medicinal materials contains five types of panaxadiol saponins (GRb 1, GRc, GRb2, GRb3 and GRd) through HPLC method determination, and the content proportion of each panaxadiol saponin meets the quality standard of 7 parameters.

Aiming at the technical problem that no medicine for effectively preventing and treating diabetes exists at present, the invention provides application of the specific active panaxadiol saponin composition (SAPDSC) or the specific active panaxadiol saponin composition prepared by the preparation method in preparing a medicine for preventing and/or treating one or more of diabetes, metabolic disorders of diabetes and complications of diabetes.

In the present invention, the diabetes preferably includes type I diabetes and type II diabetes; the medicament preferably has a pharmacological effect of slowing the insulin resistance state in type I and type II diabetic conditions.

In the present invention, the diabetes preferably includes type I diabetes and type II diabetes; the medicine has the effect of slowing down the insulin resistance state in type I and type II diabetes. The hypoglycemic agent comprises insulin or metformin.

In the invention, the specific active panaxadiol saponin composition (SAPDSC) can be used for preventing and treating diabetes and comprehensively treating diabetic metabolic disorder syndrome and various complications from the root of a disease, supports the application of the SAPDSC alone or in combination with insulin or metformin or other medicaments with coordinated action mechanisms to improve the health state of a diabetic patient, comprehensively treating the diabetic metabolic disorder syndrome and preventing and treating various complications of the diabetes, and also supports the application of the SAPDSC alone or in combination with metformin or other medicaments with coordinated action mechanisms to treat pre-diabetes so as to prevent the diabetes. Further, based on the same disease root or common pathological mechanism, SAPDSC can be used for preventing and treating other metabolic syndromes (including but not limited to atherosclerosis, hyperlipidemia, high blood viscosity, high creatinine and hypertension) by alone or in combination with insulin or metformin or other medicaments with coordinated action mechanisms, therapeutic metabolic disorders (including but not limited to hyperglycemia or lipid metabolism disorder caused by glucocorticoid medicaments, immunosuppressants or antipsychotic medicaments), metabolic disorder-related diseases (including but not limited to polycystic ovary and schizophrenia), neurodegenerative diseases with metabolic syndrome as risk factors (including but not limited to senile dementia, vascular dementia and Parkinson's syndrome) and toxic and side effects of tumor treatment characterized by metabolic disorders and/or oxidative stress injuries. The application of the SAPDSC in promoting the wound healing of the diabetic patients comprises the application of the SAPDSC in promoting the tissue and cut skin repair after medical operation, and also supports the application of the SAPDSC in promoting the skin health and promoting the skin repair after cosmetic operation as cosmeceutical.

In the present invention, the present invention also provides a panaxadiol saponin composition for skin injury repair comprising a "GRb1+ GRd" functional unit or a "GRc + GRb3" functional unit; the mass ratio of GRb1 to GRd in the functional unit of GRb1+ GRd is 1.48-1.82; the mass ratio of the GRc and GRb3 of the GRc + GRb3 functional unit is 0.69-0.85. The panaxadiol saponin composition is applied to preparation of products for repairing skin injury. The skin damage preferably comprises skin damage of healthy people or skin damage of diabetics; the product comprises a pharmaceutical or cosmetic product. The SAPDSC is composed of a functional unit GRb1+ GRd and a functional unit GRc + GRb3, the drug effect of the two functional units for independently promoting the skin damage repair of diabetic mice is equivalent to the drug effect strength of the full-composition SAPDSC, and the drug effect of the Specific Active Ginsenoside Composition (SAGC) for promoting the skin damage repair in a normal or diabetic state is also equivalent to the drug effect strength of the SAPDSC. These findings support the relative use of specific panaxadiol saponin active compositions (sapmdsc), their functional units "GRb1+ GRd" and "GRc + GRb3" and specific active total ginsenoside compositions (SAGC) containing two or one of the functional units as core active ingredients for promoting repair of skin lesions in normal or diseased states, including as active ingredients in general skin care products, as active ingredients in cosmeceutical skin care products, and as active ingredients in repair drugs for promoting cosmetic or medical surgery.

In the invention, the effect that the specific active panaxadiol saponin composition (SAPDSC, also called as GRb effective or effective composition) can comprehensively prevent and treat diabetes and the diseases related to the systemic co-morbid and metabolic disorder of the diabetes is disclosed, a novel common action mechanism of the composition is disclosed, and breakthrough progress is generated on the drug effect of preventing and treating the diabetes and the diseases related to the systemic co-morbid and metabolic disorder of the diabetes.

(one) specific active Panaxadiol saponin composition (SAPDSC)/GRb

The GRb effective or effective composition, particularly the combination of the GRb effective or effective composition and insulin or metformin or other hypoglycemic agents with coordinated action mechanisms, can safely and effectively prevent and treat diabetes and comprehensively prevent and treat diabetes systemic complications (diabetic complications).

Control of blood glucose levels has long been a central element in the treatment of diabetes, however, even effective control of blood glucose is not effective in the prevention and treatment of diabetes and its complications. In agreement, we also observed in this invention that the classic first line drug metformin and the novel first line drug dapagliflozin of type II diabetes, while effective in reducing glycosylated hemoglobin levels, i.e., lowering blood glucose levels, are not effective in alleviating the common symptoms in the early stages of diabetes, disease progression and degenerative complications including heart disease and diabetic foot, and that both metformin and dapagliflozin exacerbate the rash of the diabetic condition. In addition, it was observed that the early effects of insulin, metformin and gliflozin were not exerted continuously, but the effects gradually disappeared while the disease progressed as the course of the disease was prolonged, suggesting that controlling blood glucose levels was not effective in delaying the progression of the disease.

In sharp contrast to these hypoglycemic agents, effective or efficacious GRb compositions without acute and chronic hypoglycemic effects can significantly ameliorate diabetic complications, including: (1) Improving the early typical symptoms of type I diabetes (much eating and drinking symptoms, and the drug effect is numerically better than that of insulin), slowing down the peripheral neuropathy of diabetes (including hypersensitivity of nerve ending sensation and then passivation process and vegetative nerve hypofunction taking gastric emptying as an index, and the drug effect is numerically better than that of insulin), and relieving the rash in the diabetic state (equivalent to that of insulin); (2) Improving type II diabetes-associated skin rash and preventing dermatitis induced by metformin or dapagliflozin treatment; (3) In type I and type II diabetes models, the repair of large-area skin lesions (simulating diabetic feet) is greatly promoted, and the occurrence of heart diseases (including myocardial and macrovascular diseases and inflammatory cell infiltration of heart tissues) is completely prevented; (4) In particular, in type I and type II diabetes models, the GRb effective or efficacious compositions in combination with insulin or metformin completely prevent all selected considered diabetic complications including diabetic nephropathy and almost block disease progression, showing a powerful drug effect of both synergistically treating diabetes and its complications; (5) The combination of the GRb effective or effective composition and the dapagliflozin has no synergistic effect, but the drug effect of the GRb effective or effective composition is obviously weakened by the dapagliflozin, so that the GRb effective or effective composition can only play an excellent drug effect of preventing and treating diabetes and complications thereof by combining with a hypoglycemic drug with a coordinated or complementary action mechanism; (6) The efficacy of the functional unit GRb1+ GRd or the functional unit GRc + GRb3 of the GRb effective or effective composition for independently promoting the skin damage repair of the diabetic mice is equivalent to the efficacy of the whole composition, and the efficacy of the specific active ginsenoside composition taking the GRb effective or effective composition as a core active ingredient for promoting the skin damage repair in a normal or diabetic state is also equivalent to the efficacy of the GRb effective or effective composition.

(II) specific active Panaxadiol saponin compositions (SAPDSC)/GRb

Our findings demonstrate that GRb-PQN4 can protect or enhance/activate the cell rescue mechanism in diabetic states, including antioxidant capacity (maintaining NADPH and GSH content, SOD and catalase activity at or near normal levels and even compensatory increases beyond normal levels and avoids ROS accumulation and accumulation of macromolecular oxidative stress products and oxidative stress-mediated mitochondrial damage and inflammatory responses) and Ang 1-mediated metabolic regulation (thus compensating for insufficient PI3K-AKT pathway signaling associated with insulin resistance) and anti-inflammatory effects as well as vascular protection, functional homeostasis and neogenesis; GRb-PQN4 also enhances coupling of glycolysis to mitochondrial oxidative metabolism (facilitating the flow of sugar metabolic intermediates to mitochondrial oxidative metabolism), thus effectively fighting ATP deficiency and accumulation of advanced glycosylation products (AGE) in the diabetic state (and thus also fighting AGE-mediated inflammatory responses and insulin resistance), and slowing the inflammatory state of the circulatory system in the diabetic state. In particular, these pharmacological effects of GRb-PQN4 can be further enhanced by the combination of insulin or metformin, allowing various pathological or physiological indices to be maintained in the normal or near-normal range, and the levels or activities of various functional molecules in the antioxidant system and Ang1 levels to be compensated for and increased beyond normal resting states. It is clear that GRb-PQN4, particularly in combination with insulin or metformin, can exert a powerful role in protecting and repairing the structure and function of the vascular system and nerves of diabetic patients by protecting and enhancing/activating the cellular self-rescue mechanisms and correcting metabolic disorders in the diabetic state and avoid ATP deficiency, mitochondrial dysfunction and oxidative stress-related hypofunction or dysfunction of the body. Therefore, GRb-PQN4, particularly in combination with insulin or metformin, can prevent and treat diabetes and its systemic complications, i.e., diabetic complications, and can comprehensively improve physical performance and quality of life of patients, and also prevent and treat disorders associated with glucose metabolism disorders as well as disorders and sub-health states characterized by mitochondrial dysfunction, oxidative stress and chronic inflammation. The results of the study also support the related application of Specific Active Ginsenoside Composition (SAGC) with GRb active composition as the active center.

A novel mechanism for the systemic treatment of diabetes mellitus from the disease root in a non-hypoglycemic form by an effective or efficacious GRb composition.

1.1. A superior composition GRb-PQN4, although not capable of lowering blood glucose levels in type I and type II diabetic conditions, can significantly prevent and correct the core pathological and characteristic events of diabetes in diabetic complication sensitive tissues and organs and circulating blood, including the loss of energy (ATP), reduced coenzyme I (NADH), the antioxidant substances Glutathione (GSH), superoxide dismutase (SOD) and catalase, and angiopoietin-1 (Ang-1); accumulation of reactive oxygen radicals (ROS, superoxide anions and hydrogen peroxide) and advanced glycosylation products (AGEs); oxidative stress damage to DNA and lipids; proinflammatory factors (TNF-alpha,IL1 β and IL 6); in particular, ATP, AGEs and Ang-1 are maintained at physiological levels in the blood and diabetic vulnerable organs, and systematically protect and even enhance cellular antioxidant capacity in type I and type II diabetic conditions, including maintenance of physiological or near-physiological NAD ⁺ /NADH、NADP ⁺ NADPH and GSSG/GSH ratios and GSH levels, the amount of coenzyme I (NAD) that is compensated for and exceeds normal ⁺ + NADH) (type II diabetes) and SOD levels, thus avoiding ROS accumulation and its oxidative damage to biological macromolecules, as well as downstream pathological events triggered by ROS accumulation including AGEs accumulation and chronic inflammatory states (also known as metabolic inflammation). In addition, the advantageous composition GRb-PQN4 also slows excessive creatine degradation in diabetic conditions, which also helps to maintain cardiac ATP levels in homeostasis and relieves the kidney of the burden of creatinine clearance. It is pointed out here that it is now clear that, in the diabetic state, the cellular disorders associated with the oxidative damage of biomacromolecules (deoxyribonucleic acid, proteins and lipids) by the accumulation of ROS are the upstream pathological events in the development of diabetes and its complications, in which a decrease in glyceraldehyde-3-phosphate dehydrogenase (GAPDH/G3 PDH) activity leads to a large deviation of the glycolytic intermediates glyceraldehyde-3-phosphate and dihydroxyacetone phosphate from the energy metabolic pathway towards Methylglyoxal (MGO); detoxification of large amounts of MGO by cells consumes GSH, while overloaded MGO glycosylates phospholipids, nucleotides, and functional proteins to form AGEs, thus further exacerbating oxidative stress damage and indirectly extending the range of ROS damage to biomacromolecules, the range of cellular function reduction/dysfunction, the range of associated pathological events, including redox imbalance, oxidative stress state, mitochondrial dysfunction, cellular energy deficiency/loss, endoplasmic reticulum oxidative stress, inflammatory pathway activation, and insulin resistance. It is also to be noted that coenzyme I (NAD) ⁺ + NADH) and NAD thereof ⁺ the/NADH ratio is central to the regulation of sugar metabolism, so that the diabetes mellitus is characterized by NADH depletion and concomitant NAD ⁺ The increased/NADH ratio must lead to a corresponding dysfunction of the sugar metabolism in glycolytic pathways, the tricarboxylic acid cycle and mitochondrial electron transport and finally to an insufficient production of ATP, indeed in models of type I and type II diabetes of the heart, kidney and skinSevere loss of ATP levels occurs, so that physiological or near-physiological NAD is maintained ⁺ the/NADH ratio, compensatory increases in NADH levels, from an important perspective, reveal the biochemical mechanism by which effective or efficacious compositions of GRb improve the intracellular carbohydrate metabolism state in diabetic conditions. In conclusion, it is clear from our research results that in the diabetic state, glycolysis is decoupled from sugar oxidation metabolism, leading to a large amount of glycolysis intermediates deviating from energy metabolism pathway to toxic MGO and AGEs and leading to ATP deficiency as a cellular energy substance, thereby triggering various related pathological processes and promoting diabetes development and causing diabetic complications. Of significant therapeutic value, GRb effective or efficacious compositions can systematically enhance or/and restore cellular antioxidant capacity in diabetic conditions and avoid oxidative stress damage and various pathological processes triggered by ROS accumulation; the coupling of glycolysis and sugar oxidation metabolism in a diabetic state is strengthened or/and repaired, glycolysis intermediate products are promoted to flow to mitochondria for oxidative phosphorylation and energy production, thereby AGEs accumulation is obviously reduced, physiological ATP steady state is maintained, MGO and glycosylation biological macromolecules thereof can be slowed down, and the disease progression and diabetic complications caused by ATP deficiency can be avoided. Thus, the GRb effective or effective composition for treating diabetes can avoid ATP deficiency and slow down cell hypofunction/disorder caused by AGEs accumulation and various downstream pathological events and malignant consequences thereof.

The GRb effective or effective composition has important significance for protecting the vascular system in a diabetic state and preventing and treating diabetic complications, and directly supports the medical application of the GRb effective or effective composition in preventing and treating diabetic complications and diseases related to angiogenesis disorders of the diabetic complications.

The morbidity and mortality from diabetes is mainly due to cardiovascular complications, of which microcirculation dysfunction and vascular inflammation are the main causes of impaired wound healing, nephropathy, heart disease, retinopathy, erectile dysfunction and neuropathy. In one aspect, the disordered neovasculature is a hallmark of end-stage diabetic retinopathy, nephropathy, and atherosclerotic plaque instability; on the other hand, defects in angiogenesis can lead to impaired wound healing, skin ulceration, fibrosis and impaired collateral vessel development. It is now clear that dysfunction of angiopoietins (Ang 1 and Ang 2) is yet another important factor in the dysfunction and resultant disruption of the diabetic vasculature. Angiopoietin-1 (Ang 1) plays a key role in vascular maturation, promoting vascular endothelial cell survival, stabilizing the endothelium and supporting the interaction between pericytes and endothelial cells and limiting vascular permeability, and Ang1 helps to delay the onset of diabetic complications by restoring microvascular function and can maintain the quiescence of some adult stem cells. In contrast, ang2 exerts an effect of antagonizing Ang1 function, promoting vessel wall destabilization and disruption of the connection between endothelial cells and pericytes, and a decrease in Ang1/Ang2 ratio associated with Ang2 high expression is closely associated with diabetic vascular dysfunction and is involved in neovascular diabetic-associated retinal diseases. Therefore, ang1 has been an attractive and viable drug target for the treatment of diabetic complications. Moreover, a bispecific antibody drug, faricimab, directed against both Ang2 and VEGF, showed promising results in clinical trials in patients with diabetic retinopathy.

Our results illustrate the key issues in 4 areas below: 1. our mouse models of type I and type II diabetes mellitus used replicated the state of elevated Ang2 levels and their associated elevated Ang2/Ang1 ratios in the blood of diabetic patients, with low Ang1 levels further promoting the state of elevated Ang2/Ang1 ratios, and with reduced Ang1 levels in the heart and skin of diabetic-complication-susceptible organs. It can be seen that the severe decrease of the Ang1/Ang2 ratio in blood and organs susceptible to diabetic complications, characterized by either Ang1 deficiency or an increase in Ang2, is a common molecular feature in type I and type II diabetes, thus supporting and extending the current understanding that an imbalance in the regulation of Ang1 and Ang2 results in an increase in the Ang2/Ang1 ratio, which is the culprit for vascular disease in type II diabetes patients, and that exogenous supplementation of Ang1 or inhibition of Ang2 or its downstream pathways are promising new targets for anti-diabetes and its complications. The effect of GRb-PQN4 in correcting the Ang1 deficiency of the diabetic state and its associated increase in the Ang2/Ang1 ratio is superior to that of insulin and metformin, comparable to that of dapagliflozin. GRb-PQN4 in combination with insulin or with metformin maintains or repairs the Ang1/Ang2 ratio at or in the normal range and even increases the Ang1/Ang2 ratio over normal levels compensatory, thus increasing the protection or repair of the vascular system in diabetic conditions. 4. Based on the effect that Ang1 can be independent of an insulin activated PI3K-AKT pathway, GRb-PQN4 or combined insulin or metformin thereof corrects the shortage of Ang1 in a diabetic state and the related increase of the Ang2/Ang1 ratio, an important pathway independent of the insulin activated PI3K-AKT pathway can be provided for diabetic patients, so that the method has important therapeutic value for overcoming insulin resistance of type II diabetes or treating type I diabetes in cooperation with insulin.

In conclusion, the correction of the insufficient Ang1 and the associated low Ang1/Ang2 ratio in diabetic conditions, particularly the elevation of Ang1 levels and Ang1/Ang2 ratios in the blood, is critical to the protection and repair of the vascular system of diabetic patients by GRb-PQN4 or its combination with insulin or metformin, and is a common mechanism of action for the specific active panaxadiol saponin compositions alone or in combination with insulin or metformin to effectively prevent and treat large and small vascular complications and neuropathy including cardiac dysfunction and diabetic foot. In particular, exogenous supplementation and elevation of endogenous Ang1 is known to alleviate diabetic complications by revascularization and prevention of diabetic endothelial cell damage, and Ang1 has been an attractive and promising drug target for the treatment of diabetic complications, and a bispecific antibody drug, farmimab, directed against both Ang2 and VEGF has shown promising results in clinical trials in patients with diabetic retinopathy. Therefore, the effects of GRb-PQN4 and the combination of the GRb-PQN4 and insulin or metformin on correcting insufficient Ang1 in a diabetic state and low Ang1/Ang2 ratio related to the insufficient Ang1 or complexly increasing the Ang1 level and the Ang1/Ang2 ratio in blood directly support the medical application of the GRb effective composition and the combination of the GRb effective composition and the insulin or metformin on preventing and treating diabetic nephropathy, heart disease, retinopathy (including diabetic microangiopathy and diabetic macular edema), erectile dysfunction and neuropathy. Based on the application value and prospect of promoting Ang1 function or inhibiting Ang2 function simultaneously in the aspect of developing medicaments for preventing and treating various diseases, including pulmonary hypertension and arteriosclerosis and angiogenesis-related diseases (wound repair, rheumatoid arthritis, wet age-related macular degeneration also known as exudative or neovascular age-related macular degeneration), retinal vein occlusion and diabetic macular edema, the research results also support the application of specific active panaxadiol saponin compositions (GRb effective or effective compositions) and combined insulin or metformin thereof in preventing and treating pulmonary hypertension, arteriosclerosis and angiogenesis-related diseases (including wound repair, rheumatoid arthritis, wet age-related macular degeneration) and retinal vein occlusion.

Combining the research results of our study, the effective or effective GRb composition can systematically protect the cardiovascular system of diabetic patients from the aspects of avoiding the apoptosis of endothelial cells induced by oxidative stress injury, slowing down the pathological angiogenesis of AGEs, strengthening the protection of blood vessels by Ang1, and the like, so that the effective or effective GRb composition can effectively prevent and treat cardiovascular complications of diabetes and cardiovascular diseases caused by other reasons. Obviously, the GRb effective or effective composition has a remarkable advantage in preventing and treating diabetes cardiovascular complications and other related diseases compared with Ang1 type medicaments which are possibly on the market in the future. Thus, protection and even enhancement of Ang1 vasoprotective effects further supports the protective effects of GRb effective or beneficial compositions on the cardiovascular system in diabetic conditions, the use to promote damage repair in diabetic patients, the use to prevent cardiovascular complications and diabetic foot, and also supports the treatment of diabetic or non-diabetic related diseases characterized by angiogenic disorders or degeneration of vascular structure, including but not limited to neovascular age-related macular degeneration (nAMD) and Diabetic Macular Edema (DME), either alone or in combination with insulin or metformin or other agents coordinated with their mechanisms of action.

In combination with the above 1.1-1.2, the chronic treatment of GRb effective or effective composition maintains ATP homeostasis and redox balance, avoids ROS accumulation, reduces AGEs accumulation, and slows down chronic inflammatory conditions by enhancing the coupling of glycolysis and sugar oxidation metabolism in diabetic conditions, protects/enhances the coenzyme I-mediated metabolic regulation function, the SOD/GSH-mediated antioxidant function, and the Ang 1-mediated vascular protection function, thereby effectively preventing the formation and deep development of diabetic networks and promoting the repair of the functions and structures of the relevant cells and tissues, and thus effectively preventing and treating diabetes and its systemic co-morbidities (diabetic complications).

1.3. The results of comparative studies on hypoglycemic agents further prove that the mechanisms of protecting and repairing glycolysis coupled with glycoxygenases (to generate sufficient ATP and prevent glycolytic intermediates from deviating from metabolic pathways and flowing to glycosylation pathways) and improving/protecting self-rescue are the key points for supporting the effective or effective combination of GRb to exert excellent drug effects of preventing and treating diabetes and complications thereof.

In the type I diabetes model, chronic treatment with insulin, although having an acute hypoglycemic effect, only slowed NADH relative to NAD at the end of the treatment ⁺ Deficiency, AGEs accumulation in blood and skin, and proinflammatory factor level in blood, and the action strength of reducing blood AGEs and proinflammatory factors is not as strong as GRb-PQN 4; in particular, it is not possible to combat the loss of ATP and the decrease in cellular antioxidant capacity mediated by NADPH, GSH and SOD (skin) in the heart and/or skin of diabetic vulnerable tissues with prolonged course, nor the decrease in Ang1 levels in the heart and skin, and also aggravate the accumulation of AGEs in the heart. These biochemical results are highly consistent with the inability of chronic insulin therapy to slow diabetic cardiac lesions and promote facile repair of large skin lesions. It can be seen that chronic treatment with insulin maintains or improves the coupling effects of glycolysis and glycooxidative metabolism far less than effective or advantageous GRb compositions, and chronic treatment with insulin protects the cellular antioxidant capacity and Ang 1-mediated microvascular protection/repair capacity and its associated damage repair capacity in diabetic conditions less than effective or advantageous GRb compositions. Chronic treatment with metformin or dapagliflozin, although significantly reducing the insulin resistance index (HOMA-IR) and blood glucose levels as judged by glycosylated hemoglobin, is comparable to GRb-PQN4 in the anti-diabetic characteristics of metformin. However, protecting or/and repairing cells against oxygen In terms of chemical function, the chronic treatment of metformin and dapagliflozin is inferior to the chronic treatment of GRb-PQN4, and particularly, the metformin and dapagliflozin can not effectively resist heart SOD deficiency, superoxide anion accumulation and ROS-mediated accumulation of oxidized lipid product malondialdehyde; neither metformin nor dapagliflozin chronic treatment significantly combats the decrease in ATP levels in the tissue of the organ being tested over an extended period of time, nor does it completely decrease the accumulation of AGEs in the tested tissue, including blood and heart, and dapagliflozin appears to also exacerbate the accumulation of AGEs; metformin is also completely unable to combat the decrease in Ang1 levels. It is also noted here that chronic treatment with metformin or dapagliflozin is completely resistant to NADH relative NAD characteristic of diabetes ⁺ However, this effect does not translate efficiently into a pathological change that improves the uncoupling of glycolysis and glycooxidative metabolism in diabetic states, in terms of AGEs accumulation and ATP deficiency, while GRb-PQN4 elevating the total NAD level above normal also maintains physiological ATP levels and significantly reduces AGEs accumulation. Therefore, neither metformin nor dapagliflozin chronic therapy can effectively slow down the flow of glycolytic intermediates to glycosylation pathways in the diabetic state, nor can effectively improve the yield of glucose through oxidative phosphorylation pathways, which indicates that neither metformin nor dapagliflozin chronic therapy can promote the glycolytic intermediates to enter tricarboxylic acid cycle in the type II diabetic state and finally pass through mitochondrial oxidative phosphorylation yield to maintain ATP steady state and reduce AGEs accumulation like GRb-PQN4 does, and does not have the function of activating cell self-rescue mechanism like GRb-PQN4 does.

Of particular concern here is that, in view of the many key factors in diabetes progression and diabetes systemic co-morbidities, insulin, metformin and dapagliflozin only reduce glycosylated hemoglobin levels and metabolic inflammation, with no significant improvement in oxidative stress damage, AGEs accumulation and energy (ATP) seen. This reasonably explains why hypoglycemic drugs are not effective in slowing the progression of diabetes and treating diabetic complications.

In conclusion, our research results show that protecting or enhancing glycolysis and sugar oxidation metabolism coupling (to generate sufficient energy ATP and prevent glycolysis intermediates from deviating from metabolic pathways and flowing to glycosylation pathways), protecting and improving cellular antioxidant function and related self-rescue mechanisms in pre-diabetic and diabetic states are unique advantages of GRb effective or superior compositions compared with classical first-line hypoglycemic agents and novel first-line hypoglycemic agents, and are the key points for GRb effective or superior compositions to effectively prevent and treat diabetes and systemic co-morbidities thereof. The reason why the efficacy of the GRb effective or effective composition for preventing and treating the progress of diabetes and complications thereof is far better than that of insulin or metformin or dapagliflozin chronic treatment is scientifically explained from the action mechanism, and the wide medical application of the GRb effective or effective composition for preventing and treating other diseases related to metabolic syndrome and metabolic disorder is fundamentally supported from the aspects of strengthening body resistance and consolidating constitution and strengthening body resistance and clearing source of disease pathological mechanism. In addition, by combining the key action of AGEs on the occurrence and development of diabetes and complications thereof and the excellent drug effect of the GRb effective or effective composition without the hypoglycemic effect on the prevention and treatment of the diabetes complications, but the drug effect of insulin is poor, and neither metformin nor dapagliflozin can effectively prevent and treat common symptoms of diabetes II and chronic complications, our research results indicate that the protection and repair of glycolysis and glucose oxidation metabolism are coupled under the diabetes state to generate enough energy (ATP) and prevent the final product of glycolysis from deviating from the metabolic pathway and flowing to the glycosylation pathway, and simultaneously strengthen or protect the cell antioxidant function, which is very important for the prevention and treatment of diabetes and complications thereof, and is a new development direction for effectively preventing and treating diabetes and complications thereof.

2. Importantly, the GRb effective or superior composition in combination with insulin or metformin for treatment of type I and type II diabetes can further enhance the coupling of glycolysis and sugar oxidation metabolism and completely avoid the deviation of glycolysis end products from metabolic pathways to glycosylation pathways to generate AGEs and metabolic inflammation, and also enhance or retain the cell self-rescue mechanism activating effect of the GRb effective or superior composition, maintain the cell energy state and the redox state in a physiological range, and show the effects of reducing protein over-degradation in a diabetic state (mainly type I diabetes model) and completely avoiding lipid toxicity (mainly type II diabetes model) which are not achieved independently. The following point 5 is also to be noted here.

Chronic treatment with GRb-PQN4 in combination with metformin can completely combat the elevated total cholesterol and ldl cholesterol in the blood characteristic of type II diabetes, while GRb-PQN4 or metformin alone is either ineffective or, conversely, further increases the level of total cholesterol. Based on that the blood lipid toxicity is an important risk factor of the cardiovascular complications of diabetes, research results indicate that the combination of the GRb effective or effective composition and the metformin can synergistically prevent and treat the diabetes fat toxicity, so that the pharmacological action further explains the drug effect of the combination of the GRb effective or effective composition and the metformin on effectively preventing and treating the cardiovascular complications of diabetes, and also discloses the application of the combination of the GRb effective or effective composition and the metformin on preventing and treating atherosclerosis characterized by metabolic syndrome.

Chronic treatment of GRb-PQN4 in combination with insulin or metformin increases the total amount of ATP, NADH, NAD (NAD) in the tissues/organs targeted for diabetic complications ⁺ + NADH) (based on type II diabetes model), total amount of NADP (NADPH + NADP) ⁺ )、NADPH/NADP ⁺ The ratio, NADPH (based on type II diabetes models), GSH/GSSG ratio, SOD and angiopoietin-1 (Ang-1) to near or above normal levels show that combination therapy can further activate the body's self-rescue mechanism to cope with diabetic conditions and their adverse events. The function of improving/activating the self-rescue ability of the organism is consistent with the medicine effect of strengthening the body resistance and consolidating the constitution of the ginseng and the adaptation original effect (namely the effect of strengthening the adaptability of the organism, enhancing the nonspecific resistance of the organism to various harmful stimuli and injuries such as physics, chemistry, biology and the like and recovering the disordered function to be normal), and the function of improving/activating the self-rescue ability of the organism of the effective or effective GRb composition can be further enhanced by insulin and metformin, so the scientificity of preventing and treating diabetes and systemic co-morbidity (diabetic complication) of the diabetes by combining the effective or effective GRb composition and insulin or metformin is clarified from an important angle, and the good prospect of the medicine fusion development is shown.

2.3. chronic treatment combining GRb-PQN4 with dapagliflozin (taking sugar uptake by inhibiting cells and sugar discharge by accelerating kidney as action mechanism) has no obvious synergistic effect on the indexes, but dapagliflozin weakens various pharmacological actions of GRb-PQN4, which is highly consistent with the fact that the drug effect of the combination of the two on preventing and treating common symptoms and complications of diabetes is poor, thus the synergistic effect of insulin or metformin on GRb effective or superior composition on preventing and treating diabetes and systemic co-morbid (diabetic complication) thereof is not caused by simple blood sugar lowering effect, but the result of promoting/improving sugar metabolism, antioxidation function and activating other self-rescue mechanisms of organism of the GRb effective or superior composition is strengthened.

2.4. As a positive result of improving intracellular carbohydrate metabolism and enhancing self-rescue mechanisms including anti-oxidation, GRb effective or efficacious compositions in combination with insulin or metformin produce significant, smooth and sustainable superior blood glucose lowering effects, even repair of glucose metabolism disorders, avoid blood vessel damage, insulin resistance and diabetes progression from blood glucose excursions (including extremely harmful hypoglycemia) produced by multiple administrations of insulin or metformin before and after daily administration, and also avoid negative effects of blood glucose excursions on patient mental health and quality of life.

Blood glucose excursion or blood glucose variability (GV, which includes hypoglycemia due to drug action and blood glucose excursion during the course of a day upon administration) and uncontrolled blood glucose rise over a prolonged period of treatment and/or disease progression are common problems in hypoglycemic drug therapy for diabetes mellitus, yet unresolved and closely related to the development of diabetic complications. GV has been identified as an important cause of development of diabetic vascular complications and neuropathy independent of insulin resistance, and reduces the mental health and quality of life of patients, including reduced work efficiency, mood problems (such as postprandial depression and anxiety), cognitive symptoms and sleep disturbances associated with nocturnal hypoglycemia and nocturnal breathing disorders and prolongs hospitalizations of hospitalized diabetic patients. Moreover, current insulin treatment methods (either multiple daily injections or continuous subcutaneous infusion) still present a risk of poor glycemic control and hypoglycemia in patients of all ages, particularly in children and adolescents, severe hypoglycemia leading to seizures or significant changes in mental status, while mild hypoglycemia is associated with changes in learning and attention-cognitive functions. In particular, intermittent hyperglycemia damages vascular endothelial cells by oxidative stress much more strongly than sustained hyperglycemia exposure. The GRb effective or superior combination therapy with insulin or metformin provides good resolution of insulin resistance and blood glucose fluctuations and elevated blood glucose levels with disease progression before and after daily administration of insulin or metformin during the course of therapy and blocks disease progression. Specifically, in an acute blood glucose reduction test in the chronic treatment process of a model animal with type I diabetes, GRb-PQN4 and insulin are combined to avoid hypoglycemia caused by insulin treatment, prolong the acute blood glucose reduction time of insulin, effectively avoid blood glucose fluctuation between daily multiple times of administration of insulin and avoid acute drug resistance caused by daily multiple times of administration of insulin, so that the large blood glucose fluctuation before and after multiple times of insulin injection in one day is effectively avoided, the hypoglycemia phenomenon is avoided, and the total blood glucose reduction amount is increased; the combination of the two can not only prevent the weakening of acute glucose-reducing efficacy (or called insulin resistance) of the insulin along with the treatment time course and the disease progression in the chronic treatment process of the insulin, but also gradually enhance the sensitivity of the body to the insulin and maintain the glycosylated hemoglobin level of the terminal point basically unchanged and close to the normal level (reflecting that the combination of the two has strong and continuous glucose-reducing effect and protects the oxygen supply function of the hemoglobin in the circulatory system to target tissues, organs and cells), and the chronic treatment of the insulin with the same dose can not obviously reduce the glycosylated hemoglobin level. Moreover, the combination treatment can effectively reduce the fasting blood glucose level and can effectively delay the progressive rise of the fasting blood glucose level along with the prolongation of the disease course, and the independent treatment does not have the effect, so the medicine effect of the combination treatment on delaying or even blocking the disease progression is further reflected from an important angle. Thus, the GRb effective or efficacious composition increases the body's sensitivity to insulin and delays or blocks disease progression such that chronic treatment with low doses of insulin can produce significant and stable hypoglycemic effects, while lowering the insulin dose itself is beneficial in avoiding therapeutic hypoglycemia and blood glucose excursions. Consistent findings were obtained in a type II diabetes model, GRb-PQN4 in combination with metformin significantly potentiates the acute hypoglycaemic effect of the latter and overcomes the attenuation of fasting glucose lowering effects that occurs with prolonged treatment time course or/and disease progression.

It is specifically noted herein that the potency of dapagliflozin or its acute hypoglycemic effect in contract with GRb-PQN4, fasting plasma glucose, insulin resistance index and end-point glycosylated hemoglobin level is significantly greater than that of metformin or its combination with GRb-PQN4 during chronic treatment for 26 weeks, whereas dapagliflozin or its combination with GRb-PQN4 has no significant potency in the control of systemic co-morbidities in type II diabetes (diabetic complications), and that dapagliflozin significantly impairs the potency of GRb-effective or efficacious compositions when combined. The pharmacological action of dapagliflozin on inhibiting a sodium glucose cotransporter may damage the effect of GRb-PQN4 on improving the yield of glucose through oxidative metabolism in a diabetic state. Indeed, as previously described, dapagliflozin abolished the effects of GRb-PQN4 on oxidative stress, ATP depletion, AGEs accumulation and metabolic inflammation in the diabetic state and compensatory rise in NAD (NAD) in combination therapy ⁺ + NADH), NADH, GSH and SOD, thus leading to the destruction of GRb-PQN4 by dapagliflozin in its efficacy in the prevention and treatment of diabetic complications. This again illustrates the limitations of simply lowering blood glucose levels for the prevention and treatment of diabetes and its complications, and further demonstrates the key role of GRb effective or effective compositions in targeting intracellular carbohydrate metabolism, simultaneously increasing cellular antioxidant capacity and other self-rescue mechanisms for the prevention and treatment of diabetes and its complications.

2.5. The effect of insulin or metformin on lowering blood glucose and glycosylated hemoglobin levels and the effect of GRb effective or effective compositions on promoting intracellular carbohydrate metabolism and increasing cellular antioxidant capacity can generate continuous benign interaction, thereby protecting or repairing the healthy energy metabolism system and redox system and activating/increasing the cell self-rescue mechanism, so that the sugar metabolism disorder in the pre-diabetes stage or diabetes stage and the malignant consequences caused by the sugar metabolism disorder can be prevented or corrected, and the malignant cycle between the malignant consequences and the malignant consequences can be prevented or corrected, wherein the malignant cycle comprises energy (ATP) deficiency/deficiency, oxidative stress, AGEs accumulation, mitochondrial injury, metabolic inflammation, insulin resistance, lipid toxicity, protein excessive degradation, and the burden of glomerular filtration caused by the increase of urea nitrogen and creatinine in blood.On one hand, the GRb composition can enhance the coupling effect of glycolysis and sugar oxidation metabolism to induce the blood sugar reducing effect of insulin or metformin to flow to tricarboxylic acid cycle and mitochondrial oxidative phosphorylation energy, so that the blood sugar reducing effect of the insulin or the metformin is enhanced, the oxygen carrying function of hemoglobin is protected, and AGEs accumulation and blood sugar fluctuation in the treatment process of the insulin or the metformin are avoided; on the other hand, in turn, the substitution of a physiologically stable blood glucose level for a widely fluctuating glucose level can avoid oxidative stress induced damage to mitochondria and delicate and vital cells including vascular endothelial cells due to blood glucose variability, and the protected hemoglobin can ensure oxygen required during the oxidative metabolism of intracellular sugars and avoid hypoxia common to diabetic conditions. In diabetic patients, tissues such as retina, kidney, pancreatic islets, fat, skin and wound have an anoxic state, which suggests that hypoxia plays an important role in the development of diabetes and diabetic complications. The microvascular damage and the glycosylated hemoglobin together cause the hypoxia state of the tissues and organs related to the diabetic complication, so that the decoupling of the original glycolysis and the sugar oxidation metabolism is intensified, and the small-amplitude increase of the glycosylated hemoglobin directly participates in diabetic vasculopathy complication and neuropathy complication (such as retinopathy, nephropathy and neuropathy, gastroparesis and increase of the incidence rate of infection of a surgical site) in a sugar-independent mode. Therefore, physiological glycosylated hemoglobin levels undoubtedly promote the coupling of glycolysis and glycooxidative metabolism enhanced by effective or effective GRb compositions and compensate for the inability of GRb compositions to reduce glycosylated hemoglobin, while the microvasculature protected by GRb compositions further ensures the supply of nutrients such as oxygen and glucose to the target tissue and organ. Moreover, the powerful effect of the system in increasing intracellular antioxidant capacity and the effect of enhancing the coupling of glycolysis with sugar oxidative metabolism or correcting uncoupling can also be mutually supported. Cellular antioxidant systems rely on both the glucose metabolic pathway for normal operation and in turn regulate sugar metabolic activities such as: all 3 precursor amino acids of GSH can be derived from sugar metabolism intermediates, and especially pyruvate deficiency may be associated with diabetic glycine deletion; NADH/NAD ⁺ And NADPH/NADP ⁺ The balance of (a) is both regulated and dependent on carbohydrate metabolism, and the GSH/GSSG balance is dependent on NADPH/NADP ⁺ Balancing; SOD and GSH are important for protecting mitochondria and sugar oxidation metabolism by cooperatively eliminating ROS (reactive oxygen species) from mitochondria; GSH can avoid or reduce AGEs accumulation and the various malignant events that they cause, including metabolic disorders and metabolic inflammation, by effectively detoxifying the most important glycosylated precursor, MGO. Finally, it is pointed out that sufficient ATP from the oxidative metabolism of sugars not only avoids the excessive activation of compensatory fat metabolism and the excessive degradation of proteins and creatine in the diabetic state due to ATP deficiency, thus avoiding the subsequent lipid toxicity, excessive protein consumption and the burden of increased creatinine in the blood on glomerular filtration, but also improves ATP required for the biochemical reactions and processes targeted by insulin or metformin (neither of which are effective against the antidiabetic-associated ATP deficiency) and reactivates various physiological and biochemical processes that are impaired or even arrested by ATP deficiency, thus improving overall the health and working efficacy of the patient. In conclusion, the effective or effective composition of GRb and insulin or metformin act together to prevent and treat diabetes in a complementary and synergistic manner in the action mechanism, and can protect or repair the healthy energy metabolism system and redox system in the pre-diabetes stage or the diabetes stage and activate/improve the cell self-rescue mechanism, thereby endowing the cells with the ability to effectively exert the physiological function, damage resistance, infection resistance and damage repair promotion, so that the GRb composition not only can effectively prevent and treat diabetes and systemic co-morbid diseases (diabetic complications) thereof, but also can comprehensively improve the health state and working efficacy of diabetic patients.

The characteristic advantages of the GRb effective or effective composition in treating diabetes root (origin-righting) and effectively preventing and treating disease network formation and development can realize the breakthrough of the drug effect in effectively preventing and treating diabetes and systemic co-morbid diabetes (diabetic complication).

It is clear that in therapeutic principles, GRb effective or advantageous compositions and combinations thereof with insulin or metformin reduce blood glucose levels compared to current hypoglycemic agents (trying to inhibit absorption or production, promote excretion, etc., but neglecting the root of disease (i.e., intracellular carbohydrate metabolism disorder) and candidates that target oxidative stress or AGEs accumulation that are widely focused on carbohydrate metabolism disorders (e.g., therapeutic strategies that use exogenous antioxidants to apply antioxidant stress, inhibit polyol pathways, inflammatory pathways, downstream pathways within PKC or MAPK), are more consistent with the characteristics of disease development and progression, have the distinct advantages of positive origin (management of diabetic roots) and effective control of disease network formation and progression Diabetic peripheral neuropathy (including sensory neuropathy and autonomic neuropathy), central neuropathy (including diabetic dementia and diabetic mental disorder), diabetic cardiovascular disease, diabetic nephropathy, diabetic eye disease (including diabetic retinopathy and diabetic cataract), and diabetic foot; promoting the healing/repair of pre-diabetic patients and diabetic patients in medical operations or traumatic wounds. The effective or superior GRb composition and the medical effect of the GRb composition in combination with insulin or metformin for preventing and treating diabetes and systemic complications are also characterized in that systemic treatment can be carried out on certain complications. For example, diabetic cardiopathy generally refers to diabetic patients 'complicated or concomitant coronary atherosclerotic heart disease, diabetic cardiomyopathy and cardiac arrhythmia and cardiac dysfunction caused by microangiopathy, vegetative nerve dysfunction, and GRb effective or effective composition, especially in combination with insulin or metformin, can be used for the combined prevention and treatment of diabetic patients' coronary atherosclerotic cardiopathy, diabetic cardiomyopathy and cardiac arrhythmia and cardiac dysfunction.

4. Based on the effects of improving the glucose oxidation metabolism state, the oxidation-reduction balance, the energy state and the mitochondrial function and the high safety of the composition, the GRb effective or optimal composition has important significance for improving the reproductive capacity and the reproductive quality of diabetics, provides a safer and more effective treatment for the gestational diabetics, and can improve the quality of life of the couples of the diabetics.

The prevalence of diabetic male sexual dysfunction is close to 50%, while diabetic women seem to be slightly lower. Testicular dysfunction, impotence, decreased fertility, and retrograde ejaculation are common symptoms in diabetic men. Diabetes is also the most common cause of erectile dysfunction in men. The sperm quality of diabetic men is also poor, including decreased sperm motility and concentration, abnormal morphology, and abnormal increase in seminal plasma. In diabetic female neuropathy, vascular injury and psychological problems are associated with the pathogenesis of decreased libido, decreased vaginal lubrication, orgasmic dysfunction and dyspareunia. The relationship between the production of excess free radical oxygen in diabetic pregnancy and embryogenesis disorder has also been proposed. Indeed, maternal diabetes during pregnancy is associated with an increased risk of complications in the offspring, such as abnormal fetal growth and development, polyhydramnios, fetal loss and congenital malformations. In addition, newborns of diabetic mothers experience hypovolemia and reduced bone mineral content. Both biochemically and molecularly, mitochondrial dysfunction and oxidative stress and its triggered pathological events contribute to diabetic dysfunction and reproductive dysfunction. Therefore, the comprehensive improvement of the mitochondrial function, the carbohydrate metabolism state, the redox balance and the energy state of the gestational diabetes patient has important significance for improving the fertility and the fertility quality of the patient. Our findings suggest that hypoglycemic agents do not implement this new therapeutic strategy and that effective or efficacious GRb compositions, particularly in combination with insulin or metformin, can perform this therapeutic strategy well, creating new hopes for patients in the fertile phase. Meanwhile, the high safety of treatment is very important for the diabetics who are pregnant. The use of metformin in pregnancy has increased worldwide as evidence from random control trials has shown its safety and efficacy. Our research results show that the GRb effective or effective composition has high safety on diabetes patients under the action of regulating carbohydrate metabolism and cell self-rescue mechanism, is at least safer than metformin, is remarkably superior to metformin in the aspects of improving intracellular metabolism, reducing AGEs accumulation and metabolic inflammation and the like, and can generate excellent blood sugar reducing effect, provide enough cell energy and completely avoid oxidative stress, AGEs accumulation and inflammation states by virtue of the synergy of the GRb effective or effective composition. Therefore, an effective or effective combination of GRb or in combination with insulin or metformin would provide a safer and more effective treatment for pregnant and gestational diabetic patients, and would be an ideal choice for pregnant hyperglycemic patients. Since sex is also an important aspect in the life of couples, the efficacy of the effective or effective GRb composition in preventing and treating diabetic reproductive system complications is also of great significance in improving the quality of life of patients.

5. The popularization and application of GRb effective or effective composition based on common pathological mechanism and common pharmacological action in medicine or matter combined therapy with insulin, metformin or other action mechanism.

Mitochondrial dysfunction and its associated toxic pathological events including ATP deficiency/absence of the energetic substance, oxidative stress injury, accumulation of advanced glycosylation products (AGEs), and chronic inflammation are common pathological mechanisms involved in the functional disorders/degeneration and subsequent structural degeneration and/or damage of cells and tissues and organs in a variety of pathological conditions related to metabolic diseases, metabolic disorders, or age-related conditions. Consistently, GSH deficiency, metabolic disorders and microvascular dysfunction are common features of diabetes and neurodegenerative diseases (including senile dementia and parkinson's disease), and GSH deficiency, metabolic disorders and neuroinflammation are also characteristic changes of psychotic disorders (schizophrenia and bipolar disorder) and are also important components of the disease pathology. In particular, AGEs are derived both primarily from disorders of carbohydrate metabolism (glycolysis is decoupled from sugar via mitochondrial oxidative metabolism), which in turn is exacerbated by insulin resistance and impairment of mitochondrial function through various mechanisms that induce oxidative stress and highly expressed pro-inflammatory factors/molecules mediated inflammation, as well as by affecting transcriptome, lipidome and proteome to impair other physiological processes and trigger a variety of pathological processes, including neovascularization associated with microvascular abnormalities and fibrosis-associated extracellular matrix accumulation. Thus, AGEs accumulation plays a key role in the progression of a variety of diseases, including diabetes, obesity, cardiovascular disease (hypertension, coronary and peripheral artery disease/atherosclerosis, stroke, and microvascular complications), chronic kidney disease, liver fibrosis, chronic obstructive pulmonary disease, pulmonary fibrosis, neurodegenerative diseases, alcoholic brain injury, psychotic disorders, and unhealthy aging. Furthermore, anti-inflammatory molecules that inhibit AGEs have proven to be good candidates for improving diabetic complications and degenerative diseases, and inhibition of binding of AGEs to their receptors has also been considered as a promising new therapeutic strategy for the treatment of AGE-related diseases, including physiological aging, neurodegenerative/neuroinflammatory diseases, diabetes and its complications, autoimmune/rheumatic inflammatory diseases, bone degenerative diseases, and chronic kidney diseases. Based on the similarity of the pathological mechanisms of diabetes and senile dementia, intranasal inhalation of insulin is also an attractive treatment for moderate cognitive disorders or dementia; metformin has also been tried or used for neurodegenerative diseases including amnestic mild cognitive impairment, alzheimer's disease and parkinson's disease and for psychotic disorders including schizophrenia and autism, and its beneficial effects are associated with its anti-inflammatory and antioxidant properties. In conclusion, the research results of the GRb effective or effective composition also support the use of the GRb effective or effective composition alone and in combination with insulin or metformin or other hypoglycemic agents with coordinated action mechanisms in the following aspects.

Use of a grb effective or advantageous composition alone or in combination with insulin or metformin or other drugs/substances with a coordinated mechanism of action for pre-diabetic people to prevent the development of diabetes.

Pre-diabetes refers to patients with elevated blood sugar but not meeting the diagnostic criteria for diabetes, and a large portion of the population develops diabetes without attention to control. Insulin resistance is the main cause of type II diabetes, and type I diabetes also develops with disease progression, prolonged treatment period, intensive insulin treatment, and the like. From the pathological mechanism, the sugar metabolism disorder in the prediabetes stage is the result of insulin resistance, and MGO mediated AGEs accumulation, oxidative stress, mitochondrial dysfunction and metabolic inflammation related to abnormal sugar metabolism are all involved in insulin resistance and type I diabetes islet cell injury, because the prediabetes progress to type II diabetes and type I diabetes progresses and insulin resistance appears. Moreover, there are increasing studies demonstrating that MGO derived from glycolysis together with ROS leads to cellular dysfunction, an important factor in the progression of obesity to type II diabetes. Therefore, the research results support the application of the GRb effective or effective composition to the treatment and prevention of the type II diabetes singly or in combination with metformin or other medicaments/substances with coordinated action mechanisms from the aspects of drug effect and action mechanism, and also support the application of the GRb effective or effective composition to the treatment and prevention of the type I diabetes singly or in combination with insulin or other medicaments/substances with coordinated action mechanisms.

Use of effective or efficacious compositions of grb alone or in combination with metformin or other drugs/substances with coordinated mechanisms of action for the treatment of metabolic syndrome and the prevention of diabetes, atherosclerosis and non-atherosclerotic cardiovascular diseases.

Metabolic syndrome is manifested by a group of related diseases including, but not limited to, insulin resistance, hyperglycemia, hypertension, central obesity, hyperlipidemia, hyperviscosity, hypercreatinine, and atherosclerotic dyslipidemia, is a high risk factor for diabetes, atherosclerosis, and non-atherosclerotic cardiovascular disease, and also increases the morbidity and mortality of cerebrovascular disease. Since resistance to the effects of insulin metabolism is an important component of the pathophysiology of the metabolic syndrome, this syndrome is also referred to as "insulin resistance syndrome". However, chronic low-grade inflammatory conditions (also called metabolic inflammation) are characteristic of obesity, insulin resistance, type II diabetes mellitus and metabolic heart disease, the occurrence of which is closely related to and in turn aggravates insulin resistance and metabolic disorders, and are also central mechanisms of the pathophysiology of metabolic syndrome, and IL-1 β produced by metabolic inflammation is a key molecule in the pathogenesis of metabolic heart disease. Therefore, the GRb effective or effective composition strengthens the coupling of glycolysis and sugar oxidative metabolism and promotes the production of mitochondrial oxidative phosphorylation under the II-type diabetes state, improves the metabolic inflammation state under the diabetes state and strengthens the action of metformin on slowing down insulin resistance, particularly the action of combining the two to completely avoid the metabolic inflammation state and the insulin resistance, and respectively supports the application of the GRb effective or effective composition to the treatment of metabolic syndrome and the prevention of diabetes and metabolic cardiovascular diseases alone or in combination with metformin or other medicaments/substances with coordinated action mechanisms.

Medical use of effective or efficacious grb compositions alone or in combination with insulin or metformin or other drugs/substances with coordinated mechanisms of action to prevent and treat metabolic disorders associated with drug therapy including, but not limited to, hyperglycemia, lipid metabolism disorders and diabetes associated with glucocorticoid therapy, statin therapy, immunosuppressants and antipsychotic therapy.

Drug-induced metabolic syndrome/hyperglycemia and diabetes are an increasingly global problem in clinical practice, where glucocorticoids, immunosuppressants, antipsychotics (especially second generation) as well as antiretroviral therapy and immune checkpoint inhibitors for immunotherapy of cancer are very commonly used or are increasingly used in clinical practice, their use being at potential risk of developing severe hyperglycemia/diabetes. In addition, statins (widely used in hyperlipidemic cardiovascular diseases such as hyperlipidemia, hypertension, atherosclerotic heart disease, etc.), thiazide diuretics, and certain beta-blocker antihypertensive agents are also at risk of causing new diabetes and exacerbating preexisting diabetes. Reduced insulin sensitivity (i.e. insulin resistance) and/or reduced insulin secretion is a common cause of metabolic syndrome/hyperglycemia and diabetes with drug therapy, and metformin is currently the first drug to correct the therapeutic metabolic syndrome and diabetes, with insulin also being used for severe hyperglycemia. In summary, our findings support that effective or efficacious compositions of GRb in combination with metformin have been used in the treatment of the therapeutic metabolic syndrome characterized by insulin resistance, hyperglycemia and diabetes in combination with insulin for the treatment of related conditions characterized by decreased insulin secretion.

Medical use of an effective or advantageous combination of grb alone or in combination with insulin or metformin or other drugs/substances with coordinated mechanisms of action for the prevention and treatment of metabolic disorder-related conditions including, but not limited to, polycystic ovary syndrome, schizophrenia, autism, anxiety, bipolar disorder, major depression, attention deficit/hyperactivity disorder and post-traumatic stress disorder.

Polycystic ovary syndrome is one of the most common endocrine and metabolic disorders in premenopausal women, manifested by hyperandrogenism, ovarian dysfunction and associated metabolic disorders, including abdominal obesity, insulin resistance, obesity, metabolic disorders (such as hypertension or dyslipidemia); patients with polycystic ovary syndrome are also at high risk for type II diabetes and future emotional and psychiatric disorders. It is particularly noted here that studies conducted over the last few years have demonstrated that increased oxidative stress is associated with the progression of polycystic ovarian syndrome and associated complications, and has demonstrated a relationship between mitochondrial dysfunction and polycystic ovarian syndrome. In particular, events associated with mitochondrial dysfunction including GSH insufficiency, oxidative stress, and chronic inflammation have all been shown to be associated with the onset and progression of polycystic ovarian syndrome. Currently, the most common strategy for treating insulin resistance in polycystic ovarian syndrome is to use insulin sensitizers, particularly metformin. Metformin works similarly to lifestyle interventions in weight loss but works better in reducing androgen concentrations, and is subject to standard treatment for hypertension or dyslipidemia. In summary, our findings support the use of effective or efficacious GRb compositions alone or in combination with metformin for the treatment of polycystic ovarian syndrome.

There has been compelling evidence that psychiatric disorders characterized by an increased risk of metabolic syndrome including dyslipidemia, abdominal obesity, hypertension and hyperglycemia include major depression, bipolar disorder, schizophrenia, anxiety, autism, attention deficit/hyperactivity disorder and post-traumatic stress disorder. There is emerging evidence that there is a bidirectional longitudinal impact between psychiatric disorders and metabolic syndrome. For example, recent studies have demonstrated that metabolic co-morbidities between schizophrenia and type II diabetes are due at least in part to shared genetic mechanisms, and that genetic predisposition to psychoneuroendocrine dysfunction in patients with schizophrenia and major depression may lead to increased risk of type II diabetes and metabolic syndrome. In addition, different populations of psychotic patients have intrinsic pathophysiological characteristics of a deregulated homeostatic system, including the hypothalamic-pituitary-adrenal axis and inflammatory responses, and these pathophysiological characteristics are also associated with the development of metabolic syndrome. In particular, psychotic disorders share a metabolic disorder phenotype with metabolic syndrome/type II diabetes, including: mitochondrial dysfunction, altered redox balance (including GSH deficiency), AGEs accumulation, and chronic low grade inflammation, and these pathological events are profoundly involved in the neurological and behavioral disorders of schizophrenia and bipolar disorder. In addition, the metabolic disorder phenotype and its effects on diseases that occur in psychotic disorders are also present in neurodegenerative diseases such as alzheimer's disease, huntington's disease and parkinson's disease. It is also noted herein that certain psychotropic agents, such as second generation anti-schizophrenia agents, also have profound effects on the increased imbalance of metabolic syndrome, increase the risk of type II diabetes and cardiovascular disease in patients, exacerbate metabolic syndrome, decrease quality of life in patients, and also do not facilitate recovery of the disease in neurophysiologic functions, leading to a shortened life expectancy. Therefore, the past symptomatic treatment strategies need to be changed, the biochemical and molecular environments for treating and/or supporting the mental disorder diseases are a disease root strategy, and if the traditional symptomatic treatment strategies are combined, the curative effect of treating both symptoms and root causes can be achieved. Currently, enhancement of mitochondrial function is thought to contribute to the amelioration of neurological and behavioral impairment associated with such diseases. Due to its clear safety profile, metformin therapy has been used in neuropsychiatric practice, including schizophrenia and autism, and exhibits beneficial effects on autism. In conclusion, our findings support the use of effective or advantageous GRb compositions for the treatment of psychotic disorders, either alone or in combination with metformin or in combination with other drugs with a coordinated mechanism of action.

Medical use of effective or advantageous combinations of grb alone or in combination with insulin or metformin or other drugs/substances with coordinated mechanisms of action for the prevention and treatment of neurodegenerative diseases with metabolic syndrome as a risk factor, including but not limited to senile dementia, vascular dementia and parkinson's syndrome.

Type II diabetes is closely associated with neurodegenerative diseases. On the one hand, type II diabetes can directly lead to senile dementia, and can also be an important risk factor for senile dementia, vascular dementia, and parkinson's disease. On the other hand, type II diabetes has highly similar metabolic disorder characteristics to neurodegenerative diseases, including mitochondrial dysfunction, oxidative stress, AGEs accumulation, insulin resistance and systemic metabolic inflammation, and these pathological events, particularly the vicious circle between them, lead to the functional and structural degeneration of the relevant brain cells. Therefore, senile dementia is referred to as diabetes occurring in the brain, i.e., type III diabetes, and metformin achieves certain benefits in clinical trials for treating senile dementia and parkinson's disease. It is noteworthy here that human and animal model study data of neurodegenerative diseases suggest that mitochondrial dysfunction and oxidative stress are key factors in initiating and propagating neurodegenerative processes. Thus, we reasoned that mitochondrial dysfunction leads to inadequate oxidative breakdown of glycolytic intermediates leading to toxic MGO and ultimately to AGEs in the pre-neurodegenerative disease stage, and that cellular disinfection of MGO also consumes large amounts of GSH to exacerbate ROS accumulation that accompanies or causes mitochondrial dysfunction, both of which lead to chronic inflammatory states. It can be seen that the effective or efficacious GRb composition systematically increases cellular antioxidant capacity, particularly compensable increases in GSH and SOD levels, and potentiates the coupled effects of glycolysis and carbohydrate oxidation metabolism, strongly supporting its therapeutic use for the treatment of neurodegenerative diseases, either alone or in combination with metformin or other drugs/substances whose mechanism of action is coordinated.

Medical use of effective or advantageous grb compositions alone or in combination with insulin or metformin or other drugs/substances with a coordinated mechanism of action for the prevention and treatment of fibrosis related disorders.

Fibrosis is an abnormal deposition of extracellular matrix that can lead to organ dysfunction, morbidity, and death. The disease burden caused by fibrosis is enormous and there is currently no treatment that can prevent or reverse fibrosis. Diseases associated with fibrosis include: liver cirrhosis, non-alcoholic steatohepatitis, chronic kidney disease, heart failure, diabetes, idiopathic pulmonary fibrosis, and scleroderma. Metabolic alterations are increasingly recognized as an important pathogenic process for fibrosis in many organ types. Pathologically, fibrosis is characterized by an imbalance in extracellular matrix (ECM) hyperplastic homeostasis. There is reliable data to directly link glycolysis to extracellular matrix production. Glucose metabolism can provide energy for this anabolic process and provide a basis for the production of collagen. This is analogous to the Warburg effect in cancer, where cancer cells preferentially utilize aerobic glycolysis to generate ATP and NADPH, and provide glycolytic intermediates as carbon sources to promote proliferation. In fact, this interaction between glycolysis and extracellular matrix production has been demonstrated in various steps of extracellular matrix synthesis, including amino acid synthesis, hydroxylation, glycosylation, and extracellular matrix secretion. AGEs are consistently involved in the fibrotic process in several organs via their Receptors (RAGE), including liver fibrosis, heart fibrosis, lung fibrosis and kidney fibrosis. Therefore, a GRb effective or beneficial composition enhances the coupling of glycolysis to sugar oxidative metabolism, consumes the intermediates of glycolysis and tricarboxylic acid cycle by promoting oxidative catabolism, thereby effectively reducing the flux of metabolic intermediates to extracellular matrix synthesis, including amino acid synthesis and glycosylation, and ultimately achieving anti-fibrotic effects. Indeed, GRb-effective or efficacious compositions almost completely inhibit the occurrence of cardiac fibrosis in the type I diabetic state and very significantly reduce the extent of cardiac fibrosis in the type II diabetic state, and although metformin does not show significant efficacy, it can almost completely prevent the occurrence of cardiac fibrosis in combination with GRb-effective or efficacious compositions. Similarly, thyroid hormones, which reduce the severity of bleomycin-induced pulmonary fibrosis, have a mechanism of action associated with their increased mitochondrial function, so hormone therapy is thought to exert anti-fibrotic effects by regulating cellular respiration and oxidative catabolism, and targeting these key metabolic pathways is thought to be an exciting and promising opportunity for future fibrosis therapy. In addition, lipotoxicity due to lipid accumulation or defective fatty acid oxidation also leads to the development of fibrosis. Therefore, the GRb effective or effective composition can maintain the fat metabolism steady state and avoid the fat toxicity effect by combining with insulin or metformin, and is also beneficial to resisting tissue organ fibrosis. To date, some of the existing metabolic drugs used to treat diseases such as diabetes (metformin, insulin) and hypercholesterolemia (pravastatin) have become candidates for the treatment of fibrosis; drugs that are resistant to insulin, such as hesperidin and pentoxifylline, are also being evaluated for their potential therapeutic value for fibrosis. In summary, our findings support the use of GRb effective or efficacious compositions alone or in combination with insulin or metformin or other drugs/substances with coordinated mechanisms of action for the prevention and treatment of fibrosis-related disorders, including: liver cirrhosis, non-alcoholic steatohepatitis, chronic kidney disease, heart failure, idiopathic pulmonary fibrosis, and scleroderma.

Use of a grb effective or efficacious composition alone or in combination with metformin for delaying aging.

The aging process is characterized by a decline in the function of cells, tissues and whole organs, beginning with perturbations in critical cellular processes such as mitochondrial function, protein balance and stress-clearing systems. An increasing number of studies have demonstrated causal relationships between MGO-derived AGEs and age-related tissue dysfunction, revealing a previously underestimated role of dicarbonyl stress in determining healthy or unhealthy aging. If the defense mechanisms (e.g., GSH-mediated glyoxalase system) are not effective in counteracting the accumulation of MGO during aging, tissue damage can result, inducing metabolic disease, vascular dysfunction, and neuronal damage, leading to unhealthy aging; this will ensure healthy ageing if a good balance between MGO formation and defense mechanism activity is maintained. It can be seen that AGEs bridge the classical hypothesis of aging, including the theory of free radicals, apoptosis, and mitochondrial dysfunction. It is understood here that both glycolytic intermediate accumulation or/and GSH deficiency resulting from mitochondrial dysfunction can trigger MGO toxicity and AGEs accumulation, and that sufficient SOD and GSH can ensure mitochondrial protection from oxidative damage by the byproduct ROS produced by the respiratory chain during oxidative phosphorylation. Therefore, the effective or effective GRb composition systematically improves the antioxidant capacity of cells, and particularly enhances the powerful effects of increasing GSH and SOD, and enhances glycolysis and oxidative metabolic coupling so as to reduce the generation of MGO and enhance the detoxification effect, thereby supporting the medical application of the composition in delaying senility.

In the embodiment of the invention, the GRb-PQ2, GRb-PQN4, GRb-PQN1 and GRb-PQN3 are used together with insulin, and have the drug effects of improving polydipsia, polyphagia and polyuria symptoms of type I diabetes and reducing the blood sugar level, obviously reducing the glycosylated hemoglobin level, promoting the drug effect of repairing skin damage of diabetic mice, relieving the drug effect of renal function degradation of the diabetic mice, reducing the drug effect of the blood sugar level of the type I diabetes mice, prolonging the acute blood sugar reducing time of the insulin, avoiding the large fluctuation of the blood sugar level during multiple daily times of administration, improving the protein metabolic disorder of the diabetes and relieving the renal function degradation of the diabetic mice.

The invention provides application of the Specific Active Ginsenoside Composition (SAGC) in preparing a health product for improving one or more of insulin resistance state, diabetes metabolic disorder state and diabetes complication.

In the present invention, the specific active ginsenoside composition has pharmacological activities of widely regulating metabolism and correcting metabolic disorders and malignant consequences thereof, has novel therapeutic principles of fundamentally preventing and treating diabetes and comprehensively treating diabetic metabolic disorder syndromes and various complications thereof, SAPDSC (also the active core of SAGC) can alleviate insulin resistance state in type I and type II diabetes and improve metabolic disorder state and malignant consequences thereof in diabetes by non-hypoglycemic dependent mechanisms (including but not limited to: activating NAD (coenzyme I), i.e., the regulation mechanism of cellular metabolism and function, increasing NADP, i.e., the antioxidant mechanism of coenzyme II and glutathione, and correcting ATP deficiency to normal level in diabetes), and can exert powerful and stable hypoglycemic effects in coordination with insulin or metformin, while the hypoglycemic agent can also strengthen the effects of SAPDSC in activating NAD, NAPD and glutathione mediated metabolic regulation and antioxidant stress injury and generate more comprehensive and powerful effects in correcting type I and type II diabetes metabolic disorders (including but not limited to: glucose metabolic disorders, protein balance, protein metabolism disorders, SAPDSC can generate negative pharmacodynamic metabolic disorders: can improve the health state, life and work quality of diabetics comprehensively, can relieve diabetic metabolic disorder syndromes (including but not limited to more than three and less than one symptoms, namely diuresis, polydipsia, polyphagia and weight loss, fatigue and weakness, malaise, sleep disorder and skin itch) and prevent and treat various diabetic complications (including but not limited to diabetic skin diseases, diabetic neuropathy, diabetic cardiomyopathy and vasculopathy, diabetic nephropathy and diabetic feet), and the stability and strength of the drug effects are further improved by combining SAPDSC with insulin or metformin.

In the present invention, the population served by the nutraceutical preferably comprises at least one of: pre-diabetic populations, metabolic syndrome populations, populations with metabolic syndrome complicated with polycystic ovary, populations facing drug-induced toxicity characterized by metabolic disorders or oxidative stress damage, and elderly populations with metabolic syndrome. The population that is subject to drug-induced toxicity characterized by metabolic disorders or oxidative stress injury preferably includes, but is not limited to: anti-tumor drug therapy results in cardiotoxicity and peripheral neuropathy, drug therapy results in metabolic disorders. As a health care product, the specific active ginseng total saponin composition can improve the metabolic disorder signs (including but not limited to hyperlipidemia and hypertension) of pre-diabetic people and metabolic syndrome people and the common sub-health state and/or the individual negative experience (including but not limited to fatigue and weakness, malaise, reduction of work efficiency and capacity, dizziness and sleep disorder), prevent the appearance of the sub-health state caused by metabolic syndrome symptoms, improve the metabolic state of polycystic ovary patients and prevent the progression of the metabolic disorder, thereby playing a role of adjuvant therapy, prevent or relieve cardiotoxicity and peripheral neuropathy caused by metabolic disorder or oxidative stress injury caused by antitumor drug treatment, prevent or slow down the metabolic disorder caused by glucocorticoid drug, immunosuppressant and antipsychotic drug treatment, and reduce the risk of cardiovascular diseases and neurodegenerative diseases (including but not limited to Alzheimer's disease, vascular dementia and Parkinson's syndrome) of the aged metabolic syndrome people.

In the invention, the application is preferably that the specific active ginsenoside composition is combined with at least one of nutrients, metabolism regulating substances and medicinal and edible traditional Chinese medicines to prepare health care products. The nutrients preferably include at least one of: proteins, amino acids, vitamins, metabolic intermediates. The metabolic regulation substance preferably comprises an NAD precursor.

In the present invention, the medicament preferably further comprises a pharmaceutically acceptable excipient or carrier. The dosage form of the medicament preferably comprises at least one of: oral formulations, injectable formulations, and microneedles. The oral formulation preferably includes solid formulations and liquid formulations, the solid formulation preferably including but not limited to: capsule, common tablet, dispersible tablet, enteric coated tablet, granule, pill, enteric coated preparation, controlled release preparation, and capsule containing pellet and small tablet. The method for preparing the drug is not particularly limited, and the method for preparing the drug well known in the field can be adopted.

The invention provides a health care product for improving diabetes, diabetic metabolic disorder and diabetic complication, which comprises the specific active ginsenoside composition as the only active component or the specific active ginsenoside composition and hypoglycemic drug combined as the active components. The hypoglycemic agent comprises insulin or metformin.

In the present invention, the health product preferably further comprises an acceptable excipient or carrier. The dosage form of the health care product preferably comprises a solid preparation and/or a liquid preparation. The solid formulation forms preferably include, but are not limited to: granule, capsule, common tablet, dispersible tablet and pill.

The specific active panaxadiol saponin composition for preventing and treating diabetes and its complications, and the preparation method and application thereof provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1. Synthesis of Panax quinquefolium and its total saponins from stems and leaves two panaxadiol saponin compositions GRb-PQ1 and GRb-PQ2 with the same constituents but different content configurations were prepared.

The method comprises (1) preparing GRb-PQ1 composition from mixture of radix Panacis Quinquefolii root total saponin 1 part in 2020 and radix Panacis Quinquefolii stem and leaf total saponin 2 part in 2020; (2) The GRb-PQ2 composition is prepared from a mixture of 1 part of American ginseng root total saponin in 2016 and 2 parts of American ginseng stem and leaf total saponin in 2016.

The specific preparation method comprises the following steps: 30 g of each of the mixtures described in the dosage protocol were dissolved in 300 ml of 30% aqueous ethanol, and the solution was applied to a reversed-phase C18 silica gel (ODS, 300 g) column equilibrated with 30% ethanol. Eluting with 2.0L of 30% ethanol aqueous solution, discarding 30% ethanol eluate, eluting with 3.0L of 43% ethanol aqueous solution, collecting one part of eluate per 500 ml, collecting 6 fractions (Fr.1-Fr.6), eluting with 6.0L of 55% ethanol aqueous solution per 500 ml, collecting one part per 500 ml, and collecting 10 fractions (Fr.7-Fr.18). The components are detected by High Performance Liquid Chromatography (HPLC), and the components (Fr.7-Fr.15) containing the panaxadiol saponins GRb1, GRc, GRb2, GRb3 and GRd are combined and concentrated under reduced pressure until the mixture is completely dried, so that the panaxadiol saponins composition GRb-PQ1 (14.99 g, 49.96% yield) and GRb-PQ2 (15.77 g, 52.57% yield) are respectively obtained. The content of five ginsenoside components of GRb1, GRc, GRb2, GRb3 and GRd and the total content thereof in each panaxadiol saponin composition are measured by an HPLC method, and the measurement result (Table 1) shows that the total content of five ginsenoside components in the two panaxadiol saponin compositions is more than 93 percent, but the content of each single panaxadiol saponin in each composition is different, particularly the content of each single component in the GRb-PQ1 composition is greatly different, the highest GRb1 content is 35.37 percent, the second GRd content is 24.09 percent, and the lowest GRb2 content is only 6.6 percent; the content of five panaxadiol saponins in the GRb-PQ1 composition is relatively close to 16.13-24.46%, and particularly the content of four saponin components of GRb1, GRc, GRb2 and GRd is very close to 16.13-18.29%.

TABLE 1 content of Individual Panaxadiol saponins in Panaxadiol saponin compositions and their Total content (%)

Example 2 compositions of the same panaxadiol saponins in different proportions have different activities in resisting diabetes and its complications (the potency of GRb-PQ2 is better than that of GRb-PQ 1).

Example 2.1 in combination with insulin, GRb-PQ2 is superior to GRb-PQ1 in ameliorating polydipsia, polyphagia, and polyuria symptoms of type I diabetes and lowering blood glucose levels.

The research method comprises the following steps:

a single intraperitoneal injection of streptozotocin (150 mg/kg) is used for creating an ICR mouse type I diabetes model, the increase of blood sugar level of animals in the model is caused by the lack of insulin level due to the damage of islet beta cells, and the diabetes complications caused by the model are related to continuous hyperglycemia and insulin signal loss. This provides an opportunity to examine the effect of the test drug on the metabolic state and complications of diabetes and its mechanism of action from multiple perspectives. First, the model was used to compare the improvement effect of two GRb compositions in combination with insulin on the pathological features of type I diabetic animals, such as polydipsia, polyphagia, polyuria and weight loss. Then, 35 eligible mice were divided into a normal animal control group, a type I diabetes model group, an insulin group (2.5 IU/kg), a combination of insulin and a low dose GRb-PQ1 composition (2.5 IU/kg +10 mg/kg), a combination of insulin and a high dose GRb-PQ1 composition (2.5 IU/kg +20 mg/kg), a combination of insulin and a low dose GRb-PQ2 composition (2.5 IU/kg +10 mg/kg), and a combination of insulin and a high dose GRb-PQ2 composition (2.5 IU/kg +20 mg/kg), with 5 animals per group. Insulin is injected once by subcutaneous injection, GRb-PQ1 and GRb-PQ2 compositions are administered once by intragastric administration once every day during the period of 8. The weight, diet and water consumption of each group of animals was recorded daily and the wetness level of the animal feeding bedding was photographed to reflect the urine output of the animals. In addition, the blood glucose test strips measure blood glucose excursions within 3 hours after drug administration to understand the direct effect of drug treatment on blood glucose levels; measuring fasting blood glucose and random blood glucose of the mice after being administrated for 24 hours by using blood glucose test paper every week, and knowing the stable change of the blood glucose level possibly generated by the medicine; while monitoring the dynamic changes in blood glucose levels that may occur over the course of treatment.

Results and discussion of the study:

2.1A, when the composition is used together with insulin, the drug effect of GRb-PQ2 on improving polydipsia, polyphagia and polyuria symptoms of type I diabetes is better than that of GRb-PQ 1.

As shown in tables 2 to 6 and FIG. 1, the diabetic model animals exhibited typical symptoms of "more than three and one less" of metabolic disorders in clinical type I diabetic patients, and the mice in the diabetic model group were significantly lower in body weight than the normal group, while the water intake and urine output were significantly higher than those of the normal animals.

The combination treatment of the insulin and the GRb composition can obviously reduce the food intake, water intake and urine output of diabetic animals, but has no improvement effect on the weight loss of diabetes, wherein the treatment effect of the insulin and the GRb-PQ2 with the concentration of 10mg/kg is more durable (tables 2 to 6). Within 24 hours, the bedding for normal animals was substantially dry, while the bedding for model group animals was 80% wet. The animals bedding humidity was not significantly improved in the insulin alone group compared to the model group, whereas the animals bedding humidity was significantly improved in the combination of insulin and GRb composition compared to the model group, with the insulin being most dry with the 10mg/kg GRb-PQ2 combination bedding (fig. 1). It is emphasized that the improvement in the various indices remains effective over the course of treatment, indicating that the improvement in these indices is a stable effect of the combination therapy of GRb composition and insulin, a pharmacodynamic response in which the metabolic state is qualitatively changed. Therefore, research results support the medical application of the GRb composition and insulin in combined prevention and treatment of diabetes.

TABLE 2 GRb composition combined with insulin does not restore weight (g) in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing difference significance division between a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p <0.05, n =5 compared to the normal control group.

TABLE 3 GRb composition in combination with insulin for reducing food intake (g) in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing difference significance division between a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to normal control group<0.01; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

table 4 GRb composition in combination with insulin increases weight to feed ratio in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing difference significance division between a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to normal control group <0.01; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

TABLE 5 GRb composition in combination with insulin to lower water intake (g/mouse/24 h) in type I diabetic mice

TABLE 6 GRb composition in combination with insulin reduces urine volume (g/mouse/24 h) in type I diabetic mice

Note: all ofData are presented as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to normal control group<0.01; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

the GRb composition and insulin are used together, so that the fasting blood glucose level of a type I diabetic mouse can be reduced, the blood glucose reducing time of the insulin can be obviously prolonged, the phenomenon of insulin resistance along with the prolonging of the treatment time course can be prevented, and the drug effect of GRb-PQ2 is superior to that of GRb-PQ 1.

2.1B1, combined with insulin, the drug effect of GRb-PQ2 in reducing fasting blood glucose of type I diabetic mice is superior to that of GRb-PQ 1.

After Streptozotocin (STZ) is injected into a large dose of a mold, the average fasting blood glucose level of the model group animals is more than 16.7mmol/L, which proves that the mold is successfully formed. To determine whether the blood glucose levels of the animals in each group had improved, fasting blood glucose and random blood glucose levels were determined 24 hours after the previous dose. As shown in Table 7, the fasting blood glucose levels in the model animals gradually increased with the course of disease, and the chronic treatment with insulin did not slow down the increase, whereas each of the dose groups of GRb-PQ1 or GRb-PQ2 and insulin treatment showed a slower fasting blood glucose increase, and the fasting blood glucose levels in each of the combination treatment groups other than the high dose group of GRb-PQ1 were significantly lower than the model on average at the 3 week treatment completion; in particular, at the completion of 4 and 5 weeks of treatment, fasting blood glucose levels of the 10mg/kg GRb-PQ2 and insulin combination treatment group continued to be significantly lower than the model group and significantly lower than the insulin group. However, the randomized (actually postprandial) blood glucose levels and urine blood glucose levels of the model group and the various treatment groups did not differ significantly from those of the untreated model group, and therefore no experimental data were shown. Since a novel antidiabetic dapagliflozin tablet reduces blood glucose levels by promoting the kidney to excrete glucose from the urine, our data indicate that diabetic mice will clear a portion of their blood glucose by urination, while insulin and GRb combination, and both, will not affect the hyperglycemic organism's response to reducing blood glucose levels by urine.

TABLE 7 GRb compositions in combination with insulin for lowering fasting plasma glucose (mM) in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to normal control group<0.01; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

2.1B2.GR-PQ2 has better drug effect for prolonging the hypoglycemic time of insulin and preventing the insulin resistance along with the prolongation of the treatment time course than the drug effect of GRb-PQ 1.

In clinical application, insulin has the blood sugar fluctuation that blood sugar rapidly decreases and then rises after administration, and the phenomenon that the blood sugar reducing effect gradually weakens along with the prolonging of treatment time, so the research specially carries out experimental design aiming at the problems: after 12 hours from the previous administration, the mice were fasted for 12 hours, fasting blood glucose was measured, blood glucose was measured after 30 minutes from each administration of the composition, blood glucose was measured again after 30 minutes from feeding, and then blood glucose levels were measured every 1 hour (120 minutes after administration in the test procedure of week 5, each group was given the corresponding drug again), and the measurement was continued twice and the area under the 3-hour blood glucose curve was calculated. As shown in FIG. 2 and Table 8, the acute hypoglycemic efficiency of insulin gradually decreased with the duration of treatment, which was shown to decrease to the minimum blood glucose level from 10.95. + -. 1.09mM in the first week to 12.57. + -. 0.52mM in the 4 th week (Table 9), and from 74.23. + -. 3.37 to 87.57. + -. 3.98 in the area under the curve of relative blood glucose; in sharp contrast to insulin alone, GRb-PQ1 and GRb-PQ2 not only abolished insulin resistance over a prolonged treatment period, but also significantly prolonged blood glucose lowering duration (FIG. 2, D and Table 8), and the low dose (10 mg/kg) of GRb-PQ2 combination was best combined with insulin (FIG. 2).

TABLE 8 area under the blood glucose curve (AUC, 0-3 hours)

Note: all data are expressed as mean ± SD; analyzing the group difference of the model group or the treatment group and the normal control group by using a t test, and performing multiple groups of sample difference significance scores except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to normal control group<0.01; in comparison to the model set, ^# p<0.05；n＝5。

TABLE 9 minimum blood glucose level (mM)

Note: all data are expressed as mean ± SD, n =5. Analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing difference significance division between a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; compared with the group of insulin, the insulin-containing composition, ^& p<0.05， ^&& p<0.001。

2.1C. in combination with insulin, GRb-PQ2 significantly reduced glycosylated hemoglobin levels, whereas GRb-PQ1 only had a tendency to decrease.

The level of glycosylated hemoglobin (GHb, where HbA1c content is the largest) is directly proportional to the mean blood glucose concentration at the first 4 to 12 weeks, so the HbA1c level is used to reflect the mean plasma glucose concentration over time (4 to 8 weeks) and may also be evaluated to monitor the therapeutic effect of long-term blood glucose regulation. As shown in Table 10, 1/2 of the clinical treatment dose of insulin treatment failed to reduce HbA1c level for 5 weeks, GRb-PQ1 (10 mg/kg) tended to reduce HbA1c level when used with insulin, while both GRb-PQ2 doses (10 and 20 mg/kg) when used with insulin (2.5 IU/kg) significantly reduced HbA1c level, and were better at low doses, with HbA1c levels (52.21. + -. 2.15 mM) close to normal (49.28. + -. 1.82 mM). The results of the study further demonstrate that GRb compositions, particularly GRb-PQ2, in combination with low doses of insulin without overall hypoglycemic effects can synergistically exert their significant hypoglycemic effects. Clinical trials of chronic complications of diabetes have shown that intensive insulin treatment reduces HbA1c to 53mmol/mol (77 mmol/mol in the untreated group) in patients, reducing the risk of retinopathy, nephropathy and neuropathy by 35-70%. Therefore, the research results support the medical application of the GRb composition, particularly GRb-PQ2 and insulin in preventing and treating diabetes complicated by retinopathy, nephropathy and neuropathy.

TABLE 10 Effect of GRb-PQ1 and GRb-PQ2 in combination with insulin on glycosylated hemoglobin (HbA 1 c) levels

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and a normal control group by using a t test, and performing difference significance division among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to normal control group<0.05，**p<0.01，***p<0.005; in comparison to the model set, ^# p<0.05；n＝5。

taken together, the results of the above studies show that GRb-PQ2, in combination with 1/2 of the therapeutic dose of insulin (almost ineffective in this study), not only can lower fasting blood glucose levels in type I diabetic mice, but also can significantly prolong the acute hypoglycemic time of insulin and prevent the occurrence of insulin resistance phenomenon with the prolongation of the treatment time course, so that glycosylated hemoglobin can be maintained at a level close to normal. Insulin resistance not only hinders the clinical application of insulin to effectively treat type I diabetes, so that many patients often suffer from various complications such as retinopathy, nephropathy, neuropathy, cardiovascular disease and the like, but also often suffer from obesity and type II diabetes, and therefore, improving insulin resistance is important for preventing the occurrence of diabetes, controlling blood sugar of the diabetes and preventing the complications. Therefore, the research results support the medical application of the panaxadiol saponin composition GRb-PQ2 and other effective GRb compositions in combination with insulin to the treatment of type I diabetes mellitus and type II diabetes mellitus accompanied with the reduction of the level of insulin and complications of the diabetes mellitus, and also support the medical application of the GRb-PQ2 and other effective GRb compositions in the prevention and treatment of other types II diabetes mellitus and complications of the type II diabetes mellitus. It is also noted here that insulin resistance is a major underlying mechanism leading to "metabolic syndrome" (also known as insulin resistance syndrome, including obesity, hyperglycemia, hyperlipidemia, and hypertension), and is also an important risk factor for cardiovascular disease and increases its morbidity and mortality. Therefore, our findings also support the medical use of GRb-PQ2 and other effective GRb compositions for the prevention and treatment of "metabolic syndrome" and cardiovascular diseases.

Example 2.2 combination with insulin, GRb-PQ2 has a better effect in promoting the repair of skin lesions in diabetic mice than GRb-PQ 1.

The research method comprises the following steps:

in order to reveal the potential medicinal value of the GRb composition and the combined treatment of the GRb composition and insulin for preventing and treating diabetic foot, the wound repair condition of animals in each test group is observed by using a diabetic chronic comprehensive wound model. After 2 weeks of treatment (when the course of type I diabetes is 4 weeks), the animals were anesthetized with sodium pentobarbital, and the longer hair on the back of the mice was treated with electric hair clippers, followed by further removal of the back hair with depilatory cream. Then, the skin on the back was sterilized with iodophor, a 2 × 2cm full-thickness wound surface of skin was created with scissors under aseptic conditions while on the back, cut to the subcutaneous fascia, and the wound skin was covered with gauze after being sterilized with alcohol. Mice were allowed free diet and water after surgery, and each group of animals continued to be given the corresponding medication. Wound healing was recorded as photographs taken on the day of skin injury, i.e., before treatment (day 0) and on

days

3, 7 and 14 after treatment, and the wound area of each group was counted.

Results and discussion of the study:

as shown in FIG. 3, the healing rate of the wound was significantly higher in the 2 dose groups treated with GRb-PQ2 in combination with insulin than in the injury control group, and the drug effect of the low dose GRb-PQ2 (10 mg/kg) was better than that of the high dose GRb-PQ2 (20 mg/kg) at the start of treatment day 3; insulin alone or in combination with GRb-PQ1 is not effective in promoting wound healing. The research results support the medical application of GRb-PQ2 and other effective GRb compositions in preventing and treating diabetic foot.

In particular, the research results also show that the GRb composition consisting of the same single panaxadiol saponin has the effect of promoting wound healing only under the condition of certain or a plurality of specific content configurations, thereby further prompting the complexity of the interaction between the activities of different single panaxadiol saponin components in the GRb composition and the key effect on the overall drug effect of the composition, and scientifically utilizing the interaction between different active components to develop the potential of great medicinal value of the panaxadiol saponin composition for preventing and treating diabetes and complications thereof.

Example 2.3 combination with insulin, GRb-PQ2 has better effect in slowing the deterioration of renal function of diabetic mice than GRb-PQ 1.

The research method comprises the following steps:

in order to reveal the potential medicinal value of the combination therapy of the GRb composition and the hypoglycemic agent on the prevention and treatment of diabetic nephropathy, the influence of the tested drug on various biochemical indexes indicating the renal function in urine and blood of various groups of animals is detected. Urine from mice 24 hours after administration was taken at

weeks

2, 3, 4 and 5 of treatment to determine levels of relevant indices of renal function (urea nitrogen and creatinine); after 5 weeks of treatment, mice were euthanized and blood urea nitrogen and creatinine levels were measured.

Results and discussion of the study:

as shown in table 11, the urea nitrogen level in urine of the model animals increased sharply with the increase of the disease course, and the urea nitrogen level in blood at the end point of the experiment (i.e., the disease course at week 7) was also significantly higher than that of the normal control group; 1/2 clinical treatment dose of insulin reduced urea nitrogen levels in urine only at the 4 week treatment sampling point, and the low dose group of GRb-PQ1 in combination with insulin reduced urea nitrogen levels in urine but not blood levels at each sampling point beginning at week 3; both doses of GRb-PQ2 with insulin significantly reduced the urea nitrogen levels in urine and blood at each sampling point beginning at week 3, especially at a dose of 10mg/kg to maintain normal levels of urea nitrogen in the blood of diabetic mice. These results demonstrate that GRb-PQ2 is significantly superior to GRb-PQ1 in efficacy, and that the efficacy at low doses (10 mg/kg) is superior to that at high doses (20 mg/kg). It is also noted herein that urea nitrogen is a metabolite of protein, and that increased urea nitrogen levels in the urine and blood at the end of the test, over the course of disease in diabetic mice, reflect excessive degradation or inefficient utilization of diabetes-related proteins, and that increased urea levels in the blood at this stage are not associated with renal function. It can be seen that GRb-PQ2 combined with insulin can significantly improve the protein metabolism of type I diabetic mice.

TABLE 11 GRb compositions in combination with insulin reduce the urine and blood urea levels (mM) in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing difference significance division between a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.05,**p<0.01，***p<0.005; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

as shown in table 12, creatinine levels in urine decreased to about 1/2 of those in normal animals starting at week 2 (week 4 of disease) of the untreated model group, while creatinine levels in blood were significantly higher than those in normal animals at week 5 (i.e., week 7 of disease) of the end point of the experiment. It can be seen that at the end of the experiment, untreated type I diabetic animals had a reduced ability to undergo renal glomerular filtration and to exclude creatinine from the blood. The creatinine level in urine of each sampling point in the insulin treatment group is close to or even higher than the normal level, and the creatinine level in blood is close to the level of the model group at the end point of the experiment, so that the insulin can promote the generation of creatinine in the type I diabetic mice and protect the kidney of the type I diabetic mice to remove the creatinine in the blood through the urine to a certain extent. It is noteworthy that GRb-PQ1 and GRb-PQ2, when used in combination with insulin, significantly increased the creatinine level in urine of diabetic mice starting at 3 weeks of treatment, and in particular, the two dose groups of GRb-PQ2 also significantly decreased the creatinine level in blood, and the 10mg/kg dose maintained the blood creatinine level at that of normal animals, whereas the corresponding two dose groups of GRb-PQ1 did not significantly decrease the creatinine level in blood. It can be seen that the drug effect of GRb-PQ2 on the glomerular filtration function of the diabetic mice is superior to that of GRb-PQ 1. The research results prove that the toxin expelling function of the kidney of a diabetic mouse can be protected by combining GRb-PQ2 and insulin, and even the existing functional deficiency can be improved, so that the medical application of the GRb-PQ2 and other effective GRb compositions in combination with insulin in preventing and treating diabetic nephropathy complications is supported.

TABLE 12 combination of GRb composition and insulin for lowering creatinine levels (mM) in blood in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing difference significance division between a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.05; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

example 2.4 analysis of the relationship between the content distribution of the main ginsenoside components of GRb-PQ1 and GRb-PQ2 and the pharmacological effects.

The experimental data of examples 2.1 to 2.3 show that the efficacy of GRb-PQ2 against type I diabetes is significantly better than that of GRb-PQ 1. As can be seen from Table 1, the content difference of five ginsenosides in the two compositions of GRb-PQ1 and GRb-PQ2 is large, wherein the content of GRb1 and GRd in GRb-PQ1 is very high and is respectively 35.37% and 24.09%, the content of GRc and GRb3 is similar and is respectively 13.19% and 13.99%, and the content of GRb2 is only 6.60%; in GRb-PQ2, the content of GRb3 is the highest (24.46%), and the content of other four saponins is closer and between 16.13 and 18.29%. The increase in GRc, GRb2 and GRb3 and the decrease in GRb1 and GRd (especially GRb 1) content in GRb-PQ2 compared to GRb-PQ1 indicates that the appropriate ratio of the individual panaxadiol saponins in the GRb composition is critical to the efficacy of GRb-PQ 2. In order to further visually analyze the relationship between the content distribution of the five panaxadiol saponin components constituting the GRb composition and the antidiabetic efficacy of the composition, we performed a tabular analysis with respect to the main efficacy index (table 13). GRb1 is known to be converted to GRd in the gut by the gut flora, thus grouping GRb1 and GRd as the same functional unit; since the results of the study of example 2 have indicated that increasing the levels of GRc and GRb3 in the GRb composition significantly enhances the anti-diabetic efficacy, there is reason to group GRc and GRb3 as another functional unit. Accordingly, the applicant has set the difference and ratio of 7 parameters including (Rc + Rb 3) and (Rb 1+ Rd) and the ratio of GRb1/GRd, GRc/GRb3, GRb3/GRb1, GRc/GRb1 and GRb1/GRc/GRb2/GRb3/GRd (hereinafter referred to as 7 parameters) to reveal the relationship between the content configuration and the drug effect of the main single panaxadiol saponin in the GRb composition. Based on the mathematical statistics of the indices, p >0.05 is defined as invalid (-), p <0.05 is defined as valid (+) and p <0.01 is defined as very valid (+ +).

TABLE 13 relationship between the content of panaxadiol saponins and the drug action in different panaxadiol saponins compositions

As can be seen from tables 1 and 13, based on GRb-PQ1, when the content of GRc and GRb2 is increased to make the content of five types of panaxadiol saponins in GRb-PQ2 very close (wherein the content ratio of the four types of saponins, GRb1/GRc/GRb2/GRd, is close to 1), the GRb composition has significant anti-diabetic efficacy. Therefore, we can consider that there may be better and various content configurations for the antidiabetic, superior GRb composition, for which we carried out the study work of example 3 in hopes of confirming this possibility.

Example 3. Preparation of Panaxadiol saponin compositions GRb having the same Panaxadiol saponin content but different formulations of the total saponins from roots and stems of Panax species.

Example 3.1A Panaxadiol saponin composition GRb-PN rich in GRc and GRb3 was prepared from the total saponins of stem and leaf of Panax notoginseng.

Panax notoginseng (Panax notoginseng) stems and leaves of Panax genus contain multiple panaxadiol saponins, wherein GRc and GRb3 are the most abundant, so that the panaxadiol saponins composition rich in GRc and GRb3 is prepared from Panax notoginseng stem and leaf total saponins, and is called as GRb-PN composition. The specific preparation method comprises the following steps: 30 g of total saponins in stem and leaf of Panax notoginseng are dissolved in 300 ml of 30% ethanol aqueous solution, and the solution is loaded into a reversed phase C18 silica gel (ODS, 300 g) chromatographic column equilibrated with 30% ethanol. Eluting with 2.0L of 30% ethanol aqueous solution, discarding 30% ethanol eluate, eluting with 3.0L of 43% ethanol aqueous solution, collecting one part of eluate per 500 ml, collecting 6 fractions (Fr.1-Fr.6), eluting with 6.0L of 48% ethanol aqueous solution per 500 ml, collecting one part per 500 ml, and collecting 10 fractions (Fr.8-Fr.18). Detecting each component with High Performance Liquid Chromatography (HPLC), mixing components (Fr.8-Fr.16) containing panaxadiol saponins GRb1, GRc, GRb2 and GRb3, concentrating under reduced pressure to completely dry to obtain panaxadiol saponins GRb-PN (12.06 g, yield 40.20%). The content of the four ginsenoside components GRb1, GRc, GRb2 and GRb3 and the total content thereof in the panaxadiol saponin composition GRb-PN were measured by HPLC method, and the measurement results (Table 14) show that the total content of the four ginsenosides in the panaxadiol saponin composition GRb-PN is 91.11%, wherein the content of GRc and GRb3 is high, 32.44% and 42.90% respectively.

TABLE 14 content of Individual Panaxadiol saponins and their Total content (%)

Example 3.2 preparation of Panaxadiol saponin compositions GRb-PQN1 to GRb-PQN6 having the same ginsenoside component but different content ratios by using GRb-PQ2 and GRb-PQ1 as precursors and adding GRb-PN in different ratios.

Adding 0.1 part of GRb-PN on the basis of 1 part of GRb-PQ2 to obtain a new GRb-PQN1 composition; to 1 part of GRb-PQ1, 0.5 parts, 0.6 parts, 0.7 parts, 0.8 parts and 0.9 parts of GRb-PN were added to obtain novel compositions of GRb-PQN2, GRb-PQN3, GRb-PQN4, GRb-PQN5 and GRb-PQN6, respectively. The content of five panaxadiol saponins and their total saponins in each composition was determined by HPLC (table 15) and the content of (GRc + GRb 3) and (Rb 1+ Rd) and 7 parameters including the difference and ratio of (Rc + Rb 3) and (Rb 1+ Rd) and the ratio of GRb1/GRd, GRc/GRb3, GRb3/GRb1, GRc/GRb1 and GRb1/GRc/GRb2/GRb3/GRd in each composition were calculated (table 16).

TABLE 15 content of Individual Panaxadiol saponins and their Total content (%)

TABLE 16 content profile of individual panaxadiol saponins in each panaxadiol saponin composition

Example 3.3 Gentianol saponin compositions with 7 parameters more finely varied were prepared from Panax Ginseng, panax Ginseng stems and leaves, and Panax Notoginseng stems and leaves.

In order to determine the content characteristics of the panaxadiol saponins in the ginseng, the ginseng stem and leaf, the American ginseng stem and leaf and the pseudo-ginseng stem and leaf of the Panax genus (Panax), the content of five panaxadiol saponins (GRb 1, GRc, GRb2, GRb3 and GRd) in the total saponins of the ginseng, the ginseng stem and leaf, the American ginseng stem and leaf and the pseudo-ginseng stem and leaf is determined by an HPLC method, and the difference and the complementarity of the five panaxadiol saponins contained in the total saponins of the ginseng, the ginseng stem and leaf, the American ginseng stem and leaf and the pseudo-ginseng stem and leaf are analyzed. The content of single panaxadiol saponin is higher than or equal to 20%, higher than or equal to 10% and lower than 20%, medium than or equal to 5% and lower than 10%, and low than 5%.

The analysis results are shown in tables 17 and 18. The total content of five panaxadiol saponins in the total ginsenosides is 52.92%, the high-content saponins comprise GRb1 (16.18%), GRc (14.50%) and GRb2 (11.36%), the medium-content saponins comprise GRd (8.39%), and the low-content saponins comprise GRb3 (2.49%). The total content of five panaxadiol saponins in the total saponins of the ginseng stems and leaves is 32.32%, the high-content saponins comprise GRd (14.71%), the medium-content saponins comprise GRb1 (6.21%) and GRb2 (5.28%), and the low-content saponins comprise GRc (4.51%) and GRb3 (1.59%). The total content of five panaxadiol saponins in the American ginseng total saponins is 59.10%, the extra-high content of saponins comprises GRb1 (31.95%), the high content of saponins comprises GRd (11.01%), the medium content of saponins comprises GRc (9.81%), and the low content of saponins comprises GRb2 (3.38%) and GRb3 (3.28%). The total content of five panaxadiol saponins in the total saponin bag of the stems and leaves of the American ginseng is 40.16%, the high-content saponins comprise GRd (16.42%), the medium-content saponins comprise GRb3 (8.32%) and GRb2 (6.52%), and the low-content saponins comprise GRb1 (4.60%) and GRc (4.30%). The total content of five panaxadiol saponins in the total saponins of the stems and leaves of panax notoginseng is 46.78%, the extra-high content of saponins comprises GRb3 (20.15%), the high content of saponins comprises GRc (15.72%), and the low content of saponins comprises GRb1 (4.13%), GRb2 (4.08%) and GRd (2.71%).

TABLE 17 content of five Panaxadiol saponins in total saponins of different herbs of Panax (%, n = 3)

TABLE 18 complementation relationship of ginsenoside as main active ingredient between different herbs in Panax

In order to find out a more effective panaxadiol saponin composition, the complementary characteristics of the contents of each single panaxadiol saponin in the total saponin raw materials of each Chinese medicinal material in the panax are fully utilized, and different total saponin raw materials are prepared into the panaxadiol saponin composition according to the following mixing configuration scheme: (1) Preparing a panaxadiol saponin composition GRb-PGN1 from a mixture of 2 parts of total saponins of panax ginseng, 1 part of total saponins of panax ginseng stems and leaves and 2 parts of total saponins of panax notoginseng stems and leaves; (2) The panaxadiol saponin composition GRb-PGN2 is prepared from the mixture of 2 parts of total ginsenoside, 1.5 parts of total ginsenoside from stem and leaf of Panax ginseng and 1.5 parts of total saponin from stem and leaf of Panax notoginseng.

The specific preparation method comprises the following steps: 30 g of each of the mixtures described in the dosage regimen were dissolved in 300 ml of 30% aqueous ethanol and applied to a column of reversed-phase C18 silica gel (ODS, 300 g) equilibrated with 30% ethanol. The eluate was eluted with 2.0 l of 30% aqueous ethanol, and the 30% ethanol eluate was discarded, followed by elution with 3.0 l of 43% aqueous ethanol, wherein 6 fractions (fr.1 to fr.6) were collected in one portion per 500 ml of eluate, followed by elution with 6.0 l of 55% aqueous ethanol (fr.7 to fr.18) in one portion per 500 ml of eluate, and 10 fractions (fr.7 to fr.18) were collected. The components are detected by High Performance Liquid Chromatography (HPLC), and the components (Fr.7-Fr.15) containing the panaxadiol saponins GRb1, GRc, GRb2, GRb3 and GRd are combined and concentrated under reduced pressure until the mixture is completely dried, so that the panaxadiol saponins composition GRb-PGN1 (13.87 g, the yield is 46.23%) and GRb-PQ2 (12.99 g, the yield is 43.30%) are respectively obtained. The content of five ginsenoside components, i.e. GRb1, GRc, GRb2, GRb3 and GRd, in the ginsenoside compositions GRb-PGN1 and GRb-PGN2 and the total content thereof were measured by HPLC method, and the measurement results (Table 15 and Table 16) showed that the total content of five ginsenosides in the ginsenoside compositions GRb-PGN1 and GRb-PGN2 was 94.78 and 95.25%, respectively, and the content of each single ginsenoside was more than 12%, wherein the content of GRb1, GRc and GRb3 was more than 18.8%, and the content of GRb1 and GRc was more than 21.8%. It can be seen that the content configuration of the panaxadiol saponins in the GRb-PGN1 and the GRb-PGN2 further enriches the diversity of the content configuration of each single active saponin in the panaxadiol saponins composition, so that the panaxadiol saponins composition with better efficacy for preventing and treating diabetes and more effective compositions which may exist are found in the follow-up process.

Example 4 search for possible GRb compositions with better anti-diabetic efficacy and more highly effective compositions.

Example 4.1 combination with insulin, GRb-PQN4 has superior efficacy in ameliorating polydipsia, polyphagia, and polyuria symptoms of type I diabetes and lowering blood glucose over other compositions.

The research method comprises the following steps:

modeling, grouping and dosing: an ICR mouse type I diabetes model is formed by one-time intraperitoneal injection of streptozotocin (150 mg/kg), 40 diabetes mice with qualified blood sugar levels are randomly and uniformly divided into a type I diabetes model control group (model group for short) and a drug treatment group, and the method comprises the following steps: insulin (2.5 IU/kg, corresponding to 1/2 of the clinical treatment dose), insulin and GRb-PQN3 (2.5 IU/kg +10 mg/kg), insulin and GRb-PQN4 (2.5 IU/kg +10 mg/kg), insulin and GRb-PQN5 (2.5 IU/kg +10 mg/kg), insulin and GRb-PQN6 (2.5 IU/kg +10 mg/kg), insulin and GRb-PGN1 (2.5 IU/kg +10 mg/kg), and insulin and GRb-PGN2 (2.5 IU/kg +10 mg/kg), with 5 animals per group, and a normal control group was set. The insulin is injected subcutaneously once and the GRb composition is administered intragastrically once in the morning for 30-30 periods of 8.

Observation of drug efficacy in improving typical symptoms of diabetes and fasting blood glucose: at 2 time points, pre-treatment (week 0) and day 14 of treatment (week 2), specimens were collected for determination of the following experimental data, including: body weight, food intake, water intake and urine output over 24 hours, and random blood glucose, fasting blood glucose was measured after 12 hours of fasting at night. "three more (polydipsia, polyphagia and polyuria) and one less (weight loss)" are typical common symptoms of diabetes, are external manifestations of metabolic disorders, especially glucose metabolism disorders, and the effect of glucose-lowering drugs on improving the symptoms is limited. Therefore, the efficacy of a drug in ameliorating these symptoms also reflects the effect of a drug in ameliorating metabolic disorders of diabetes.

Preparing and adjusting a treatment scheme: the subgroup of GRb compositions was adjusted according to the 2 week treatment effect, with the following specific adjustment schedule: treatment of the active subgroup was continued for 3 weeks; for the non-effective subgroup, treatment was terminated and the corresponding animals were used to observe whether one or all of the 3 effective GRb components (including GRb-PQN4, GRb-PQN1 and GRb-PQ 2) were still effective in the higher-order intervention of diabetes, or to further clarify the pharmacodynamic advantages of PQN4 over PQN1 and PQ2, with the GRb component at constant doses and for a treatment period of 3 weeks. On the last day of 5 or 3 weeks following completion of the continuous treatment, specimens were collected for determination of the following experimental data, including: body weight, food intake, water intake and urine output over 24 hours and random blood glucose levels, fasting blood glucose levels were determined after 12 hours of overnight fasting.

The direct hypoglycemic effect of the effective GRb composition in combination with insulin was observed: in order to determine the effectiveness of the GRb composition in combination with insulin to overcome the rapid onset of acute hypoglycemic effects of insulin leading to hypoglycemia and the rapid rebound of blood glucose after failure, fasting blood glucose levels were resumed immediately after fasting blood glucose level determination and each group of therapeutic agents were administered to determine blood glucose levels in each group of animals at 30, 90 and 150 minutes after administration as compared to observations of insulin alone and an effective GRb composition in combination with insulin.

The observation of the effect of an effective GRb composition in combination with insulin to overcome acute resistance to insulin: it is to be noted here that in example 2.1 we observed the development of resistance phenomena in chronic insulin treatment, which are expressed as: with the prolonging of the treatment course, the acute hypoglycemic effect, namely the maximum hypoglycemic drug effect within 3 hours after the administration is weakened and the maintenance time of the hypoglycemic drug effect is shortened (figure 2), the drug resistance phenomenon of the chronic treatment of the insulin can be remarkably relieved by combining the GRb-PQ1 and the insulin; in contrast, the acute hypoglycemic effect of GRb-PQ2 in combination with insulin was gradually and greatly enhanced with the course of treatment. Therefore, the phenomenon of acute insulin resistance that may occur within the same day is further observed here, as well as the slowing effect of effective GRb compositions on this acute insulin resistance. For this purpose, the blood glucose level was scheduled at 1 hour (210 and 330 minutes in continuous timing) and 2 hours (270 and 390 minutes in continuous timing) after each administration and 3 hours (450 minutes in continuous timing) after the last administration, respectively, at 150 and 270 minutes after the first administration (the last experimental purpose); the area under the curve of blood glucose levels was calculated at 9 time points within 7.5 hours from the start of the first dose to 2.5 hours after the third dose. To see if insulin or combination therapy resulted in blood glucose levels falling below normal (i.e., a hypoglycemic state), a normal control group was also set.

Results and discussion of the study:

4.1A. GRb-PQN4, in combination with insulin, has superior efficacy in ameliorating polydipsia, polyphagia and polyuria symptoms of type I diabetes than GRb-PQN3 and GRb-PQ2, other GRb compositions being ineffective.

Compared with normal animals, diabetic model animals show "more than three and one less" symptoms that are typical clinically for diabetic patients: and the three means: much eaten (table 19), much drunk (table 20) and much urine (table 21), and little means weight loss (table 22) and a reduced ratio of weight to food intake (table 23). Insulin treatment equivalent to a clinical 1/2 therapeutic dose failed to improve the "three more or one less" symptoms for both 2 and 5 weeks; in combination with this dose of insulin, GRb-PQN3 and GRb-PQN4 significantly improved the "three more or one less" symptom in the second week of treatment, and then continued to be effective as the time of administration was extended. Although not able to combat the weight loss in animals, the ratio of body weight to food intake was significantly increased (table 23), indicating that GRb-PQN3 and GRb-PQN4 could increase food availability, which indicates the effect of GRb-PQN3 and GRb-PQN4 in improving the metabolic activity of the ability in type I diabetes. Furthermore, the pharmacological strength of GRb-PQN4 is numerically superior to that of GRb-PQN 3. However, no significant effect was observed for GRb-PQN5, GRb-PQN6, GRb-PGN1 and GRb-PGN2 as a result of various detection indexes. Thus, from the third week on, the animals of these groups received no more treatment than previously but 3 weeks of treatment with GRb-PQN4, GRb-PQN1 and GRb-PQ2, respectively, in combination with insulin to determine whether these compositions were still effective at the higher stage of disease intervention and to further clarify the pharmacodynamic advantages of GRb-PQN4 over GRb-PQN1 and GRb-PQ 2. As shown in Table 24, GRb-PQN4, GRb-PQN1 and GRb-PQ2, respectively, in combination with ineffective doses of insulin significantly improved the "more than three and one less" symptoms in diabetic mice with higher disease grade, and GRb-PQN4 was the most potent, followed by GRb-PQN1. The research results support the medical application of the GRb effective compositions GRb-PQN4, GRb-PQN1 and GRb-PQ2 to the prevention and treatment of 'more than three and one less than three' symptoms of diabetes patients independently, particularly in combination with insulin, and also show that the GRb compositions, particularly the GRb compositions which are respectively combined with insulin can obviously improve the metabolic disorder in the type I diabetes state from an important view point.

TABLE 19 GRb compositions in combination with insulin to reduce food intake (g/mouse/24 h) in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.05，**p<0.01; in comparison to the model set, ^# p<0.05；n＝5。

TABLE 20 GRb compositions in combination with insulin to reduce water intake (g/mouse/24 h) in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p vs control group<0.005；In comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

TABLE 21 GRb compositions in combination with insulin to reduce urine volume (g/mouse/24 h) in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p in comparison with control group <0.01，***p<0.005; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

TABLE 22 GRb compositions in combination with insulin fail to restore weight (g) in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with Duncan multi-range test; p <0.05 compared to control; n =5.

TABLE 23 GRb composition in combination with insulin to increase weight (g) to food intake (g) ratio in type I diabetic mice

Note: all data are expressed as mean ± SD; by usingAnalyzing the difference between the model group or the treatment group and the normal control group by using t test, and performing the difference significance analysis among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) and combining with Duncan multi-range test; p compared to control group<0.01，***p<0.005; in comparison to the model set, ^# p<0.05；n＝5。

TABLE 24 GRb-PQN4 and other effective compositions are still effective in type I diabetic mice treated with ineffective compositions

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group <0.05,**p<0.01，***p<0.005; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

4.1B, combined with insulin, GRb-PQN4 has better effect in lowering blood sugar level of type I diabetic mice than GRb-PQN3 and GRb-PQ2, and other GRb compositions are ineffective.

4.1B1, combined with insulin, the drug effect of GRb-PQN4 in reducing fasting blood glucose of type I diabetes mice is better than that of other compositions.

Before treatment (week 0), the fasting plasma glucose levels in the animals of each group averaged more than 16.7mmol/L, indicating that these animals are in type I diabetes (Table 25). To determine whether the blood glucose levels were improved in the animals of each administration group, fasting blood glucose and random blood glucose levels (i.e., postprandial blood glucose) were measured 24 hours after the last administration. As shown in Table 25, GRb-PQN3 and GRb-PQN4 were both effective in reducing fasting blood glucose levels in type I diabetic mice at the time points of treatment of 2 weeks and 5 weeks, respectively, in combination with a non-effective dose of insulin, and the effect of GRb-PQN4 was numerically stronger than that of GRb-PQN3, while GRb-PQN5 and GRb-PQN6 showed only a trend of efficacy, but neither GRb-PGN1 nor GRb-PGN2 showed any efficacy. However, when these groups received no-treatment for 2 weeks instead of GRb-PQN4, GRb-PQN1 or GRb-PQ2 in combination with insulin at a non-effective dose for 3 weeks, their fasting blood glucose levels were also significantly reduced (Table 26), and the potency of GRb-PQN4 and GRb-PQN1 was comparable. These findings, in combination with the results of improvement of "three more one less" symptoms, demonstrate that GRb-PQN1, GRb-PQN3, GRb-PQN4 and GRb-PQ2 are effective compositions for improving typical symptoms of diabetes and lowering fasting blood glucose levels. Therefore, only these effective compositions are of interest in the subsequent observation of pharmacodynamic indices.

TABLE 25 GRb component in combination with insulin to lower fasting plasma glucose (mM) in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p in comparison to control group<0.005; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the insulin-resistant composition, ^& p<0.05；n＝5。

TABLE 26 GRb-PQN4 and other efficacious compositions remain efficacious in type I diabetic mice treated with ineffective compositions

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p vs control group<0.005; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

4.1B2.GRb-PQN3, GRb-PQN4 and GRb-PQ2 can prolong the acute hypoglycemic time of insulin and avoid the large fluctuation of blood sugar level during multiple daily administration.

To reveal whether effective compositions improve the acute hypoglycemic effect of insulin and slow the acute insulin resistance phenomenon, we designed the following tests: at the end of the trial, i.e. at

week

5 or 3 of treatment, the mice fasted for 12 hours after the last administration, fasting blood glucose (scored as 0 minutes) was determined, and then each group was fed and administered separately, and blood glucose levels were determined 30, 90 and 150 minutes after administration; at 150 and 270 minutes, one more time administration was performed, respectively, with blood glucose level being scheduled at 1 hour (210 and 330 minutes on a continuous basis) and 2 hours (270 and 390 minutes on a continuous basis) after each administration and 3 hours (450 minutes on a continuous basis) after the last administration; the area under the curve of blood glucose levels was calculated for 9 time points from the start after the first dose to 7.5 hours after the third dose. To see if insulin or combination therapy resulted in blood glucose levels falling below normal (i.e., a hypoglycemic state), a normal control group was also set. As shown in FIG. 4, for the insulin monotherapy group, the maximal hypoglycemic effect was obtained 30 minutes after the first dose, but blood glucose had already risen back to the pre-dose level by 150 minutes (2.5 hours), had dropped below normal (5.02 mM) 60 minutes after the second dose (210 minutes on a continuous basis), had risen back to 17.2mM over 60 minutes (270 minutes on a continuous basis), but had only a small drop in blood glucose and then a rapid rise in blood glucose 60 minutes after the third dose (330 minutes on a continuous basis). In contrast, the combination treatment group of insulin with GRb-PQN4, GRb-PQN3, GRb-PQN1 and GRb-PQ2, respectively, did not show a decrease in blood glucose level below the normal level after administration, while the minimum blood glucose level after the two subsequent administrations was also closer to the normal level, and both the blood glucose rate and the peak of the rise were significantly lower than those of the insulin group (a in fig. 4). Consistently, the area under the blood glucose curve was significantly higher for the insulin group than for the combination treatment group (B in fig. 4). Moreover, the drug effect of the effective composition is not obviously different in the whole treatment period of 5 weeks and the treatment period of 3 weeks, and the drug effect of the effective composition for treating the diabetes is not limited by the disease degree.

Combining the above research results, it can be seen that the diabetic patients have large blood sugar level fluctuation during multiple daily insulin administrations, and the problem cannot be solved by increasing the administration frequency, and the combination of the GRb effective composition and insulin can obtain a more stable and longer-lasting blood sugar reducing effect, thereby effectively supporting the use of the GRb effective composition in combination with insulin for treating type I diabetes and type II diabetes with low insulin levels.

Example 4.2 efficacy of GRb-effective composition in combination with insulin for promoting skin lesion repair in diabetic mice, wherein GRb-PQN4 has the strongest efficacy and GRb-PQN1, GRb-PQN3 and GRb-PQ2 have the same efficacy.

The research method comprises the following steps:

the study was carried out on the basis of example 4.1. After 2 weeks of treatment with the drug, the animals were anesthetized with sodium pentobarbital, and the longer hair on the back of the mice was treated with electric hair clippers, followed by further removal of the back hair with depilatory cream. Then, the skin on the back was sterilized with iodophor, a 1.5 × 1.5cm full-thickness skin wound surface was created with scissors under aseptic conditions while on the back, and the wound surface was cut to the subcutaneous fascia, and covered with gauze after alcohol sterilization of the skin around the wound. Mice were allowed free diet and water after surgery, and each group of animals continued to be treated or treated with the corresponding medication. Wound healing was recorded and wound area was counted by taking photographs immediately after skin excision (day 0), on

days

3, 6, 9, 12 and 15, respectively.

Results and discussion of the study:

as shown in FIG. 5, it can be seen from the third day that the skin wound repair rate of the untreated diabetes model group mice is significantly lower than that of the normal control group animals, the healing rate of the skin lesion of the diabetes mice can be significantly accelerated by the single treatment of insulin and the combined treatment of the insulin and each effective composition, and the drug effect of the combined treatment is generally better than that of the single treatment of insulin. From the data on

days

6, 9 and 12, the healing rates were the same for the GRb-PQN4 full treatment group (which had been treated for 2 weeks at the time of skin lesion modeling) and the 3-week post treatment group (which was administered beginning on the day of skin lesion modeling, i.e., day one), and were numerically superior to insulin alone and other efficacious compositions including GRb-PQN1, GRb-PQN3 and GRb-PQ2, but no significant difference was seen between the other efficacious compositions. It is specifically noted that, based on the above-mentioned 4 effective compositions and insulin combination, the same acute hypoglycemic effect (fig. 4) is obtained, which indicates that the reason why the effect of GRb-PQN4 in promoting the skin wound repair of diabetic mice is superior to that of other effective components is independent of the blood sugar level, or that the non-hypoglycemic mechanism is involved in the effect of the effective compositions in promoting the skin wound repair. The results of the studies again show that GRb-PQN1, GRb-PQN3, GRb-PQN4 and GRb-PQ2 are effective compositions for treating diabetes and that GRb-PQN4 has the best efficacy.

Example 4.3.GRb effective compositions in combination with insulin can ameliorate diabetic protein metabolism disorders and slow the deterioration of renal function in diabetic mice.

The research method comprises the following steps:

the study was carried out on the basis of example 4.1. At the end of treatment, at 24 hours after the last dose, urine samples were taken for the determination of urea nitrogen (associated with protein metabolism) and renal function-related indicators including albumin and creatinine levels. At the treatment end point, some groups received 5 weeks of full treatment and some received 3 weeks of treatment (table 27).

Results and discussion of the study:

as shown in Table 27, the urea nitrogen level in the urine of untreated diabetes model group mice was 7.5 times that of the normal control group animals, indicating that these diabetes model mice exhibited protein metabolism disorder. From the urea nitrogen level, insulin treatment can significantly improve protein metabolism disorder of diabetes, and the improvement effect in combination with GRb-PQN4 is further enhanced.

The creatinine level in urine of untreated diabetes model mice was significantly lower than that of normal control mice, but the urine protein level was not different, but the albumin/creatinine ratio was significantly increased. Therefore, the diabetic mice have reduced glomerular creatinine filtration function, and the insulin treatment alone can obviously protect the glomerular creatinine filtration capability of the diabetic mice, but the combined treatment with the effective composition does not show further improvement of creatinine filtration capability.

TABLE 27 GRb effective composition in combination with insulin for improving protein metabolism and kidney function in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.01，***p<0.005; in comparison to the model set, ^# p<0.05；n＝5。

example 4.4. Analysis of the content profile of the major individual components in GRb compositions as a function of potency to obtain quality control criteria for efficacious/effective compositions.

On the basis of the above research results, the 7 parameters proposed in example 2.4, including the difference and ratio of (Rc + Rb 3) and (Rb 1+ Rd) and the ratio of GRb1/GRd, GRc/GRb3, GRb3/GRb1, GRc/GRb1 and GRb1/GRc/GRb2/GRb3/GRd, were used as the characteristics for describing the content configuration of active ingredients in the composition, and the pharmacodynamic data lists (table 28) of examples 2 and 4 were used to visually analyze the relative content configuration among the four types of panaxadiol saponins (GRb 1, GRc, GRb3 and GRd) and the pharmacodynamic relationship of the composition. Judging the effective level, p is greater than 0.05, and is invalid (-) according to the statistical result of experimental data; p <0.05, effective (+); p <0.01, very potent (++).

TABLE 28 correlation of content profile and potency of individual Panaxadiol saponins in GRb compositions

Combining all the research results of the above example 4, it is demonstrated that GRb-PQN1, GRb-PQN3, GR-PQN4 and GRb-PQ2 have significant activity for relieving typical symptoms (three more or one less) of type I diabetes, strengthening and improving the acute hypoglycemic drug effect of insulin, promoting the repair of skin lesions of diabetic animals, improving the metabolic state of diabetic proteins, and protecting the renal function of diabetic mice; GRb-PQN4 is better than other effective compositions in most indexes, so that GRb-PQN4 is further called as an effective composition.

As can be seen from table 28, the formulation of the panaxadiol saponins content in the effective compositions GRb-PQ2, GRb-PQN1, GRb-PQN3 and GRb-PQN4 has the following characteristics: the difference between (GRc + GRb 3) and (GRb 1+ Rd) is positive and between 5.06 and 19.36, and the ratio of (GRc + GRb 3)/(GRb 1+ GRd), GRb1/GRd, GRc/GRb3, GRb3/GRb1 and GRc/GRb1 is respectively between 1.13 and 1.64, 0.94 and 1.65, 0.66 and 0.78, 1.03 and 1.91 and 0.80 and 1.29. However, within these 6 parameters, the content distribution of the ineffective composition occurs, i.e. the effective values of the 6 parameters of the content distribution in the effective composition are not continuous, and no other effective or effective content distribution can be inferred from the 6 parameters of an effective or effective composition. Furthermore, from the ratios of GRb1/GRc/GRb2/GRb3/GRd of these effective compositions, the relative ratio of GRb2 content to GRc content in the composition is between 0.32 and 1.11, so that GRb2 content in the composition can be present in accordance with the natural ratios of the raw materials. Here we provide, but not limited to, the values of GRb1/GRc/GRb3/GRd for the 7 parameter pool of 4 groups, 1.07/1.00/1.52/1.13, 0.77/1.00/1.48/0.73, 1.25/1.00/1.29/0.78 and 1.10/1.00/1.30/0.66, respectively, as content allocation and quality control criteria for the preparation of effective compositions.

In summary, we found that 4 effective compositions, GRb-PQ2, GRb-PQN1, GRb-PQN3 and GRb-PQN4, are active panaxadiol saponin compositions with independent content configuration characteristics, and therefore we subsequently called specific panaxadiol saponin active compositions (SPDSAC, also called GRb effective or effective compositions). Clearly our findings break the conventional wisdom of the skilled person for the use of ginsenoside activities (i.e. concerning the content of total saponins in the product and the additive effect of the activities of different saponins), and reveal that the appropriate ratio between different saponins is the key to the effective use of different saponin activities. The discovery has great significance for the utilization of the medicine and health care which effectively utilizes the biological activity and pharmacological action of the ginsenoside, and can guide us to develop new medicine and health care application of the ginsenoside.

Example 4.5 Synthesis of Panax species for the preparation of GRb effective composition and Specific Active Ginsenoside Composition (SAGC).

Example 4.5A. GRb effective compositions were prepared by controlling the raw material charge ratios and continuously collecting the fractions containing the target components (hereinafter referred to as direct process).

With the effective composition GRb-PQN4 as a preparation target, the following four feeding schemes, but not limited to, are predicted according to the content of each single panaxadiol saponin GRb1, GRc, GRb3 and GRd in each original medicinal material to obtain GRb-PQN4 and an active panaxadiol saponin composition close to GRb-PQN4, namely that the panaxadiol saponin compositions GRb-PQN4 and PQN4 are respectively prepared from a mixture of (1) ' 1 part of ginseng stem and leaf total saponin + 1 part of American ginseng root total saponin + 1 part of American ginseng stem and leaf total saponin + 1.75 parts of panax notoginseng stem and leaf total saponin ', (2) ' 1 part of ginseng stem and leaf total saponin + 1.5 parts of American ginseng root total saponin + 1 part of stem and leaf total saponin + 3 parts of panax notoginseng stem and leaf total saponin ', (3) ' a mixture of ' American ginseng root total saponin 1 part of saponin + 1 part of American ginseng stem and leaf total saponin + 1 part of panax notoginseng saponin + 1 part of stem and leaf total saponin + 1.5 parts of American ginseng stem and leaf total saponin '.

The specific preparation method comprises the following steps: 30 g of each of the mixtures described in the dosage protocol were dissolved in 300 ml of 30% aqueous ethanol, and the solution was applied to a reversed-phase C18 silica gel (ODS, 300 g) column equilibrated with 30% ethanol. Eluting with 2.0L of 30% ethanol aqueous solution, discarding 30% ethanol eluate, eluting with 3.0L of 43% ethanol aqueous solution, collecting one part of eluate per 500 ml, collecting 6 fractions (Fr.1-Fr.6), eluting with 6.0L of 55% ethanol aqueous solution per 500 ml, collecting one part per 500 ml, and collecting 10 fractions (Fr.7-Fr.18). The fractions were analyzed by High Performance Liquid Chromatography (HPLC), and fractions (Fr.7-Fr.15) containing the panaxadiol saponins GRb1, GRc, GRb2, GRb3 and GRd were combined, concentrated under reduced pressure to dryness to give the panaxadiol saponin compositions GRb-PQN4a (13.38 g, yield 44.59%), GRb-PQN4b (13.10 g, yield 43.68%), GRb-PQN4c (13.41 g, yield 44.69%) and GRb-PQN4d (12.34 g, yield 41.14%), respectively. The content of five ginsenoside components, i.e., GRb1, GRc, GRb2, GRb3 and GRd, in each of the panaxadiol saponin compositions GRb-PQN4a, GRb-PQN4b, GRb-PQN4c and GRb-PQN4d and the total content thereof were measured by HPLC method, and the results (table 29 and table 30) showed that the total content of five panaxadiol saponins in the panaxadiol saponin compositions GRb-PQN4a, GRb-PQN4b, GRb-PQN4c and GRb-PQN4d was 90% or more, respectively, with the highest content of GRb3 (25.05 to 26.95%), followed by GRb1 (21.59 to 23.37%), GRc (19.43 to 21.38), GRd (14.08 to 15.75%) and GRb2 (6.57 to 8.08%), which were identical to the content of single ginsenoside in GRb-PQN4 composition and the content of GRb-PQN saponin in the GRb-PQN4 composition was completely identical to that of GRb-PQN4, and the formulated with the highest content of GRb-PQN saponin composition and the same as that of GRb-PQN saponin composition of GRb-PQN4 b-PQN4 and the high-PQN 4 and the formulated with the high content of single saponin composition and the formulated in composition were very similar to the advantageous composition of GRb-PQN4 composition.

TABLE 29 content of individual panaxadiol saponins and total content of five panaxadiol saponins (%)

TABLE 30 content profile of individual panaxadiol saponins in GRb-PQN4 and GRb-PQN4 a-GRb-PQN 4g compositions

Example 4.5B. GRb effective compositions were prepared by mixing 4 targets prepared from mixed charges, according to the respective panaxadiol saponins content of the 4 targets (hereinafter indirect).

The method is characterized in that total ginsenosides derived from ginseng roots, ginseng stems and leaves, american ginseng roots, american ginseng stems and leaves and pseudo-ginseng stems and leaves of Panax genus are used as selectively usable raw materials, the availability and economic cost of the current raw materials, the content of each single saponin of GRb1, GRc, GRb3 and GRd in the raw materials and the content complementation characteristics of the 4 saponins are comprehensively utilized, two or more than two 3 types of total ginsenosides are used as the raw materials, the respective feeding amount is determined by simple calculation and is mixed, and 4 target objects are firstly prepared, namely: GRb1 target, GRc target, GRb3 target and GRd target, which mainly contained GRb1, GRc, GRb3 and GRd, respectively, and the contents of GRb1, GRc, GRb3 and GRd in these 4 targets were analyzed by HPLC. The specific preparation method comprises the following steps: 30 g of the mixture of the determined batch portions were dissolved in 300 ml of 30% ethanol aqueous solution, respectively, and the solution was applied to a reversed phase C18 silica gel (ODS, 300 g) column equilibrated with 30% ethanol. Eluting with 2.0L of 30% ethanol aqueous solution, discarding the 30% ethanol eluate, eluting with 40% ethanol aqueous solution, collecting one part of eluate per 500 mL, eluting with 43% ethanol aqueous solution until the amount of GRb1 or GRb1 is not very low in HPLC analysis, collecting one part of eluate per 500 mL, eluting with 47% ethanol aqueous solution until the amount of GRc or GRc is not very low in HPLC analysis, collecting one part of eluate per 500 mL, eluting with 55% ethanol aqueous solution until the amount of GRb3 or GRb3 is not very low in HPLC analysis, and collecting one part per 500 mL until the amount of GRd or GRd is not very low in HPLC analysis. Detecting the fractions eluted and collected by 40%, 43%, 47% and 55% ethanol by HPLC, combining the fractions mainly containing GRb1, GRc, GRb3 and GRd, concentrating under reduced pressure to completely dry to obtain four target substances, namely GRb1, GRc, GRb3 and GRd.

Then, these four objects mentioned above were quantitatively mixed according to the quality standard of the advantageous composition GRb-PQN4 or other effective composition, i.e., GRb1/GRc/GRb3/GRd ratio, to prepare a GRb effective composition having the same or similar GRb1/GRc/GRb3/GRd ratio as that of the advantageous composition GRb-PQN4 or other effective composition.

GRb-PQN4e, GRb-PQN4f and GRb-PQN4g are three GRb compositions prepared according to the indirect method described above, and HPLC analysis shows: the content of individual panaxadiol saponins in each of the three GRb compositions GRb-PQN4e, GRb-PQN4f and GRb-PQN4g (table 29) and the configuration of the content of individual panaxadiol saponins (table 30) are very similar to those of the advantageous composition GRb-PQN4 and the compositions GRb-PQN4a, GRb-PQN4b, GRb-PQN4c and GRb-PQN4d prepared by the direct method, indicating that indirect methods as described are also viable methods for preparing advantageous or effective compositions of GRb.

Example 4.5C preparation of specific active Total Saponin composition (SAGC)

A Specific Active Ginsenoside Composition (SAGC) is prepared according to four but not limited to these four compounding schemes described in example 4.5A; or mixing a certain amount of Panax Chinese medicinal material (kg) according to a mixing scheme, percolating with 10-1510 times of 60-80% ethanol water solution (liter) for 3 times, and recovering solvent under reduced pressure to obtain Specific Active Ginsenoside Composition (SAGC); then, the contents of two panaxatriol saponins (GRg 1 and GRe) and five panaxadiol saponins (GRb 1, GRc, GRb2, GRb3 and GRd) in the Specific Active Ginsenoside Composition (SAGC) were measured by HPLC analysis method, and the relative proportion of their contents, the total content of five panaxadiol saponins and the total content of seven ginsenosides were calculated. The specific active ginsenoside compositions SAGC-a, SAGC-b, SAGC-c and SAGC-d are four composition products prepared by mixing and formulating according to the four mixing schemes described in example 4.5A. As shown in tables 31 and 32, the total content of seven ginsenosides in the four specific active total ginsenoside compositions was between 52.19-57.58%, wherein the total content of five ginsenosides was between 43.56-46.73%; the content of GRc + GRb3 is always larger than that of GRb1+ GRd, and the respective (GRc + GRb 3)/(GRb 1+ GRd) ratios of SAGC-a, SAGC-b, SAGC-c and SAGC-d, GRb1/GRd ratio, GRc/GRb3 ratio, GRb3/GRb1 ratio, GRc/GRb1 ratio and GRb1/GRc/GRb3/GRd ratio are respectively identical with the respective ratios of GRb effective or effective compositions GRb-PQN4a, GRb-PQN4b, GRb-PQN4c and GRb-PQN4d, and are also very close to the respective ratios of GRb effective composition GRb-PQN 4. The analysis result shows that: the content of each core active panaxadiol saponin contained in the specific active panaxadiol saponin composition (SAGC) is the same as or very close to the content of each corresponding core active panaxadiol saponin in the specific active panaxadiol saponin composition (SAPDSC), so that the specific active panaxadiol saponin composition (SAGC) and the specific active panaxadiol saponin composition (SAPDSC) have the same or similar medical and health care applications in preventing and treating diabetes and diabetic complications and metabolic disorder related diseases thereof.

TABLE 31 Total Saponin compositions with specific activities SAGC-a, SAGC-b, SAGC-c and SAGC-d the content of each individual ginsenoside and the total content of five ginsenoside (%)

TABLE 32 content configuration of Individual Panaxadiol Saponin in specific active ginsenoside compositions SAGC-a, SAGC-b, SAGC-c and SAGC-d

Example 4.5 is summarized and discussed

(1) Compared with the method for preparing the active panaxadiol saponin composition by only using the American ginseng root total saponin and the American ginseng stem leaf total saponin, the method for preparing the active panaxadiol saponin composition by comprehensively utilizing the ginseng and the stem leaf total saponin thereof, the American ginseng and the stem leaf total saponin thereof and the panax notoginseng stem leaf total saponin has remarkable advantages of effectively utilizing limited resources, reducing the preparation cost and obtaining the active panaxadiol composition with excellent effect. The concrete points are as follows: obtaining an active product meeting the product quality standard, wherein the former must be at the cost of discarding a considerable amount of GRb1 and GRd, and the latter skillfully utilizes GRc and GRb3 which are discarded at high proportion in the unused or low-price panax notoginseng stem and leaf total saponin to flush the high content of GRb1 in the panax notoginseng root total saponin and the high content of GRd in the panax notoginseng stem and leaf total saponin; the combined use of the ginseng stem and leaf total saponin, the American ginseng stem and leaf total saponin and the panax notoginseng stem and leaf total saponin can further reduce the use amount of the American ginseng root, thereby further saving limited medicinal material resources and greatly reducing the preparation cost of the product; more importantly, the total saponins of all the medicinal materials of the ginseng are comprehensively utilized, and the product of the high-quality GRb effective composition with the same content and quality as the GRb-PQN4 effective composition is easier to prepare.

(2) The indirect method not only increases the steps of the preparation process but also does not remarkably increase the preparation cost, but also further increases the interconnection and intercommunication among different varieties of the ginseng medicinal materials and between stems, leaves and roots, thereby improving the flexibility of comprehensively utilizing the resources, further being beneficial to improving the economic and medicinal values of the medicinal materials, reducing the raw material cost of the GRb effective composition and overcoming the limitation of resource shortage on the preparation of the GRb effective composition. In particular, the ratio between each of the major effective individual panaxadiol saponins of the target GRb effective or efficacious composition can be precisely achieved.

Example 5 GRb-PQN4 alone or in combination with insulin ameliorates the polydipsia, polyphagia, and polyuria symptoms and lowers blood glucose levels in type I diabetes.

Based on the effective compositions GRb-PQ2, GRb-PQN1, GRb-PQN3 and GRb-PQN4, the effective compositions have the characteristics and advantages of systematically treating metabolic disorders and various symptoms of diabetes compared with hypoglycemic drugs, particularly have great potential value of jointly treating diabetes with the hypoglycemic drugs, in subsequent researches, the effective compositions are further systematically researched by taking GRb-PQN4 as an example, the effective compositions are independently and jointly systematically treating the diabetes with the commonly used hypoglycemic drugs, so that the unique antidiabetic effect of the effective compositions is further confirmed, and a novel action mechanism of the effective compositions is attempted to be disclosed. In that

Thus, GRb-PQN4 alone or in combination with insulin was previously observed to improve the symptoms of polydipsia, polyphagia and polyuria and to reduce blood glucose levels in type I diabetes.

The research method comprises the following steps of modeling, grouping and administration: an ICR mouse type I diabetes model is created by one-time intraperitoneal injection of streptozotocin (150 mg/kg), 40 mice with qualified blood sugar level are randomly and uniformly divided into a type I diabetes model control group, an insulin group (2.5 IU/kg), GRb-PQN4 (10 mg/kg) and a combination group of insulin and GRb-PQN4 (2.5 IU/kg +10 mg/kg), and a normal control group is set, wherein each group comprises 10 animals. The insulin is injected and administered once by subcutaneous injection, the GRb-PQN4 is administered once by intragastric administration, the same amount of physiological saline is injected subcutaneously and the same amount of sterilized water is injected by intragastric administration in 2 control groups every day during the period of 8.

The effect of improving typical symptoms of diabetes and fasting blood glucose levels was observed: at

weeks

0, 2, 4, 7 and 10, the animal weight, diet and water consumption were recorded for each group and the wetness level of the animal feeding bedding was photographed to reflect the animal's urine output. In order to reflect the effect of drug therapy on improving or reversing the metabolic state of diabetic mice rather than the acute effect of drugs on the metabolic state, the fasting blood glucose and the random blood glucose after 24 hours of administration (at the moment, insulin injection is completely ineffective, and the half-life period of GRb-PQN4 is over) are measured by a blood glucose test strip, so that the effect of drug therapy on improving the glucose metabolism of the diabetic mice can be known.

The observation of GRb-PQN4 enhances the acute hypoglycemic effect of insulin and overcomes the blood sugar fluctuation and the drug effect of acute insulin resistance at the interval of daily administration: the blood sugar fluctuation phenomenon that hypoglycemia occurs after the common administration of the clinical insulin treatment and then the blood sugar rapidly rises is achieved, and the insulin dosage required by long-term insulin treatment or disease progression is increased. The results of the studies of example 2 and example 4.1 predict that GRb-PQN4 in combination with insulin can overcome the drastic fluctuation of blood glucose levels and the gradual decrease of drug efficacy due to chronic treatment or disease progression during the first and subsequent dosing intervals of insulin, and to confirm this prediction, the insulin or GRb-PQN4 or both are administered immediately after fasting blood glucose is measured at week 0, i.e., the first day when treatment is started and at day 7 when treatment is 2, 4, 7 and 10 weeks, and then administered once more at

minutes

150 and 270, respectively, and blood glucose levels before administration (0 min), at

minutes

30, 90 and 150 and 60 minutes after the first administration (210 min continuous timing) and at minutes 120 (270 min continuous timing) and at

minutes

60, 120 and 180 minutes after the third administration (330 min continuous timing, 390 min and 450 min continuous timing) are observed and the area under the blood glucose level curve is calculated for the above 9 time points.

End-point sampling and glycosylation level determination: the glycosylated hemoglobin level is directly proportional to the mean blood glucose concentration from the first 4 weeks to 3 months, and thus reflects the mean plasma glucose concentration over a period of time (4 to 12 weeks). Therefore, in order to determine the total blood glucose exposure experienced by the animals during the 5 and 10 weeks of treatment, after the 5 and 10 weeks of measurement of the above-mentioned GRb-PQN4 effect on acute insulin hypoglycaemia, blood was collected and serum samples were prepared for the determination of glycosylated haemoglobin and the associated indices in the subsequent examples. Meanwhile, heart, kidney and muscle samples are collected for the measurement of relevant indexes in the subsequent examples according to the requirements of biochemical measurement and histological analysis.

The research results are as follows:

GRb-PQN4 alone or in combination with insulin ameliorates the polydipsia, polyphagia, and polyuria symptoms of type I diabetes.

As shown in tables 33 to 37 and fig. 6, the diabetic model animals exhibited typical symptoms of "more than three and one less" of metabolic disorders of clinical type I diabetic patients, and the body weights of the mice of the diabetic model group were significantly lower than those of the normal group, while the water intake and urine output were significantly higher than those of the normal group. As shown in table 34, treatment with insulin and GRb-PQN4 alone significantly reduced food intake in diabetic mice during the chronic treatment period of week 10 only at

weeks

2 and 4, while combined treatment significantly reduced food intake during the entire treatment period beginning at week two and also significantly reduced the ratio of food intake to body weight at weeks 7 and 10 (table 35). The same study results as for reduction of food intake were obtained for diabetic mice, with insulin and GRb-PQN4 alone significantly reducing water intake only at

weeks

2 and 4, while the combination treatment significantly reduced water intake throughout (table 36); for polyuria symptoms, insulin and GRb-PQN4 treatment alone showed a tendency to reduce urine output only at

weeks

2 and 4, while both combination treatments significantly reduced urine output throughout (table 37 and fig. 6). In conclusion, the single treatment of insulin and GRb-PQN4 with 1/2 clinical dose can only partially improve the typical symptoms of more than three and one less diabetes mellitus in the early stage of the disease, and the combined treatment of the insulin and the GRb-PQN4 can completely and obviously improve the typical symptoms of more than three and one less diabetes mellitus and is effective continuously for a long time, which reflects the excellent efficacy of the combined treatment in improving the typical symptoms of diabetes mellitus and inhibiting the development of the disease.

TABLE 33 GRb-PQN4 in combination with insulin fails to restore weight (g) in type I diabetic mice

TABLE 34 GRb-PQN4 in combination with insulin reduces food intake (g/mouse/24 h) in type I diabetic mice

Note: all data are expressed as mean ± SD; differences between the model or treated groups and the normal control groups were analyzed using the t-test, and differences between groups of samples other than the normal control groups were performed using one-way analysis of variance (ANOVA) in combination with the Duncan multiple-range testDistinguishing the significance; p compared to control group<0.05，**p<0.01; in comparison to the model set, ^# p<0.05；n＝5。

TABLE 35 GRb-PQN4 in combination with insulin increases the weight (g) to food intake (g) ratio in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with Duncan multi-range test; p compared to control group <0.05，**p<0.01; in comparison to the model set, ^# p<0.05；n＝5。

TABLE 36 GRb-PQN4 in combination with insulin reduces type I diabetic mice water intake (g/mouse/24 h)

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and a normal control by using a t test, and performing the difference significance analysis among a plurality of groups of samples except the normal control by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.01，***p<0.005; in comparison to the model set, ^# p<0.05， ^## p<0.01; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

TABLE 37 GRb-PQN4 in combination with insulin reduces urine volume (g/mouse/24 h) in type I diabetic mice

Note: all data are expressed as mean ± SD; analysis of differences between model or treated groups and normal controls by t-test, analysis of one-way variance (ANOVA) in combinationThe Duncan multi-range test performs a significance analysis of differences between groups of samples except for normal controls; p compared to control group<0.05,**p<0.01; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

the combination of GRb-PQN4 and insulin not only can reduce the fasting blood glucose level of type I diabetic mice, but also can obviously prolong the hypoglycemic time of the insulin, prevent the phenomenon of insulin resistance along with the prolongation of the treatment time course and obviously reduce the glycosylated hemoglobin level.

The combination of 5.2A.GRb-PQN4 and insulin can effectively delay the progressive rise of fasting blood glucose level of type I diabetic mice, and can also effectively improve the blood glucose fluctuation after the insulin is taken and the drug resistance generated by the repeated use of insulin.

As shown in Table 38, fasting blood glucose levels in untreated diabetes model groups increased from 24.71mmol/L at week 4 (week 0) to 32.17mmol/L at week 4 and 34.27mmol/L at week 7 nearly as close to week 10, indicating progression of disease progression with increasing course of disease; neither insulin nor GRb-PQN4 alone can delay the progression of the disease; the blood glucose level of the combination treatment group was significantly lower than that of the model control group at week 4, and the fasting blood glucose level at week 10 was not significantly different from that before the treatment, so that the combination treatment group was effective against the progress of the decrease in the glucose metabolism of diabetes. As shown in table 39, the randomized blood glucose levels were not significantly different between the model group and each treatment group, suggesting that GRb-PQN4 alone or in combination with insulin did not affect the absorption of carbohydrates and sugars in the food by the digestive tract.

TABLE 38 GRb-PQN4 in combination with insulin reduces fasting blood glucose levels (mM) in type I diabetic mice

Note: all data are expressed as mean ± SD; the differences between the model group or the treated group and the normal control group were analyzed by t-test, and the normal control was divided by one-way analysis of variance (ANOVA) combined with Duncan's multiple range test Analyzing the significance of the difference among other groups of samples; p vs control group<0.005; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

TABLE 39 GRb-PQN4 in combination with insulin does not reduce postprandial blood glucose levels (mM) in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with Duncan multi-range test; p <0.005, compared to control; n =5.

The combination of 5.2B.GRb-PQN4 and insulin can avoid hypoglycemia, prolong the hypoglycemic time of insulin, effectively avoid blood sugar fluctuation between daily multiple administrations of insulin and avoid acute drug resistance caused by daily multiple administrations of insulin; the combination of the two can not only prevent the insulin resistance along with the treatment time course and/or the disease progression in the chronic treatment process of the insulin, but also gradually enhance the sensitivity of the body to the insulin so as to reflect the drug effect of blocking the disease progression and/or repairing the disease, thereby leading the combination of the GRb-PQN4 and the low dose of the insulin to generate obvious, stable and gradually enhanced hypoglycemic effect.

As shown in fig. 7, the complete coincidence of the 2 curves (a in fig. 7) and the same area under the blood glucose curve (table 40) of blood glucose levels at 0, 30, 90, 150 and 6 time points after 150 and 270 minutes of re-administration, which occurred after the first (week 0) administration of insulin or its combination GRb-PQN4, indicates that the acute effect of GRb-PQN4 does not affect the acute hypoglycemic effect of insulin, or that the absence of acute hypoglycemic activity of GRb-PQN4 does not affect the sensitivity of the body to insulin; however, for 2 weeks of treatment, the 2 curves showed significant separation at several time points (B in fig. 7): one is that blood glucose levels in the insulin group decreased below normal levels (normal 6.78 for 4.78mM insulin; normal 9.19 for 2.70mM insulin) at 30 minutes after the first dose and 60 minutes after the second dose (210 minutes on a continuous basis), but blood glucose levels 60 minutes after the third dose (330 minutes on a continuous basis) were higher than normal, whereas the combination group decreased to below normal levels (9.07 and 5.73 mM) only in small increments at 30 minutes after the 3 rd dose (390 minutes on a continuous basis); secondly, the blood glucose level in the insulin group animals returned to the pre-dose high blood glucose level (24.79 mM) 120 minutes after the second dose (which was continuously timed at 270 minutes), while the blood glucose level in the combination treatment group animals remained at a lower level (15.75 mM); the blood glucose levels returned to substantially pre-dose high blood glucose levels in the insulin group 120 minutes after the third dose (continuous timing 390), while the combination group remained at near normal blood glucose levels. Consistently, the mathematical value of the area under the blood glucose curve (93) for the animals of the combination treatment group was less than the mathematical value of the insulin group (112). It can be seen that the GR-PQN4 in combination with insulin can effectively avoid the phenomenon of hypoglycemia caused by rapid decrease of blood sugar after the insulin is given and the subsequent rapid rise of blood sugar level to the hyperglycemia state, so that the duration of stable near-normal blood sugar level is obviously prolonged.

This effect of GR-PQN4 was more pronounced with increasing duration of treatment (C-E in FIG. 7), specifically: starting at week 4, the separation of the two curves was further separated in the original pattern as the treatment time course was extended, and the overall appearance was: the upward shift of the blood glucose nadirs, the upward shift of the rebound peaks and the significant increase in the area under the blood glucose curve in the insulin group mice (table 40), indicate a drug resistance phenomenon of chronic treatment with insulin or insulin resistance associated with disease progression; the mice in the combination treatment group did not show such a decrease in the degree of hypoglycemic effect and a decrease in the duration of hypoglycemic effect with a prolonged treatment period, but rather a tendency to gradually increase the effect, which is summarized as the values of the area under the blood glucose curve at

weeks

4, 7 and 10 were all significantly smaller than the area under the blood glucose curve in the insulin group (Table 40), and the area under the curve at week 10 was also significantly smaller than the area under the curve at the first dose (week 0). In particular, insulin failed to reduce blood glucose levels to the normal range (D-F in FIG. 7, 330 minutes) in the third consecutive day from week 4, while combination therapy could be reduced to the normal range, showing that multiple doses of insulin during the course of chronic therapy resulted in insulin resistance and that GRb-PQN4 in combination with insulin was effective in preventing the development of such insulin resistance. It can be seen that the combination of GRb-PQN4 and insulin not only prevents the drug resistance of chronic treatment of insulin, but also gradually enhances the sensitivity of the body to insulin, thus reflecting the drug effect of blocking disease progression and/or repairing disease, so that the combination of GRb-PQN4 and low-dose insulin can produce significant, stable and gradually enhanced hypoglycemic effect.

TABLE 40 area under blood glucose Curve (AUC, 0-7.5 h)

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.01，***p<0.005; in comparison to the model set, ^# p<0.05， ^## p<0.01; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05; compared with the period of 0 week, the method has the advantages that, ⁺ p<0.05；n＝5。

the above results of the study taken together illustrate the following three key issues: (1) The combination of GRb-PQN4 and insulin can overcome the intense fluctuation of blood sugar before and after the daily multiple medication of insulin treatment and the acute drug resistance (or called acute insulin resistance) after multiple injections, effectively avoid the hypoglycemic phenomenon and stably maintain the blood sugar level at the normal level or close to the normal level; (2) The combination of GRb-PQN4 and insulin can not only prevent the drug resistance phenomenon which is presented along with the treatment time course and/or the drug resistance phenomenon which is presented along with the disease progression and takes week as a unit and the drug resistance phenomenon which is presented after a plurality of times of administration in one day in the chronic treatment process of insulin, but also can gradually enhance the sensitivity of the organism to the insulin in the diabetic state, thereby leading the combination of the GRb-PQN4 and the insulin with low dose to generate obvious, stable and gradually enhanced hypoglycemic effect; (3) GRb-PQN4 in combination with insulin not only reduces and/or blocks the progression of type I diabetes but also restores the body's sensitivity to insulin, thus reflecting the effect of blocking disease progression and/or restoring disease. Based on that insulin resistance and blood sugar level fluctuation (GV) are 2 independent important factors which can predict or cause various common symptoms and various complications of diabetes patients, the research conclusion not only strongly supports the excellent efficacy of the GRb effective composition disclosed in the previous embodiment on improving typical symptoms of diabetes alone, particularly in combination with insulin, from the scientific principle of treatment or pharmacological effects, but also shows the wide application prospect of the GRb effective composition and insulin and other hypoglycemic agents in treating diabetes and preventing and treating other common symptoms and various complications of diabetes. The efficacy of GRb-effective compositions alone or in combination with insulin for the systemic treatment of diabetic complications and their pharmacological effects in common will be further demonstrated in the examples which follow.

The combination of 5.2c.grb-PQN4 and insulin can significantly reduce glycosylated hemoglobin levels.

As shown in Table 41, the glycosylated hemoglobin level (HbA 1 c) of the model group mice increased by 31.65% from 80.66mmol/M at the start of the test to 106.19mmol/M at week 10; the glycosylated hemoglobin levels of the animals of the combination treatment group were significantly lower than those of the mice of the model group at

weeks

5 and 10, and the level was only numerically increased by 4.11% at week 10 compared to week 5, but the two were not statistically different; insulin treatment alone showed only a trend towards decline, whereas GRb-PQN4 was completely ineffective. It can be seen that GRb-PQN4 in combination with insulin synergistically produced a potent effect of lowering glycosylated hemoglobin levels. The glycosylated hemoglobin level is directly proportional to the mean blood glucose concentration from the first 4 weeks to 3 months, and thus reflects the mean plasma glucose concentration over a period of time (4 to 8 weeks). Therefore, the effect of GRb-PQN4 combined with chronic insulin treatment on reducing the glycosylated hemoglobin level of diabetic mice further proves the effect of GRb-PQN4 on prolonging the hypoglycemic time of insulin and preventing drug resistance (or insulin resistance) caused by chronic insulin treatment.

TABLE 41 GRb-PQN4 in combination with insulin reduces glycosylated hemoglobin (HbA 1 c) levels in blood of type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p in comparison with control group<0.05，**p<0.01; in comparison to the model set, ^# p<0.05; compared with the 5-week period, the method has the advantages that, ⁺ p<0.05；n＝5。

it is specifically noted herein that glycated hemoglobin (HbA 1 c) is considered the gold standard for predicting the blood glucose-related risk of diabetic microvascular and macrovascular complications within 5 to 10 years, and recent studies have shown that a low level hemoglobin-related hypoxic state accompanied by elevated glycated hemoglobin can be directly involved in the common symptoms and various complications of diabetes as a non-blood glucose risk factor. Clinical trials of chronic complications of diabetes have shown that intensive insulin treatment reduces HbA1c to 53mmol/mol (77 mmol/mol in the untreated group) in patients, reducing the risk of retinopathy, nephropathy and neuropathy by 35-70%. Gastroparesis is a common complication of long-term diabetes, attributable to vagal dysfunction, and part of systemic autonomic neuropathy. Recent studies have demonstrated that glycosylated blood glucose levels can also predict the severity of gastroparesis in diabetic patients. In particular, for non-diabetic patients, a slight increase in glycosylated hemoglobin levels can significantly increase the incidence of surgical site infections, as well as factors that are associated with low-grade proteinuria in adults in china, and increase the risk of mild proteinuria in the elderly in china, independent of blood glucose levels. This suggests that elevated glycosylated hemoglobin levels may be directly related to impaired repair in diabetic patients independently of blood glucose levels, perhaps associated with lowered hemoglobin levels that may provide oxygen to the injury site that accompany elevated glycosylated hemoglobin levels. Consistently, there is emerging new evidence that tissues such as retina, kidney, pancreatic islets, fat, skin and wounds present a state of hypoxia in diabetic patients, suggesting that hypoxia plays an important role in the development of diabetes and diabetic complications (Diabetologia, 2021,64, 709-716), and that the efficacy of a novel glucose-lowering agent, sodium glucose co-transporter 2 (SGLT 2), in reducing diabetic nephropathy complications is linked to its role in promoting erythrocytosis (am.j.kidneydis., 2021, 77.

In conclusion, the GRb effective composition or the 1/2 clinical dose of insulin alone for chronic treatment can not significantly reduce the glycosylated hemoglobin level in the blood of type I diabetic mice, while the GRb effective composition and the insulin combine to generate a strong effect of maintaining the glycosylated hemoglobin level in the type I diabetic state substantially unchanged and approaching a normal level, which not only reflects that the GRb effective composition and the insulin combine to have a stable and strong blood sugar reducing effect, but also can protect the oxygen supply function of hemoglobin in a circulatory system to target tissue organs and cells. Obviously, maintaining the glycosylated hemoglobin level of the diabetic patient close to the normal low level is beneficial to avoid or relieve the tissue hypoxia state of the diabetic patient and the diabetes metabolic disorder aggravated by hypoxia (including aggravating the lactic acid rise and the mitochondrial oxidation generation deficiency related to the anaerobic glycolysis of glucose) and the acute symptoms related to hypoxia including the common symptoms of weakness after activity, dizziness, even mental lassitude, dizziness, headache, tinnitus, dim eyesight and diabetes of the diabetic patient. Therefore, the functions of lowering the blood sugar level of diabetes and protecting the oxygen carrying capacity of hemoglobin are important pharmacological actions of the GRb effective composition and insulin for systematically treating diabetes and complications thereof.

Example 6 GRb-PQN4 in combination with insulin can prevent peripheral neuropathy in diabetic mice.

The research method comprises the following steps:

the experiment was carried out on the basis of example 5. In order to reveal potential medicinal value of GRb-PQN4 and combined treatment of the GRb-PQN4 and hypoglycemic on preventing and treating diabetic peripheral neuropathy, the change of peripheral nerve sensitivity of a diabetic mouse along with the development of the disease is determined by using a hot plate experiment and a tail immersion experiment. On the day of grouping, 5 mice with relatively uniform heat-sensitive reactions are screened in each group for experiment and the latent period of pain caused by heat stimulation felt before treatment of each group is obtained; then, from day 3 of treatment, every 3 days until day 15; later, every 7 days until day 35 to understand the effect of drug treatment on hypersensitivity of peripheral sensory nerve endings in early stage of diabetes; finally, another test was performed on

days

49 and 70, respectively, to understand the effect of drug treatment on sensory inactivation associated with the deterioration of sensory nerve terminal function around diabetes. To avoid interfering with the observation of the target drug effect by the direct effect of the therapeutic drug on the neurosensory function, both the hot plate experiment and the tail dip experiment were performed 24 hours after the last administration. The specific method comprises the following steps: firstly, carrying out a hot plate experiment (55 +/-0.3 ℃), placing the hindpaw of the mouse on a hot plate instrument, when the animal feels pain caused by thermal stimulation, the animal licks the hindpaw or jumps, recording the latency period of licking the hindpaw or jumping by the mouse, and indicating that the lower the pain threshold value is, the shorter the latency period is. If no response occurred within 60 seconds, the latency was recorded as 60 seconds. The mean was taken in triplicate, each time at 20 min intervals. And then carrying out a tail immersion experiment, immersing 30-40% of the far end of the mouse tail into a water bath at 50 ℃, and measuring the tail flicking incubation period. If no response occurred within 15 seconds, the latency was recorded as 15 seconds. Each animal was replicated three times, each time 15 minutes apart, and the average was taken.

The research results are as follows:

as shown in table 42 and a in fig. 8, compared with the normal control group, diabetic mice in the diabetes model group exhibited reduced thermal pain sensitivity after plantar thermal hypersensitivity (which is an important reason for diabetic peripheral neuralgia) which reflects reduced ability of plantar sensory nerve to sense harmful stimulation and is an important reason for causing diabetic feet to be injured), and the treatment with insulin or GRb-PQN4 alone only delayed the process to some extent, but the insulin combined with GRb-PQN4 treatment can avoid early desensitization of diabetic mice and significantly slow down the subsequent process of sensory nerve terminal function degradation. As shown in table 43 and B in fig. 8, the tail thermal sensitivity of diabetic mice did not show early hypersensitivity, but gradually decreased, the pain latency was significantly longer than that of normal animals (indicating the decrease of the tail sensory nerve of diabetic mice in the ability to sense external noxious stimuli) starting on day 10, the GRb-PQN4 alone treatment did not show protective effect, the insulin alone treatment showed a certain protective effect only before week 5, but the GRb-PQN4 in combination with insulin effectively protected the nerve endings at the tail of diabetic mice throughout the whole 70-day treatment and observation process. The results of the above studies show that the treatment of insulin or GRb-PQN4 alone can only delay the sensory disorder of peripheral nerve endings of type I diabetic mice from hypersensitivity to passivation to a certain extent, but the combined treatment of GRb-PQN4 and insulin can not only avoid the early desensitization of diabetic mice caused by pain sensation, but also obviously slow down the process and severity of the subsequent sensory nerve endings functional degeneration. The above experimental results support the medicinal value of GRb-PQN4 and other effective/effective GRb compositions in combination with insulin for the prevention and treatment of peripheral neuropathy-related symptoms in diabetic patients, including pain and paresthesia (e.g., burning discomfort, crawling, numbness, itchy skin, etc.) caused by sensory neuropathy, and also support the medicinal value of GRb effective compositions alone or in combination with insulin for the prevention of diabetic foot from the perspective of preventing the diabetic patients from sensory nerve hypofunction or loss of the ability to perceive external risks as a result of this loss. Based on the fact that diabetic neuropathy has a common mechanism, research results also support the use of GRb effective compositions alone or in combination with insulin or other hypoglycemic agents for the prevention and treatment of conditions of diabetic autonomic dysfunction or/degeneration, including cardiovascular system symptoms (including but not limited to heart rate >90 beats/minute at rest, possibly with palpitations; patients are susceptible to significant reduction in their cardiac vascular irritation to ischemia, and patients are therefore susceptible to painless myocardial infarction; orthostatic hypotension), digestive system symptoms (typical symptoms are gastroparesis, diarrhea, constipation, and most patients show diarrhea alternating with constipation), urinary system symptoms (urinary retention or incontinence), and other system symptoms (including but not limited to abnormal skin sweating, such as reduced sweating, and symptoms such as sweating of the left body skin, and dryness of the right body skin).

TABLE 42 GRb-PQN4 and its Effect on the latency (in seconds) of the plantar sensation of pain by thermal stimulation in mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and a normal control group by using a t test, and performing difference significance division among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.05; in comparison to the model set, ^# p<0.05；n＝5。

TABLE 43 GRb-PQN4 and its Effect on mouse Tail Heat sensitivity (sec.) in combination with insulin

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing difference significance division between a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.05; in comparison to the model set, ^# p<0.05；n＝5。

example 7 GRb-PQN4 alone or in combination with insulin can prevent diabetic autonomic neuropathy related complications.

The research method comprises the following steps:

diabetic gastroparesis is characterized by delayed gastric emptying and is a common symptom of diabetic gastrointestinal vegetative neuropathy, 50-76% of diabetic patients suffer from gastroparesis, typical symptoms of the gastroparesis include abdominal distension, early satiety, anorexia, eructation, nausea and vomiting, and symptoms are usually serious after meals, so that appetite loss, malnutrition, malabsorption and weight loss can be caused. Therefore, the effect of GRb-PQN4, alone or in combination with insulin, on diabetic autonomic neuropathy is observed here for gastroparesis. The test was carried out on the basis of example 5. Animals were fasted for 12h at week 10 of treatment and then euthanized, immediately the stomach was removed and the empty stomach, i.e. stomach tissue and its contents, were weighed.

The research results are as follows:

as shown in table 44, compared with the normal animals, the weight of the stomach tissue in the diabetes model group did not change significantly, and the weight of the stomach content and the ratio of the weight of the stomach tissue to the weight of the stomach tissue were significantly increased, which indicates that the mice with type I diabetes with an untreated course of 12 to 13 weeks exhibited a decrease in the gastric emptying capacity, i.e., a complication of gastroparesis; the weight of stomach tissue of the GRb-PQN4 chronic treatment group is obviously higher than that of the model control group, the ratio of the weight of the stomach content to the weight of the stomach tissue is obviously lower than that of the model control group, and the drug effect of the combination of the GRb-PQN4 chronic treatment group and the insulin is further enhanced to be shown in the way that the weight of the stomach content is obviously lower than that of the model control group, and the ratio of the weight of the stomach content to the weight of the stomach tissue is lower than that of the GRb-PQN4 single treatment group; clinical 1/2 dose insulin treatment failed to significantly improve gastroparesis complications. The research results prove that GRb-PQN4 can effectively prevent and treat diabetic gastroparesis complications alone or in combination with insulin. The research results support the clinical application value of GRb-PQN4 alone or in combination with insulin in preventing and treating diabetic gastroparesis complications and other vegetative neuropathy related diseases, including myocardial infarction, malignant arrhythmia, sudden death, constipation, erectile dysfunction, neurogenic bladder dysfunction and the like.

TABLE 44 effects of GRb-PQN4 and its combination with insulin on diabetic gastroparesis in mice

Note: all ofData are presented as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing difference significance division between a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.05; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

example 8 grb-PQN4 composition alone or in combination with insulin significantly improved diabetic mouse skin rash.

The research method comprises the following steps:

the study was carried out on the basis of example 5. In order to further disclose potential medicinal values of GRb-PQN4 and the combination treatment of the GRb-PQN4 and hypoglycemic drugs for preventing and treating diabetic skin complications. For this purpose, diabetic mice were induced to develop skin rash by applying non-pathogenic stimulation to normal skin, i.e., 4% sodium sulfide in combination with 5% formaldehyde, to the dorsal skin surface to observe the incidence of skin rash and the recovery rate of dermatitis in each group of animals in comparison. Since diabetic dermatological complications may occur at various stages of the course of the disease, after 2 weeks of this treatment, the animals were anesthetized with sodium pentobarbital, the longer hair on the back of the mice was treated with electric hair clipper, the back of the mice was further depilated with 4% sodium sulfide, and the next day 5% formaldehyde was applied to the back to induce rash. Mice had free diet and water after surgery, and each group of animals continued the corresponding medication or treatment. Wound healing was recorded by photographing on the day of formaldehyde application (day 0) and on

days

3, 6, 9, 12 and 15, respectively, and the wound area was counted.

The research results are as follows: as shown in fig. 9 and table 45, the rash onset was most severe in the diabetic model group animals on

days

3 and 6 after formaldehyde application, and then slowly recovered but not completely recovered by 15 days (fig. 9), with a total incidence of 100% (table 45); the rash severity in the animals treated with insulin or GRb-PQN4 alone was less severe and recovery was significantly accelerated in terms of morbidity than in the model animals (Table 45), whereas no rash was evident in the combined treatment of GRb-PQN4 and insulin. Therefore, the combination of GRb-PQN4 and insulin can effectively prevent and treat diabetic skin disease complications. Acute metabolic disorders and chronic degenerative complications of diabetes mellitus lead to a high incidence of skin disorders in diabetic patients, including vitiligo and psoriasis, pruritus, desquamation, skin rashes, seborrheic dermatitis, dry scabs, warts, eczema, skin pigmentation, and candida infections. The research result of the embodiment directly supports the medical application of GRb-PQN4 combined with insulin or other hypoglycemic drugs to prevent and treat diabetic skin disease complications, and the excellent hypoglycemic effect and the hemoglobin oxygen carrying capacity protection generated by combining the GRb-PQN4 combined with the insulin or other hypoglycemic drugs indirectly support the application of the GRb effective composition and the insulin or other hypoglycemic drugs to prevent and treat diabetic skin disease complications.

TABLE 45 GRb-PQN4 in combination with metformin or dapagliflozin significantly reduced the incidence of type 2 diabetic mouse skin rash (%)

Note: all data are expressed as mean ± SD; carrying out significance analysis on differences among a plurality of groups of samples by using a rank sum test; p compared to control group<0.05; in comparison to the model set, ^# p<0.05; n =5; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

example 9 GRb-PQN4 alone or in combination with insulin significantly promoted the repair of skin lesions in diabetic mice.

The research method comprises the following steps:

the study was carried out on the basis of example 5. In order to reveal the potential medicinal value of GRb-PQN4 and the combined treatment of the GRb-PQN4 and hypoglycemic drugs for preventing and treating diabetic foot, the wound repair condition of each group of animals is observed by utilizing a diabetic chronic comprehensive wound model. At week 5 of treatment (when the course of type I diabetes is 7 weeks), the animals were anesthetized with pentobarbital sodium, and the longer hair on the back of the mice was treated with electric hair clippers, followed by further removal of the back hair with depilatory cream. Then, the back skin was sterilized with iodophor, a 2 × 2cm full-thickness skin wound surface was created with scissors under aseptic conditions while the back was facing, the wound surface was cut to the subcutaneous fascia, and the skin around the wound was covered with gauze after alcohol sterilization. Mice were allowed free diet and water after surgery, and each group of animals continued to be treated or treated with the corresponding medication. Wound healing was recorded by shooting on the day of full wound modeling (day 0) and on

days

3, 6, 9, 12 and 15 after treatment, respectively, and the wound area was counted at each time point.

The research results are as follows:

as shown in fig. 10, the skin wound healing rate was significantly slower in the diabetic model group mice than in the normal control group mice; on day 6 of treatment, the wound area of GRb-PQN4 mice was significantly smaller than that of diabetic model mice (153.86mm 2 vs. 120.70mm 2), while the wound area of GRb-PQN4 and insulin combination treated mice further decreased (94.99mm 2), and the wound area of the combination treated mice was significantly smaller than that of the model mice at each of the subsequent time points, but insulin alone failed to promote wound repair at any time point. It can be seen that GRb-PQN4 alone or in combination with insulin can significantly promote wound healing, and GRb-PQN4 is a major contributor to the efficacy of combined therapy. Research results support the medical application of GRb-PQN4 and other effective compositions in preventing and treating diabetic foot alone or in combination with insulin and other hypoglycemic agents, and also support the medical application of the GRb-PQN4 and other effective compositions in promoting various postoperative rehabilitation of diabetic patients.

Example 10 GRb-PQN4 in combination with insulin for the prevention and treatment of chronic diabetic complications cardiac and renal complications.

The research method comprises the following steps:

the study was carried out on the basis of example 5. In order to reveal the potential medicinal value of the combined treatment of the GRb-PQN4 and the hypoglycemic agent for preventing and treating the diabetic nephropathy, various biochemical indexes indicating the renal function in urine and blood of various groups of animals are detected. Urine of mice 24 hours after the last administration was taken at

weeks

2, 4, 7 and 10 of treatment administration to determine the levels of creatinine and albumin in the urine; at

weeks

5 and 10 of treatment, blood samples of 5 mice per group were taken under deep anesthesia for measuring creatinine levels in the blood and urine albumin to creatinine ratio and urine creatinine to blood creatinine ratio were analyzed to reveal the efficacy of GRb-PQN4 alone or in combination with insulin for the prevention and treatment of renal complications. Cardiac specimens prepared at the treatment end-point (week 10) for example 5 were analyzed for histological lesions of the cardiac vessels and myocardium by HE staining and Mass staining, respectively, to understand the protective effect of each treatment group on the heart of type I diabetic mice undergoing approximately 13 weeks of disease progression.

The research results are as follows:

the GRb-PQN4 and the insulin can obviously protect the renal function of the type I diabetic mice, and the combined drug effect of the GRb-PQN4 and the insulin is better.

Abnormal elevation of blood creatinine is a common index for determining renal insufficiency, and here, in order to analyze the functional state of glomerular filtered creatinine, creatinine levels in urine and blood and the ratio of urine creatinine level to blood creatinine level of each group of animals are comparatively analyzed. As shown in table 46, prior to treatment (week 0), creatinine levels in urine in the diabetes model group and the treatment group animals assigned to insulin, GRb-PQN4 or a combination thereof were at the same level and significantly lower than urine creatinine levels in normal control group animals (/ p < 0.05); however, by the time of 2 weeks after completion of the treatment (at which the course of disease is 4-5 weeks), the urine creatinine levels in the model animals decreased to about 1/2 of the level before 2 weeks and remained at this level throughout the subsequent increased 8-week course, while the creatinine levels in the blood appeared to increase significantly over the blood creatinine levels in the normal control animals with the course of disease; at week 10 the urinary creatinine/blood creatinine ratio was less than 1/4 of that of normal animals. These data indicate that type I diabetic mice develop a decrease in glomerular creatinine filtration function from 4 to 5 weeks into the course of disease, which is severely reduced by 12 to 13 weeks into the course of disease. GRb-PQN4 and insulin treatment alone can significantly protect the kidney filtration function of diabetic mice, but the two appear in 2 distinct protective forms: during chronic treatment for 10 consecutive weeks, insulin first increases the urinary creatinine level manyfold above the normal control value and then falls back below the normal control value; GRb-PQN4 is the level of maintaining the treatment, or GRb-PQN4 can block the decline of the filtering function of glomeruli to creatinine along with the progress of diabetes, but the drug effect of insulin is better than that of GRb-PQN4 in terms of the ratio of urine creatinine/blood creatinine at the 10 th week; in particular, chronic combination therapy can restore the original missing glomerular filtration function, mainly as indicated by the increase in the initial severely reduced urinary creatinine level from week 4 to a level indistinguishable from normal control, and by the proximity of the urinary creatinine/blood creatinine ratio to normal control at week 10. The results of the above studies show that chronic treatment with both insulin and GRb-PQN4 can counteract to some extent the decrease in glomerular filtration function that occurs with the prolongation of the course of diabetes, and that the combined chronic treatment of both can not only block the progressive decrease in glomerular filtration function but also correct the existing functional deficiencies.

TABLE 46 GRb-PQN4 in combination with insulin to lower and raise the blood creatinine levels and the ratio of urine creatinine to blood creatinine in diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and a normal control group by using a t test, and performing difference significance division among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.05，**p<0.01; in comparison to the model set, ^# p<0.05， ^## p<0.01; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05; compared with 0 week or 5 weeks of the present group, ⁺ p<0.05， ⁺⁺ p<0.01， ⁺⁺⁺ p<0.001；n＝5。

urine albumin to creatinine ratio is another common indicator for kidney function, and a portion of the protein is filtered into urine in the silk spheres of the kidney, but is absorbed back into the blood in the renal tubules. Therefore, when disease or dysfunction of the kidney occurs, the glomerular basement membrane is destroyed and its permeability is increased, and albumin in the blood is discarded/leaked through the glomerular basement membrane to become proteinuria, which is also caused by the decrease of the renal tubular resorption function. As shown in table 47, the urine albumin to creatinine ratio was significantly lower in the diabetic model group and the treatment group animals assigned to insulin, GRb-PQN4, or a combination of both, than in the normal control group before treatment (week 0), indicating that the basement membrane of the glomeruli of diabetic mice is intact; however, by the time of 2 weeks after completion of the treatment (at this time, the course of disease is 4-5 weeks), the ratio of the model control group was significantly higher than that of the normal control group, and the ratio increased with the prolongation of the course of disease, indicating that type I diabetic mice with a 4-5 week course had progressive glomerular basement membrane injury or reduced tubular reabsorption function; GRb-PQN4 and insulin can obviously reduce the ratio of urine albumin to creatinine at each time point by single treatment, and can completely block the phenomenon that the ratio is increased along with the prolongation of the course of disease from the 4 th week; and the drug effect of the combination therapy is further improved, which is particularly characterized in that the ratio of urine albumin to creatinine is maintained at a level close to that of a normal control from the 2 nd week. The research data show that insulin and GRb-PQN4 can remarkably protect the intact glomerular basement membrane and the tubular reabsorption function of diabetic mice, and the combined treatment of the insulin and the GRb-PQN4 can almost maintain the glomerular basement membrane and the tubular reabsorption function of the diabetic mice at a level close to normal.

TABLE 47 GRb-PQN4 and insulin in combination reduce the albumin to creatinine ratio (μ g/mg) in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and a normal control group by using a t test, and performing difference significance division among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.05,**p<0.01; in comparison to the model set, ^# p<0.05, ^## p<0.01; compared with the 2 weeks of the present group, ⁺⁺ p<0.01；n＝5。

the combination of the above results shows that: the independent administration of GRb-PQN4 and insulin can protect the kidney function of type I diabetic mice to a certain extent, and the combination of the GRb-PQN4 and the insulin can synergistically generate more remarkable protective effect, so that the medicinal application of the GRb-PQN4 and other effective/effective compositions to prevention and treatment of diabetic nephropathy is supported, wherein the GRb-PQN4 and other effective/effective compositions are independent, and particularly combined with combined insulin or other hypoglycemic agents.

grb-PQN4 alone or in combination with insulin significantly protected the heart of type I diabetic mice, but insulin did not.

As shown in fig. 11, HE staining results showed that the heart of type I diabetic mice with a disease course of 12-13 weeks exhibited unclear structural boundaries of cardiomyocytes, broken myofibers and a large amount of inflammatory cell infiltration; MS staining also showed pathological characterization of cardiac tissue fibrosis and fibrosis of the vessel wall in cardiac tissue; GRb-PQN4 treatment significantly prevented the appearance of these pathological changes, and the protective effects of combination therapy with insulin were further enhanced, but no significant protective effects were seen with insulin alone. Therefore, research results support the medical application of GRb-PQN4 alone, particularly in combination with insulin or other hypoglycemic agents, in preventing and treating diabetic cardiac complications. In particular, despite strict control of blood glucose levels in long-term clinical practice, cardiovascular complications remain a major cause of morbidity and mortality in diabetic patients, and our findings also show that insulin alone does not have a significant protective effect on heart damage/degeneration in type I diabetic mice. Therefore, the significant medical value of the GRb effective composition for preventing and treating the cardiovascular complications of the diabetes is further highlighted, and the GRb effective composition is independent, and particularly combined with insulin or other hypoglycemic drugs.

Example 11 GRb-PQN4 and its use with insulin can improve the energy metabolic state and redox balance of type I diabetes.

The research method comprises the following steps:

the animals of example 5 were used to observe the changes in urea nitrogen levels in urine of affected mice with the course of disease extending and the effects of GRb-PQN4, insulin and combination thereof on urea nitrogen levels in urine of affected mice, and then the blood levels of sorbitol, urea nitrogen, uric acid, creatine and creatinine were observed in each group of animals for 5 weeks of treatment (7-8 weeks of course) and 10 weeks of treatment (12-13 weeks of course), and the levels of creatine and creatinine in muscle were also observed, as well as the comparison of Adenosine Triphosphate (ATP), coenzyme I (NAD) oxidized forms (NAD) in muscle, heart and kidney ⁺ ) And reduced form (NADH)) Coenzyme II (NADP) in oxidized form (NADP) ⁺ ) And the levels of reduced form (NADPH), glutathione (GSH) and its oxidized form (GSSG). In order to further clarify the redox state of the cells, the activities of superoxide dismutase (SOD) and catalase in heart and skin of the diabetic vulnerable organs, the levels of superoxide anions and hydrogen peroxide in blood and the vulnerable organs, and the content of oxidative stress products (including lipid oxidative stress product malondialdehyde and nucleic acid DNA oxidative stress product 8-hydroxydeoxyguanosine) were further determined.

The research results are as follows:

the compensatory effect of GRb-PQN4 in combination with insulin on intracellular high levels of glucose shunting by the polyol pathway was not altered.

In diabetic conditions, intracellular glucose metabolism disorders lead to elevated intracellular glucose levels, and the polyol pathway is activated as a compensatory response to shunting of intracellular accumulated glucose. However, when this pathway is excessively activated, a large amount of NADPH, which is an antioxidant substance, is consumed and NADP is produced at the step of converting glucose into sorbitol ⁺ If NADP is present ⁺ Failure to reduce or not replenish NADPH in time from other sources can lead to depletion of NADPH and oxidative stress; the conversion of sorbitol to fructose consumes large amounts of NAD ⁺ And produces NADH and fructose, thereby reducing NAD ⁺ NADH ratio and possibly a decrease in the glycolytic metabolic pathway NAD ⁺ Depends on the activity of key enzymes and physiological glycolysis activity, and fructose triggers related pathological changes through glycosylation. Our findings (table 48) indicate that none of GRb-PQN4, insulin and both combination therapies attenuated the compensatory activation of the polyol pathway in the diabetic state, and we will focus on the effects of these therapies on NADPH levels and glycosylation products in vivo in the diabetic state in subsequent studies to fully understand the effects of therapeutic drugs on the diabetic polyol pathway and their pharmacological significance.

TABLE 48 combination of GRb-PQN4 with insulin does not affect sorbitol levels in blood of type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and a normal control by using a t test, and performing the difference significance analysis among a plurality of groups of samples except the normal control by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p <0.05 compared to control; n =5.

Treatment with grb-PQN4 in combination with insulin ameliorates protein metabolism disturbance or maintains protein metabolism homeostasis in the type I diabetic state.

Urea nitrogen is the main end product of human protein metabolism, and is mainly filtered by the glomerulus and discharged with urine. Abnormal rapid protein breakdown can lead to elevated urea nitrogen levels in the blood and urine, as well as renal dysfunction in filtering urea nitrogen. Therefore, observing the urea nitrogen level of the drug in blood and urine and the ratio of the urea nitrogen level in urine to the urea nitrogen level in blood of type I diabetic mice can reflect the effects of the drug in improving protein metabolism disorder and protecting glomerular filtration function in diabetic states. As shown in table 49, from the 4-week treatment point (disease course 6-7 weeks), the urea nitrogen level in urine of untreated diabetes model group mice was close to 4 times the urea nitrogen level of normal control group animals, and increased to 5 times the treatment 10-week (disease course 12-13 weeks), and the urea nitrogen level in blood was also significantly higher than that of normal control group, and the ratio of urine urea nitrogen level to blood urea nitrogen level was also significantly higher than that of normal control group. The combination of these studies reflects severe protein over-degradation or protein metabolism disturbance in untreated diabetic model animals. However, insulin treatment can only alleviate the excessive protein breakdown of the diabetic state at an early stage, as shown by: the insulin treatment group significantly reduced the urea nitrogen level in urine only at week 4, whereas GRB-PQN4 did not reduce the urea nitrogen level in urine during the entire course of treatment. Importantly, the combined treatment of GRb-PQN4 and insulin can remarkably reduce the urea nitrogen level in urine and blood in the whole course, which shows that the combined treatment of GRb-PQN4 and insulin can improve the protein metabolism disorder in the diabetic state for a long time. The protein metabolism disorder is closely related to the deterioration of the life quality of diabetics, such as emaciation and fatigue, the deterioration of work and life quality, the deterioration of cellular immunity and humoral immunity, the deterioration of resistance, infection and the like, and the research result supports the medical application of GRb-PQN4 and other effective/effective compositions in combination with insulin or other hypoglycemic drugs to improve the life quality of the diabetics from the action mechanism.

TABLE 49 GRb-PQN4 in combination with insulin reduces urea nitrogen levels in urine and blood of type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.05，***p<0.005; in comparison to the model set, ^# p<0.05， ^## p<0.01; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

GRb-PQN4 and its combination with insulin maintain creatine metabolic homeostasis in the state of type I diabetes.

Creatine is synthesized and released into the blood primarily by the kidneys and liver and is taken up and stored by muscle cells. Creatine, which receives a source of ATP and is an important storage form of energy in muscle, is delivered to Adenosine Diphosphate (ADP) to produce ATP when needed, so that creatine plays a key role in maintaining ATP level homeostasis in skeletal muscle, and is also a key factor in cardiac contraction and energy metabolism. In particular, mitochondrial dysfunction is associated with decreased oxidative phosphorylation activity in diabetic conditions, and thus the role of creatine in maintaining ATP homeostasis is more important in diabetic patients, and decreased renal function in diabetic conditions can lead to decreased creatine levels and thus impaired creatine in maintaining ATP homeostasis. Indeed, serum creatinine levels are low in type II diabetes as an indicator of muscle mass, and low serum creatinine levels can predict the development of type II diabetes, provided that the effects of diabetes and pre-diabetic glomerular hyperfiltration are excluded. In view of the above, it is necessary to further observe the effect of GRb-PQN4 in combination with insulin on the protein metabolic homeostasis in the diabetic state, in order to maintain the protein metabolic homeostasis in the diabetic state.

As shown in table 50, creatinine levels were significantly higher in blood of untreated type I diabetic mice of 7-8 weeks of age (5 weeks) than in normal control group with a concomitant decrease in creatine/creatinine ratio; by the time the disease progresses to 12-13 weeks of age (10 weeks), the creatine levels decline and creatinine levels continue to rise, and the decrease in the creatine/creatinine ratio is further amplified. Neither insulin nor GRb-PQN4 alone was effective in treating elevated blood creatinine and reduced creatine/creatinine ratio in anti-diabetic mice, but the combination of both parameters maintained the levels of these parameters close to those of normal control animals.

Table 50 GRb-PQN4 in combination with insulin increases creatine and decreases creatinine levels in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.05; in comparison to the model set, ^# p<0.05；n＝5。

as shown in table 51, in muscle tissue, the levels of creatine, particularly creatinine, in the untreated diabetic model group mice were significantly lower than those in the normal control group animals at 2 time points of 5 weeks and 10 weeks, and the creatine/creatinine ratio was greatly reduced. Since creatinine is a non-functional total metabolite of creatine, a decrease in this ratio may be a protective compensatory response by the body. In combination with the above 2 time points where the diabetic mice had less than 1/2 of the normal animal urinary creatinine levels (Table 46), these data illustrate several points: type I diabetic mice have a lower capacity to synthesize creatine than normal animals and may be associated with reduced kidney function; the substantial non-reduction of creatine in the blood with a reduction in muscle approaching 1/2 accompanied by a profound reduction in creatinine indicates a reduced creatine uptake capacity or a reduced microvascular circulation in type I diabetic conditions; creatine functions in maintaining ATP homeostasis in muscle and other related tissues such as the heart are reduced in type I diabetes mellitus. Advantageously, treatment with GRb-PQN4 alone can significantly increase the creatine and creatinine levels throughout the muscle of diabetic mice, indicating that GRb-PQN4 can increase creatine synthesis in the type I diabetic state; insulin can only improve creatinine level, and the combined treatment of the insulin and the insulin maintains the drug effect of GRb-PQN 4. It can be seen that GRb-PQN4 has a unique advantage of maintaining creatine metabolic homeostasis in the type I diabetes state, as compared to insulin.

TABLE 51 GRb-PQN4 in combination with insulin increases creatine and creatinine levels (μmol/mg) in the muscle of type I diabetic mice

Note: all data are expressed as mean ± SD; differences between the model group or treatment group and the normal control group were analyzed using t-test, and significance analysis of differences among groups of samples other than the normal control was performed using one-way analysis of variance (ANOVA) in combination with the Duncan multi-range test. P compared to control group<0.05,**p<0.01，***p<0.005; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

combining the results of the studies in tables 50 and 51, GRb-PQN4 alone or in combination with insulin is effective in maintaining creatine homeostasis in the diabetic state and thus is beneficial in maintaining ATP homeostasis in the diabetic state. Research results support the medical application of GRb-PQN4 and creatine precursor amino acids (arginine, glycine and methionine) in improving the metabolic disorder of diabetes. Furthermore, a decrease in creatine level is considered to be one of the major causes of sarcopenia, and therefore, the above findings also support the medical use of GRb-PQN4 alone or in combination with insulin or other metabolic regulators or nutritional agents such as creatine pro-amino acids for the prevention and treatment of sarcopenia. Due to the key action of the kidney on creatine synthesis, the research results further support the medical application of GRb-PQN4 combined with insulin in preventing and treating diabetic nephropathy from the aspect of function. It is also noted that the results of the study reveal from a new perspective the role of effective GRb compositions in protecting diabetic kidney and/or liver function.

GRb-PQN4 and its combination with insulin maintain ATP levels and energy metabolism homeostasis in type I diabetes.

Energy metabolism involves a complex and tightly regulated network to produce sufficient ATP and maintain its level homeostasis, so ATP levels may also reflect the health of the metabolic network. Specifically, ATP production is concentrated on mitochondria, which are associated with energy metabolism, oxidative stress, cell and tissue functions. In the relevant organs in the diabetic state, this mechanism can be disturbed at multiple levels and multiple pathways, including: substrate utilization, mitochondrial oxidative metabolism, redox regulation, transfer of high-energy phosphate bonds between creatine and ATP, ultimately leading to ATP deficiency, redox imbalance, mitochondrial damage, chronic inflammation, and diabetic complications. Although we have made substantial progress in understanding the pathophysiology of metabolic disorders of diabetes, this knowledge has not been translated into a viable therapeutic approach. Here we further determined the protective effects that type I diabetic mice may have as the course of disease increases, ATP levels in muscle, kidney and heart change and GRb-PQN4, insulin or a combination of both.

As shown in table 52, the ATP levels in the muscle, kidney and heart tissues of the diabetic control animals did not change significantly by 5 weeks of treatment (course 7-8 weeks) compared to the normal control group, but significantly decreased by 10 weeks of treatment (course extended to 12-13 weeks), with the greatest muscle decrease (56.67% compared to the control group) and the least heart decrease (31.58%). Consistent with the finding of unique advantage of GRb-PQN4 in maintaining creatine metabolic homeostasis over insulin in type I diabetes, GRb-PQN4 could not only completely counteract the decrease in ATP levels in the above tissues in type I diabetes, but also mathematically elevate ATP levels at various time points above that of the normal control group, showing the potent ability of GRb-PQN4 to enhance ATP synthesis in type I diabetes, but insulin could not elevate ATP levels in these tissues and organs in diabetes, while the effect of both insulin and GRb-PQN4 in combination in elevating ATP levels was further enhanced. These findings again show the unique advantage of chronic treatment of GRb-PQN4 over chronic treatment with insulin for the correction of diabetic metabolic disorders, which not only completely combats ATP deficiency in type I diabetes but also maintains it at levels approximately higher than in normal animals.

Based on the association of ATP level with metabolic homeostasis and functions of cells and tissues and organs, the research results further support the medical application of GRb-PQN4 and combined insulin thereof in improving the overall health state of diabetic patients and preventing and treating diabetic sarcopenia, diabetic heart disease and diabetic nephropathy, and further suggest the unique effect of GRb-PQN4 in correcting diabetic metabolic disorders and redox imbalance. In summary, the GRb-PQN4 composition completely reverses ATP deficiency in diabetic patients enough to provide energy support for the work, daily activities and functional maintenance and execution of various organs of diabetic patients, and avoids various pathological events and vicious cycles induced by energy deficiency. The synergistic effect of the insulin on the GRb-PQN4 composition not only accords with the theory that the combination of two pharmacological active substances with different action mechanisms can generate the synergistic effect, but also supports the medical application of the GRb-PQN4 composition and a hypoglycemic agent in the combined prevention and treatment of diabetic complications.

TABLE 52 GRb-PQN4 and its use in combination with insulin to increase ATP levels (nmol/mg protein) in muscle, kidney and heart in type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group <0.05; in comparison to the model set, ^# p<0.05， ^## p<0.01; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05， ^&& p<0.001；n＝5。

GRb-PQN4 and its combination with insulin maintain redox balance in type I diabetic conditions.

To reveal the potential role and mechanism of GRb-PQN4 alone or in combination with insulin to more broadly maintain metabolic homeostasis in the diabetic state, we further determined the three most important pairs of redox substances in vivo: oxidized coenzyme I (NAD) ⁺ ) And reduced coenzyme I (NADH), oxidized coenzyme II (NADP) ⁺ ) And the changes in reduced coenzyme II (NADPH), glutathione (GSH) and oxidized glutathione (GSSG) with the progression of the disease process in type I diabetes and the effects of chronic treatment with GRb-PQN4 and insulin and both on such changes.

Combination treatment of 11.5A.GRb-PQN4 with insulin slowed down the decrease in NADH levels and maintained NAD in muscle and heart of diseased mice ⁺ NADH ratio at normal level.

As shown in Table 53, untreated type I diabetes model groups mice had total amounts of muscle, kidney and heart NAD (NAD) between 7 and 8 weeks of disease duration (5 weeks of treatment) and between 12 and 13 weeks of disease duration (10 weeks of treatment) ⁺ And NADH) is not obviously different from the total amount of NAD of normal control group animals, GRb-PQN4 or insulin and the combination treatment of the two do not influence the total amount, which indicates that the functions of various NAD syntheses are the same.

TABLE 53 GRb-PQN4 or insulin and their combination did not affect the total amount of NAD (nmol/mg protein) in muscle, kidney and heart of type I diabetic mice

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Note: all data are expressed as mean ± SD; the differences between the model group or the treatment group and the normal control group are analyzed by using a t test, and the significance analysis of the differences among a plurality of groups of samples except the normal control group is carried out by using one-way analysis of variance (ANOVA) and combining a Duncan multi-range test, and no significant difference is found in each group.

However, at the 2 time points, NADH levels in muscle and heart were significantly lower in mice in the type I diabetes model group than in normal control animals (Table 54), while NAD ⁺ the/NADH ratio was significantly increased (Table 56), while the kidney was maintained within the normal range for both parameters. This inter-tissue difference indicates that the muscle and cardiac metabolic characteristics are homogeneous, while the kidney has its own metabolic characteristics. Neither GRb-PQN4 nor insulin alone was able to significantly combat the decreased NADH levels and NAD in muscle and heart of type I diabetic mice ⁺ The NADH ratio is obviously increased, but the NADH level and NAD of the muscle and heart of the diabetic mouse can be combined and treated ⁺ the/NADH ratio is maintained in the normal range; notably, the combination of GRb-PQN4 and insulin decreased NADH levels and increased NAD in diabetic kidneys ⁺ NADH ratio, and significantly improves renal NAD ⁺ Horizontal but reduced heart NAD ⁺ Level (table 55). The above results are combined to illustrate two key problems: first, chronic treatment of GRb-PQN4 in combination with insulin can maintain NAD in diabetic state ⁺ And NADH, which is indicative of the health of glycolytic activity and the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation. This pharmacological effect supports the effect of GRb-PQN4 in combination with insulin to increase ATP levels in muscle and heart in diabetic conditions. Secondly, the combination treatment of GRb-PQN4 and insulin can generate different regulation phenotypes according to the own metabolic characteristics of tissues and organs, and the specific action mechanism of the combination treatment is worthy of further disclosure.

TABLE 54 GRb-PQN4 in combination with insulin increases NADH levels (nmol/mg protein) in muscle and heart of type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.05，**p<0.01; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

table 55 GRb-PQN4 in combination with insulin increases renal but decreases cardiac NAD in type I diabetic mice ⁺ Horizontal (nmol/mg protein)

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; in comparison to the model set, ^# p<0.05；n＝5。

table 56 GRb-PQN4 in combination with insulin decreases NAD in muscle and heart of type I diabetic mice ⁺ Ratio to NADH

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with Duncan multi-range test; p compared to normal control group<0.05，**p<0.01; in comparison with the set of models, ^# p<0.05， ^## p<0.01; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05， ^&& p<0.001；n＝5。

11.5B.GRb-PQN4 and its combination treatment with insulin increases total amount of NAPD and maintains NADP in muscle and heart of diabetic mice ⁺ the/NADPH ratio is at or near normal levels.

As shown in Table 57, the hearts of untreated diabetes model group mice exhibited total NADP (NADPH + NADP) at weeks 7 to 8 (5 weeks of treatment) of the disease course ⁺ ) The levels were significantly lower than those of the normal control animals, and were further reduced by weeks 12 to 13 of the course (10 weeks of treatment), at which time the total NADP in the muscle was also significantly reduced, but the total NADP in the kidney was not reduced, again showing similarities in metabolic characteristics of the muscle and heart and differences from the kidney. Insulin and GRb-PQN4 only showed a tendency to counteract the decrease in total NADP, while the combination of chronic treatment of both showed that diabetic mice maintained their total NADP at normal levels even at higher mathematical values, but did not affect the total NADP in the kidneys at normal levels. It can be seen that the combination of insulin and GRb-PQN4 can selectively and greatly improve the NADP-producing ability of the muscle and heart of diabetic mice. The combination of insulin and GRb-PQN4 remarkably increases NAPD of muscle and heart of diabetic mice ⁺ And NADPH levels (tables 58 and 59), and decreased NADP ⁺ The opposite result was produced for the kidney as compared to the NADPH ratio (Table 60). The above results are combined to illustrate two key problems: first, chronic treatment with GRb-PQN4 in combination with insulin increases total NADP in muscle and heart and maintains NADP in type I diabetic mice ⁺ And NADPH. It can be seen that the combined treatment of GRb-PQN4 and insulin can improve the capacity of NADP-mediated antioxidant damage from two aspects of expanding NADP antioxidant capacity and the conversion rate of reduction type and oxidation type, and the effect predicts that the combined treatment of GRb-PQN4 and insulin can improve the antioxidant capacity of glutathione. Secondly, the combination treatment of GRb-PQN4 and insulin can generate different regulation phenotypes according to the own metabolic characteristics of tissues and organs, and the specific action mechanism of the combination treatment is worthy of further disclosure.

TABLE 57 GRb-PQN4 in combination with insulin increases total NADP (nmol/mg protein) in muscle and heart of type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.05; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

TABLE 58 GRb-PQN4 in combination with insulin increases NADP in kidney and heart of type I diabetic mice ⁺ Horizontal (nmol/mg protein)

TABLE 59 GRb-PQN4 in combination with insulin increases NADPH levels (nmol/mg protein) in muscle and heart of type I diabetic mice

Note: all data are expressed as mean ± SD; differences between the model group or treatment group and normal control group were analyzed using t-test, and significant differences between groups of samples other than normal control were performed using one-way analysis of variance (ANOVA) in combination with Duncan's multiple-range test Performing sexual analysis; p compared to control group<0.05，**p<0.001,***p<0.005; in comparison to the model set, ^# p<0.05; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

TABLE 60 GRb-PQN4 in combination with insulin decreases NADP in muscle and heart of type I diabetic mice ⁺ Ratio to NADPH

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.05; in comparison to the model set, ^# p<0.05, ^## p<0.001; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

11.5C.GRb-PQN4 and its combination treatment with insulin increased the total amount of glutathione in the muscle and heart of type I diabetic mice and maintained the GSH/GSSG ratio above normal levels.

The results of the experiment showed that the total amount of glutathione [ reduced Glutathione (GSH) + oxidized glutathione (GSSG) ] in the muscle and heart of the untreated type I diabetes model group mice had a decreased tendency compared to the total amount of glutathione in the normal control group animals in the course of 7 to 8 weeks (treatment 5 weeks) and in the course of 12 to 13 weeks (treatment 10 weeks) (table 61), and that the levels of GSH and the ratio of GSH/GSSG in the muscle and heart were significantly decreased (tables 62 and 63). The treatment with GRb-PQN4 in combination with insulin increased total glutathione levels in muscle and heart throughout the treatment period, wherein both GSH levels and GSH/GSSG ratios exceeded those of normal control animals; GRb-PQN4 and insulin alone only increased GSH levels and GSH/GSSG ratios in animal muscle throughout the course of administration; while GRb-PQN4 treatment for 10 weeks also increased GSH levels and GSH/GSSG ratios in the animal hearts. These results demonstrate that GRb-PQN4 in combination with insulin synergistically increases glutathione antioxidant capacity and efficiency of use, where the role of GRb-PQN4 is indispensable.

TABLE 61 GRb-PQN4 and its use in combination with insulin to increase the total glutathione content (nmol/mg protein) in the vital organs of type I diabetic mice

note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group<0.05; in comparison to the model set, ^# p<0.05， ^## p<0.01; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05；n＝5。

TABLE 63 GRb-PQN4, insulin or a combination thereof for increasing the GSH to GSSG ratio in the important visceral organs of type I diabetic mice

Note: all data are expressed as mean ± SD; analyzing the difference between the model group or the treatment group and the normal control group by using a t test, and performing the analysis of the difference significance among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to control group <0.05; in comparison to the model set, ^# p<0.05, ^## p<0.01; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05, ^&& p<0.01；n＝5。

here, it is also noted that the total amount of glutathione, the GSH level and the GSH/GSSG ratio in the kidney of the diabetes model group mice did not see any change compared to the normal control, which again reveals that the kidney is different from the muscle and the heart. In combination with the results of the related studies obtained in the previous experiments, i.e., total amount of NAD in kidney, NAD in diabetic model group mice ⁺ NADH ratio, total NADP and NADP ⁺ the/NADPH ratio is kept at the normal level of each corresponding relevant parameter in the kidney of the mice in the normal control group of animals, which shows that in the state of type I diabetes, the kidney of the mice is damaged by oxidative stress more slowly or later than the muscle and heart, and the ATP level in the kidney is reduced independent of NAD. Therefore, GRb-PQN4 and its effect on increasing renal ATP levels in diabetic mice in combination with insulin also works with NAD ⁺ NADH-mediated glycolysis, tricarboxylic acid cycle and electron transport of Complex enzyme I were not involved.

grb-PQN4 and its combination treatment with insulin increases superoxide dismutase activity above normal levels in the heart and skin and maintains levels of oxygen free radicals and oxidative stress products in the blood, heart and skin in type I diabetic mice in the normal range.

In order to further clarify the redox state of the cells, the activities of superoxide dismutase (SOD) and catalase in heart and skin of the diabetic vulnerable organs, the levels of superoxide anions and hydrogen peroxide in blood and the vulnerable organs, and the content of oxidative stress products (including lipid oxidative stress product malondialdehyde and nucleic acid DNA oxidative stress product 8-hydroxydeoxyguanosine) were further determined.

The results of the experiment (table 64) show that the activity of superoxide dismutase and catalase in the heart and superoxide dismutase in the skin of mice in the type I diabetes model group is significantly lower than that of normal animals (control group) (. Star.) (. Sup.p)<0.05 or x p<0.001 ); positive control insulin significantly increased the activity of superoxide dismutase only in mouse hearts compared to the model group: ( ^# p<0.05 GRb-PQN4 alone or in combination with insulin) significantly increased superoxide dismutase and catalase in mouse hearts and skinActivity of dismutase (A) ^# p<0.05 ^## p<0.01 or ^### p<0.001 And out of control (. Star.p.)<0.05)。

TABLE 64I type diabetic mice different tissues antioxidase Activity (units/mg protein)

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except for the normal by using one-way analysis of variance (ANOVA) and combining a Duncan multi-range test; p compared to normal control group <0.05,***p<0.001,; in comparison to the model set, ^# p<0.05， ^## p<0.01， ^### p<0.001；n＝5。

further, as shown in tables 65 and 66, the contents of superoxide anion and hydrogen peroxide as oxidative stress agents and malondialdehyde and 8-hydroxydeoxyguanosine as oxidative stress products were significantly increased in the blood, heart and skin of type I diabetic mice as compared with normal mice (control group) (. About.p)<0.05 or x p<0.01 ); the positive control drug insulin significantly reduced the level of the oxidative stress product 8-hydroxydeoxyguanosine only in the blood of the mice compared to the model group: ( ^# p<0.05 GRb-PQN4 significantly reduces the levels of all of said oxidative stress agents and products except malondialdehyde in heart and skin ((II)) ^# p<0.05 or ^## p<0.01 And GRb-PQN4 in combination with insulin significantly reduces the level of all said oxidative stress agents and products of oxidative stress: ( ^# p<0.05 or ^## p<0.01 And both have a stronger reducing effect on superoxide anions in the skin and malondialdehyde as well as 8-hydroxydeoxyguanosine in the blood and heart.

TABLE 65.I diabetes mice different tissue oxidative stress substance content

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except for the normal by using one-way analysis of variance (ANOVA) and combining a Duncan multi-range test; p compared to normal control group <0.05; in comparison to the model set, ^# p<0.05， ^## p<0.01；n＝5。

TABLE 66.I diabetes mice different tissue oxidative stress product content

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except for the normal by using one-way analysis of variance (ANOVA) and combining a Duncan multi-range test; p in comparison with normal control group<0.05，**p<0.01; in comparison to the model set, ^# p<0.05， ^## p<0.01; n =5. The combination of the research results proves that: GRb-PQN4 alone or in combination with insulin can significantly improve superoxide dismutase and catalase activity in mouse heart and superoxide dismutase activity in skin, significantly reduce the content of superoxide anion and hydrogen peroxide as oxidative stress substances and malondialdehyde and 8-hydroxydeoxyguanosine as oxidative stress products, while insulin has much less antioxidant effect than GRb-PQN 4. It can be seen that GRb-PQN4 alone can well protect the antioxidant capacity of cells in type I diabetes, so that oxidative stress damage in type I diabetes can be remarkably alleviated, and especially, the combined treatment of GRb-PQN4 and insulin can further improve the antioxidant capacity of cells mediated by superoxide dismutase and catalase in a compensatory way and basically avoid oxidative stress damage.

It is specifically noted herein that superoxide dismutase catalyzes the formation of superoxide anions into oxygen and hydrogen peroxide, and appears to be the first line of defense against oxygen-derived free radicals. Subsequently, catalase and GSH can decompose hydrogen peroxide into non-toxic molecular oxygen and water, and both the Glutathione (GSH) antioxidant system (consisting of GSH, GSSG and related enzymes) and the thioredoxin antioxidant system (consisting of NADPH and thioredoxin) can defend against oxidative stress by effectively removing various ROS.

So far, the research results prove that GRb-PQN4 has good protection effect on a cell antioxidant system under the type I diabetes state, including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) antioxidant system (consisting of GSH, GSSG and related enzymes) and thioredoxin antioxidant system (consisting of NADPH and thioredoxin), and the combination of the GRb-PQN4 and insulin can improve the systemic antioxidant capacity of cells to exceed the normal level in a compensatory way. Based on the important role of oxidative stress injury on the occurrence and development of diabetes and its complications, our research results strongly support the use of GRb effective compositions alone, especially in combination with insulin, for the prevention and treatment of type I diabetes and its complications.

Example 11.6 GRb-PQN4 and its combination with insulin treatment reduces advanced glycation end products (AGEs) in type I diabetic mice.

As shown in table 67, the levels of advanced glycosylation end products (AGEs) in circulating blood, heart and skin were significantly higher in untreated type I diabetic mice (model controls) with a course of 12-13 weeks than in normal controls, where AGEs levels in heart were 3 times higher and AGEs levels in blood were 1.80 times higher than in normal controls; AGEs levels in blood and skin of animals treated chronically with GRb-PQN4 were very significantly lower than those of model controls and maintained normal levels of AGEs in skin; the chronic treatment group of insulin had significantly lower AGEs levels in skin only than the model control, and in particular had significantly higher AGEs levels in heart than the model control; AGEs levels in the chronic treatment group of GRb-PQN4 in combination with insulin were maintained in a near normal or normal range in blood, heart and skin, especially at values below normal controls in skin. The results of the above studies show that chronic treatment with GRb-PQN4 can effectively reduce the levels of advanced glycosylation end products in circulating blood, skin and vital organs of type I diabetes, and chronic treatment with insulin can reduce AGEs levels less strongly than GRb-PQN4, and the combined treatment can exert the drug effect synergistically and almost completely prevent or eliminate the level of hyperglycosylation in type I diabetes.

It is particularly pointed out here that, in the diabetic state, advanced glycosylation end products (AGEs) are end products which are toxic and lose their original physiological functions as a result of glucose efflux from the polyol pathway and the glyceraldehyde 3-phosphate/dihydroxyacetone phosphate-Methylglyoxal (MGO) pathway in the glycolysis process and binding to proteins, phospholipids and nucleic acids in the form of fructose and MGO, respectively, and which are highly sugar-rich and sugar metabolism disorders, and thus AGEs are considered to be important contributors to the reduction of the quality of life of diabetic patients and the initiation and progression of diabetic complications, in particular life-threatening chronic complications. Therefore, in combination with the fact that GRb-PQN4 is unable to lower blood glucose levels but maintain ATP levels within a normal range in type I diabetic mice (Table 52), the above results indicate that GRb-PQN4 is effective in reducing the deviation of glycolytic intermediates from the energy metabolism pathway to AGEs and promoting oxidative phosphorylation of glycolytic intermediates to mitochondria in type I diabetic conditions and high glucose environments, while insulin is far less active than GRb-PQN4, but the combination completely blocks the deviation of glycolytic intermediates from the energy metabolism pathway to AGEs. In summary, GRb-PQN4 can improve and/or correct glucose metabolism disorder in type I diabetes, and the effect of correcting diabetes metabolism disorder is further enhanced in combination with insulin, so that GRb-PQN4, particularly in combination with insulin, can delay or block the development of diabetes, improve the quality of life of diabetes patients and prevent and treat diabetic symptoms and various chronic complications fundamentally.

TABLE 67 effects of GRb-PQN4, insulin or a combination of both on AGEs levels in blood and tissue of type I diabetic mice

Note: all data are expressed as mean ± SD; analysis of the appearance of the model group or the treated group and the Normal control group by t-testSignificant differences, significant analysis of differences among groups of samples other than normal controls was performed using one-way analysis of variance (ANOVA) in combination with the Duncan multi-range test; p compared to control group<0.05，**p<0.01，***p<0.005; in comparison to the model set, ^# p<0.05， ^## p<0.01; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05, ^&& p<0.01；n＝5。

example 11.7 GRb-PQN4 and its combination therapy with insulin reduces the level of proinflammatory factors in the blood of type I diabetic mice.

As shown in table 68, the levels of pro-inflammatory factors including the pro-inflammatory transcription factor TNF α and the inflammatory factors IL1 β and IL6 in the circulating blood of untreated type I diabetic mice with a disease course of 12-13 weeks (model controls) were significantly higher than those of the normal controls, approximately 2 and 3 times higher than those of the normal controls, respectively; the level of the 3 inflammatory factors of the animals in the GRb-PQN4 chronic treatment group is very remarkably lower than that of the model control group; the insulin chronic treatment group significantly reduced the levels of IL1 β and IL6, but increased TNF α; the levels of 3 inflammatory factors in the chronic treatment group of GRb-PQN4 in combination with insulin were all maintained within normal ranges. Taken together, the results of the above studies indicate that chronic treatment with GRb-PQN4 is effective in reducing the chronic inflammatory state of type I diabetes, and chronic treatment with insulin reduces the proinflammatory factors less comprehensively and with less intensity than GRb-PQN4, and that combined treatment with both is almost completely effective in preventing or eliminating the chronic inflammatory state of type I diabetes.

TABLE 68 effects of GRb-PQN4, insulin or a combination of both on proinflammatory factor levels in blood of type 1 diabetic mice

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except the normal control by using one-way analysis of variance (ANOVA) in combination with Duncan multi-range test; p compared to normal control group<0.05，**p<0.01，***p<0.005; andthe ratio of the model sets is determined, ^# p<0.05， ^## p<0.01; compared with the group of insulin, the medicine composition has the advantages that, ^& p<0.05， ^&& p<0.01；n＝5。

non-infectious chronic low-level inflammatory states are one of the features of type II diabetes, and endoplasmic reticulum stress, hypoxia, cell hypertrophy, death and fibrosis caused by high levels of free fatty acids associated with sugar metabolism disorders and mitochondrial dysfunction are all involved in the development and progression of chronic inflammation in diabetes, and thus, this inflammatory state is also referred to as metabolic inflammation (metaflumation). In addition, acute glucose excursions may cause significant oxidative stress and inflammation of rat aortic endothelial cells, increase adhesion of monocytes to rat aortic endothelial cells, and increase endothelial apoptosis, resulting in severe cardiovascular injury. Importantly, metabolic inflammation, a product of metabolic disorders, in turn exacerbates metabolic disorders by inducing multiple mechanisms such as insulin resistance, damaging islet cells, and the like, and promotes the development of diabetes and its complications. Therefore, inflammation is widely recognized as a key etiology that plays an important role in the development of diabetes and diabetic complications, and thus anti-inflammatory strategies help not only control but also treat the symptoms of type II diabetes, but classical anti-inflammatory drugs do not produce satisfactory results in preventing diabetes progression and cardiovascular events.

In the context of this context, our findings illustrate several key issues: (1) Metabolic inflammation also occurs in type I diabetes, and a 1/2 therapeutic dose of insulin only slows this chronic inflammatory state; (2) GRb-PQN4 can remarkably improve chronic inflammation in the type I diabetes, and the effect is not related to blood sugar level but related to improvement of intracellular metabolic disorder, or the effect of GRb-PQN4 on improvement of chronic inflammation related to type I diabetes further proves that GRb-PQN4 can improve the metabolic disorder in the type I diabetes and slow down or avoid vicious circle between the metabolic disorder and the chronic inflammation; (3) The combination of GRb-PQN4 and insulin can act synergistically and prevent and/or eliminate chronic inflammation in the type I diabetes and avoid or terminate vicious circle between metabolic disturbance and chronic inflammation, so that the combination of the GRb-PQN4 and insulin can synergistically correct the metabolic disturbance in the type I diabetes and provide a reasonable explanation for the GRb-PQN4 to improve the insulin sensitivity of a type I diabetes mouse; (4) The combined treatment of GRb-PQN4 and insulin can provide a treatment which is different from a classical anti-inflammatory drug and is effective in relieving metabolic inflammation for a patient with type I diabetes, and can effectively block the development of type I diabetes and the occurrence and the development of common symptoms and chronic complications thereof. It is further demonstrated by combining the above research results that GRb-PQN4 can improve and/or correct the metabolic disorders of sugar in type I diabetes, and the action of correcting the metabolic disorders of diabetes is further enhanced in combination with insulin, so that GRb-PQN4, particularly in combination with insulin, can delay or block the development of diabetes from the disease root, improve the quality of life of diabetic patients and prevent and treat common symptoms and chronic complications.

Example 12 GRb-PQN4 in combination with metformin or dapagliflozin improves polydipsia, polyphagia and polyuria symptoms and lowers blood glucose levels in type II diabetes.

The research method comprises the following steps:

a mouse model of type II diabetes was prepared using a conventional method: multiple intraperitoneal injections of streptozotocin (30 mg/kg) in combination with 60% high fat diet resulted in a model of type II diabetes in C57BL/6 mice.

Grouping and dosing regimens: 49 diabetic mice meeting the hyperglycemic criteria were divided into: a normal animal control group (a normal control group for short), a type II diabetes model group (a model group for short), a GRb-PQN4 group (10 mg/kg), a metformin group (200 mg/kg), a metformin + GRb-PQN4 group (200 mg/kg +10 mg/kg), a dapagliflozin group (1 mg/kg) and a dapagliflozin + GRb-PQN4 group (1 mg/kg +10 mg/kg), wherein each group comprises 7 animals. The administration is carried out once by intragastric administration in 8-9 morning; the set dose of the metformin and the dapagliflozin is equivalent to the clinical treatment dose, and according to the clinical medication requirements of the metformin and the dapagliflozin, 1/2 of the initial dose of the two medicaments is increased at the 14 th week of treatment, 1/2 of the existing dose of the two medicaments is increased again at the 22 th week of treatment, and the treatment is continued for 26 weeks until the experiment is finished.

And (3) observation of drug effect: the weight, diet, water intake, and urine output of each group were recorded before treatment (week 0) and at 2 weeks, 4 weeks, 7 weeks, 10 weeks, 14 weeks, 18 weeks, 22 weeks, and 26 weeks of treatment, and the wetness of the animal feeding bedding was photographed to further visually reflect the urine output of the animals. In addition, random blood glucose and fasting blood glucose 24 hours after the last administration were measured by a blood glucose test strip to understand the effect that the drug may have on blood glucose levels; insulin resistance (12 hours fasting followed by intraabdominal injection of 0.75U/kg insulin, measurement of blood glucose levels at 30, 60, 90 and 120 minutes) and glucose tolerance (12 hours fasting followed by gavage of 1mg/kg glucose, measurement of blood glucose levels at 30, 60, 90 and 120 minutes post-glucose administration) were also monitored as the treatment time extended.

The research results are as follows:

12.1 GRb-PQN4 in combination with metformin or dapagliflozin can ameliorate the symptoms of polydipsia, polyphagia and polyuria in type II diabetic mice.

The body weights of the animals of the respective test groups did not differ significantly throughout the test, and thus no relevant data was shown. As shown in table 69, the food intake of the model group mice was significantly higher in the course of 10 weeks than that of the normal control group animals, and was significantly reduced from the beginning of 14 weeks; GRb-PQN4 and metformin combination treatment can maintain the food intake lower than that of the model group within 2-10 weeks, and then maintain the food intake higher than that of the model group within 14-26 weeks, which shows that the two combination treatments are beneficial to maintain the body weight of the sick mice close to the normal level.

TABLE 69 Effect of GRb-PQN4 in combination with metformin or dapagliflozin on food intake in type II diabetic mice (g/only/24 h)

Note: all data are expressed as mean ± SD; by usingAnalyzing the significance difference of the model group or the treatment group and the normal control group by using t test, and performing significance analysis of the difference among a plurality of groups of samples except the normal control group by using one-way analysis of variance (ANOVA) in combination with Duncan multi-range test; p compared to normal control group<0.05; in comparison to the model set, ^# p<0.05；n＝7。

as shown in Table 70, the water intake of the model group mice was significantly higher than that of the normal control group animals at the early stage of the disease course (4 to 10 weeks), and decreased from the beginning of the disease course of 14 weeks to the range of the water intake of the normal control group animals; the urine output of the model mice varied uniformly (Table 71), i.e., at 2-10 weeks, the urine output was almost 1.5-2 times (p < 0.05) that of the normal control animals, and then (14-26 weeks) was close to that of the normal control animals. GRb-PQN4 in combination with metformin significantly reduced animal water intake at 4-18 weeks (/ p < 0.05) and significantly reduced animal urine intake at 14-18 weeks (/ p < 0.05) compared to model group animals. In combination with the above results, the use of GRb-PQN4 and other efficacious or effective compositions in combination with metformin to ameliorate the symptoms of polydipsia, polyphagia and polyuria in type II diabetics is supported.

TABLE 70 GRb-PQN4 Effect on Water intake in type II diabetic mice in combination with metformin or dapagliflozin (g @) Only/24 h)

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except for the normal by using one-way analysis of variance (ANOVA) and combining a Duncan multi-range test; p compared to normal control group<0.05; in comparison to the model set, ^# p<0.05；n＝7。

TABLE 71 Effect of GRb-PQN4 in combination with metformin or dapagliflozin on urine volume in type II diabetic mice (g/only/24 h)

GRb-PQN4 has no hypoglycemic effect but can enhance the hypoglycemic effect of metformin or dapagliflozin to a certain extent.

12.2a.grb-PQN4 prolongs the effective hypoglycemic treatment period of chronic treatment with metformin and dapagliflozin.

It is a common clinical phenomenon that the effect of hypoglycemic agents is weakened with the prolongation of the treatment time course or the progression of diseases, and therefore, we observed whether GRb-PQN4 can prolong the effective treatment period of metformin and dapagliflozin. As shown in table 72, fasting blood glucose levels of the mice of the model and each administered group exceeded 2 times of the blood glucose levels of the normal control group animals before the treatment (week 0), blood glucose levels of the animals of the model group further increased until reaching a peak at week 26 as the course of disease extended, GRb-PQN4 alone did not decrease fasting blood glucose levels, metformin decreased blood glucose levels of the mice only at

weeks

4, 7 and 18 (# p < 0.05), and elevated blood glucose levels with the progress of the course were slowed down at each time point except for

weeks

10, 14 and 26 (# p < 0.05). While the combination of GRb-PQN4 with metformin or dapagliflozin extended the hypoglycemic shelf-life of metformin or dapagliflozin, respectively, but did not improve their hypoglycemic strength. It can be seen that GRb-PQN4 can prolong the effective treatment period of metformin and dapagliflozin for reducing blood sugar.

TABLE 72 Effect of GRb-PQN4 treatment with metformin or dapagliflozin on fasting plasma glucose (mM) in type II diabetic mice

Note: all data areExpressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis on the difference between a plurality of groups of samples except for normal by using one-way analysis of variance (ANOVA) in combination with Duncan multi-range test; p compared to normal control group<0.01; in comparison to the model set, ^# p<0.05；n＝7。

for postprandial blood glucose levels, the model group animals also exhibited further increases in blood glucose levels with increasing course of disease until reaching a peak at week 26, as shown in table 73. Except for the early few time points, metformin alone or in combination with GRb-PQN4 did not significantly reduce postprandial blood glucose levels in diabetic mice, and dapagliflozin alone or in combination with GRb-PQN4 also reduced postprandial blood glucose levels only at

weeks

2, 4, and 7.

TABLE 73 GRb-PQN4 in combination with metformin or dapagliflozin able to reduce postprandial blood glucose (mM) in type II diabetic mice

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control by using a t test, and performing significance analysis on the difference between a plurality of groups of samples except the normal group by using one-way analysis of variance (ANOVA) in combination with Duncan multi-range test; p vs. normal control group <0.01; in comparison to the model set, ^# p<0.05；n＝7。

the combined treatment of 12.2B.GRb-PQN4 and metformin or dapagliflozin strengthens the acute hypoglycemic effect of the two.

To further determine the acute hypoglycemic effect of GRb-PQN4 potentiating metformin or dapagliflozin, if present, the blood glucose levels of the mice were determined 2 hours after the last day of administration at weeks 15, 18 and 22 of treatment, respectively. As shown in table 74, both metformin and dapagliflozin significantly reduced mouse blood glucose levels at 2 hours post-dose; although GRb-PQN4 alone did not affect blood glucose levels, at week 15 and week 18, 2 time points, the blood glucose levels in the GRb-PQN4 treatment group in combination with metformin or dapagliflozin were significantly lower than those in the metformin or dapagliflozin treatment group alone, indicating that GRb-PQN4 potentiated their acute hypoglycemic effects. Since GRb-PQN4 alone does not have a tendency to lower blood sugar, it is reasonable to assume that this potentiation is not the sum of 2 hypoglycemic effects but that GRb-PQN4 potentiates the sensitivity of type II diabetic organisms to metformin and dapagliflozin or that GRb-PQN4 has a complementary mechanism of action to metformin or dapagliflozin.

TABLE 74 hypoglycemic agent effect 2h after administration of GRb-PQN4 alone or in combination with metformin or dapagliflozin

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis on the difference between a plurality of groups of samples except for normal by using one-way analysis of variance (ANOVA) in combination with Duncan multi-range test; p compared to normal control group<0.01; in comparison to the model set, ^# p<0.05；n＝7。

chronic treatment of GRb-PQN4 in combination with metformin or dapagliflozin enhanced their overall glucose lowering/GRb-PQN 4 in combination with insulin significantly reduced glycosylated hemoglobin levels.

As shown in table 75, chronic treatment for 26 weeks both metformin and dapagliflozin significantly reduced the glycosylated hemoglobin levels in the affected mice, and GRB-PQN4 in combination with metformin further reduced the glycosylated hemoglobin levels, but did not potentiate the effect of dapagliflozin. The glycosylated hemoglobin level reflects the mean plasma glucose concentration over time (4-8 weeks), so the reduced glycosylated hemoglobin levels demonstrate that the treatment with metformin or dapagliflozin set forth in example 11 effectively reduced the blood glucose levels in the affected mice, and that GRb-PQN4 further potentiates the hypoglycemic effect of metformin. The results of the study support the clinical use of GRb-PQN4 to control type II diabetes blood glucose, preferably in combination with metformin.

TABLE 75 GRb-PQN4 in combination with metformin or dapagliflozin significantly reduced glycosylated hemoglobin levels and insulin resistance coefficients

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except for the normal by using one-way analysis of variance (ANOVA) and combining a Duncan multi-range test; p compared to normal control group<0.05,**p<0.01，***p<0.005; in comparison to the model set, ^# p<0.05, ^## p<0.01；n＝7。

12.2D.GRb-PQN4 combined with metformin or dapagliflozin can obviously improve insulin resistance state

Insulin resistance is a characteristic of type II diabetes, from which we further observed the effect of chronic treatment with GRb-PQN4 in combination with metformin or dapagliflozin on insulin resistance in diabetic mice. Blood glucose levels were measured at 30, 60, 90 and 120 minutes 24 hours after dosing at

weeks

0, 2, 4, 7, 10, 14, 18, 22 and 26 of treatment, with 12 hours fasting, and with 0.75U/kg i.p. insulin. The experimental results are shown in table 75, metformin and dapagliflozin after being treated for 26 weeks both significantly reduce the insulin resistance index (HOMA-IR) assessed in the steady-state model, GRb-PQN4 alone does not reduce the insulin resistance index, but GRb-PQN4 in combination with metformin enhances the insulin resistance improving effect, and the effect strength is not as strong as that of dapagliflozin.

Insulin resistance has become a major pathophysiological factor for the development and progression of diabetes, particularly type II diabetes, and is associated with metabolic syndrome (referring to the accumulation of at least three of the following diseases: hypertension, abdominal obesity, hyperglycemia, low high density lipoprotein levels, and high serum triglycerides) and polycystic ovary syndrome. Therefore, our findings support the medical use of GRb-PQN4 and other potent or potent compositions in combination with metformin for the treatment of metabolic syndrome and polycystic ovary syndrome.

Example 13 GRb-PQN4 and its combination with metformin or dapagliflozin for the treatment of diabetic complications

Diabetes can cause various complications, including acute or chronic complications of heart, brain, liver, lung, kidney, eyes, limbs, skin, nerves and the like, which are more than 80, wherein the diabetic cardiovascular and cerebrovascular diseases are the most common chronic complications in clinic and cause great pain to the mind and body of a patient and even take the life of the patient. The existing diabetes treatment drugs slow down complications by controlling blood sugar level, but have limited clinical effects, so that the drug effect of GRb-PQN4 and the combination of the GRb-PQN4 and metformin (the classic II type diabetes is also the first drug) or dapagliflozin (a novel glucose cotransporter 2/SGLT2 anti-type II diabetes drug) in preventing and treating common diabetes complications is emphatically observed.

Example 13.1 GRb-PQN4 can significantly improve skin rash in type II diabetic mice and prevent dermatitis induced by treatment with metformin or dapagliflozin.

The research method comprises the following steps:

in order to reveal the potential medicinal value of the GRb-PQN4 composition and the combined treatment of the GRb-PQN4 composition and the hypoglycemic agent for preventing and treating the diabetic rash, a diabetic chronic rash model is constructed by using 4% of sodium sulfide and 5% of formaldehyde, and the recovery condition of the rash of each group of animals is observed. After 2 weeks of treatment, the animals were anesthetized with pentobarbital sodium, the mice were treated with hair longer on their backs by electric hair clippers, then they were further depilated by applying 4% sodium sulfide on their backs, and the next day 5% formaldehyde was applied on their backs to induce skin rash. Mice were allowed free diet and water after surgery, groups of animals continued to the respective drug treatments or treatments, and the incidence and severity of dermatitis (rash) were observed on the day after surgery (day 0), on

days

3, 6 and 9 and the skin area was photographed.

The research results are as follows:

as shown in table 76 and fig. 12, on

days

3, 6, and 9 of formaldehyde application, the diabetic model group animals developed almost all rashes, and the normal control group animals also developed a high incidence of rashes but were significantly less severe than the model group animals; in the treatment groups, the incidence rate of the rash of the sick mice is obviously reduced by GRb-PQN4 in the whole course, the incidence rates of 3 days and 6 days are reduced by the combined treatment of GRb-PQN4 and metformin, the severity degree of the rash is also obviously reduced, and the treatment of other groups has no efficacy of reducing the incidence rate and the severity degree; in contrast, metformin and dapagliflozin treated alone exacerbate the degree of rash in the affected mice. Acute metabolic disorders and chronic degenerative complications of diabetes, including vitiligo and psoriasis, pruritus, desquamation, skin rashes, seborrheic dermatitis, dry scabs, warts, eczema, skin pigmentation, candida infections, etc., lead to a high incidence of skin disorders in diabetic patients, which complications, although not fatal, severely reduce the quality of life of the patients. Our findings suggest that first-line hypoglycemic agents have limited therapeutic value for diabetic skin complications and may exacerbate skin rash. This further highlights the unique clinical utility value of GRb-PQN4 and other efficacious or effective compositions for the prevention and treatment of diabetic skin complications, either alone or in combination with metformin.

TABLE 76 GRb-PQN4 in combination with metformin or dapagliflozin significantly reduced the incidence of type 2 diabetic mouse skin rash (%)

Note: all data are expressed as mean ± SD; carrying out significance analysis on differences among a plurality of groups of samples by using a rank sum test; p compared to control group<0.05; in comparison to the model set, ^# p<0.05；n＝5。

example 13.2 GRb-PQN4 significantly promoted skin lesion repair in type II diabetic mice without this effect by both metformin and dapagliflozin.

The research method comprises the following steps:

in order to reveal the potential medicinal value of GRb-PQN4 and the combined treatment of the GRb-PQN4 and hypoglycemic drugs on the prevention and treatment of diabetic foot, the wound repair condition of each group of animals is observed by utilizing a diabetic chronic comprehensive wound model. At week 5 of the treatment, the animals were anesthetized with sodium pentobarbital, and the longer hair on the back of the mice was treated with electric hair clippers, followed by further removal of the back hair with depilatory cream. Then, the skin on the back was sterilized with iodophor, a 1.5 × 1.5cm whole skin wound surface was created with scissors under aseptic conditions on the back, the skin was cut to the subcutaneous fascia, and the skin around the wound was covered with gauze after alcohol sterilization. Mice were allowed free diet and water after surgery, and each group of animals continued to be treated or treated with the corresponding medication. Wound healing was recorded as photographs taken on

days

0, 3, 6, 9, 12, and 15 after molding (and also after wound treatment), respectively, and wound area was counted.

The research results are as follows:

as shown in fig. 13, on day 3 after the treatment, it was seen that the wound area of the mice of the diabetic model group was significantly larger than that of the mice of the normal control group; GRb-PQN4 alone or in combination with metformin or dapagliflozin can remarkably promote wound healing of diabetic mice, and the effect of GRb-PQN4 and metformin combined therapy is optimal; however, metformin and dapagliflozin alone showed only a trend of efficacy. With different time points of prolonged administration time, the metformin and dapagliflozin which are used alone do not have any effect of promoting wound healing, but GRb-PQN4 alone or combined with metformin and dapagliflozin can promote wound healing obviously, and the wound healing speed of the GRb-PQN4 and metformin combined treatment group mice reaches the wound healing level of normal control group animals. The results of the study illustrate two key issues: firstly, the metformin and dapagliflozin can not effectively promote the skin damage repair of diabetic mice, and the result is consistent with the limited drug effect that a plurality of drug treatments including metformin can not ensure a rapid and definite repair process observed in long-term clinical practice, which indicates that the reduction of blood sugar can not effectively promote the wound repair of diabetic patients; secondly, although GRb-PQN4 can not reduce the blood sugar level of the diabetic mouse, the composition has the strong effect of promoting the repair of the skin lesion of the diabetic mouse, and the drug effect of the composition combined with metformin can be enhanced to a certain extent, so that the GRb-PQN4 and other effective or effective compositions can provide a brand-new effective treatment method for various injuries of diabetic patients.

Example 13.3 GRB-PQN4 alone and in combination with metformin or dapagliflozin is useful for the treatment of cardiovascular disease in type II diabetes.

The research method comprises the following steps:

in addition to disorders of carbohydrate metabolism and their associated mitochondrial dysfunction, hyperlipidemia-associated atherosclerosis is also implicated in cardiovascular complications of diabetes mellitus. Accordingly, we observed the pharmacodynamic observation endpoint, i.e., levels of total cholesterol, low density lipoprotein cholesterol, and Triglycerides (TG) in blood at 26 weeks of treatment, and stained cardiac sections with hematoxylin-eosin (HE) and Masson (MS) to observe the health status of cardiomyocytes, blood vessels, and fibrosis, respectively; MS staining blue indicates collagen positive, deeper staining indicates more severe fibrosis, and the percentage of blue area to total tissue area was calculated by ImageJ software. Taking a plasma sample 24 hours after the last administration, and measuring the blood fat index by using a conventional biochemical method; histochemical cardiac specimens were prepared by conventional methods and stained by conventional methods.

The research results are as follows:

as shown in Table 77, total cholesterol and LDL cholesterol in blood were significantly increased in type II diabetes model group mice compared to normal control group mice (p)<0.05 And no increase in triglycerides was seen; GRb-PQN4 alone does not reduce any of the lipid profiles in the affected mice, and metformin further increases total cholesterol levels (C) ^# p<0.05 ); importantly, the combination of GRb-PQN4 and metformin can significantly reduce the levels of total cholesterol and low density lipoprotein cholesterol in diabetic mice, but the combination of GRb-PQN4 and dapagliflozin has no effect.

TABLE 77 GRb-PQN4 in combination with metformin reduces the level (mM) of an index associated with lipid metabolism in blood of type II diabetic mice for 26 weeks

alterations in lipid metabolism are an important pathogenesis in the association of cardiovascular diseases, diabetes, obesity and other metabolic diseases. In particular, cholesterol accumulation plays an important role in the pathogenesis of cardiovascular diseases, diabetes, obesity and other metabolic diseases by driving inflammation and endoplasmic reticulum stress, and excess cholesterol and cholesterol crystals also trigger activation of inflammasome, which underlies the development and progression of atherosclerosis. Total cholesterol, especially low density lipoprotein cholesterol, is an important risk factor for cardiovascular diseases, so the research result predicts the effect of GRb-PQN4 in combination with metformin on protecting the cardiovascular vessels of diabetic mice.

Indeed, cardiac HE and MS staining results show that GRb-PQN4 combined with metformin or dapagliflozin can protect myocardial cells and cardiac vessels of diabetic mice, and the drug effect of the combination with metformin is better than that of the combination with dapagliflozin, particularly the GRb-PQN4 alone has the best treatment effect. As shown in fig. 14, HE staining results indicated: hypertrophy of myocardial cells in some regions of model mice resulted in unclear structural boundaries (blue arrows) and rupture of muscle fibers (black arrows), and inflammatory cell infiltration around blood vessels (yellow arrows) and marked thickening of arteriole walls (green arrows). Consistently, as shown in fig. 15, MS staining and its semi-quantitative results showed that the collagen volume fraction of the heart of the untreated diabetes model group mice was significantly higher than that of the normal control group animals, indicating severe myocardial fibrosis. It can be seen that untreated type II diabetic mice, which had suffered from 26 weeks, developed typical diabetic cardiac lesions in both myocardium and blood vessels. The dapagliflozin mice also exhibited various lesions in the model group of mice: cardiomyocyte hypertrophy, unclear structural boundaries (blue arrows), myofiber rupture (black arrows), small inflammatory cell infiltration (yellow arrows), significant thickening of arteriole walls (green arrows) (fig. 14), and significantly high collagen integral numbers (fig. 15); although the metformin group mice showed hypertrophy of cardiac muscle cells, unclear structural boundaries (blue arrows), slight inflammatory cell infiltration (yellow arrows), significant thickening of arteriole walls and significantly high collagen volume, the degree of lesion was alleviated compared with the model group animals. The cardiac muscle and vascular morphology of the GRb-PQN4 group mice are close to those of the normal control group mice, and the following results can be seen: intact cell morphology, well-arranged muscle fibers, no cellular hypertrophy and inflammatory cell exudation, normal arteriole walls (fig. 14), and significantly slowed the degree of myocardial fibrosis in affected mice (fig. 15). However, the effect of the combined treatment of GRb-PQN4 and metformin or dapagliflozin on the prevention of myocardial fibrosis and vascular wall thickening is equivalent to the effect of GRb-PQN4, but the effect of protecting the myocardium is not as good as the effect of GRb-PQN 4. It is necessary here to review the results of the relevant studies in the type I diabetes model (example 10): GRb-PQN4 alone almost completely combats type I diabetic cardiac disease. Since GRb-PQN4 does not lower the blood glucose level and glycosylated hemoglobin level in type I and type II diabetic mice (examples 2 and 12), nor total cholesterol and LDL cholesterol level, it can be seen that the mechanism of GRb-PQN4 for protecting diabetic cardiac complications is independent of blood glucose and blood lipid levels, which further illustrates that GRb-PQN4 has a completely different mechanism of action from the existing antidiabetic drugs, thus providing a completely new strategy for preventing and treating diabetes and its complications, and the related mechanism of action will be disclosed in example 14.

The above results of the study taken together illustrate two key issues: first, although metformin and dapagliflozin (SGLT 2 inhibitor) are the first hypoglycemic agents for patients with cardiovascular disease complicated by diabetes, the protective effect on the myocardium and cardiovascular disease of type II diabetes is limited. Consistent with our findings are: intensive type II diabetes treatment aimed at controlling blood glucose levels above the normal range reduces the risk of major macrovascular and microvascular events only to a slightly greater than 10% [ Future cardio. 2018,14 (6): 491-509]. There are also clinical research reports indicating whether metformin reduces the risk of cardiovascular disease in type II diabetic patients and remains uncertain [ Diabetologia.2017,60 (9): 1620-1629]. GRb-PQN4 can play a significant better drug effect than first-line hypoglycemic drugs for protecting diabetic hearts through a non-hypoglycemic mechanism, and GRb-PQN4 alone or in combination with metformin or dapagliflozin has an important therapeutic value for preventing and treating diabetic cardiovascular complications.

Finally, it is also noted that creatinine levels in urine and blood of diabetic mice showed no renal complications in type II diabetic mice at the age of 26 weeks (and therefore no study data is shown).

Example 14 chronic treatment of GRb-PQN4 and its combination with metformin can ameliorate or correct intracellular energy metabolism disorders and redox imbalances in type II diabetic mice, reduce or completely prevent the production of advanced glycosylation end products (AGEs) and metabolic inflammatory states, but there is no significant protective effect of dapagliflozin itself or in combination with GRb-PQN4, which has as a major mechanism the inhibition of sodium-glucose cotransporter 2 (SGLT 2), the promotion of blood glucose excretion through the urine.

The research idea is as follows:

the heart can be the tissue and organ most vulnerable to glucose metabolism disorder in diabetic condition, so that cardiovascular complications are the first cause of life quality reduction and death of diabetic patients, and the research result of example 12 shows that untreated type II diabetic mice have obvious pathological changes of cardiac muscle and local blood vessel of heart. Therefore, the effect of GRb-PQN4, metformin and dapagliflozin alone and the chronic treatment of GRb-PQN4 in combination with metformin and dapagliflozin respectively on the energy metabolism state of the heart is observed in comparison with the heart as an example; the simultaneous determination of 2 products or results as metabolites of diabetes is also an important reason for the promotion of diabetes development and complications, namely: advanced Glycosylation Endproducts (AGEs) and metabolic inflammation. AGEs accumulation and metabolic inflammatory states are characteristic pathological events of type II diabetes, which in turn, are derived from metabolic disorders that further exacerbate the diabetic metabolic disorders through a variety of known and/or unknown mechanisms including induction of insulin resistance and inflammatory degeneration of islet cells, among others, and promote the development of diabetes and the development and progression of its degenerative complications. AGEs are intrinsically linked to metabolic inflammatory states, mediate a variety of biological effects through their cell-binding receptors, including activation of endoplasmic reticulum stress and inflammatory stress pathways (including proinflammatory signaling pathway activation such as NF- κ B mediated by the proinflammatory factor TNF- α, production of proinflammatory cytokines such as IL-1 and IL-6, and cell adhesion molecules), and endoplasmic reticulum stress leads to oxygen radical accumulation and is involved in the pathophysiology of cardiovascular disease. Therefore, slowing down AGEs formation and metabolic inflammation is considered to have the effect of treating both symptoms and root causes for the II diabetes. In conclusion, the following studies revealed that GRb-PQN4 may reverse intracellular carbohydrate metabolism disorder and avoid the occurrence of the linked malignant events and the malignant cycle caused by the disorder.

The research method comprises the following steps:

at 24 hours after the last administration of the last week of the experiment in example 12, i.e., the 26 th week of the administration treatment, blood and heart were rapidly collected by a conventional method, and serum was prepared and samples for measuring each set index in the global, cytoplasmic and mitochondrial regions of the heart tissue were prepared, and each set index was detected by a commercial kit. The set index comprises ATP and NAD ⁺ NADH, NADP +, NADPH, GSH, and GSSG, advanced glycosylation end products (AGEs), and proinflammatory factors including TNF- α, IL1 β, and IL6.

The research results are as follows:

example 14.1 GRb-PQN4 in combination with metformin significantly improved cardiac energy metabolism in diabetic mice.

GRb-PQN4 in combination with metformin maintained ATP homeostasis.

As shown in table 78, in type II diabetic mice, ATP levels in the heart tissue global, cytosolic, and mitochondrial were significantly reduced compared to normal mice (p < 0.05), and GRb-PQN4 alone or in combination with metformin significantly increased ATP levels to levels close to those in normal animals. The result shows that GRb-PQN4 has the effects of promoting energy metabolism and maintaining the redox stability of an organism, and not only can the drug effect be enhanced, but also the disease course progress of type II diabetic heart disease can be more comprehensively improved by combining with metformin. Example 13.3 the results in fig. 14 also demonstrate that GRb-PQN4 in combination with metformin can significantly improve pathological characteristics such as myocardial cell swelling, unclear structural boundaries, myofiber rupture, infiltration with a small amount of inflammatory cells, significant thickening of arteriole walls, and cardiac tissue fibrosis in model mice.

TABLE 78 GRb-PQN4 and its use with metformin significantly elevated ATP levels in cardiac mitochondria and cytoplasm (nmol/mg protein)

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of difference between a plurality of groups of samples comprising the model control group and the treatment group by using one-way analysis of variance (ANOVA) in combination with Duncan multi-range test; p compared to normal control group<0.05，**p<0.01; in comparison to the model set, ^# p<0.05， ^## p<0.01；n＝5。

GRb-PQN4 alone and in combination with metformin can significantly increase the total cardiac NAD (NAD) ⁺ + NADH) and maintenance NAD ⁺ The balance of NADH.

As shown in Table 79, the total amount of NAD in heart tissue global (total tissue), mitochondria and cytoplasm of the model group mice with type II diabetes for at least 26 weeks was not significantly different from that of the normal control group mice. Importantly, NADH is significantly reduced in its total tissues and mitochondria, whereas NAD ⁺ And NAD ⁺ the/NADH ratio was significantly increased (Table 80), NADH and NADH being present in the cytoplasm ^NAD+ Decrease and NAD ⁺ Change in the increase of the NADH ratio. It can be seen that NAD is present in the cytoplasm and mitochondria of the heart in diabetic mice ⁺ The reactions that accept the reduction of one electron from glycolysis and tricarboxylic acid cycle production, respectively, to NADH are hindered. It is to be noted in particular that in mitochondria NADH transfers one of the electrons to the respiratory chain via mitochondrial complex enzyme I and contributes to oxidative phosphorylation and returns itself to NAD ⁺ The state is ready to accept electrons generated by the tricarboxylic acid cycle. Since ATP levels in diabetic mice were significantly lower than those in normal control mice in this state, it was suggested that mitochondrial oxidative phosphorylation activity was significantly impaired. Therefore, the data from the above studies indicate that cardiac tricarboxylic acid cycle activity was impaired in diabetic mice, thus resulting in a decrease in ATP production and a decrease in ATP levels mediated by oxidative phosphorylation (Table 78).

TABLE 79 GRb-PQN4 alone and in combination with metformin significantly elevated total cardiac NAD (nmol/mg protein)

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of difference between a plurality of groups of samples comprising the model control group and the treatment group by using one-way analysis of variance (ANOVA) in combination with Duncan multi-range test; p compared to normal control group<0.05; in comparison to the model set, ^# p<0.05；n＝5。

TABLE 80 GRb-PQN4 and its use with metformin or dapagliflozin, respectively, reduces NADH content (nmol/mg protein) and maintains NAD levels in heart tissue of mice resistant to type II diabetes ⁺ NADH ratio at normal level

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control by using a t test, and performing significance analysis on the difference between a plurality of groups of samples comprising the model group and the treatment group by using one-way analysis of variance (ANOVA) in combination with Duncan multi-range test; p in comparison with normal control group <0.05; in comparison to the model set, ^# p<0.05， ^## p<0.01；n＝5。

in the treatment group, metformin can significantly increase NADH in total tissues and mitochondria but cannot increase the level of NADH in cytoplasm, while dapagliflozin has no significant efficacy; GRb-PQN4 or its combination therapy with metformin can completely fight the decrease of NADH level and NAD in total tissue, cytoplasm and mitochondria of diseased heart ⁺ And NAD ⁺ the/NADH ratio is increased, GRb-PQN4 mainly contributes to the drug effect of the combination group, but the drug effect of the combination group and dapagliflozin is not as good as that of GRb-PQN4 aloneEffect (table 80). It can be seen that GRb-PQN4 corrects low intracellular NADH levels and NAD in type II diabetic conditions ⁺ The increased NADH ratio imbalance effect was greater than that of metformin, and GRb-PQN4 consistently enhanced ATP potency than metformin (Table 78). In particular, both treatments also increase the total amount of NAD, among others ⁺ And NADH levels were 10-20% above the normal control. Thus, increased intracellular NAD capacity and maintenance of NAD ⁺ Dynamic equilibrium with NADH is an important mechanism of action of GRb-PQN4 and its combination metformin for increasing ATP levels in type II diabetic hearts. In other words, GRb-PQN4 and its combination with metformin can treat type II diabetes mellitus at the root of the disease. However, the combination of GRb-PQN4 and dapagliflozin only improves some of the above-mentioned indications, while metformin and dapagliflozin alone are almost ineffective, which further supports that the combination of GRb-PQN4 and metformin is superior to the combination of GRB-PQN4 and dapagliflozin for the treatment of type II diabetes.

Example 14.2 GRb-PQN4 or use with metformin can significantly improve or restore cardiac redox balance in diabetic mice.

14.2A.GRb-PQN4 and metformin can obviously improve NADP of diabetic mice ⁺ NADPH is balanced, and the combined drug effect of the two is better; the combination of GRb-PQN4 and dapagliflozin has no obvious therapeutic value of the combined drug.

As shown in tables 81 and 82, the total amount of NADP (NADP) in the total cardiac tissue, mitochondria and cytoplasm of the model group mice with type II diabetes for at least 26 weeks ⁺ + NADPH) was not significantly different from the total amount of NADP in the normal control group, but NADPH and the NADP +/NADPH ratio were significantly decreased and increased, respectively (. P)<0.05 In total tissue with NADP ⁺ The significant increase indicates that NADP synthesis in the heart of the diabetic mice is not affected yet, but the NADP is not affected yet ⁺ The ability to reduce back to NADPH is significantly reduced. Further, NADP in cytoplasm and mitochondria in model group mice ⁺ NADPH ratio is NADP of normal control mice respectively ⁺ 1.79 and 1.33 times the/NADPH ratio, which indicates that diabetic mice develop NADPH from NADP ⁺ Regeneration and damageIs to appear in the cytoplasm. In combination with II diabetic model mice in which plasma sorbitol levels were significantly higher than those of normal control animals (table 75), it can be further assumed that the rate of NADPH consumption by the polyol pathway in the heart of diabetic mice exceeded the rate of NADPH regeneration by the pentose phosphate shunt. The results of this study are consistent with known characteristics of diabetic metabolic disorders.

Importantly, GRb-PQN4 alone and in combination with metformin significantly reduced NADP in total tissues, mitochondria and cytoplasm of the heart of diabetic mice ⁺ (NADPH ratio) ^# p<0.05 And also significantly increases NADPH levels in total tissue and cytoplasm: ( ^# p<0.05 And each parameter was close to the level of the normal control (table 82). Based on the drug effect, the combination of GRb-PQN4 and metformin further increases the level of NADPH in mitochondria and the NADP of the mitochondria ⁺ the/NADPH ratios were maintained at the levels of normal controls. In combination with the results of studies in which none of GRb-PQN4, metformin, and both combinations thereof reduced sorbitol levels in the blood of diabetic II mice (table 75), the above data demonstrate that GRb-PQN4, metformin, and both combinations thereof enhance the ability of diabetic mice to regenerate NADPH. However, dapagliflozin only significantly reduced NADP in total tissue and cytoplasm ⁺ (NADPH ratio) ^# p<0.05 And the potency of the drug is not as strong as GRb-PQN4 and metformin, especially the drug effect of the combination of dapagliflozin and GRb-PQN4 is not as strong as that of GRb-PQN4 alone. The research results comprehensively support the medical application of GRb-PQN4 and metformin to the treatment of II type diabetes mellitus in principle.

TABLE 81 GRb-PQN4 and its use with metformin or dapagliflozin respectively did not alter the total cardiac NADP (nmol/mg protein)

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except for the normal by using one-way analysis of variance (ANOVA) and combining a Duncan multi-range test; there were no significant differences between groups; n =5.

TABLE 82 GRb-PQN4 and its use in combination with metformin or dapagliflozin for NADP and NADPH content (nmol/mg protein) and NADP in cardiac tissue of type II diabetic mice ⁺ Influence of the/NADPH ratio

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except for the normal by using one-way analysis of variance (ANOVA) and combining a Duncan multi-range test; p compared to normal control group<0.05; in comparison to the model set, ^# p<0.05， ^## p<0.01；n＝5。

14.2B.GRb-PQN4 and metformin can obviously improve the GSH/GSSG balance of sick mice independently, and the combined drug effect of the two is better; the combination of GRB-PQN4 dapagliflozin has no obvious therapeutic value of the combined drug.

As shown in tables 83 and 84, the total amount of glutathione (GSH + GSSG) in the heart total tissue, mitochondria and cytoplasm of the type II diabetic mice with the least 26 weeks of disease was not significantly different from that of the mice in the normal control group, but the GSH and GSH/GSSG ratio in the total tissue and the GSH/GSSG ratio in the mitochondria and cytoplasm were significantly lower than the corresponding values (p) in the normal control group<0.05 Indicating that glutathione synthesis in the heart of diabetic mice is not affected, but its ability to reduce GSSG back to GSH is reduced. Since GSSG reduction back to GSH is NADPH dependent, the results of this study demonstrate the previously observed low NADPH levels in the diabetic mouse heart. And maintaining NADP ⁺ The effects of NADPH balance are consistent, and the GRb-PQN4 and the metformin can be used for treating diabetic mice completelyThe reduction of the GSH/GSSG ratio in tissues, mitochondria and cytoplasm and the GSH level in the whole tissue, and the maintenance of these parameters at normal or even higher levels, indicates that both have a potent effect of up-regulating the antioxidant capacity of glutathione in diabetic mice. Particularly, GRb-PQN4 also remarkably increases the GSH level in cardiac mitochondria of diabetic mice, and the level and GSH/GSSG ratio are both significantly higher than those of a normal control ( ^# p<0.05 It further highlights the advantage of GRb-PQN4 in protecting mitochondria of diabetic animals from oxidative stress damage compared with metformin. Based on the drug effect, the drug effect strength of the GRb-PQN4 and metformin combined treatment is further enhanced, and the measured parameters are obviously superior to or tend to be superior to the parameter indexes of normal control. Although dapagliflozin slowed the diabetic mouse heart NADP to some extent ⁺ The imbalance of the/NADPH ratio (Table 82), but it did not improve the functional state of glutathione in diabetic mice, and the efficacy of the combination therapy of GRb-PQN4 and dapagliflozin in improving the functional state of glutathione in diabetic mice was less potent than that of GRb-PQN4 alone. The results of the above studies combined demonstrate that chronic treatment with GRb-PQN4 and metformin can maintain the antioxidant function of glutathione in the heart of type II diabetic mice at a level close to normal, and that the combination of the two can increase this function beyond normal.

Glutathione plays an important role in the regulation of antioxidant defenses, nutrient metabolism and cellular events including gene expression, DNA and protein synthesis, cell proliferation and apoptosis, signal transduction, cytokine production and immune responses and protein glutathionylation. Glutathione deficiency causes oxidative stress and chronic inflammation to play a key role in the pathogenesis of many diseases including diabetes, obesity, and cardiovascular disease. Therefore, the effect of GRb-PQN4 and the combination of GRb-PQN4 and metformin on glutathione of the heart of a diabetic mouse further supports the medical application of GRb-PQN4 and metformin, particularly the combination of GRb-PQN4 and metformin, in treating type II diabetes and complications thereof from the treatment principle.

TABLE 83 GRb-PQN4 and its combination with metformin or dapagliflozin did not affect the total amount of glutathione (GSH + GSSG) (nmol/mg protein) in the heart tissue of type II diabetic mice

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except for the normal by using one-way analysis of variance (ANOVA) and combining a Duncan multi-range test; no significant difference was seen for each group, n =5.

TABLE 84 GRb-PQN4 and its use in combination with metformin or dapagliflozin to increase GSH content (nmol/mg protein) and GSH/GSSG ratio in cardiac tissue of type II diabetic mice

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except for the normal by using one-way analysis of variance (ANOVA) and combining a Duncan multi-range test; p in comparison with normal control group<0.05; in comparison to the model set, ^# p<0.05， ^## p<0.01；n＝5。

14.2C.GRb-PQN4 can obviously improve the antioxidant activity in the heart of a type II diabetic mouse, the drug effect of metformin is lower than that of GRb-PQN4, and the enzyme activity can be further improved to exceed the normal level by combining the two drugs; the combination of GRb-PQN4 and dapagliflozin counteracts the respective therapeutic effects.

The results (Table 85) show that the activity of superoxide dismutase and catalase in heart tissue of type II diabetic mice is significantly reduced compared with that of normal animals in the control group<0.05 or x p<0.001 GRb-PQN4, dapagliflozin and metformin can obviously improve the activity of antioxidant enzymes (superoxide dismutase and catalase) in the heart of a type II diabetic mouse by single treatment: ( ^# p<0.05 or ^## p<0.01)However, GRb-PQN4 had the best efficacy and Rb-PQN4 in combination with metformin further increased the enzyme activity above the average of normal controls, but GRB-PQN4 in combination with dapagliflozin counteracted the respective enzyme activity-increasing effects.

TABLE 85 type II diabetic mice Heart tissue antioxidase Activity (units/mg protein)

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except for the normal by using one-way analysis of variance (ANOVA) and combining a Duncan multi-range test; p compared to normal control group<0.05,***p<0.001; in comparison to the model set, ^# p<0.05， ^## p<0.01, ^### p<0.001；n＝5。

the chronic treatment of GRb-PQN4 can obviously reduce the level of oxygen free radicals and oxidative stress products in the tissues and organs of type II diabetic mice, the efficacy of metformin is lower than that of GRb-PQN4, and the combination of GRb-PQN4 and metformin can maintain the level of the oxygen free radicals and the oxidative stress products at normal or nearly normal level, but the combination of the GRb-PQN4 and dapagliflozin cancels the therapeutic efficacy of the GRb-PQN 4.

As shown in tables 86 and 87, the levels of superoxide anion and hydrogen peroxide as oxygen free radicals and malondialdehyde and 8-hydroxydeoxyguanosine as oxidative stress products were significantly increased in the blood and heart of type II diabetic mice as compared with those of normal animals in the control group ([ p ])<0.05，**p<0.01 or x p<0.001 GRb-PQN4 significantly reduced superoxide anion, hydrogen peroxide and malondialdehyde levels in blood and heart of type II diabetic mice (A) ((B)) ^# p<0.05 ); metformin significantly reduces superoxide anion levels in the hearts of type II diabetic mice toAnd hydrogen peroxide levels in blood and heart: ( ^# p<0.05 or ^## p<0.01 But not reducing the levels of the oxidative stress products malondialdehyde and 8-hydroxydeoxyguanosine in the blood and heart; the combination of GRb-PQN4 with metformin significantly reduced the levels of all of the oxygen free radicals and oxidative stressor products to levels close to those of normal controls. However, dapagliflozin not only fails to reduce the levels of various oxidative stress agents and oxidative stress product in blood and heart of type II diabetic mice, but in combination with GRb-PQN4 counteracts the therapeutic efficacy of GRb-PQN 4.

Table 86. Oxygen free radical content in blood and heart of type ii diabetic mice (n = 5)

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except for the normal by using one-way analysis of variance (ANOVA) and combining a Duncan multi-range test; p compared to normal control group <0.05，*p<0.05; in comparison to the model set, ^# p<0.05， ^## p<0.01；n＝5。

TABLE 87 oxidative stressor product content in blood and heart of type II diabetic mice

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except for the normal by using one-way analysis of variance (ANOVA) and combining a Duncan multi-range test; p compared to normal control group<0.05，**p<0.01，***p<0.001; in comparison to the model set, ^# p<0.05， ^## p<0.01；n＝5。

the results of the experiments described in combination with 14.2C and 14.2D show that: GRb-PQN4 alone significantly reduced the levels of most of the oxygen free radicals and oxidative stressor products in the blood and heart of type II diabetic mice, and metformin had less antioxidant potency than GRb-PQN4, while GRb-PQN4 in combination with metformin significantly reduced and maintained normal or near normal levels of all of the oxidative stressor and oxidative stressor products in the blood and heart of type II diabetic mice. It can be seen that GRb-PQN4 alone has a good effect of protecting the antioxidant capacity of cells in type II diabetes, and is combined with metformin.

It can be seen that GRb-PQN4 alone can well protect the cell antioxidant capacity in type II diabetes, so that oxidative stress damage in type II diabetes can be significantly slowed down, and particularly, the combined treatment of GRb-PQN4 and metformin can further improve the superoxide dismutase and catalase mediated cell antioxidant capacity in a compensatory manner and basically avoid oxidative stress damage.

It is specifically noted herein that superoxide dismutase catalyzes the superoxide dismutase to oxygen and hydrogen peroxide, and appears to be the first line of defense against oxygen-derived free radicals. Subsequently, catalase or GSH can decompose hydrogen peroxide into molecular oxygen and water, and both a Glutathione (GSH) antioxidant system (consisting of GSH, GSSG, and related enzymes) and a thioredoxin antioxidant system (consisting of NADPH and thioredoxin) can defend against oxidative stress by effectively removing various ROS. There is increasing evidence that overproduction of mitochondrial ROS is causally related to diabetes and diabetic complications. ROS play an important role in the development of insulin resistance in type II diabetes (T2 DM) and in the reduction of pancreatic beta-cell function (anions, maize Cellular Redox Homeostasis by Elimination of Reactive Oxygen species, cell Physiol biochem.2017;44 (2): 532-553.), and more studies have demonstrated that ROS-mediated oxidative stress is an important cause of type I and type II diabetes and its complications.

So far, the research results prove that GRb-PQN4 has good protection effect on cell antioxidant systems in I and II diabetes states, including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) antioxidant systems (consisting of GSH, GSSG and related enzymes) and thioredoxin antioxidant systems (consisting of NADPH and thioredoxin), and can improve the cell systemic antioxidant capacity to exceed the normal level in a compensatory way by combining with insulin or metformin. Based on the important role of oxidative stress injury in the development of diabetes and its complications, our findings strongly support the use of GRb effective compositions alone, particularly in combination with insulin or metformin, for the prevention and treatment of type I and II diabetes and its complications.

Example 14.3 chronic treatment of GRb-PQN4 was effective in reducing advanced glycation end products (AGEs) in type II diabetic mice, GRb-PQN4 in combination with metformin completely prevented the production of AGEs, neither metformin nor dapagliflozin alone reduced AGEs levels.

As shown in table 88, advanced glycosylation end products (AGEs) levels in circulating blood and heart were significantly higher in untreated type II diabetic mice (model controls) over 26 weeks of disease course than in normal controls; the AGEs levels of blood and heart of animals treated by GRb-PQN4 are obviously lower than those of animals treated by model control group, and the AGEs of heart are maintained at normal level, which is highly consistent with the drug effect that GRb-PQN4 treated by chronic therapy can effectively protect heart of II diabetic mice; notably, chronic treatment with metformin and dapagliflozin, while showing significant efficacy in lowering blood glucose and slowing insulin resistance (table 75), did not show any reduction in the increase in AGEs levels in blood and heart in diabetic mice. AGEs levels in GRb-PQN4 and its chronic treatment group combined with metformin were maintained within normal ranges in both blood and heart, but in combination with dapagliflozin, the efficacy of GRb-PQN4 was impaired. The research results are combined to show that the GRb-PQN4 chronic treatment without hypoglycemic drug effect can effectively reduce the levels of advanced glycosylation end products (AGEs) in circulating blood and important organs in the II diabetes state, the hypoglycemic drug chronic treatment capable of effectively reducing the blood sugar level can not reduce the levels of the advanced glycosylation end products, the metformin can cooperate with the GRb-PQN4 to play a role in preventing the AGEs, but the dapagliflozin can eliminate the drug effect of the GRb-PQN 4. The research result further supports the medical application of the GRb effective composition and metformin to prevention and treatment of diabetes and common symptoms and chronic complications thereof from the action mechanism, and also scientifically explains the phenomenon of poor drug effect of the combination of GRb-PQN4 and dapagliflozin on improvement of type II diabetes complications.

Based on the important contribution of AGEs to diabetic complications, the research result provides an important point for the scientific basis that GRb-PQN4 and the combination of the GRb-PQN4 and metformin can effectively prevent and treat common symptoms and chronic complications of II diabetes, and further predicts the efficacy of GRb-PQN4, particularly the combination of the GRb-PQN4 and metformin, in preventing and treating other chronic complications.

TABLE 88 Effect of GRb-PQN4 and hypoglycemic Agents or combinations thereof on AGEs levels in blood and cardiac tissue of type II diabetic mice

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control group by using a t test, and performing significance analysis on the difference between a plurality of groups of samples except for normal by using one-way analysis of variance (ANOVA) in combination with Duncan multi-range test; p in comparison with normal control group<0.05; in comparison to the model set, ^# p<0.05；n＝5。

based on the fact that AGEs are intracellular glycometabolism disorder under diabetic condition, which causes glucose to flow out from polyol pathway and glyceraldehyde 3-phosphate/dihydroxyacetone phosphate-Methylglyoxal (MGO) pathway and combine with protein, phospholipid and nucleic acid in the form of fructose and MGO, respectively, to form final products which lose original physiological functions and have toxicity in glycolysis link, and the effect of GRb-PQN4 on reversing ATP in type II diabetes state to be insufficient to normal level, the research result reflects the effect that GRb-PQN4 chronic treatment can remarkably improve or correct intracellular glycometabolism disorder in type II diabetes state, namely, the effect that glycolysis intermediate product deviates from energy metabolism pathway to AGEs and promotes the flow to mitochondria to be oxidized and phosphorylated can be effectively reduced, and metformin or dacomin does not have the effect or has the effect to be insufficient, but the combination of metformin and GRb-PQN4 can completely block glycolysis intermediate from deviating from energy metabolism pathway and flowing to AGEs.

Example 14.4 GRb-PQN4 and its combination with insulin treatment to reduce proinflammatory factor levels in blood in type I diabetic mice

Metabolic inflammation is a characteristic pathological event of type II diabetes, which results from metabolic disorders that in turn further exacerbate diabetic metabolic disorders and promote the development of diabetes and its complications by inducing mechanisms such as insulin resistance and inflammatory degeneration of islet cells. Therefore, the effect of treating both principal and secondary aspects of diabetes mellitus II is considered to be achieved by slowing down the metabolic inflammation. As shown in table 89, the levels of proinflammatory factors including the proinflammatory transcription factor TNF α and the inflammatory factors IL1 β and IL6 in the circulating blood of untreated type II diabetic mice (model controls) over 26 weeks of disease were 2-fold higher than those of normal controls, and the increase was similar to that of type I diabetic mice (table 68); the levels of 3 inflammatory factors of the GRb-PQN4 or metformin chronic treatment group are obviously lower than those of the model control group, and the two groups have equivalent drug effect intensity, and particularly the combination of the two groups can control the level of the inflammatory factors at a normal level; however, dapagliflozin has no effect of reducing the level of inflammatory factors, and GRb-PQN4 and dapagliflozin have no protective efficacy. Several clinical studies have shown that metformin reduces the levels of systemic inflammatory markers (such as serum TNF α, IL6 and PAI1 levels and neutrophil to lymphocyte ratios) in type II diabetic patients, and that metformin also exhibits an ameliorating effect on metabolic inflammation in obese mice and cell models. As can be seen, our findings demonstrate therapeutic effects on metformin to improve metabolic inflammatory states.

Taken together, the above results indicate that chronic treatment with GRb-PQN4 without hypoglycemic effect or metformin with hypoglycemic effect is effective in reducing the chronic inflammatory state of type II diabetic condition, and that the two in combination act synergistically and can completely prevent or eliminate the metabolic chronic inflammatory state of type II diabetic condition. However, dapagliflozin, which has been shown to inhibit sodium-glucose cotransporter 2 (SGLT 2) and promote the excretion of blood glucose through the urine as the main mechanism of action, is not only unable to slow down metabolic inflammation by itself but also completely eliminates the pharmacological effects of GRb-PQN 4. In combination with the results of the study in the type I diabetes model (table 68), it can be seen that extracellular high blood glucose levels do not directly cause, or lower blood glucose does not improve, metabolic inflammation in type I or type II diabetes; the drug effect of GRb-PQN4 in improving the metabolic inflammation of type I or type II diabetes and the strong drug effect of GRb-PQN4 in preventing or eliminating the metabolic inflammation in combination with insulin or metformin reflect the effect that GRb-PQN4 can effectively improve and/or correct the intracellular metabolic disorders in the type I and type II diabetes states, and reflect the strong pharmacological effect that GRb-PQN4 in combination with insulin or metformin can synergistically reverse the intracellular metabolic disorders in the type I and type II diabetes states.

TABLE 89 effects of GRb-PQN4 and hypoglycemic agents and combinations thereof on proinflammatory factor levels in blood of type II diabetic mice

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except the normal by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to normal control group<0.05; in comparison to the model set, ^# p<0.05， ^## p<0.01；n＝5。

non-infectious chronic low-level inflammatory states, also known as metabolic inflammation, are characteristic pathological events of type II diabetes, and our data suggest that metabolic inflammation is also present in type I diabetes. The development of metabolic inflammation involves a multifactorial co-action, including lipid toxicity associated with disturbances of carbohydrate metabolism and pathological events arising from mitochondrial dysfunction including endoplasmic reticulum stress, hypoxia, cellular hypertrophy, death and fibrosis. In addition, acute glucose excursions may cause significant oxidative stress and inflammation of rat aortic endothelial cells, increase adhesion of monocytes to rat aortic endothelial cells, and increase endothelial apoptosis, resulting in severe cardiovascular injury. In particular, metabolic inflammation can in turn exacerbate metabolic disturbances in the diabetic state and promote the development of diabetes and its complications through multiple mechanisms, including induction of insulin resistance and damage to islet cells. It can be seen that, although the mechanisms that cause metabolic inflammation are complex and not yet fully understood, the fact that they lead to the escalation of glucose metabolism disorders and promote the development of diabetes and complications as a result of diabetic metabolic disorders and in turn by inducing insulin resistance and other multiple, yet unexplained mechanisms is undoubted. Therefore, anti-inflammatory strategies are thought to be helpful not only in controlling the symptoms of type II diabetes, but also in treating the causes leading to type II diabetes, and our data from studies show that anti-inflammatory strategies are also appropriate for type I diabetes. Classical anti-inflammatory drugs do not produce satisfactory results in the prevention of diabetes progression and cardiovascular events, and our findings indicate that the hypoglycemic effects of hypoglycemic agents are not effective in preventing and treating metabolic inflammation of diabetes, nor in preventing diabetes progression and cardiovascular events.

In the context of this context, our findings illustrate several key issues: (1) Metabolic inflammation also occurs in type I diabetes, and a 1/2 therapeutic dose of insulin only slows this chronic inflammatory state; (2) GRb-PQN4 can remarkably improve chronic inflammation in the type I diabetes, and the effect is not related to blood sugar level but related to improvement of intracellular metabolic disorder, or the effect of GRb-PQN4 on improvement of chronic inflammation related to type I diabetes further proves that GRb-PQN4 can improve the metabolic disorder in the type I diabetes and slow down or avoid vicious circle between the metabolic disorder and the chronic inflammation; (3) The combination of GRb-PQN4 and insulin can act synergistically and prevent or eliminate chronic inflammation in the type I diabetes state and avoid or terminate vicious circle between metabolic disturbance and chronic inflammation, so that the combination of the GRb-PQN4 and insulin can synergistically correct the metabolic disturbance state in the type I diabetes state and provide a reasonable explanation for the GRb-PQN4 to improve the insulin sensitivity of a type I diabetes mouse; (4) The combined treatment of GRb-PQN4 and insulin can provide a treatment which is different from a classical anti-inflammatory drug and is effective in relieving metabolic inflammation for a type I diabetes patient, and can effectively block the development of type I diabetes and the common symptoms and the occurrence and development of chronic complications of the type I diabetes. As further explained by the above research results, GRb-PQN4 can improve or correct the glucose metabolism disorder in the type I diabetes, and the effect of correcting the diabetes metabolism disorder is further enhanced in combination with insulin, so that GRb-PQN4, particularly in combination with insulin, can delay or block the development of diabetes, improve the quality of life of the diabetic patients and prevent and treat common symptoms and chronic complications.

Example 15 GRb-PQN4 alone or in combination with insulin or metformin reduced levels of angiopoietin-1and increased levels of angiopoietin-1in anti-diabetic mice.

The research aims are as follows:

the morbidity and mortality of diabetes is primarily due to cardiovascular complications, with microcirculation dysfunction and vascular inflammation being the major causes of impaired wound healing, nephropathy, heart disease, retinopathy, erectile dysfunction and neuropathy in diabetic patients (Angiopoietin-1 and Angiopoietin-2in metabolic disorders. Despite the tremendous advances in glycemic control, antidiabetic drugs fail to restore or prevent vascular damage caused by metabolic disorders. The research results of the previous examples prove that the specific active panaxadiol saponin composition (SAPDSC) represented by GRb-PQN4 can remarkably protect heart blood vessels of mice with type I and type II diabetes, promote the healing of large-area skin lesions of diabetic foot models and prevent neuropathy. It is now clear that there are various mechanisms for inducing vascular endothelial injury in diabetes, including the accumulation of ROS and advanced glycosylation products (AGEs) leading to endothelial cell apoptosis and dysfunction and the dysfunction of angiopoietins (Ang 1and Ang 2) mediating vascular injury repair and angiogenesis disorders. The results of the previous examples have shown that GRb-PQN4 alone or in combination with insulin or metformin enhances the antioxidant capacity of the cells themselves in the diabetic state, including maintaining NADPH and GSH levels, SOD (superoxide dismutase) and catalase activity at or near normal levels and even compensatory increases beyond normal levels, and avoids the accumulation of ROS and the accumulation of malonaldehyde and 8-hydroxydeoxyguanosine, which are macromolecular oxidative stress products. Angiopoietin-1 (Ang 1) plays a key role in vascular maturation, promoting vascular endothelial cell survival, stabilizing the endothelium and supporting the interaction between pericytes and endothelial cells and limiting vascular permeability, and Ang1 helps to delay the onset of diabetic complications by restoring microvascular function and can maintain the quiescence of some adult stem cells (organic interactions-1 in vascular regeneration. Trends mol. Med.,2013, 19. In contrast, angiopoietin-2 (Ang 2) acts against Ang1 function, promoting vascular wall destabilization and disruption of the junctions between endothelial cells and pericytes, and a decrease in Ang2 over-expression-associated Ang1/Ang2 ratio is closely associated with diabetic vascular dysfunction and is involved in neovascular diabetes-associated retinal disease (Angiopoietin-1 and Angiopoietin-2in metabolic disorders. Thus, ang1 has been an attractive and promising drug target for The treatment of diabetic complications (angiopietin-1 and angiopietin-2 in metabolic disorders, both of which are The following therapeutic strategies to The human being and logues in diabetes mellitus.j. Endocronol. Invest.,2016, 39.

The research method comprises the following steps:

using the specimen at the 10-week end point of the mice treated for type I diabetes (T1D) in example 5 and the specimen at the 26-week end point of the mice treated for type II diabetes (T2D) in example 12, the content of Ang1 and Ang2 and the ratio of both in the target tissues and organs of each test group were determined using a commercial kit. Since platelet-derived growth factor (PDGF) dysfunction may also be involved in diabetes-related atherosclerosis, we also determined PGGF levels in the target specimens from each test group.

Results and discussion of the study:

as shown in tables 90 and 91, in the type I and type II diabetic mouse models, the measured samples included that the level of Ang1 in the circulating blood, heart and skin was severely deficient relative to the normal control, whereas Ang2 was significantly higher in the blood than the normal control but did not show changes in the heart and skin, but the levels of platelet-derived growth factor (PDGF) were not deviated from the normal range everywhere, and the type I and type II mice showed highly consistent changes. Consistently, as shown at 92 and table 93, the ratio of Ang1 to Ang2 (Ang 1/Ang 2) was very significantly reduced in blood (compared to normal controls, ^*** p<0.001 Is significantly reduced in heart and skin (compared to normal controls, ^* p<0.05). Consistent with the maintenance or compensatory increase of the effects of endogenous antioxidants in diabetic conditions, chronic treatment with GRb-PQN4 alone could significantly counteract the decrease in blood Ang1 levels in mice in type I and type II diabetic conditions and completely counteract the decrease in heart and skin, respectively, and even compensatory increase in the heart Ang1 levels in type II diabetic mice beyond normal controls while completely counteracting the increase in blood Ang2 levels (tables 90 and 91), so that the Ang1/Ang2 ratio in blood was maintained within normal ranges significantly higher than that in untreated diabetic animals, heart and skin (tables 92 and 93). In significant contrast, insulin only partially antagonizes the increase in Ang1 levels in blood in type I diabetic mice and the intensity of action is less than that of GRb-PQN4 (table 90), but no significant antagonism is seen in the decrease of Ang1/Ang2 ratio in blood, heart and skin (table 92); dapagliflozin significant pairThe decrease in Ang1 levels in blood and heart of mice resistant to type II diabetes, while metformin only significantly resisted the decrease in Ang1 in blood (table 90) with a corresponding change in Ang1/Ang2 ratio (table 93). Importantly, GRb-PQN4 in combination with insulin or metformin maintained Ang1 and Ang2 levels and their ratios in blood, heart and kidney within normal ranges, particularly significantly elevated Ang1/Ang2 ratios in blood of type I diabetic mice beyond normal controls (table 92), but no further enhancement of the respective effects was seen with GRb-PQN4 in combination with dapagliflozin.

The combination of the above results illustrates the key problems in the following 4 aspects: 1. our type I and type II diabetic mouse models used replicated the increased Ang2 levels and their associated increased Ang2/Ang1 ratios in the blood of diabetic patients, with lower Ang1 levels further promoting increased Ang2/Ang1 ratios, and with decreased Ang1 levels in the heart and skin of the susceptible organs of diabetic complications. It can be seen that a severe decrease in the Ang1/Ang2 ratio, characterized by either loss of Ang1 or elevation of Ang2, in the blood and organs susceptible to diabetic complications is a common molecular feature of type I and type II diabetes, thus supporting and expanding the current understanding: an imbalance in Ang 1and Ang2 regulation results in an increase in Ang2/Ang1 ratio, which is the culprit for vascular disease in type II diabetic patients, and exogenous supplementation of Ang1 or inhibition of Ang2 or its downstream pathways is a promising new target for antidiabetic and its complications (antioxidant-1 and angiotoxin-2 in diabetes disorders. The effect of GRb-PQN4 in correcting the Ang1 deficiency of the diabetic state and its associated increase in the Ang2/Ang1 ratio is superior to that of insulin and metformin, comparable to that of dapagliflozin. GRb-PQN4 in combination with insulin or with metformin maintains or repairs the Ang1/Ang2 ratio at or to the normal range and even increases the Ang1/Ang2 ratio over normal levels compensatory, thus increasing the protection or repair of the vascular system in diabetic conditions. 4. Based on the effects that Ang1 can be independent of an insulin activated PI3K-AKT pathway (organic actions of angiopoietin-1in insulin regeneration. Trends mol. Med.,2013, 19.

In conclusion, the correction of the Ang1 deficiency and the associated low Ang1/Ang2 ratio in diabetic conditions, particularly the elevation of Ang1 levels and Ang1/Ang2 ratios in blood, is critical to the protection and repair of the vascular system of diabetic patients by Rb-PQN4 or its combination with insulin or metformin, and is a common mechanism of action of specific active ginsenoside compositions alone or in combination with insulin or metformin to effectively prevent and treat large and small vascular complications and neuropathy, including cardiac dysfunction and diabetic foot. In particular, exogenous supplementation and elevation of endogenous Ang1 is known to alleviate diabetic complications by remodeling microvessels and preventing diabetic EC damage, and Ang1 has been an attractive and promising drug target for The treatment of diabetic complications (endothelial protein-1 and endothelial protein-2 in metabolic disorders. Therefore, the effects of GRb-PQN4 and the combination of the GRb-PQN4 and insulin or metformin on correcting insufficient Ang1 in a diabetic state and low Ang1/Ang2 ratio related to the insufficient Ang1 or complexly increasing the Ang1 level and the Ang1/Ang2 ratio in blood directly support the medical application of the GRb effective composition and the combination of the GRb effective composition and the insulin or metformin on preventing and treating diabetic nephropathy, heart disease, retinopathy (including diabetic microangiopathy and diabetic macular edema), erectile dysfunction and neuropathy. Development of a medicament for preventing and treating pulmonary hypertension, arteriosclerosis (Intra and arterial remodeling of angiopathy-1-Tie 2 receptor in health and disease. J. Cell mol. Med.,2008, 12) and angiogenesis-related disorders including wound repair, rheumatoid arthritis, wet age-related macular degeneration (w-AMD, also known as exudative or neovascular age-related macular degeneration), retinal vein occlusion (RVO, which is one of fundus diseases that are very common to The elderly and easily cause vision loss, and are referred to as "blindness, blurred vision" in traditional Chinese medicine), and diabetic macular edema (The observing roll of The ocular hypertension-tissue disease for thermal surgery, 26. 2022. For example, 145). Our findings also support the use of GRb effective compositions and their use in combination with insulin or metformin for the prevention and treatment of pulmonary hypertension, arteriosclerosis and angiogenesis-related disorders including wound repair, rheumatoid arthritis, wet age-related macular degeneration and retinal vein occlusion.

To this end, our findings demonstrate that GRb-PQN4 can protect or enhance/activate the cellular self-rescue mechanism in diabetic states, including antioxidant capacity (maintaining NADPH and GSH levels, SOD and catalase viability at or near normal levels or even compensatory enhancement beyond normal levels and avoiding ROS accumulation and macromolecular oxidative stress product accumulation and oxidative stress-mediated mitochondrial damage and inflammatory responses) and Ang 1-mediated metabolic regulation (thus compensating for the insufficient PI3K-AKT pathway signaling associated with insulin resistance) and anti-inflammatory effects as well as vascular protection, functional homeostasis and neogenesis; GRb-PQN4 also enhances the coupling of glycolysis to mitochondrial oxidative metabolism (facilitating the flux of carbohydrate metabolism intermediates to mitochondrial oxidative metabolism), effectively fighting ATP deficiency and the accumulation of advanced glycosylation products (AGE) in the diabetic state (and thus also fighting AGE-mediated inflammatory responses and insulin resistance), and slowing the inflammatory state of the circulatory system in the diabetic state. In particular, these pharmacological effects of GRb-PQN4 may be further potentiated by the combination of insulin or metformin. It is evident that GRb-PQN4, particularly in combination with insulin or metformin, exerts a powerful action of protecting and repairing the structure and function of the vascular system of diabetic patients and avoiding ATP deficiency, mitochondrial dysfunction and oxidative stress related hypofunction or dysfunction of the body by protecting or/and enhancing/activating the cell rescue mechanism and correcting metabolic disorders in the diabetic state. Therefore, GRb-PQN4, particularly in combination with insulin or metformin, can prevent and treat diabetic systemic co-disease, i.e. diabetic complications, can comprehensively improve physical performance and quality of life of patients, and can prevent and treat disorders associated with glucose metabolism disorders as well as disorders and sub-health states characterized by mitochondrial dysfunction, oxidative stress and chronic inflammation.

TABLE 90I type diabetes mellitus (T1D) mice angiogenesis-related factor content (pg/mL or pg/mg protein) in different tissues and organs

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except the normal by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p in comparison with normal control group<0.05，**p<0.01; in comparison to the model set, ^# p<0.05， ^### p<0.001；n＝5。

TABLE 91 content of angiogenesis-related factors in different tissues and organs of type II diabetic (T2D) mice

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control by using a t test, and performing significance analysis of the difference among a plurality of groups of samples except the normal by using one-way analysis of variance (ANOVA) in combination with a Duncan multi-range test; p compared to normal control group<0.05，**p<0.01；In comparison to the model set, ^# p<0.05， ^## p<0.01；n＝5。

TABLE 92 diabetes mellitus (T1D) mice type I angiopoietin-1 to angiopoietin-2 ratio (Ang 1/Ang 2)

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control by using a t test, and performing significance analysis on the difference between a plurality of groups of samples except the normal group by using one-way analysis of variance (ANOVA) in combination with Duncan multi-range test; p compared to normal control group <0.05，***p<0.001; in comparison to the model set, ^# p<0.05， ^## p<0.01， ^### p<0.001；n＝5。

TABLE 93 diabetes mellitus type II (T2D) mice angiopoietin-1 to angiopoietin-2 ratio (Ang 1/Ang 2)

Note: all data are expressed as mean ± SD; analyzing the significance difference of the model group or the treatment group and a normal control by using a t test, and performing significance analysis on the difference between a plurality of groups of samples except the normal group by using one-way analysis of variance (ANOVA) in combination with Duncan multi-range test; p compared to normal control group<0.05，***p<0.001; in comparison to the model set, ^# p<0.05， ^## p<0.01， ^### p<0.001；n＝5。

example 16 efficacy of the composition GRb-PQN4 for promoting the skin damage repair of diabetes was resolved, and it was found that GRb2 was an irrelevant pharmacodynamic component, and "GRb1+ GRd" and "GRc + GRb3" were functional units of the GRb-PQN4 composition.

Purpose of study

The research results of the previous examples prove that GRb-PQN4 can remarkably promote the skin damage repair of mice with type I and type II diabetes mellitus alone or together with insulin or metformin, and the drug effect resolution research is carried out on the composition of the GRb-PQN4 to reveal the drug effect contribution of GRb2 and the components of GRb1+ GRd and GRc + GRb3 to GRb-PQN4 respectively.

Experimental methods

Inducing an I-type diabetes model for an adult ICR mouse by using STZ, and comparing and observing GRb-PQN4, GRb-PQN4 excluding GRb2 (namely GRb-PQN4-GRb 2), GRb1+ GRd forming parts and GRc + GRb3 to promote the drug effect of repairing the skin lesion of the diabetic mouse by using a model animal; dosages of the moieties "GRb1+ GRd" and "GRc + GRb3" include dosages equivalent to and actual dosages in the GRb-PQN4 composition. The dosages of each test group were designed according to the weight ratio of GRb2, "GRb1+ GRd" and "GRc + GRb3" to GRb-PQN4, respectively, as shown in Table 94.

Results of the study

As shown in table 94, the efficacy of GRb-PQN4 excluding GRb2 in promoting diabetic mouse skin lesion repair was not reduced at all compared with the efficacy of GRb-PQN4, indicating that GRb2 and GRb-PQN4 do not promote diabetic mouse skin lesion repair; it is noted that the components of the composition such as the GRb1+ GRd, the GRc + GRb3 and the GRb-PQN4 composition can remarkably promote skin damage repair at the same dosage and the actual dosage in the composition, but the drug effect of the actual dosage in the composition is better than that of the GRb-PQN4 composition at the same dosage at low dosage, and the drug effect strength is equivalent to that of the GRb-PQN4 composition, which indicates that the drug effect of the GRb-PQN4 composition is not simply added to that of the GRb1+ GRd and the GRc + GRb3 composition, and neither functional unit can replace the composition. The above studies indicate that GRb2 is not an active ingredient of GRb-PQN4, and that "GRb1+ GRd" and "GRc + GRb3" are two functional units of GRb-PQN 4.

TABLE 94 influence of GRb-PQN4, GRb-PQN4 deficient in GRb2, functional units "GRc + GRb3" and "GRb1+ GRd" on wound area during skin lesion repair in type I diabetic mice.

Example 17 GRb-PQN4 and Specific Active Ginsenoside Composition (SAGC) containing GRb-PQN4 as core active ingredient significantly promote wound healing in skin lesions of normal animals

Experimental methods

A Specific Active Ginsenoside Composition (SAGC) containing GRb-PQN4 as core active ingredient means that the composition contains two functional units of PQN4 and other ginsenosides. The ICR mice of middle-aged (11-12 months old) and adult (3-4 months old) are respectively used for preparing a model of large-area skin-missing wounds, the specific method is the same as the previous method, and the model is used for comparatively observing the drug effect of GRb-PQN4 and the Specific Active Ginsenoside Composition (SAGC) containing two functional units as core active ingredients for promoting wound healing. After administration, the condition of the wound surface of the mouse is photographed and sampled every 3 to 5 days, the healing condition is observed, the length and the width of the wound are measured by a ruler, the wound surface area is estimated by CAD software, and the wound surface healing rate = [ (initial wound surface area S) ₀ Wound surface area S at each time point _t ) Initial wound surface area S ₀ ]×100％。

Results of the study

As shown in tables 95 and 96, the Specific Active Ginsenoside Composition (SAGC) containing GRb-PQN4 and GRb-PQN4 as core active ingredients significantly promoted the healing of mouse skin lesions in middle-aged mice at a dose of 5mg/kg administered once a day from day 3 to day 14, and had equivalent potency to the middle-aged mice, but did not show significant potency at a low dose (2.5 mg/kg) administered once a day (table 95); for skin injury of young mice, GRb-PQN4 can also completely and remarkably promote skin injury healing until the healing rate reaches 92.76%, while the pharmacodynamic strength of a Specific Active Ginsenoside Composition (SAGC) is relatively weak, but the pharmacodynamic strength cannot be further improved by increasing the dosage of the SAGC (table 96). Research results show that the drug effects of GRb-PQN4 and the Specific Active Ginsenoside Composition (SAGC) taking the GRb-PQN4 as the core active component can remarkably promote the skin damage repair of normal animals of different age groups, and the effective dose of the GRb-PQN4 is consistent with the dose of the GRb-PQN4 for promoting the skin damage repair of diabetic mice.

In combination with the results of the study in example 16: the drug effect of the functional units GRb1+ GRd and GRc + GRb3 of GRb-PQN4 for promoting the skin damage of the diabetic mice is equivalent to the drug effect strength of the GRb-PQN4 composition; at equal dosage, GRb-PQN4 has equivalent efficacy strength to SAGC. The research results support the relative application of the specific panaxadiol saponin active composition (SAPDSC), the functional unit 'GRb 1+ GRd' and the functional unit 'GRc + GRb 3' thereof and the specific active total ginsenoside composition (SAGC) containing two functional units or one of the two functional units as a core active ingredient in promoting the repair of skin lesions in normal or disease states, and comprise the application of the active ingredient as a common skin care product, the application of the active ingredient as a cosmeceutical product and the application of the active ingredient as an active ingredient of a repair medicament for promoting cosmetology and medical surgery.

TABLE 95 time varying rate of wound healing in middle aged mice in each group

Note: p <0.05 compared to saline control group; * P <0.01; n =5.

TABLE 96 time-dependent rate of wound healing in mice of each component year

Note: p <0.05 compared to saline control group; * P <0.01; n =5.

Summary and discussion of the efficacy and mechanism of action of all examples

(first) study of drug efficacy

The GRb effective/effective composition, particularly the combination of the GRb effective/effective composition and insulin or metformin or other hypoglycemic drugs with coordinated action mechanisms, can safely and effectively prevent and treat diabetes and comprehensively prevent and treat diabetes systemic complications (diabetic complications).

Diabetes is a chronic metabolic disease, and is a disease with the most complications (also called diabetes systemic co-morbidity), which comprises common symptoms of diabetes (such as ' three more one less ' symptoms appearing in early stage of the disease and poor physical strength, poor mental state and low working efficiency appearing later), diabetic skin diseases, peripheral neuropathy (including vegetative neuropathy and sensory neuropathy), central neuropathy, microvascular complications (including diabetic nephropathy and retinopathy), macrovascular complications including coronary heart disease, peripheral vascular disease, cerebrovascular disease, diabetic foot and wound healing difficulty of diabetic patients (which are common malignant results of vasculopathy, nervous system disorders and metabolic disorders), and diabetes is also a high risk factor of neurodegenerative diseases such as senile dementia and parkinson's disease. Diabetes has therefore posed a serious threat to global public health. However, there is no effective drug for preventing diabetes at present, and treating diabetes systemic comorbidity remains one of the largest clinical challenges in diabetes treatment. Control of blood glucose levels has long been a central element in the treatment of diabetes, however, even effective control of blood glucose is not effective in the prevention and treatment of diabetes and its complications. In agreement, we also observed in this invention that the classic first line drug metformin and the novel first line drug dapagliflozin for type II diabetes, while effective in reducing glycosylated hemoglobin levels, i.e., lowering blood glucose levels, were not effective in alleviating the common symptoms of early stages of diabetes, disease progression and degenerative complications including heart disease and diabetic foot, and that both metformin and dapagliflozin exacerbate the rash in the diabetic state. In addition, it was observed that the early-stage effects of insulin, metformin and gliflozin were not exerted continuously, but the effects gradually disappeared while the disease progressed as the course of the disease was prolonged, suggesting that controlling blood glucose levels was not effective in delaying the progression of the disease.

In sharp contrast to these hypoglycemic agents, effective or efficacious GRb compositions without acute and chronic hypoglycemic effects can significantly ameliorate diabetic complications, including: (1) Improving the early typical symptoms of type I diabetes (much eating and drinking symptoms, and the drug effect is numerically better than that of insulin), slowing down the peripheral neuropathy of diabetes (including hypersensitivity of nerve ending sensation and then passivation process and vegetative nerve hypofunction taking gastric emptying as an index, and the drug effect is numerically better than that of insulin), and relieving the rash in the diabetic state (equivalent to that of insulin); (2) Improving type II diabetes-associated skin rash and preventing dermatitis induced by metformin or dapagliflozin treatment; (3) In type I and type II diabetes models, the repair of large-area skin lesions (simulating diabetic feet) is greatly promoted, and the occurrence of heart diseases (including myocardial and macrovascular diseases and inflammatory cell infiltration of heart tissues) is completely prevented; (4) In particular, in type I and type II diabetes models, the GRb effective or efficacious compositions in combination with insulin or metformin completely prevent all selected considered diabetic complications including diabetic nephropathy and almost block disease progression, showing a powerful drug effect of both synergistically treating diabetes and its complications; (5) The combination of the GRb effective or effective composition and the dapagliflozin does not have synergistic effect, but the efficacy of the GRb effective or effective composition is obviously weakened by the dapagliflozin, which indicates that the GRb effective or effective composition can only play excellent efficacy for preventing and treating diabetes and complications thereof by combining with hypoglycemic drugs with coordinated or complementary action mechanisms.

(II) mechanism of action

Our findings demonstrate that GRb-PQN4 can protect or enhance/activate the cell rescue mechanism in diabetic states, including antioxidant capacity (maintaining NADPH and GSH content, SOD and catalase activity at or near normal levels and even compensatory increases beyond normal levels and avoids ROS accumulation and accumulation of macromolecular oxidative stress products and oxidative stress-mediated mitochondrial damage and inflammatory responses) and Ang 1-mediated metabolic regulation (thus compensating for insufficient PI3K-AKT pathway signaling associated with insulin resistance) and anti-inflammatory effects as well as vascular protection, functional homeostasis and neogenesis; GRb-PQN4 also enhances coupling of glycolysis to mitochondrial oxidative metabolism (facilitating the flow of sugar metabolic intermediates to mitochondrial oxidative metabolism), thus effectively fighting ATP deficiency and accumulation of advanced glycosylation products (AGE) in the diabetic state (and thus also fighting AGE-mediated inflammatory responses and insulin resistance), and slowing the inflammatory state of the circulatory system in the diabetic state. In particular, these pharmacological actions of GRb-PQN4 can be further potentiated by the combination of insulin or metformin, allowing various pathological or physiological indices to be maintained in the normal or near-normal range, and the levels or activities of various functional molecules in the antioxidant system and the Ang1 level to be compensated for and increased beyond the normal resting state. It is evident that GRb-PQN4, particularly in combination with insulin or metformin, exerts a powerful action of protecting and repairing the structure and function of the vascular system and nerves of diabetic patients by protecting and enhancing/activating the cell-rescue mechanism and correcting metabolic disorders in the diabetic state and avoids ATP deficiency, mitochondrial dysfunction and oxidative stress-related hypofunction or dysfunction of the body. Therefore, GRb-PQN4, particularly in combination with insulin or metformin, can prevent and treat diabetes and its systemic complications, i.e., diabetic complications, and can comprehensively improve physical performance and quality of life of patients, and also prevent and treat disorders associated with glucose metabolism disorders as well as disorders and sub-health states characterized by mitochondrial dysfunction, oxidative stress and chronic inflammation. The results of the study also support the related application of Specific Active Ginsenoside Composition (SAGC) with GRb active composition as the active center.

1.1. A highly effective composition GRb-PQN4, while not capable of lowering blood glucose levels in type I and type II diabetic conditions, can significantly prevent and correct the core pathological and characteristic events of diabetes in diabetic complication-sensitive tissues and organs and circulating blood, including: the absence of energy (ATP), reduced coenzyme I (NADH), the antioxidant Glutathione (GSH), superoxide dismutase (SOD) and catalase, and angiopoietin-1 (Ang-1); accumulation of reactive oxygen radicals (ROS, superoxide anions and hydrogen peroxide) and advanced glycosylation products (AGEs); oxidative stress damage to DNA and lipids; elevated proinflammatory factors (TNF-alpha, IL1 beta and IL 6); in particular, ATP, AGEs and Ang-1 are maintained at physiological levels in the blood and diabetic vulnerable organs, and systematically protect and even enhance cellular antioxidant capacity in type I and type II diabetic conditions, including maintenance of physiological or near-physiological NAD ⁺ /NADH、NADP ⁺ NADPH and GSSG/GSH ratio and GSH level, the amount of coenzyme I (NAD) compensated for and above normal ⁺ + NADH) (type II diabetes) and SOD levels, thus avoiding ROS accumulation and its oxidative damage to biological macromolecules, as well as downstream pathological events triggered by ROS accumulation including AGEs accumulation and chronic inflammatory states (also known as metabolic inflammation). In addition, the advantageous composition GRb-PQN4 also slows excessive creatine degradation in diabetic conditions, which also helps to maintain cardiac ATP levels in homeostasis and relieves the kidney of the burden of creatinine clearance. It is pointed out here that it has been shown that ROS accumulation in the diabetic state is an important common mechanism for the development of the upstream pathological events of diabetes and its complications, which are related to the oxidative damage of the biological macromolecules (deoxyribonucleic acid, proteins and lipids), in which a decrease in glyceraldehyde-3-phosphate dehydrogenase (GAPDH/G3 PDH) activity leads to a large deviation of the glycolytic intermediates glyceraldehyde-3-phosphate and dihydroxyacetone phosphate from the energy metabolism pathway towards Methylglyoxal (MGO); detoxification of large amounts of MGO by cells consumes GSH, while overloaded MGO glycosylates phospholipids, nucleotides, and functional proteins to form AGEs, thus further exacerbating oxidative stress damage and indirectly extending the range of ROS damage to biological macromolecules, the range of cellular functional decline/disorders, the range of associated pathological events, including redox imbalance, oxidative stress status, mitochondrial dysfunction, cellular energy deficiency/loss, endoplasmic reticulum oxidative stress, inflammatory pathway activation, and insulin resistance. It is also to be noted that coenzyme I (NAD) ⁺ + NADH) and NAD thereof ⁺ the/NADH ratio is central to the regulation of sugar metabolism, so that the diabetes mellitus is characterized by NADH depletion and concomitant NAD ⁺ The increase of the/NADH ratio will cause the corresponding sugar metabolism to be in the glycolysis path, tricarboxylic acid cycle and mitochondrial electron transfer dysfunction and finally generate ATP insufficiency, and the ATP level is seriously lacked in the heart, kidney and skin in the type I and type II diabetes models, so that the physiological or near-physiological NAD is maintained ⁺ the/NADH ratio, compensatory increases in NADH levels, from an important perspective, reveal the biochemical mechanism by which effective or efficacious compositions of GRb improve the intracellular carbohydrate metabolism state in diabetic conditions. From the above, it is possible to use the results of our studyIt is clearly seen that in the diabetic state, uncoupling of glycolysis from the sugar oxidative metabolism results in a large number of glycolytic intermediates deviating from the pathway of energy metabolism towards toxic MGO and AGEs and in the loss of cellular energy substance ATP, which in turn triggers a variety of related pathological processes and promotes diabetes progression and leads to diabetic complications. Of significant therapeutic value, GRb effective or efficacious compositions can systematically enhance or/and restore cellular antioxidant capacity in diabetic conditions and avoid oxidative stress damage and various pathological processes triggered by ROS accumulation; the coupling of glycolysis and sugar oxidation metabolism in a diabetes state is strengthened or/and repaired, glycolysis intermediate products are promoted to flow to mitochondria for oxidative phosphorylation, thereby AGEs accumulation is obviously reduced, physiological ATP steady state is maintained, MGO and glycosylation biomacromolecule accumulation can be slowed down, ATP deficiency can be avoided, and diseases progress and diabetic complications are caused by the MGO and the glycosylation biomacromolecule accumulation. Thus, the GRb effective or effective composition for treating diabetes can avoid oxidative stress and ATP deficiency and slow down cell hypofunction/disorder caused by AGEs accumulation and various downstream pathological events and malignant consequences thereof.

The GRb effective or effective composition has important significance for protecting the vascular system in a diabetic state and preventing and treating diabetic complications, and directly supports the medical application of the GRb effective or effective composition in preventing and treating diabetic complications and diseases related to angiogenesis disorders of the diabetic complications. The morbidity and mortality from diabetes is mainly due to cardiovascular complications, of which microcirculation dysfunction and vascular inflammation are the main causes of impaired wound healing, nephropathy, heart disease, retinopathy, erectile dysfunction and neuropathy. In one aspect, the disordered neovasculature is a hallmark of end-stage diabetic retinopathy, nephropathy, and atherosclerotic plaque instability; on the other hand, defects in angiogenesis can lead to impaired wound healing, skin ulceration, fibrosis and impaired collateral vessel development. It is now clear that dysfunction of angiopoietins (Ang 1and Ang 2) is yet another important factor in the dysfunction and resultant disruption of the diabetic vasculature. Angiopoietin-1 (Ang 1) plays a key role in vascular maturation, promotion of vascular endothelial cell survival, stabilization of endothelium and interactions between pericytes and endothelial cells and limitation of vascular permeability, and Ang1 helps to delay the onset of diabetic complications by restoring microvascular function and can maintain the quiescence of some adult stem cells (organic actions of angiopoietin-1in vascular permeability. Trends mol. Med.,2013, 19. In contrast, ang2 acts against Ang1 function, promoting vascular wall destabilization and disruption of the junctions between endothelial cells and pericytes, and a decrease in Ang2 high expression-associated Ang1/Ang2 ratio is closely associated with diabetic vascular dysfunction and is involved in neovascular diabetes-associated retinal disease (Angiopoietin-1 and Angiopoietin-2in metabolic disorders. Therefore, ang1 has been an attractive and feasible drug target for the treatment of diabetic complications (Angiopoietin-1 and Angiopoietin-2in metabolic disorders. Also a bispecific antibody drug, faricimab, directed against Ang2 and VEGF simultaneously showed promising results in clinical trials in patients with diabetic retinopathy (The role of angiopoietins in neovenous diabetes-related diseases. Diabetes ther.,2022, 13.

Our results illustrate the key issues in 4 areas below: 1. our type I and type II diabetic mouse models used replicated the increased Ang2 levels and their associated increased Ang2/Ang1 ratios in the blood of diabetic patients, with lower Ang1 levels further promoting increased Ang2/Ang1 ratios, and with decreased Ang1 levels in the heart and skin of the susceptible organs of diabetic complications. It can be seen that the severe decrease in the Ang1/Ang2 ratio, characterized by either Ang1 deletion or elevated Ang2, in the blood and in the susceptible organs of diabetic complications is a common molecular feature of type I and type II diabetes, thus supporting and extending the current understanding that an imbalance in the regulation of Ang 1and Ang2 results in an increase in the Ang2/Ang1 ratio, which is the leading cause of vascular disease in patients with type II diabetes, and that exogenous supplementation of Ang1 or inhibition of Ang2 or its downstream pathways as a promising new target for anti-diabetes and its complications (antioxidant-1 and angiotoxin-2 in diabetic disorders to the diabetes mellitus and its complications, the human of diabetes in diabetes mellitus j. Endocrinol, 2016, 39-1236 diabetes mellitus 1241, 1822, 2022 et seq. Et al, 1. The effect of GRb-PQN4 in correcting the Ang1 deficiency of the diabetic state and its associated increase in the Ang2/Ang1 ratio is superior to that of insulin and metformin, comparable to that of dapagliflozin. GRb-PQN4 in combination with insulin or with metformin maintains or repairs the Ang1/Ang2 ratio at or in the normal range and even increases the Ang1/Ang2 ratio over normal levels compensatory, thus increasing the protection or repair of the vascular system in diabetic conditions. 4. Based on the effects that Ang1 can be independent of an insulin activated PI3K-AKT pathway (organic actions of angiopoietin-1in insulin regeneration. Trends mol. Med.,2013, 19.

In conclusion, the correction of the insufficient Ang1 and the associated low Ang1/Ang2 ratio in diabetic conditions, particularly the elevation of Ang1 levels and Ang1/Ang2 ratios in blood, is critical to the protection and repair of the vascular system of diabetic patients by GRb-PQN4 or its combination with insulin or metformin, and is a common mechanism of action of specific active ginsenoside compositions alone or in combination with insulin or metformin to effectively prevent and treat large and small vascular complications and neuropathy, including cardiac dysfunction and diabetic foot. In particular, exogenous supplementation and elevation of endogenous Ang1 is known to alleviate diabetic complications by revascularization and prevention of diabetic endothelial cell damage, and Ang1 has been an attractive and promising drug target for the treatment of diabetic complications, and a bispecific antibody drug, farmimab, directed against both Ang2 and VEGF has shown promising results in clinical trials in patients with diabetic retinopathy. Therefore, the effects of GRb-PQN4 and the combination of the GRb-PQN4 and insulin or metformin on correcting insufficient Ang1 in a diabetic state and low Ang1/Ang2 ratio related to the insufficient Ang1 or complexly increasing the Ang1 level and the Ang1/Ang2 ratio in blood directly support the medical application of the GRb effective composition and the combination of the GRb effective composition and the insulin or metformin on preventing and treating diabetic nephropathy, heart disease, retinopathy (including diabetic microangiopathy and diabetic macular edema), erectile dysfunction and neuropathy. Based on the application value and prospect of promoting Ang1 function or inhibiting Ang2 function simultaneously in the development of drugs for preventing and treating various diseases, the composition comprises effective prevention and treatment results of pulmonary hypertension and arteriosclerosis (Intra and arterial remodeling diagnosis of Angiopoietin-1-Tie2 receptor in health and disease. J. Cell mol. Med.,2008, 12.

In combination with the above 1.1 and 1.2, chronic treatment with GRb effective or effective composition maintains ATP homeostasis and redox balance, avoids ROS accumulation and reduces AGEs accumulation and slows down chronic inflammatory states by enhancing coupling of glycolysis and sugar oxidation metabolism in diabetic states, protects/improves the coenzyme I-mediated metabolic regulation function, the SOD/GSH-mediated antioxidant function and the Ang 1-mediated vascular protection function as representative of cellular self-protection/self-rescue mechanisms, thus effectively preventing formation and deep development of diabetic disease networks and promoting functional and structural repair of relevant cells and tissues, and thus effectively preventing and treating diabetes and its systemic co-morbid (diabetic complications).

1.3. The results of our comparative studies on hypoglycemic agents further demonstrate that protection and repair of glycolytic and glycooxidative metabolic coupling (to generate sufficient ATP and prevent glycolytic intermediates from deviating from metabolic pathways and flowing to glycosylation pathways) and enhancement/protection of self-rescue mechanisms are key points supporting effective or efficient combination of GRb in exerting superior efficacy in preventing and treating diabetes and its complications.

In the type I diabetes model, chronic treatment with insulin, although having an acute hypoglycemic effect, only slowed NADH relative to NAD at the end of the treatment ⁺ Insufficiency, accumulation of AGEs in blood and skin, and bloodThe level of proinflammatory factors is increased, and the action strength of blood AGEs and proinflammatory factors is reduced to be lower than that of GRb-PQN 4; in particular, it is not possible to combat the loss of ATP and the decrease in cellular antioxidant capacity mediated by NADPH, GSH and SOD (skin) in the heart and/or skin of diabetic vulnerable tissues with prolonged course, nor the decrease in Ang1 levels in the heart and skin, and also aggravate the accumulation of AGEs in the heart. These biochemical results are highly consistent with the inability of chronic insulin therapy to slow diabetic cardiac lesions and promote facile repair of large skin lesions. It can be seen that chronic treatment with insulin maintains or improves the coupling effects of glycolysis and glycooxidative metabolism far less than effective or advantageous GRb compositions, and chronic treatment with insulin protects the cellular antioxidant capacity and Ang 1-mediated microvascular protection/repair capacity and its associated damage repair capacity in diabetic conditions less than effective or advantageous GRb compositions. Chronic treatment with metformin or dapagliflozin, although significantly reduced the insulin resistance index (HOMA-IR) and the blood glucose level as judged by glycosylated hemoglobin, in the type II diabetes model, and the effect of metformin on elevated proinflammatory factor levels in the blood characteristic of diabetes is comparable to the GRb-PQN4 effect. However, in terms of protecting or/and repairing cellular antioxidant function, the chronic treatment of metformin and dapagliflozin is inferior to the chronic treatment of GRb-PQN4, and particularly cannot effectively resist heart SOD deficiency, superoxide anion accumulation and ROS-mediated accumulation of oxidized lipid product malondialdehyde; neither metformin nor dapagliflozin chronic treatment significantly combats the decrease in ATP levels in the tissue of the organ being tested over an extended period of time, nor does it completely decrease the accumulation of AGEs in the tested tissue, including blood and heart, and dapagliflozin appears to also exacerbate the accumulation of AGEs; metformin is also completely unable to combat the decrease in Ang1 levels. It is also noted here that chronic treatment with metformin or dapagliflozin is completely resistant to NADH relative NAD characteristic of diabetes ⁺ However, this effect does not translate effectively into a pathological change that ameliorates the uncoupling of glycolysis and glycooxidative metabolism in the diabetic state, as seen by AGEs accumulation and ATP deficiency, while elevation of the total NAD level above normal by GRb-PQN4 also maintains physiological ATP levels and significantly reducesAGEs accumulate. Therefore, neither metformin nor dapagliflozin chronic therapy can effectively slow down the flow of glycolytic intermediates to glycosylation pathways in the diabetic state, nor can effectively improve the yield of glucose through oxidative phosphorylation pathways, which indicates that neither metformin nor dapagliflozin chronic therapy can promote the glycolytic intermediates to enter tricarboxylic acid cycle in the type II diabetic state and finally pass through mitochondrial oxidative phosphorylation yield to maintain ATP steady state and reduce AGEs accumulation like GRb-PQN4 does, and does not have the function of activating cell self-rescue mechanism like GRb-PQN4 does.

2. Importantly, the GRb effective or superior composition in combination with insulin or metformin for the treatment of type I and type II diabetes can further enhance the coupling of glycolysis and glycoxymetabolism and completely avoid the deviation of glycolysis end products from metabolic pathways to glycosylation pathways to generate AGEs and metabolic inflammation, and also enhance or retain the cell self-rescue mechanism activating effect of the GRb effective or superior composition, maintain the cell energy state and the redox state within physiological ranges, and show the effects of reducing protein over-degradation in a diabetic state (mainly type I diabetes model) and completely avoiding lipid toxicity (mainly type II diabetes model) which are not individually existed. The following point 5 is also to be noted here.

Chronic treatment with GRb-PQN4 in combination with metformin can completely combat the elevated total cholesterol and ldl cholesterol in the blood characteristic of type II diabetes, while GRb-PQN4 or metformin alone is either ineffective or, conversely, further raises the level of total cholesterol. Based on the fact that the toxicity of blood fat is an important risk factor of cardiovascular complications of diabetes, research results indicate that the combination of the effective or effective GRb composition and metformin can synergistically prevent and treat the lipotoxicity of diabetes, so that the pharmacological action further explains the drug effect of the combination of the effective or effective GRb composition and metformin on the prevention and treatment of the cardiovascular complications of diabetes, and also discloses the application of the combination of the effective or effective GRb composition and metformin on the prevention and treatment of atherosclerosis characterized by metabolic syndrome.

Blood glucose excursion or blood glucose variability (GV, which includes hypoglycemia due to drug action and blood glucose excursion during this day of administration) and uncontrolled blood glucose rise over a prolonged period of treatment or/and disease progression are common, unsolved problems associated with hypoglycemic therapy and development of diabetic complications. GV has been identified as an important cause of development of diabetic vascular complications and neuropathy independent of insulin resistance, and reduces the mental health and quality of life of patients, including reduced work efficiency, mood problems (such as postprandial depression and anxiety), cognitive symptoms and sleep disturbances associated with nocturnal hypoglycemia and nocturnal breathing disorders and prolongs hospitalizations of hospitalized diabetic patients. Moreover, current insulin treatment methods (either multiple daily injections or continuous subcutaneous infusion) still present a risk of poor glycemic control and hypoglycemia in patients of all ages, particularly in children and adolescents, severe hypoglycemia leading to seizures or significant changes in mental status, while mild hypoglycemia is associated with changes in learning and attention-cognitive functions. In particular, intermittent hyperglycemia damages vascular endothelial cells by oxidative stress much more strongly than sustained hyperglycemia exposure (Glycemic vascular, oxidative stress, and impact on the compatibility related to type 2Diabetes mellitus. Curr Diabetes Rev,2021, 17. The GRb effective or superior combination therapy with insulin or metformin provides good resolution of insulin resistance and blood glucose fluctuations and elevated blood glucose levels with disease progression before and after daily administration of insulin or metformin during the course of therapy and blocks disease progression. Specifically, in an acute blood glucose reduction test in the chronic treatment process of a model animal with type I diabetes, GRb-PQN4 and insulin are combined to avoid hypoglycemia caused by insulin treatment, prolong the acute blood glucose reduction time of insulin, effectively avoid blood glucose fluctuation between daily multiple times of administration of insulin and avoid acute drug resistance caused by daily multiple times of administration of insulin, so that the large blood glucose fluctuation before and after multiple times of insulin injection in one day is effectively avoided, the hypoglycemia phenomenon is avoided, and the total blood glucose reduction amount is increased; the combination of the two can not only prevent the weakening of acute glucose-reducing efficacy (or called insulin resistance) of the insulin along with the treatment time course and the disease progression in the chronic treatment process of the insulin, but also gradually enhance the sensitivity of the body to the insulin and maintain the glycosylated hemoglobin level of the terminal point basically unchanged and close to the normal level (reflecting that the combination of the two has strong and continuous glucose-reducing effect and protects the oxygen supply function of the hemoglobin in the circulatory system to target tissues, organs and cells), and the chronic treatment of the insulin with the same dose can not obviously reduce the glycosylated hemoglobin level. Moreover, the combination treatment can effectively reduce the fasting blood glucose level and can effectively delay the progressive rise of the fasting blood glucose level along with the prolongation of the disease course, and the independent treatment does not have the effect, so the medicine effect of the combination treatment on delaying or even blocking the disease progression is further reflected from an important angle. Thus, the GRb effective or efficacious composition increases the body's sensitivity to insulin and delays or blocks disease progression such that chronic treatment with low doses of insulin can produce significant and stable hypoglycemic effects, while lowering the insulin dose itself is beneficial in avoiding therapeutic hypoglycemia and blood glucose excursions. Consistent findings were obtained in a type II diabetes model, GRb-PQN4 in combination with metformin significantly potentiates the acute hypoglycaemic effect of the latter and overcomes the attenuation of fasting glucose lowering effects that occurs with prolonged treatment time course or/and disease progression.

It is specifically noted herein that the potency of dapagliflozin or its acute hypoglycemic effect in contract with GRb-PQN4, fasting plasma glucose, insulin resistance index and end-point glycosylated hemoglobin level is significantly greater than that of metformin or its combination with GRb-PQN4 during chronic treatment for 26 weeks, whereas dapagliflozin or its combination with GRb-PQN4 has no significant potency in the control of systemic co-morbidities in type II diabetes (diabetic complications), and that dapagliflozin significantly impairs the potency of GRb-effective or efficacious compositions when combined. The pharmacological action of dapagliflozin on inhibiting a sodium glucose cotransporter may damage the effect of GRb-PQN4 on improving the yield of glucose through oxidative metabolism in a diabetic state.Indeed, as previously described, dapagliflozin abolished the effects of GRb-PQN4 on oxidative stress, ATP depletion, AGEs accumulation and metabolic inflammation in the diabetic state and compensatory rise in NAD (NAD) in combination therapy ⁺ + NADH), NADH, GSH and SOD, thus leading to the destruction of GRb-PQN4 by dapagliflozin in its efficacy in the prevention and treatment of diabetic complications. This again illustrates the limitations of simply lowering blood glucose levels for the prevention and treatment of diabetes and its complications, and further demonstrates the key role of GRb effective or effective compositions in targeting intracellular carbohydrate metabolism, simultaneously increasing cellular antioxidant capacity and other self-rescue mechanisms for the prevention and treatment of diabetes and its complications.

2.5. Continuous benign interaction can be generated between the effects of reducing blood sugar and glycosylated hemoglobin levels of insulin or metformin and the effects of promoting intracellular carbohydrate metabolism and improving the antioxidant capacity of cells of the GRb effective or efficient composition, so that a healthy energy metabolism system and a redox system can be protected or repaired and a cell self-rescue mechanism can be activated/improved, and therefore, the vicious circle between sugar metabolism disorder in the pre-diabetes stage or the diabetes stage and the malignant consequences caused by the sugar metabolism disorder and the malignant consequences can be prevented or corrected, wherein the vicious circle comprises energy (ATP) deficiency/loss, oxidative stress, AGEs accumulation, mitochondrial injury, metabolic inflammation, insulin resistance, lipid toxicity, protein excessive degradation, and burden of glomerular filtration caused by increase of urea nitrogen and creatinine in blood. On one hand, the coupling effect of the GRb composition on enhancing glycolysis and sugar oxidation metabolism can induce the hypoglycemic effect of insulin or metformin to flow to tricarboxylic acid cycle and mitochondrial oxidative phosphorylation energy, so that the hypoglycemic effect of the insulin or metformin is enhanced, the function of hemoglobin carrying oxygen is protected, and the accumulation of AGEs and the blood sugar fluctuation in the treatment process of the insulin or metformin are avoided; on the other hand, in turn, the near physiological plateau blood glucose levels instead of the widely fluctuating glucose levels may prevent oxidative stress induced by blood glucose variability from damaging mitochondria and delicate and vital cells including vascular endothelial cells, and the protected hemoglobin may ensure oxygen required during the oxidative metabolism of intracellular sugars and may avoid hypoxia common to diabetic conditions. In diabetic patients, the retina, Hypoxic conditions exist in tissues such as kidney, pancreatic islets, fat, skin and wounds, suggesting that Hypoxia plays an important role in the development of diabetes and diabetic complications (Hypoxia and diabetes-induced factors in diabetes and its complications. Diabetologia,2021, 64. The microvascular damage and the glycosylated hemoglobin together cause the hypoxia state of tissues and organs related to the diabetic complication, so that the decoupling of the original glycolysis and the sugar oxidation metabolism is intensified, and the small-amplitude increase of the glycosylated hemoglobin can be directly involved in diabetic vasculopathy complication and neuropathy complication (such as retinopathy, nephropathy and neuropathy, gastroparesis and increase of the infection incidence rate of a surgical site) in a sugar-independent mode. Therefore, physiological glycosylated hemoglobin levels undoubtedly promote the coupling of effective or effective GRb compositions to glycolysis and carbohydrate oxidation metabolism and compensate the defects that GRb compositions cannot reduce glycosylated hemoglobin, and the microvasculature protected by GRb compositions can further ensure the supply of nutrients such as oxygen and glucose to target tissues and organs. Moreover, the powerful effect of the system in increasing intracellular antioxidant capacity and the effect of enhancing the coupling of glycolysis with sugar oxidative metabolism or correcting uncoupling can also be mutually supported. Cellular antioxidant systems rely on both the glucose metabolic pathway for normal operation and in turn regulate sugar metabolic activities such as: all 3 precursor amino acids of GSH can be derived from sugar metabolism intermediates, and especially pyruvate deficiency may be associated with diabetic glycine deletion; NADH/NAD ⁺ And NADPH/NADP ⁺ The balance of (A) is both regulated and dependent on carbohydrate metabolism, and the GSH/GSSG balance is dependent on NADPH/NADP ⁺ Balancing; SOD and GSH are important for protecting mitochondria and sugar oxidation metabolism by synergistically eliminating ROS from mitochondria; GSH can avoid or reduce AGEs accumulation and the various malignant events that they cause, including metabolic disorders and metabolic inflammation, by effectively detoxifying the most important glycosylated precursor, MGO. Finally, it should be pointed out that sufficient ATP from the oxidative metabolism of sugars is sufficient not only to avoid excessive activation of the compensatory fat metabolism and excessive degradation of proteins and creatine in the diabetic state due to ATP deficiency, thus avoiding the subsequent occurrence of lipid toxicity, excessive protein consumption and creatinine rise in the bloodThe burden of glomerular filtration is high and ATP required for biochemical reactions and physiological processes targeted by insulin or metformin (both of which are not effective against ATP deficiency associated with diabetes mellitus) can be improved and various physiological and biochemical processes weakened or even arrested by ATP deficiency can be reactivated, thus the health status and working efficacy of patients can be improved comprehensively. In conclusion, the effective or effective composition of GRb and insulin or metformin act together to prevent and treat diabetes in a complementary and synergistic manner in the action mechanism, and can protect or repair the healthy energy metabolism system and redox system in the pre-diabetes stage or the diabetes stage and activate/improve the cell self-rescue mechanism, thereby endowing the cells with the ability to effectively exert the physiological function, damage resistance, infection resistance and damage repair promotion, so that the GRb composition not only can effectively prevent and treat diabetes and systemic co-morbid diseases (diabetic complications) thereof, but also can comprehensively improve the health state and working efficacy of diabetic patients.

3. The characteristic advantages of treating diabetes root (origin righting and clearing) and effectively preventing the network formation and the progress of the diseases can realize the breakthrough of the drug effect of effectively preventing and treating the diabetes and the systemic co-morbid diseases (diabetic complications).

It is clear that in therapeutic principles, GRb effective or advantageous compositions and combinations thereof with insulin or metformin reduce blood glucose levels compared to current hypoglycemic agents (trying to inhibit absorption or production, promote excretion, etc., but neglecting the root of disease (i.e., intracellular carbohydrate metabolism disorder) and candidates that target oxidative stress or AGEs accumulation that are widely focused on carbohydrate metabolism disorders (e.g., therapeutic strategies that use exogenous antioxidants to apply antioxidant stress, inhibit polyol pathways, inflammatory pathways, downstream pathways within PKC or MAPK), are more consistent with the characteristics of disease development and progression, have the distinct advantages of positive origin (management of diabetic roots) and effective control of disease network formation and progression Diabetic peripheral neuropathy (including sensory neuropathy and autonomic neuropathy), central neuropathy (including diabetic dementia and diabetic neuropathy), diabetic cardiovascular disease, diabetic nephropathy, diabetic eye disease (including diabetic retinopathy and diabetic cataract) and diabetic foot; promoting the healing/repair of pre-diabetic patients and diabetic patients in medical operations or traumatic wounds. The effective or superior GRb composition and the medical effect of the GRb composition in combination with insulin or metformin for preventing and treating diabetes and systemic complications are also characterized in that systemic treatment can be carried out on certain complications. For example, diabetic cardiopathy generally refers to diabetic patients 'complicated or concomitant coronary atherosclerotic heart disease, diabetic cardiomyopathy and cardiac arrhythmia and cardiac dysfunction caused by microangiopathy, vegetative nerve dysfunction, and GRb effective or effective composition, especially in combination with insulin or metformin, can be used for the combined prevention and treatment of diabetic patients' coronary atherosclerotic cardiopathy, diabetic cardiomyopathy and cardiac arrhythmia and cardiac dysfunction.

The prevalence of diabetic male sexual dysfunction is close to 50%, while diabetic women seem to be slightly lower. Testicular dysfunction, impotence, decreased fertility, and retrograde ejaculation are common symptoms in diabetic men. Diabetes is also the most common cause of erectile dysfunction in men. The sperm quality of diabetic men is also poor, including decreased sperm motility and concentration, abnormal morphology, and abnormal increase in seminal plasma. In diabetic female neuropathy, vascular injury and psychological problems are associated with the pathogenesis of decreased libido, decreased vaginal lubrication, orgasmic dysfunction and dyspareunia. The relationship between the production of excess free radical oxygen in diabetic pregnancy and embryogenesis disorder has also been proposed. Indeed, maternal diabetes during pregnancy is associated with an increased risk of complications in the offspring, such as dysplasia in the fetus, polyhydramnios, fetal loss and congenital malformations. In addition, newborns of diabetic mothers experience hypovolemia and reduced bone mineral content. Both from a biochemical and molecular mechanism, mitochondrial dysfunction and oxidative stress and its causative pathological events contribute to diabetic dysfunction and reproductive dysfunction (Diabetes and the immunological function: porous organic oxidative species. Current Diabetes Rev,2008, 4. Therefore, the comprehensive improvement of the mitochondrial function, the carbohydrate metabolism state, the redox balance and the energy state of the diabetes patient with pregnancy has important significance for improving the fertility and the fertility quality of the patient. Our findings suggest that hypoglycemic agents do not implement this new therapeutic strategy and that effective or efficacious GRb compositions, particularly in combination with insulin or metformin, can perform this therapeutic strategy well, creating new hopes for patients in the fertile phase. Meanwhile, the high safety of the treatment is very important for both pregnant women and diabetics. The use of metformin in pregnancy is increasing worldwide as random control trials demonstrate its safety and effectiveness. Our research results show that the GRb effective or effective composition has high safety on diabetes patients under the action of regulating carbohydrate metabolism and cell self-rescue mechanism, is at least safer than metformin, is remarkably superior to metformin in the aspects of improving intracellular metabolism, reducing AGEs accumulation and metabolic inflammation and the like, and can generate excellent blood sugar reducing effect, provide enough cell energy and completely avoid oxidative stress, AGEs accumulation and inflammation states by virtue of the synergy of the GRb effective or effective composition. Therefore, an effective or effective combination of GRb or in combination with insulin or metformin would provide a safer and more effective treatment for pregnant and gestational diabetic patients, and would be an ideal choice for pregnant hyperglycemic patients. Since sex is also an important aspect in the life of couples, the efficacy of the effective or effective GRb composition in preventing and treating diabetic reproductive system complications is also of great significance in improving the quality of life of patients.

Mitochondrial dysfunction and its associated toxic pathological events including energy substance ATP deficiency/absence, oxidative stress injury, accumulation of advanced glycosylation products (AGEs), and chronic inflammation are common pathological mechanisms involved in metabolic disease, dysfunction/degeneration and subsequent structural degeneration and/or injury of cells and tissues and organs under a variety of pathological conditions related to metabolic disorder or age-related disease. Consistently, GSH deficiency, metabolic disorders and microvascular dysfunction are common features of diabetes and neurodegenerative diseases including senile dementia and Parkinson's disease, and GSH deficiency, metabolic disorders and neuroinflammation are also characteristic changes of psychotic disorders (schizophrenia and bipolar disorder) and also important components of the disease pathology (Redox dynamics in the pathobiology of schizophrenia and bipolar disorder: errors from animal models for affected Red Signal,2013, 18. In particular, AGEs are both primarily derived from sugar metabolism disorders (glycolysis uncoupled from sugars by mitochondrial oxidative metabolism) and in turn aggravate sugar metabolism disorders by inducing oxidative stress and by inducing various mechanisms such as high expression of proinflammatory factors/molecules mediated inflammation leading to insulin resistance and impairment of mitochondrial function, as well as by affecting geno-, lipo-and proteome damage and other physiological processes and triggering various pathological processes including microvascular abnormality-related neovascularization and fibrosis-related extracellular matrix accumulation (methyliglyoxal, a high reactive dicarboyl complex, in diabetes, its vaso complexes, and other such diseases. Physiological Rev,2020, 100. Thus, AGEs accumulation plays a key role in The progression of a variety of diseases, including diabetes, obesity, cardiovascular disease (hypertension, coronary and peripheral artery disease/atherosclerosis, stroke and microvascular complications), chronic kidney disease, liver fibrosis, chronic obstructive pulmonary disease, pulmonary fibrosis, neurodegenerative disease, alcoholic brain damage, psychiatric disorders and unhealthy aging (The role of advanced diabetes end-products in imaging and metabolic disorders: brightening association and utilization, cell, 2018, 28. Moreover, anti-inflammatory molecules that inhibit AGEs have proven to be good candidates for improving diabetic complications and degenerative diseases (Advanced glycation end-products produced systems and by macropathologies: a common distributor to infection and genetic diseases, pharmaceutical therapeutics, 2017,177, 44-55), inhibition of binding of AGEs to their receptors is also considered as a new therapeutic strategy for AGE-related diseases (including physiological aging, neurodegenerative/neuroinflammatory diseases, diabetes and its complications, autoimmune/rheumatic inflammatory diseases, bone degenerative diseases and chronic kidney diseases) (The described later of mail reaction, and Advanced glycation end product (AGE) -Receptor for AGE (RAGE) as a novel therapeutic strategy for clinical therapeutic ingredients for AGE-related diseases, tissue-related diseases, etc., 3, 25, 5591, therapeutic connectivity of The promising therapeutic ingredients, mouse, 3, 2022, 3, n.e.. Based on the similarity of the pathological mechanisms of diabetes and senile dementia, intranasal inhalation of insulin is also an attractive treatment for moderate cognitive impairment or dementia (Efficacy of oral administration in improving cognition in cognitive impairment or dementias: a systematic review and meta-analysis. Front Aging Neurosci,2022,14 963933); metformin is also tried or used in neurodegenerative diseases including amnestic mild cognitive impairment, alzheimer's disease and parkinson's disease and in psychiatric disorders including schizophrenia and autism, and its beneficial effects are related to its anti-inflammatory and antioxidant properties (metal-a future therapy for neurodegenerative diseases: the same: drug discovery, development and delivery in Alzheimer's disease. Phase Res,2017, 34. In summary, the results of the study of effective or efficacious GRb compositions also support the use of effective or efficacious GRb compositions alone and in combination with insulin or metformin or other hypoglycemic agents with a coordinated mechanism of action in several ways.

Metabolic syndrome is manifested by a group of related diseases including, but not limited to, insulin resistance, hyperglycemia, hypertension, central obesity, hyperlipidemia, hyperviscosity, hypercreatinine, and atherosclerotic dyslipidemia, is a high risk factor for diabetes, atherosclerosis, and non-atherosclerotic cardiovascular disease, and also increases the morbidity and mortality of cerebrovascular disease. Since resistance to the effects of Insulin metabolism is an important component of the pathophysiology of the metabolic syndrome, this syndrome is also known as "Insulin resistance syndrome" (Insulin signaling, resistance, and the metabolic syndrome: errors from motor models in discrete mechanisms.J Endocrinol,2014, 220T 1-T23; insulin resistance, prediabetes, metabolic syndrome: at short skin elevation tangent J Fine Clin Res Peditator Endocrinol,2017, 9S 49-S57. However, chronic low-grade inflammatory states (also called metabolic inflammation) are characteristic of obesity, insulin resistance, type II diabetes and metabolic heart disease, the occurrence of which is closely related to and in turn aggravates insulin resistance and metabolic disorders, and is also the core mechanism of the pathophysiology of metabolic syndrome, and IL-1 β produced by metabolic inflammation is a key molecule in the pathogenesis of metabolic heart disease (Targeting inflammation in metabolic syndrome trans Res,2016, 167-280, guest microbial as a trigger for metabolic inflammation in vivo and type 2diabetes. Therefore, insulin resistance and chronic/Metabolic inflammation are considered to be major factors in the 3 links of initiation, progression and transition of the Metabolic syndrome to cerebrovascular disease (Metabolic syndrome: stroke, management, and modulation by natural composition, the term Adv cardiovascular Dis,2017, 11. Therefore, the GRb effective or effective composition strengthens the coupling of glycolysis and sugar oxidative metabolism and promotes the production of mitochondrial oxidative phosphorylation under the II-type diabetes state, improves the metabolic inflammation state under the diabetes state and strengthens the action of metformin on slowing down insulin resistance, particularly the action of combining the two to completely avoid the metabolic inflammation state and the insulin resistance, and respectively supports the application of the GRb effective or effective composition to the treatment of metabolic syndrome and the prevention of diabetes and metabolic cardiovascular diseases alone or in combination with metformin or other medicaments/substances with coordinated action mechanisms.

Medical use of effective or efficacious grb compositions alone or in combination with insulin or metformin or other drugs/substances with coordinated mechanisms of action to prevent and treat metabolic disorders induced by drug therapy including, but not limited to, hyperglycemia, lipid metabolism disorders and diabetes induced by glucocorticoid, statin, immunosuppressant and antipsychotic drug therapy.

Drug-induced metabolic syndrome/hyperglycemia and diabetes are an increasingly serious problem in clinical practice (Drug-induced hyperglycaemia and diabetes. Drug Saf,2015, 38. In addition, statins (widely used in hyperlipidemic cardiovascular diseases such as hyperlipidemia, hypertension, atherosclerotic heart disease, etc.), thiazide diuretics, and certain beta-blockers antihypertensive agents are also at risk of causing new diabetes and exacerbating preexisting diabetes. Reduced insulin sensitivity (i.e., insulin resistance) and/or reduced insulin secretion is a common cause of metabolic syndrome/hyperglycemia and diabetes in drug therapy, and metformin is the first drug currently used to correct the therapeutic metabolic syndrome and diabetes, with insulin also being used for severe hyperglycemia (pharmaceutical management of glucose regulation in nutritional diabetes with controlled-production anti-diabetes drugs,2020, 80. In conclusion, our findings support that an effective or efficacious composition of GRb in combination with metformin has been used in the treatment of the metabolic syndrome characterized by insulin resistance, hyperglycemia and diabetes in combination with insulin for the associated conditions characterized by decreased insulin secretion.

Polycystic ovarian syndrome is one of the most common endocrine and metabolic disorders in premenopausal women, manifested as hyperandrogenism, ovarian dysfunction and related metabolic disorders, including abdominal obesity, insulin resistance, obesity, metabolic disorders such as hypertension or dyslipidemia (Polycystic ovarian syndrome: definition, biology, diagnosis and treatment. Nat Rev endocrine, 2018, 14; patients with polycystic ovary syndrome are also at high risk for type II diabetes and future emotional and psychiatric disorders. It is particularly noted here that studies carried out over the last years have demonstrated that increased oxidative stress is associated with the progression of Polycystic ovarian syndrome and associated complications, and have demonstrated a relationship between mitochondrial dysfunction and Polycystic ovarian syndrome (Polycystic ovarian syndrome: reprod Biol Endocrinol,2019, 17. In particular, events associated with mitochondrial dysfunction including GSH insufficiency, oxidative stress, and chronic inflammation have all been shown to be associated with the onset and progression of polycystic ovarian syndrome. Currently, the most common strategy for treating insulin resistance in polycystic ovarian syndrome is to use insulin sensitizers, particularly metformin. Metformin is similar in weight loss to lifestyle interventions but is more effective in reducing androgen concentration, and is subject to standard treatment for hypertension or dyslipidemia (polymeric overview syndrome: definition, aeration, diagnosis and treatment. Nat. Rev endothelial, 2018, 14. In summary, our findings support the use of effective or efficacious GRb compositions alone or in combination with metformin for the treatment of polycystic ovarian syndrome.

There has been compelling evidence that psychiatric disorders characterized by an increased risk of Metabolic syndrome including dyslipidemia, abdominal obesity, hypertension and hyperglycemia include major depression, bipolar disorder, schizophrenia, anxiety, autism, attention deficit/hyperactivity disorder and post-traumatic stress disorder (Metabolic syndrome in psychiatric disorders: overview, mechanisms, and pathologies. Clinical Clin neurosis, 2018, 20. There is emerging evidence that there is a bidirectional longitudinal impact between psychiatric disorders and metabolic syndrome. For example, recent studies have demonstrated that metabolic comorbidities between schizophrenia and type II diabetes are due at least in part to shared genetic mechanisms (Evidence for genetic compatibility to the involved diabetes mellitus. Transport psychic, 2018, 8. In addition, different populations of psychotic patients have intrinsic pathophysiological characteristics of a deregulated homeostatic system, including the hypothalamic-pituitary-adrenal axis and inflammatory responses, and these pathophysiological characteristics are also associated with the development of metabolic syndrome. In particular, psychotic disorders share a metabolic disorder phenotype with metabolic syndrome/type II diabetes, including: mitochondrial dysfunction, altered redox balance (including GSH deficiency), AGEs accumulation, and chronic low grade inflammation, and these pathological events are deeply involved in the expression of neurological and behavioral disorders of schizophrenia and bipolar affective disorders (mitochondrion dysfunction in schizohrenia: pathways, mechanisms and modalities. Neurosci biotahav, 2015, 48-21 mitochondrion impact. In addition, the metabolic disorder phenotype and its effects on psychotic disorders are also present in neurodegenerative disorders such as Alzheimer's disease, huntington's disease and Parkinson's disease (Mitochondrial interference: A common motif in neuropsychiatric presentations the link to the tryptophan-kynurine metabolic system. Cells,2022, 11. It is also noted herein that certain psychotropic agents, such as second generation anti-schizophrenia agents, also have profound effects on the increased imbalance of metabolic syndrome, increase the risk of type II diabetes and cardiovascular disease in patients, exacerbate metabolic syndrome, decrease quality of life in patients, and also do not facilitate recovery of the disease in neurophysiologic functions, leading to a shortened life expectancy. Therefore, the existing symptomatic treatment strategies need to be changed, the biochemical and molecular environment for treating and breeding and/or supporting the mental disorder diseases is a disease-based strategy, and the treatment effect of treating both symptoms and root causes can be achieved if the traditional symptomatic treatment strategies are combined with the existing symptomatic medicines. Currently, enhancement of Mitochondrial function is thought to contribute to the amelioration of neurological and behavioral impairment associated with such diseases (mitochondrion dysfunction in schizophrenia: pathways, mechanisms and indications. Neurosci Biobehav Rev,2015, 48. Due to its clear safety, metformin therapy has been used in neuropsychiatric practices including schizophrenia and autism (Major mental deformation, and neurological forms associated with a method of using a shock damping mechanism. J. Clin mental damage, 2016, 77. In conclusion, our findings support the use of effective or advantageous GRb compositions for the treatment of psychotic disorders, either alone or in combination with metformin or in combination with other drugs with a coordinated mechanism of action.

Type II diabetes is closely associated with neurodegenerative diseases. On the one hand, type II diabetes can lead directly to senile dementia, but also to important risk factors for senile dementia, vascular dementia and parkinson's disease. On the other hand, type II diabetes has highly similar metabolic disorder characteristics to neurodegenerative diseases, including mitochondrial dysfunction, oxidative stress, AGEs accumulation, insulin resistance and systemic metabolic inflammation, and these pathological events, particularly the vicious circle between them, lead to the functional and structural degeneration of the relevant brain cells (inflammation-forming and brain injury: new injuries and role of life-style strains on cognitive and social decisions in-forming and neurologic degradation. Front Neurosci,2021, 618395. Senile dementia is therefore known to be a type III diabetes occurring in the brain, and Metformin has achieved some benefit in clinical trials for the treatment of senile dementia and parkinson's disease (metalformin for clinical of diagnosis X syndrome and other neurological disorders, annu Rev Med,2019, 70. It is noteworthy here that human and animal model study data of neurodegenerative diseases indicate that Mitochondrial dysfunction and oxidative stress are key factors in initiating and propagating the neurodegenerative process (mitochonddriver dynamics and oxidative stress in neurological disorders. Nature,2006,443, 787-795 cable and sequence. Thus, we have reasoned that mitochondrial dysfunction leads to inadequate oxidative breakdown of glycolytic intermediates leading to toxic MGO and ultimately to AGEs in the pre-neurodegenerative disease stage, and that cellular disinfection of MGO also consumes large amounts of GSH to exacerbate ROS accumulation that accompanies or causes mitochondrial dysfunction, both of which lead to chronic inflammatory states. Thus, the GRb effective or advantageous composition systematically increases cellular antioxidant capacity, particularly compensatory increases in GSH and SOD levels, and potentiates the coupling effects of glycolysis and carbohydrate oxidative metabolism, strongly supporting its therapeutic use for the treatment of neurodegenerative diseases, either alone or in combination with metformin or other drugs/substances with coordinated mechanisms of action.

Fibrosis is an abnormal deposition of extracellular matrix that can lead to organ dysfunction, morbidity, and death. The disease burden caused by fibrosis is enormous and there is currently no treatment that can prevent or reverse fibrosis. Diseases associated with fibrosis include: liver cirrhosis, non-alcoholic steatohepatitis, chronic kidney disease, heart failure, diabetes, idiopathic pulmonary fibrosis, and scleroderma. Metabolic alterations are increasingly recognized as an important pathogenic process for fibrosis in many organ types. Pathologically, fibrosis is characterized by an imbalance in extracellular matrix (ECM) hyperplastic homeostasis. There is reliable data to directly link glycolysis to extracellular matrix production. Glucose metabolism can provide energy for this anabolic process and provide a basis for the production of collagen. This is similar to the Warburg effect in cancer, where cancer cells preferentially utilize aerobic glycolysis to generate ATP and NADPH, and provide glycolytic intermediates as carbon sources to promote proliferation. Indeed, this interaction between glycolysis and extracellular matrix production has been demonstrated in multiple steps of extracellular matrix synthesis, including amino acid synthesis, hydroxylation, glycosylation, and extracellular matrix secretion (Targeting metabolic regulation for fibrosis therapy. NatRev Drug Discov,2020, 19. AGEs are consistently involved in the fibrotic process in several organs via their Receptors (RAGE), including liver fibrosis, heart fibrosis, lung fibrosis and kidney fibrosis (pathobiology of RAGE in inflammatory diseases front Immunol,2022,13 931473). Therefore, the effective or advantageous combination of grbs enhances the coupling of glycolysis to sugar oxidative metabolism, consumes intermediates of glycolysis and tricarboxylic acid cycle by promoting oxidative catabolism, thereby effectively reducing the flow of metabolic intermediates to extracellular matrix synthesis, including amino acid synthesis and glycosylation, and finally achieving the goal of anti-fibrosis. Indeed, GRb effective or efficacious compositions almost completely inhibit the development of cardiac fibrosis in type I diabetic conditions and very significantly reduce the extent of cardiac fibrosis in type II diabetic conditions, and although metformin does not show significant efficacy, its combination with GRb effective or efficacious compositions can almost completely prevent the development of cardiac fibrosis. Similarly, thyroid hormones can reduce the severity of bleomycin-induced pulmonary fibrosis, their mechanism of action is linked to their increased mitochondrial function, so hormonal therapy is thought to exert anti-fibrotic effects by modulating cellular respiration and oxidative catabolism, and Targeting these key metabolic pathways is thought to be an exciting and promising opportunity for future fibrosis therapy (Targeting metabolic dysfunction for fibrosis therapy. NatRev Drug Discov,2020, 19. In addition, lipotoxicity caused by excessive accumulation of lipids or defects in fatty acid oxidation also leads to the development of fibrosis (Targeting fatty acid metabolism for fibrous disorders. Arch Pharm Res,2021, 44. Thus, the combination of an effective or efficacious GRb composition with insulin or metformin can maintain fat metabolism homeostasis, avoid lipotoxicity, and also contribute to its anti-tissue-organ fibrosis effect. To date, some of the existing metabolic drugs used to treat diseases such as diabetes (metformin, insulin) and hypercholesterolemia (pravastatin) have become candidates for the treatment of fibrosis; drugs against insulin resistance, such as hesperidin and pentoxifylline, are also being evaluated for their potential therapeutic value against fibrosis (Targeting metabolic dysregulation for fibrosis therapy. Nat Rev Drug Discov,2020, 19. In summary, our findings support the use of GRb effective or efficacious compositions alone or in combination with insulin or metformin or other drugs/substances with coordinated mechanisms of action for the prevention and treatment of fibrosis-related disorders, including: liver cirrhosis, non-alcoholic steatohepatitis, chronic kidney disease, heart failure, idiopathic pulmonary fibrosis, and scleroderma.

The aging process is characterized by a decline in the function of cells, tissues and whole organs, beginning with perturbations in critical cellular processes such as mitochondrial function, protein balance and stress-clearing systems. An increasing number of studies have demonstrated causal relationships between MGO-derived AGEs and age-related tissue dysfunction, revealing a previously underestimated role of Dicarbonyl stress in determining healthy or unhealthy ageing (dicarboyl stress exercise of health and health aging. Cells,2019, 8. If the defense mechanisms (e.g., GSH-mediated glyoxalase system) are not effective in counteracting the accumulation of MGO during aging, tissue damage can result, inducing metabolic disease, vascular dysfunction, and neuronal damage, leading to unhealthy aging; this will ensure healthy aging if a good balance between MGO formation and defense mechanism activity is maintained. It can be seen that AGEs bridge the classical hypothesis of aging, including the theory of free radicals, apoptosis, and mitochondrial dysfunction. It is understood here that both glycolytic intermediate accumulation or/and GSH deficiency resulting from mitochondrial dysfunction can trigger MGO toxicity and AGEs accumulation, and that sufficient SOD and GSH can ensure mitochondrial protection from oxidative damage by the byproduct ROS produced by the respiratory chain during oxidative phosphorylation. Therefore, the effective or effective GRb composition systematically improves the antioxidant capacity of cells, and particularly enhances the powerful effects of increasing GSH and SOD, and enhances glycolysis and oxidative metabolic coupling so as to reduce the generation of MGO and enhance the detoxification effect, thereby supporting the medical application of the composition in delaying senility.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims

1. A specific active panaxadiol saponin composition, which is characterized by comprising the following panaxadiol saponins: GRb1, GRc, GRb3 and GRd;

in the specific active panaxadiol saponin composition, the total mass percentage of GRc and GRb3 is more than that of GRb1 and GRd, and the difference between the total mass percentage of GRc and GRb3 and the total mass percentage of GRb1 and GRd is 4.55-21.30%;

the mass ratio of GRb1 to GRd is 0.85-1.82;

the mass ratio of GRc to GRb3 is 0.60-0.86;

the mass ratio of GRb3 to GRb1 is 0.93-2.10;

the mass ratio of GRc to GRb1 is 0.72-1.42.

2. The specific active panaxadiol saponin composition of claim 1, wherein the mass ratio of GRb1, GRc, GRb3 and GRd comprises: 0.96-1.18: 0.90-1.10: 1.37 to 1.67: 1.02-1.24, 0.69-0.85: 0.90-1.10: 1.33 to 1.63: 0.66-0.80, 1.13-1.38: 0.90-1.10: 1.16 to 1.42: 0.70-0.86 or 0.99-1.21: 0.90-1.10: 1.17 to 1.43:0.59 to 0.73;

Further preferably, the mass ratio of GRb1, GRc, GRb3 and GRd is 1.07:1.00:1.52:1.13, 0.77:1.00:1.48:0.73, 1.25:1.00:1.29:0.78 or 1.10:1.00:1.30:0.66;

3. a method for preparing a specific active panaxadiol saponin composition according to claim 1 or 2, comprising the steps of:

dissolving ginsenoside in 30 vol% ethanol water solution, and subjecting to reversed phase C ₁₈ Separating by silica gel column chromatography, eluting with 27-33% ethanol water solution by volume percentage, eluting with 37-44% ethanol water solution by gradient volume percentage, collecting eluate, and stopping eluting when ginsenoside GRb1 is detected in the eluate; eluting with 50-60% ethanol water solution, collecting eluate, stopping eluting when ginsenoside GRd is not detected in the eluate, mixing the collected eluates, and concentrating and drying the eluate to obtain specific active panaxadiol saponin composition;

The total ginsenoside comprises commercial total ginsenoside or total ginsenoside extracted by taking ginseng as a raw material and adopting 30-80% ethanol water solution by volume percentage;

the Panax Chinese medicinal materials comprise two or more of radix Panacis Quinquefolii root, caulis Panacis Quinquefolii, radix Ginseng root, caulis Ginseng and folium Notoginseng.

4. The preparation method according to claim 3, wherein the commercial ginsenoside comprises at least one of the following compositions: 0.8 to 1.2 parts by mass of American ginseng root total saponin and 1.6 to 2.4 parts by mass of American ginseng stem and leaf total saponin, 0.8 to 1.2 parts by mass of American ginseng root total saponin, 0.8 to 1.2 parts by mass of American ginseng stem and leaf total saponin and 1.4 to 2.1 parts by mass of pseudo-ginseng stem and leaf total saponin, 0.8 to 1.2 parts by mass of ginseng stem and leaf total saponin, 1.2 to 1.8 parts by mass of American ginseng root total saponin, 0.8 to 1.2 parts by mass of American ginseng stem and leaf total saponin and 2.4 to 3.6 parts by mass of pseudo-ginseng stem and leaf total saponin, 0.8 to 1.2 parts by mass of American ginseng root total saponin, 0.8 to 1.2 parts by mass of American ginseng stem and leaf total saponin and 0.8 to 1.2 parts by mass of pseudo-ginseng stem and leaf total saponin, and 0.8 to 1.2 parts by mass of pseudo-ginseng stem and leaf total saponin;

The ginseng traditional Chinese medicinal materials comprise at least one of the following raw materials: 0.8 to 1.2 parts by mass of American ginseng root and 1.6 to 2.4 parts by mass of American ginseng stem leaf, 0.8 to 1.2 parts by mass of American ginseng root, 0.8 to 1.2 parts by mass of American ginseng stem leaf and 1.4 to 2.1 parts by mass of pseudo-ginseng stem leaf, 0.8 to 1.2 parts by mass of ginseng stem leaf, 1.2 to 1.8 parts by mass of American ginseng root, 0.8 to 1.2 parts by mass of American ginseng stem leaf and 2.4 to 3.6 parts by mass of pseudo-ginseng stem leaf, 0.8 to 1.2 parts by mass of American ginseng root, 0.8 to 1.2 parts by mass of American ginseng stem leaf and 0.8 to 1.2 parts by mass of pseudo-ginseng stem leaf, and 0.8 to 1.2 parts by mass of American ginseng root, 0.8 to 1.2 parts by mass of American ginseng stem leaf and 1.2 to 1.8 parts by mass of pseudo-ginseng stem leaf;

preferably, the panaxadiol saponins GRb1, GRc, GRb3 and GRd are obtained by the following extraction steps: dissolving the mixed total ginsenoside in 30 percent ethanol water solution by volume percentage, combining with on-line analysis, and performing reversed phase C ₁₈ And (2) performing column chromatography separation by using silica gel, eluting by using 30% ethanol aqueous solution by volume percentage, then eluting by using 40% ethanol aqueous solution and collecting the eluent until GRb1 or GRb1 does not exist in the eluent or the amount of GRb1 is extremely low, eluting by using 43% ethanol aqueous solution and collecting the eluent until GRc or GRc does not exist in the eluent, eluting by using 47% ethanol aqueous solution and collecting the eluent until GRb3 or GRb3 does not exist in the eluent or the amount of GRb3 is extremely low, and eluting by using 55% ethanol aqueous solution and collecting the eluent until GRd or GRd does not exist in the eluent or the amount of GRd is extremely low.

5. A specific active ginseng total saponin composition is characterized by comprising more than two of American ginseng root total saponin, american ginseng stem leaf total saponin, ginseng root total saponin, ginseng stem leaf total saponin and panax notoginseng stem leaf total saponin, or is obtained by taking ginseng traditional Chinese medicinal materials as raw materials, extracting the raw materials by adopting an ethanol water solution with the volume percentage content of 0-80%, and removing a solvent; the Panax Chinese medicinal materials comprise two or more of radix Panacis Quinquefolii root, caulis Panacis Quinquefolii, radix Ginseng root, caulis Ginseng and folium Notoginseng;

the specific active panaxadiol saponin composition of claim 1 or 2 contains 35% by weight or more of the specific active panaxadiol saponin composition.

6. The specific active ginsenoside composition of claim 5, comprising at least one of the following: 0.8 to 1.2 parts by mass of American ginseng root total saponin and 1.6 to 2.4 parts by mass of American ginseng stem and leaf total saponin, 0.8 to 1.2 parts by mass of American ginseng root total saponin, 0.8 to 1.2 parts by mass of American ginseng stem and leaf total saponin and 1.4 to 2.1 parts by mass of pseudo-ginseng stem and leaf total saponin, 0.8 to 1.2 parts by mass of ginseng stem and leaf total saponin, 1.2 to 1.8 parts by mass of American ginseng root total saponin, 0.8 to 1.2 parts by mass of American ginseng stem and leaf total saponin and 2.4 to 3.6 parts by mass of pseudo-ginseng stem and leaf total saponin, 0.8 to 1.2 parts by mass of American ginseng root total saponin, 0.8 to 1.2 parts by mass of American ginseng stem and leaf total saponin and 0.8 to 1.2 parts by mass of pseudo-ginseng stem and leaf total saponin, and 0.8 to 1.2 parts by mass of pseudo-ginseng stem and leaf total saponin;

7. Use of the specific active panaxadiol saponin composition of claim 1 or 2 or the specific active panaxadiol saponin composition prepared by the preparation method of claim 3 or 4 alone or in combination with a hypoglycemic agent in the preparation of a medicament for preventing and/or treating one or more of diabetes, metabolic disorder-related diseases, diabetic complications, angiogenesis disorder-related diseases and delaying aging.

8. The use of claim 7, wherein the diabetes mellitus comprises type I diabetes and type II diabetes;

preferably, the medicament has the efficacy of improving and relieving metabolic disorder states and insulin resistance states under the states of type I diabetes and type II diabetes;

preferably, the metabolic disorder states in type I and type II diabetes include, but are not limited to: disorders of carbohydrate metabolism, disorders of lipid metabolism, disorders of protein metabolism in negative balance, disorders of creatine metabolism and redox imbalances.

9. The use according to claim 7, wherein the disease associated with metabolic disorders comprises at least one of: metabolic syndrome, therapeutic metabolic disorders, polycystic ovary, psychotic disorders associated with metabolic disorders, neurodegenerative disorders with metabolic syndrome as a risk factor, and tumor therapeutic toxic side effects characterized by metabolic disorders and/or oxidative stress damage;

preferably, the neurodegenerative disease with metabolic syndrome as a risk factor comprises at least one of the following diseases: amnestic mild cognitive impairment, senile dementia, vascular dementia and parkinsonism/symptoms characterized by cerebrovascular pathologies;

preferably, the diabetic complication comprises at least one of the following complications: diabetic skin disease, diabetic neuropathy, difficulty in healing of medical operation or accidental trauma for diabetic patients, diabetic cardiovascular complications, diabetic nephropathy, diabetic eye disease, diabetic foot, diabetic male sexual dysfunction, and diabetic female reproductive dysfunction;

10. The use of any one of claims 7 to 9, wherein the hypoglycemic agent comprises insulin or metformin.

11. Use of the specific active ginsenoside composition of claim 5 or 6 in the preparation of a health product for improving one or more of insulin resistance state, diabetic metabolic disorder state and diabetic complication.

12. The use as claimed in claim 11, wherein the use is the use of the specific active ginsenoside composition in combination with at least one of a nutrient, a metabolic regulation substance and a Chinese medicinal herb having homology of medicine and food for the preparation of a health product;

Preferably, the metabolism-regulating substance includes an NAD precursor, coenzyme Q10, vitamin B1, vitamin B2 and vitamin B6.

13. A medicament for the prevention and treatment of diabetes, metabolic disorders of diabetes and diabetic complications, which comprises the specific active ginsenoside composition of claim 1 or 2 or the specific active ginsenoside composition prepared by the preparation method of claim 3 or 4 as the only active ingredient, or the specific active ginsenoside composition and a hypoglycemic agent in combination as the active ingredients;

the hypoglycemic agent comprises insulin or metformin.

14. The medicament of claim 13, further comprising a pharmaceutically acceptable excipient or carrier;

further preferably, the oral preparation comprises a solid preparation and a liquid preparation, and the solid preparation comprises: tablet, dispersible tablet, granule, pill, controlled release preparation, pellet and capsule.

15. A health product for improving diabetes, metabolic disorders of diabetes and diabetic complications, comprising the specific active ginsenoside composition of claim 5 or 6 as the only active ingredient or the specific active ginsenoside composition in combination with at least one of nutrients, metabolic regulation substances and medicinal and edible Chinese medicines as the active ingredient;

The hypoglycemic agent comprises insulin or metformin.

16. The nutraceutical of claim 15, further comprising an acceptable excipient or carrier;

further preferably, the solid formulation form comprises: granule, capsule, tablet, dispersible tablet and pill.

17. A panaxadiol saponin binary composition is characterized by comprising a GRb1+ GRd functional unit or a GRc + GRb3 functional unit;

the mass ratio of GRb1 to GRd in the functional unit of GRb1+ GRd is 1.48-1.82;

18. Use of a specific active panaxadiol saponin composition of claim 1 or a panaxadiol saponin binary composition of claim 17 in the preparation of a medicament and/or health product for repairing skin injury;

the skin lesions include skin lesions in the following cases: skin damage in healthy people, skin damage in diabetics, skin damage caused by face-lifting, beauty and surgery;

preferably, the product comprises a pharmaceutical, cosmeceutical, cosmetic or cosmetology product.