KR102292059B1 - Composition comprising of the extract of Cissus verticillata and Cyclopia Intermedia as an active ingredient for preventing or treating diabetic complication and diabetes - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of diabetes or diabetic complications comprising a combination extract consisting of insulina (Cissus verticillata ) and honeybush (Cyclopia intermedia) as an active ingredient. In terms of reducing fat weight, lowering blood glucose and glycated hemoglobin, and suppressing low molecular weight cholesterol through db/db mice, which are diabetes model mice (Experimental Example 1), the insulin and honeybush combination extract was unexpected and markedly antidiabetic, which cannot be predicted by those skilled in the art. By confirming the effect, it can be usefully used for the prevention or treatment of diabetes or diabetic complications.

Description

Composition comprising of the extract of Cissus verticillata and Cyclopia Intermedia as an active ingredient for preventing or treating diabetic complication and diabetes, comprising a combination extract of insulin and honeybush as an active ingredient

The present invention relates to a composition for treating or preventing diabetes and diabetic complications comprising a combination extract of insulina and honeybush as an active ingredient.

[Document 1] O'Rahilly S, et al., Diabetologia , 31 pp792-797, 1988

[Document 2] Barnett AH, et al., Diabetologia , 20 , pp87-93, 1981

[Document 3] Smith U., et al., Int. J. Obes. Relat. Metab. Disord. , 26 , pp897-904, 2002

[Document 4] Mayerson AB, et al., Am. Fam. Physician , 55 pp817-824, 1997

[Document 5] Vannasaeng S, et al., J. Med. Assoc. Thai. , 78 , pp578-585, 1995

[Document 6] Kannel WB and McGee DL, JAMA , 241, pp2035-2038, 1979

[Document 7] Fuller JH, Lancet, 1, pp1373-1376, 1980

[Document 8] Drobnik J and Oliveira AB, J. Ethnopharmacol., 171 , pp317-329, 2015

[Document 9] Miller AB, et al., Pharm. Biol., 53 , pp548-554, 2015

[Document 8] Japanese Patent Laid-Open No. 2001-238649

[Document 9] International Patent Publication No. PCT/WO 2001/15716 A1

[Document 10] International Patent Publication No. PCT/WO 2008/110552 A2)

[Document 11] wang IK et al., Effects of grape seed extract and its ethylacetate/ethanol fraction on blood glucose levels in a model of type 2 diabetes. Phytother. Res., 23, pp1182-1185, 2009

The present invention relates to a composition for the treatment or prevention of diabetes and diabetic complications comprising a combination extract of insulina and honeybush as an active ingredient.

Diabetes mellitus is characterized by persistent hyperglycemia in which the concentration of glucose in the blood remains abnormally high. In order for glucose from the blood to enter the cells and be used, the help of a hormone called insulin secreted by pancreas beta (β) cells is needed. Even if it does not play a role in putting glucose into the cells (type 2 diabetes), the glucose cannot enter the cells and remains in the blood, resulting in abnormally elevated blood sugar, resulting in excess glucose in the urine.

Type 1 diabetes mellitus is called insulin dependent diabetes mellitus, and the beta cells of pancreas are destroyed by virus infection or autoimmune disease. unable to control blood sugar Such insulin-dependent diabetes mellitus appears genetically or appears in young people, and the incidence is known to account for about 10-15% of all diabetes mellitus. Considering that diabetes is observed in more than 70% of cases with a family history of diabetes or identical twins, it can be seen that genetic factors play a very important role (O'Rahilly S, et al., Diabetologia , 31 pp792). -797, 1988; Barnett AH, et al., Diabetologia , 20 , pp87-93, 1981).

In type 2 diabetes mellitus, insulin-secreting pancreatic beta cells are in a normal form, but it is caused by insulin resistance in the body, which is called non-insulin dependent diabetes mellitus. do. In general, non-insulin-dependent diabetes mellitus is common in older adults or obese young adults, and accounts for 85-90% of diabetes patients worldwide. In non-insulin-dependent diabetes mellitus, the disease is promoted by obesity. The reason is that when food is consumed excessively, an increase in blood sugar is induced. At this time, the blood induces hyperinsulinemia by the excessively secreted insulin, and the target organ such as the liver overreacts due to hyperinsulinemia, and the insulin receptor decreases in the liver to reduce the excessive reaction. . Since this decrease in insulin receptor eventually reduces the response to insulin, even if insulin is normally secreted from the pancreas, the effect of insulin eventually decreases sharply. (Smith U., et al., Int. J. Obes. Relat. Metab. Disord. , 26 , pp897-904, 2002).

Until now, various approaches have been tried in research for the treatment of diabetes, and among them, diet and exercise, direct administration of insulin or blood sugar-lowering drugs are known to be effective. Sulfonylureas, thiazolidinediones, and biguanides are known as blood sugar-lowering drugs, and although they effectively lower blood sugar, they are known to have very large side effects. Sulfonylurea is known to cause mild symptoms such as hypoglycemia, indigestion, dizziness, and somnolence, as well as an increase in kidney and liver disease and cardiovascular mortality, so its use is very limited. On the other hand, since thiazolidinedione enhances the response of insulin in the presence of insulin, it can be used only when insulin is secreted from the body, and causes erythrocyte abnormalities, such as dizziness and headache. , is effective only in the presence of insulin and can cause erythrocyte abnormalities and headaches (Mayerson AB, et al., Am. Fam. Physician , 55 pp817-824, 1997; Vannasaeng S, et al., J. Med. Assoc). Thai. , 78 , pp578-585, 1995). Because of these side effects, the use of synthetic substances is very restricted, so it is thought that the use of natural substances rather than synthetic substances will play a very important role in reducing side effects. In addition, since obesity is the basis of metabolic diseases that cause various diseases, when controlling obesity that causes insulin secretion abnormality, it is not only for the treatment and prevention of diabetes, but also for various metabolic diseases and dementia related to insulin secretion abnormality. is expected to be effective.

The production of final glycated products due to high blood sugar is known to cause diabetic arteriosclerosis, peripheral nerve palsy, and kidney disease. In particular, the glycated hemoglobin level is high in diabetic patients, such as heart disease, retinopathy, nephropathy, blood circulation disorder, and retinal damage. , nerve cell damage, renal dysfunction, and serious chronic complications such as vascular complications (Kannel, WB and McGee, DL JAMA, 241, pp2035-2038, 1979). In particular, the incidence of cardiovascular diseases such as arteriosclerosis, cerebral infarction, cerebral thrombosis, and myocardial infarction is higher in diabetic patients than in normal persons (Fuller, J.H., Lancet, 1, pp1373-1376, 1980).

Insulin ( Cissus verticillata ) was first described in historical sources in the 16th century, and was historically prescribed for stomach weakness, fever, or convulsions (Drobnik J, Oliveira AB, " Cissus verticillata (L.) Nicolson & CE Jarvis (Vitaceae): its identification and usage in the sources from 16th to 19th century". J. Ethnopharmacol., 171 , pp317-329, 2015).

In addition, through a recent paper, insulin has been reported to have a high antibacterial effect against Streptococcus mutans (Miller AB et al., The antibacterial and antifungal activity of essential oils extracted from Guatemalan medicinal plants. Pharm. Biol., 53 , pp548-554, 2015), the blood sugar lowering effect of insulin tea is known (Japanese Patent Laid-Open No. 2001-238649, published on September 14, 2001), a plant belonging to the genus Cissus (Cissus quadrangularis), A weight-suppressing effect of a combination of Vernonia glabra and chitosan has been disclosed (International Patent Publication No. PCT/WO 2001/15716 A1).

Honeybush ( Cyclopia intermedia , honeybush) is an evergreen shrub belonging to the family Melianthaceae, known as a plant that grows wild along the cliffs of South Africa. It has been used to treat colds, insomnia, and stomach upset as a therapy, and an antidiabetic effect of a plant extract of the genus Cyclopia subternata has been disclosed (International Patent Publication No. PCT/WO 2008/110552 A2)

However, none of the above documents discloses or teaches a synergistic therapeutic effect of a combination extract of insulina and honeybush on diabetic diseases.

Accordingly, the present inventors performed the development of a therapeutic agent using natural material resources effective for obesity/diabetes disease, and as a result of conducting the experiment by administering a combination extract of insulina and honeybush to db/db mouse, a type 2 diabetes model animal, In diabetic mice, it showed a reduction in fat weight, a decrease in blood sugar and HbA1c. , Prevention or treatment of obesity/diabetes by confirming that the present invention exerts a synergistic anti-diabetic or diabetic complication effect that cannot be predicted by those skilled in the art in terms of low molecular cholesterol suppression and diabetes suppression efficacy through the optimal combination condition of insulin and honeybush By confirming that it can be usefully used, the present invention was completed.

As one aspect for achieving the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of diabetes or diabetic complications containing a combination extract consisting of insulina (Cissus verticillata ) and honeybush (Cyclopia intermedia) as an active ingredient to provide.

In addition, the present invention provides a health functional food for preventing and improving diabetes or diabetic complications containing a combination extract consisting of insulin (Cissus verticillata ) and honeybush (Cyclopia intermedia) as an active ingredient.

Insulin as defined herein ( Cissus verticillata ) is characterized in that it is imported, such as Costa Rita, Chile, Argentina, Paraguay, Mexico, the United States.

Honeybush as defined herein ( Cyclopia intermedia ), characterized in that it is imported from Germany and South Africa.

The combination extract composed of insulina (Cissus verticillata ) and honeybush (Cyclopia intermedia ) as defined herein is preferably in a dry weight relative mixing ratio of 1: 0.01 to 100 (w/w) by weight, preferably, 1: 0.5 to 50 (w/w) parts by weight, more preferably 1: 0.2 to 20 (w/w) parts by weight, even more preferably 1 to 15: 1 (w/w) , even more preferably from 1 to 10:1 (w/w), most preferably from 1 to 5:1 (w/w).

Insulin as defined herein ( Cissus verticillata ) and / or honeybush ( Cyclopia intermedia ) is a sample of the outpost, stem, leaf, root, etc. of each sample, water including purified water, methanol, ethanol, butanol, etc. A solvent selected from the lower alcohol of 4, alcohol, or a mixture thereof, preferably a mixed solvent of water and ethanol, more preferably, 10 to 80% alcohol or ethanol, even more preferably 20 to 50% alcohol or ethanol soluble extracts.

The combination of insulina and honeybush is

(1) an extract of a combination of insulina and honeybush, or

(2) a mixture of individual insulin extracts and honeybush extracts, or

(3) including combinations of these.

In one embodiment, the combination extract of the present invention may be a mixture of insulina extract and honeybush extract.

In the present specification, "extract" means not only extracts obtained by adding an extraction solvent to herbal medicines, but also concentrates and dilutions of the extracts, and dried products of the extracts, concentrates, and dilutions. Unless otherwise specified, "extract" means at least one selected from the group consisting of an extract obtained by adding an extraction solvent to a crude drug, a dried product of the extract, a concentrate or dilution of the extract, and a dried product of the concentrate or dilution. can be interpreted as meaning

The composition containing the extract of the present invention as an active ingredient contains 0.1 to 50% by weight of the extract based on the total weight of the composition.

As defined herein, "obesity" means excessive accumulation of body fat in the body, including, but not limited to, obesity that causes various diseases such as angina, myocardial infarction, stroke, arteriosclerosis and pancreatitis.

"Diabetes" as defined herein is selected from the group consisting of type 1 diabetes, also known as insulin-dependent or juvenile diabetes, and type 2 diabetes, known as non-insulin-dependent or adult diabetes, which accounts for about 90% of diabetic patients. It may be any one, but is not limited thereto.

As defined herein, “diabetic complications” means cardiovascular diseases, such as heart disease, retinopathy, nephropathy, blood circulation disorders, retinal damage, nerve cell damage, renal dysfunction, vascular complications, arteriosclerosis, cerebral infarction, cerebral thrombosis, or myocardial infarction. It may be any one selected from the group consisting of related diseases, but is not limited thereto.

As used herein, the term "prevention" refers to any action of inhibiting or delaying diabetes or diabetic complications by administration of a composition containing the extract.

In addition, the term "treatment" used in the present invention means any action in which the symptoms of a disease are improved or beneficially changed by administration of a composition containing the extract.

Hereinafter, the present invention will be described in more detail.

The extracts of the present invention can be obtained by the following preparation method.

For example, the present invention will be described in detail below.

In addition, the combination extract of the present invention can be prepared as follows.

After washing and shredding the dried insulin and honey bushes, respectively, 1 to 20 times the weight of the dried material, preferably, about 4 to 8 times the volume of purified water containing purified water, methanol, ethanol, butanol, etc. having 1 to 4 carbon atoms of a solvent selected from a lower alcohol, alcohol, or a mixture thereof, preferably a mixed solvent of water and ethanol, is mixed several times at 10 to 150° C., preferably 20 to 120° C. for 1 hour to 1 week, preferably 14 a first step of repeatedly performing ultrasonic extraction, hot water extraction, room temperature extraction, or reflux extraction, preferably room temperature extraction, about 1 to 20 times, preferably 2 to 10 times for a period of time to 52 hours; a second step of obtaining individual extracts obtained by filtration, concentration under reduced pressure, and drying of the extract obtained in step 1; Each dry extract powder of the second step has a dry weight relative blending ratio of 1: 0.01 to 100 (w/w) by weight, preferably 1: 0.5 to 50 (w/w) by weight, more preferably Preferably, 1: 0.2 to 20 (w/w) parts by weight, even more preferably 1 to 15:1 (w/w) parts by weight, even more preferably 1 to 10:1 ( w/w), most preferably 1 to 5:1 (w/w), so that the combined extract of the present invention can be obtained through the third step process.

As a result of performing animal experiments on db/db mice, which are type 2 diabetic mice, the combined extract of the present invention was significantly increased in body weight in type 2 diabetic mice compared to the normal group, insulin and honeybush In the group administered with the combination extract, there was no significant difference in body weight with the diabetic group. However, it was confirmed that the weight of fat was significantly reduced compared to the type 2 diabetic mouse, and thus it was confirmed that the fat reduction effect was excellent. Meanwhile, blood glucose and glycated hemoglobin (HbA1c) levels were sharply increased in type 2 diabetic mice, and decreased in the group administered with the insulin and honeybush combination extract. It was confirmed that the efficacy was remarkable in the combination extract. Together with this, it was confirmed that the total cholesterol, low molecular cholesterol (LDL-cholesterol) and triglyceride levels increased by type 2 diabetes were reduced by the administration of the insulin and honeybush combination extract. , it was confirmed that the combination extract of insulin and honeybush had the best efficacy. The present invention confirmed that the insulin and honeybush combination extract exhibits unexpected and excellent antidiabetic effects that cannot be predicted by those skilled in the art in terms of low molecular cholesterol inhibition and diabetes prevention and low molecular cholesterol inhibition efficacy than individual extracts.

Accordingly, the present invention provides a pharmaceutical composition and health functional food for the prevention or treatment of diabetes or diabetic complications containing a combination extract consisting of insulina (Cissus verticillata ) and honeybush (Cyclopia intermedia) obtained by the above manufacturing method as an active ingredient to provide.

The composition containing the extract of the present invention as an active ingredient contains 0.1 to 50% by weight of the extract based on the total weight of the composition.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes or diabetic complications, and health comprising a combination extract composed of insulina (Cissus verticillata ) and honeybush (Cyclopia intermedia ) obtained by the above manufacturing method and manufacturing method as an active ingredient Provides functional foods.

In addition, insulin and honeybush do not have problems such as toxicity and side effects as drugs that have been edible or used as herbal medicines for a long time.

The pharmaceutical composition of the present invention comprises 0.1 to 50% by weight of the extract based on the total weight of the composition.

The pharmaceutical composition comprising the extract of the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition containing the extract of the present invention is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, and sterile injection solutions, respectively, according to a conventional method. can be used for

Specifically, in the case of formulation, it can be prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant usually used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations include at least one excipient in the compound, for example, starch, calcium carbonate, sucrose ), lactose, and gelatin may be mixed and prepared.

In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. Non-aqueous solvents and suspending agents include vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, and glycerogelatin may be used.

The preferred dosage of the extract of the present invention varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the extract of the present invention can be administered in an amount of (0.0001-100) mg/kg, preferably (0.001-100) mg/kg divided once or several times a day. In the composition, the extract of the present invention may be formulated in an amount of (0.0001-50)% by weight based on the total weight of the total composition.

The pharmaceutical composition of the present invention may be administered to mammals such as mice, mice, livestock, and humans by various routes. All modes of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural and intracerebroventricular injections.

In addition, the present invention provides a health functional food for preventing and improving diabetes or diabetic complications containing a combination extract consisting of insulin (Cissus verticillata ) and honeybush (Cyclopia intermedia) as an active ingredient.

"Health functional food" as defined herein means a food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6727 of the Health Functional Food Act. It refers to ingestion for the purpose of obtaining useful effects for health purposes such as regulating nutrients with respect to structure and function or physiological effects.

The health functional food of the present invention contains the extract in an amount of 0.01 to 95%, preferably 1% to 80% by weight based on the total weight of the composition.

In addition, for the purpose of preventing or improving the disease of the present invention, pharmaceutical dosage forms such as powders, granules, tablets, capsules, pills, suspensions, emulsions, and syrups, or tea bags, leached teas, health functional foods in the form of health drinks, etc. can be manufactured and processed.

In addition, the present invention provides a dietary supplement or food additive for preventing and improving diabetes or diabetic complications containing a combination extract consisting of insulina (Cissus verticillata ) and honeybush (Cyclopia intermedia) as an active ingredient.

In addition, the health functional food may additionally contain food additives, and the suitability as a "food additive" is determined according to the general rules and general test methods of the Food Additives Code approved by the Food and Drug Administration, unless otherwise specified. It is judged according to the standards and standards.

Items listed in the "Food Additives Codex", for example, chemical synthetic products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as depigmentation, licorice extract, crystalline cellulose, guar gum, L- Mixed preparations such as sodium glutamate preparations, noodles-added alkalis, preservatives, and tar dye preparations may be mentioned.

Functional foods containing the extract of the present invention include bread, rice cakes, dried fruits, and nuts. Confectionery products such as candy, chocolate, chewing gum, and jam products Ice cream products such as ice cream, ice cream, and ice cream powder Milk, low-fat milk, lactose-digested milk, processed milk, goat milk, fermented milk, buttermilk, concentrated milk, milk cream , Butter oil, natural cheese, processed cheese, milk powder, milk products such as whey processed meat products, processed eggs, meat products such as hamburgers Fish meat products such as fish cakes, ham, sausage, bacon, etc. Noodles such as raw noodles, fried noodles, luxury dried noodles, improved noodles, frozen noodles, pasta, etc. Fruit drinks, vegetable drinks, carbonated drinks, soy milk, yogurt, etc. lactic acid bacteria drinks, mixed drinks, such as soy sauce, soybean paste, red pepper paste , Chunjang, Cheonggukjang, mixed soybean paste, vinegar, sauces, tomato ketchup, curry, seasoning foods such as dressing, margarine, shortening, and pizza, but is not limited thereto.

The health functional beverage composition of the present invention is not particularly limited in other ingredients except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. Examples of the aforementioned natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); disaccharides, (eg maltose, sucrose, etc.); and conventional sugars such as polysaccharides (eg, dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have. The proportion of the natural carbohydrate is generally about (1-20) g, preferably about (5-12) g per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts. salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the extract of the present invention may be added to food or beverage for the purpose of preventing the target disease. At this time, the amount of the extract in the food or beverage may be added in an amount of 0.01 to 15% by weight of the total food weight, and the health drink composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 mL. can

The extract according to the present invention, which is added to food including beverages in the process of manufacturing the health functional food, may appropriately increase or decrease its content as necessary.

The extract according to the present invention, which is added to food including beverages in the process of manufacturing the health functional food, may appropriately increase or decrease its content as necessary.

Insulin and honeybush combination of the present invention in terms of fat weight reduction, blood sugar lowering, glycated hemoglobin reduction, and low molecular cholesterol suppression efficacy through db/db mice, which are type 2 diabetes model mice for the samples according to the present invention By confirming that the extract exerts an unexpected synergistic antidiabetic effect that cannot be predicted by those skilled in the art in terms of low molecular cholesterol suppression and antidiabetic effect than other combination extracts, it can be usefully used for the prevention or treatment of diabetes and diabetic complications.

1 is a chart measuring the change in body weight while administering a combination extract of insulin and honeybush to a normal group, db/db mouse, a type 2 diabetes genetically engineered model, and db/db mouse for 4 weeks;
Figure 2 is a table of measuring blood glucose in blood while sacrificing animals after administration of a combination extract of insulin and honeybush to normal group, db/db mouse, db/db mouse, which is a genetically engineered model of type 2 diabetes; is,
Figure 3 is a normal group, type 2 diabetes genetically engineered db / db mice, db / db mice after administration of a combination extract of insulin and honeybush for 4 weeks, the animals are sacrificed in the blood while glycated hemoglobin at the time of sacrifice in the blood. It is a chart measuring (glycated hemoglobin, HbA1c),
Figure 4 is a normal group, type 2 diabetes genetically engineered db / db mice, db / db mice after administration of insulin and honeybush combination extract for 4 weeks, hematoxylin & hematoxylin & It is a diagram dyed with eosin,
Figure 5 is a normal group, type 2 diabetes genetically engineered db / db mouse, db / db mouse insulin and honeybush combination extract was administered for 4 weeks, then the pancreas tissue was collected at the time of animal sacrifice and glucagon ( Immunostaining with an antibody against glucagon)
Figure 6 is a normal group, type 2 diabetes genetically engineered db / db mice, db / db mice after administration of the insulin and honeybush combination extract for 4 weeks, after collecting the pancreas tissue at the time of animal sacrifice, insulin ( Immunostaining with an antibody to insulin).

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the content of the present invention is not limited by the examples.

Reference Example 1: Preparation of insulin extract

Insulina ( Cissus verticillata, imported from Costa Rica) leaves and stems are collected and dried, and 42 g of dried insulin leaves and stems are ground finely with a grinder, then 1 L of 30% ethanol is added thereto and immersed at room temperature for 48 hours. After filtration, an extract was obtained.

The filtered extract was completely removed from ethanol using a vacuum rotary evaporator (N-1000, Tokyo Rikakikai, Tokyo, Japan) and concentrated, followed by a freeze dryer (DRC-100+FDU-2100, Rep Corporation) for 48 hours. During the period, moisture was completely removed to obtain an insulina extract (11.96 g) in a dry powder state.

Reference Example 2: Preparation of honeybush extract

After collecting and drying honeybush (Cyclopia intermedia , honeybush, imported from Germany) leaves, 42 g of dried honeybush leaves are finely ground with a grinder, 1 L of 30% ethanol is added thereto, and immersed at room temperature for 48 hours. Filtration gave an extract.

The filtered extract was completely removed from ethanol using a vacuum rotary evaporator (N-1000, Tokyo Rikakikai, Tokyo, Japan) and concentrated, followed by a freeze dryer (DRC-100+FDU-2100, Rep Corporation) for 48 hours. During the period, moisture was completely removed to obtain a honeybush extract (10.85 g) in a dry powder state.

Example 1: Preparation of mixed extracts of various combination ratios

1-1. Mixed Extracts (0.3:1)

Extracted in the same manner as above, except that the insulin extract and honeybush extract are 0.3:1 (insulin extract: honeybush extract) on a dry weight basis, and the individual extracts are completely mixed in the above mixing ratio and mixed. An extract (hereinafter referred to as “CB1”) was prepared.

1-2. Mixed Extracts (1:1)

Extracted in the same manner as in the above preparation method except that the insulin extract and honeybush extract are 1:1 (insulin extract: honeybush extract) by weight, and the individual extracts are completely mixed in the above mixing ratio to obtain a mixed extract (hereinafter referred to as “CB2”) was prepared.

1-3. Mixed Extracts (3:1)

Extracted in the same manner as in the above preparation method except that the insulin extract and honeybush extract are 3:1 by weight (insulin extract: honeybush extract), and the individual extracts are completely mixed in the above mixing ratio to obtain a mixed extract (hereinafter referred to as “CB3”) was prepared.

1-4. Mixed Extracts (10:1)

Extracted in the same manner as in the above manufacturing method, except that the insulin extract and honeybush extract are 10:1 by weight (insulin extract: honeybush extract), and the individual extracts are completely mixed in the above mixing ratio to obtain a mixed extract (hereinafter referred to as “CB4”) was prepared.

Experimental Example 1. Blood sugar and metabolism-related lowering effect of insulin extract and insulin and honeybush combination extract in type 2 diabetes model mice

In order to confirm the hypoglycemic, glycated hemoglobin and metabolic-related lowering effects of the insulin extract and the insulin and honeybush combination extract of the above Example in diabetic model mice, the experiment was performed in accordance with the method described in the existing literature as follows (Hwang IK et al., Effects of grape seed extract and its ethylacetate/ethanol fraction on blood glucose levels in a model of type 2 diabetes. Phytother. Res., 23, pp1182-1185, 2009).

1-1. Method of breeding and administration of laboratory animals

As experimental animals, 30 male db/db mice weighing around 40 g (Jackson Laboratories, Maine, USA) and 6 male C57BL/KsJ mice weighing around 27 g were used as negative controls. Experimental animals were bred at a temperature of 22 ± 2 °C and a relative humidity of 55 ± 10% under a constant light-dark cycle from 7 am to 7 pm, and food and water were freely ingested.

1-2. Weight, blood sugar and metabolism-related lowering effect experiments

In order to confirm the blood sugar and weight-lowering effect of the insulin extract and honeybush combination extract of Example 1, the weight of the animal was measured at 10 am at the same time as the start of the experiment, and the tail was cut off to obtain blood and then an automatic blood glucose meter ( Blood glucose was measured using ACCU-CHEK, Roche, Mannheim, Germany). No substance was administered to the negative control group and 6 db/db mice, and the insulin and honeybush combination extract (60 mg/kg) of Example 1 was administered to 30 db/db mice using an esophageal needle. Orally administered. After administering the combination extract of insulin and honeybush for 4 weeks, alfaxalone (Alfaxan, 75 mg/kg Careside, Seongnam, South Korea) and xylazine (10 mg/kg; Bayer Korea, Seoul, South Korea) were administered. Animals were euthanized, weighed, and blood was drawn.

The results are shown in Table 1 and FIG. 1, and as shown in FIG. 1, it was confirmed that the continuous increase in body weight was observed in all animals with increasing age, and in the case of the negative control group, the weight was significantly lower than that of diabetic mice. On the other hand, in the case of the group administered with the insulin extract and honeybush combination extract of Example 1, the weight was observed less than that of the type 2 diabetic mice that were not, but no significant difference could be confirmed.

After sacrificing the animal 4 weeks after administration, the body fat was collected and confirmed. As a result, a significantly higher fat weight was observed in diabetic animals than in the control group. It was observed that little was observed, and it was confirmed that there was an unexpected synergistic body fat reduction effect that could not be predicted by those skilled in the art, especially in the 3:1 combination (Table 1).

Experimental results on the effect of fat weight in type 2 diabetes model mice division fat weight (g) Inhibition rate (%) normal group 1.594±0.1913 - Type 2 diabetes group (db/db) 2.472±0.1696 - Example 1-1 (0.3:1 combination agent) 2.343±0.2441 5.207 Example 1-2 (1:1 combination agent) 2.139±0.1486 13.467 Example 1-3 (3:1 combination agent) 1.997±0.1170 19.185 Examples 1-4 (10:1 combination agent) 2.227±0.1711 9.906 *Inhibition rate (%) is based on type 2 diabetes group (db/db).

As shown in FIG. 2 , in the case of the type 2 diabetes group (db/db), it was confirmed that blood sugar was observed at a 95% confidence interval compared to the normal group, and in the case of diabetic animals, the blood sugar decreased with the lapse of weeks. It was observed that there was a sharp increase. In the group administered with insulin (Cissus verticillata ) and honeybush (Cyclopia intermedia ), it was confirmed that blood sugar decreased at the 3rd week of administration of the extract, and in particular, the significant decrease continued in the group administered with the 3:1 combination.

Also, as shown in FIG. 3 , the glycated hemoglobin (HbA1c) level measured by collecting blood at the time of sacrificing the animal was significantly higher in the type 2 diabetic group (db/db) than in the normal group. In the case of the insulin and honeybush combination extract administration group, the HbA1c level was lower than that of the type 2 diabetes group (db/db), and in particular, a significant decrease was confirmed in the group administered with the 3:1 combination extract. there was.

It was confirmed that the level of total cholesterol measured in the serum was significantly increased in the type 2 diabetes group compared to the control group. Meanwhile, in the case of the insulin and honeybush combination extract administration group, it was confirmed that the total blood cholesterol level was significantly reduced compared to the type 2 diabetes group (db/db). Similarly, it was confirmed that the levels of low molecular cholesterol (LDL-cholesterol) and triglyceride were significantly higher in the diabetic group than in the control group. It was confirmed that the triglyceride level was rapidly decreased, and in particular, excellent inhibitory activity was confirmed in terms of low molecular cholesterol level. The reduction of these metabolic-related indicators was particularly highest in the group administered with the 3:1 insulin and honeybush combination extract, confirming the inhibitory activity of diabetes and diabetic complications (Table 2).

Effect of blood total cholesterol level, high molecular cholesterol level, low molecular cholesterol level and triglyceride level division Metabolism related indicators (unit: mM) total cholesterol in the blood high molecular cholesterol low molecular cholesterol triglycerides normal group 0.9303±0.2889 0.7162±0.1484 0.1012±0.0197 0.2403±0.0615 Type 2 diabetes group (db/db) 1.7865±0.3138 0.7523±0.2646 0.3326±0.0801 0.6256±0.1782 Example 1-1 (0.3:1 combination agent) 1.6204±0.2311 0.7636±0.1190 0.3064±0.0682 0.5751±0.1257 Example 1-2 (1:1 combination agent) 1.2767±0.1994 0.7973±0.1862 0.1964±0.0495 0.4914±0.1383 Examples 1-3 (3:1 combination agent) 1.0614±0.2978 0.8184±0.1818 0.1705±0.0346 0.4190±0.0831 Examples 1-4 (10:1 combination agent) 1.4284±0.2873 0.7508±0.1733 0.3014±0.0734 0.5136±0.0920 *

1-3. Liver and pancreatic morphology change confirmation experiment

After euthanasia for the animals of Experimental Example 1, livers and pancreas were collected from the animals, and tissues were fixed with 10% neutral buffered formalin for 24 hours. After the usual dehydration and clarification process, paraffin tissue embedding was performed. The embedded tissue was used in experiments by making 3-㎛ sections and attaching them to silane-coated slides (Muto-Glass, Muto Pure Chemicals, Tokyo, Japan). In order to observe the general shape, hematoxylin & eosin (HE) staining was performed by a conventional method, and immunohistochemical staining for glucagon and insulin was performed to confirm the tissue distribution of glucagon and insulin in the pancreas.

As a result of performing HE staining on liver tissue as shown in FIG. 4 , almost no fat was observed in the cytoplasm of hepatocytes in the normal group, and a pattern of uniformly staining in the cytoplasm was confirmed. On the other hand, in the case of the type 2 diabetes group (db/db), it was confirmed that the staining property was somewhat decreased due to the accumulation of fat in the cytoplasm of the hepatocytes. However, compared to the diabetic group that was not administered, the accumulation of fat in the liver was somewhat less, and it was confirmed that the cytoplasmic staining property was higher.

As a result of confirming the immunostaining for glucagon and insulin in the pancreatic tissue as shown in FIGS. 5 and 6, cells stained with glucagon in the control group were observed in the periphery of the pancreatic islet, Insulin-stained cells were identified throughout the islets. In the case of diabetic mice, cells containing glucagon were observed in the form of spreading widely in the islets, and cells containing insulin were observed sparsely compared to the control group. On the other hand, in the case of diabetic mice administered with the insulin and honeybush combination extract, glucagon-stained cells were only partially observed in the marginal area as in other groups, and insulin-stained cells were strongly observed throughout the pancreatic islets, and the shape of the pancreatic islets was different. In the 3:1 combination extract of insulinina and honeybush, it was observed to have a round shape, and it was confirmed that the insulin staining property was also strong.

As described above, in terms of the efficacy of reducing fat weight, lowering blood sugar, lowering glycated hemoglobin and inhibiting low molecular weight cholesterol through db/db mice, which are type 2 diabetes model mice, the combination extract of the present invention showed excellent efficacy, and it was confirmed that it exerts unexpected synergistic anti-obesity and anti-diabetic effects that cannot be predicted by those skilled in the art in terms of such anti-diabetic efficacy.

An example of the formulation of the composition containing the extract of the present invention will be described, but the present invention is not intended to limit it, but to describe it in detail.

Formulation Example 1. Preparation of powder

CB1 Extract ------------------------------------------ 20 mg

Lactose ----------------------------------------------- 100 mg

talc -------------------------------------------------------------- 10 mg

The above ingredients are mixed and filled in an airtight bag to prepare a powder.

Formulation Example 2. Preparation of tablets

CB2 Extract ----------------------------------------- 10 mg

Corn Starch --------------------------------------------------- 100 mg

Lactose ----------------------------------------------- 100 mg

Magnesium Stearate ---------------------------------- 2 mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional manufacturing method of tablets.

Formulation Example 3. Preparation of capsules

CB3 Extract ------------------------------------------ 10 mg

crystalline cellulose ------------------------------------- 3 mg

Lactose ----------------------------------------------------- 14.8 mg

Magnesium Stearate ------------------------------- 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled in a gelatin capsule to prepare a capsule.

Formulation Example 4. Preparation of injection

CB4 Extract -------------------------------------- 10 mg

mannitol -------------------------------------------- 180 mg

Sterile distilled water for injection ------------------------------ 2974 mg

Na ₂ HPO ₄ 12H ₂ O ------------------------------------------------- 26 mg

According to a conventional method for preparing injections, it is prepared with the content of (2) above ingredients per 1 ampoule.

Formulation Example 5. Preparation of liquid formulation

CB1 Extract ----------------------------------------- 10 mg

Isomerized sugar --------------------------------------------- 10 g

Mannitol -------------------------------------------------------------- 5 g

Purified water ----------------------------------------------- Appropriate amount

According to a conventional method for preparing a liquid, each component is added to purified water to dissolve, an appropriate amount of lemon flavor is added, the above components are mixed, and purified water is added to adjust the total to 100, then fill a brown bottle and sterilize to prepare a liquid. do.

Formulation Example 6. Preparation of health food

CB2 Extract ------------------------------------------------- 1000 mg

Vitamin mixture ----------------------------------- Appropriate amount

Vitamin A Acetate -------------------------------- 70 μg

Vitamin E ---------------------------------------------------- 1.0 mg

Vitamin B1 -------------------------------------------------- 0.13 mg

Vitamin B2 ---------------------------------------- 0.15 mg

Vitamin B6 ----------------------------------------- 0.5 mg

Vitamin B12 ---------------------------------------- 0.2 μg

Vitamin C ------------------------------------------------------ 10 mg

Biotin --------------------------------------------- 10 μg

Nicotinamide ------------------------------------ 1.7 mg

Folic acid ----------------------------------------------- 50 μg

Calcium Pantothenate ------------------------------------- 0.5 mg

Mineral mixture ----------------------------------- Appropriate amount

ferrous sulfate ---------------------------------------- 1.75 mg

Zinc Oxide --------------------------------------------------- 0.82 mg

Magnesium carbonate ------------------------------------- 25.3 mg

Potassium Phosphate ---------------------------------------- 15 mg

Dicalcium Phosphate ------------------------------------ 55 mg

Potassium Citrate ----------------------------------------- 90 mg

Calcium carbonate ---------------------------------------------------- 100 mg

Magnesium Chloride ----------------------------------------------- 24.8 mg

The composition ratio of the vitamin and mineral mixture is a composition that is relatively suitable for health food in a preferred embodiment, but the mixing ratio may be arbitrarily modified. , to prepare granules, and can be used for preparing health food compositions according to a conventional method.

Formulation Example 7. Preparation of a health drink

CB2 Extract ------------------------------------------------- 1000 mg

citric acid ---------------------------------------------------- 1000 mg

Oligosaccharide ------------------------------------------ 100 g

Plum Concentrate ------------------------------------------ 2 g

Taurine ---------------------------------------------- 1 g

Add purified water ----------------------------- Total 900 mL

After mixing the above ingredients according to a conventional health drink manufacturing method, after stirring and heating at 85° C. for about 1 hour, the resulting solution is filtered and obtained in a sterilized container, sealed and sterilized, and then refrigerated. It is used in the manufacture of health beverage compositions.

Claims (7)

Insulin ( Cissus verticillata ) Leaf or stem and honeybush ( Cyclopia intermedia ) Dry weight relative mixing ratio of leaves 1: 0.2 to 20 (w / w) of water and ethanol mixed solvent of a combination composed of parts by weight of an extract soluble in a solvent A pharmaceutical composition for preventing or treating diabetes or diabetic complications, including as an active ingredient. delete delete The method of claim 1,
The diabetes is a pharmaceutical composition, characterized in that type 1 diabetes or type 2 diabetes. Insulin ( Cissus verticillata ) Leaf or stem and honeybush ( Cyclopia intermedia ) Dry weight relative mixing ratio of leaves 1: 0.2 to 20 (w / w) of water and ethanol mixed solvent of a combination composed of parts by weight of an extract soluble in a solvent A health functional food for preventing or improving diabetes or diabetic complications, including as an active ingredient. delete Insulin ( Cissus verticillata ) Leaf or stem and honeybush ( Cyclopia intermedia ) Dry weight relative mixing ratio of leaves 1: 0.2 to 20 (w / w) of water and ethanol mixed solvent of a combination composed of parts by weight of an extract soluble in a solvent A dietary supplement for preventing or improving diabetes or diabetic complications, including as an active ingredient.

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