KR20070108248A - Plant extracts and method and uses therefore - Google Patents

Abstract

The invention relates to compositions such as pharmaceutical compositions and comestibles comprising extracts of Terminalia arjuna and methods for preventing or treating diabetes, obesity, cardiovascular disease, cancer and related disease conditions in mammals, such as humans, and uses therefore.

Description

PLANT EXTRACTS AND METHOD AND USES THEREFORE}

The present invention relates to extracts from the Terminalia plant species which can be used in methods for managing diseases and related diseases such as diabetes, obesity, cardiovascular disease and cancer. In particular, the present invention provides a method for extracting the extract from the terminalia arjuna , their use as a medicament for the treatment of diseases such as diabetes, obesity, cardiovascular disease and cancer in mammals, in particular humans, methods of preparing these agents And to a delivery format thereof.

diabetes

Diabetes is a common and serious disease characterized by hyperglycemia. The disease can be classified into two main classes. That is, it can be classified into insulin dependent diabetes mellitus (IDDM) as known as type 1 diabetes and insulin independent diabetes mellitus (NIDDM) as known as type 2 diabetes (World Health Organization Study Group. Diabetes mellitus. WHO Tech. Rep. Ser. 727: 1-113. 1985). IDDM is caused by insulin deficiency caused by cell-mediated autoimmune destruction of pancreatic beta cells and is usually present in children (Yoon J W., Insulin-dependent diabetes mellitus.In: Roitt IM and Delves P J. (Eds.) Encyclopedia of Immunology, Second Edition.Academic Press Ltd., London, pp. 1390-1398, 1998; Bach J F., Insulin-dependent diabetes mellitus as a beta cell targeted disease of immunoregulation.J. Autoimm. 8: 439-463, 1995). IDDM accounts for about 10-15% of the world's diabetic population (World Health Organization Study Group. Diabetes mellitus. WHO Tech. Rep. Ser. 727: 1-113. 1985). In contrast, NIDDM is caused by various combinations of insulin resistance and insulin deficiency and is usually present in adults (Jun HS, et al., Pathogenesis of non-insulin-dependent (Type II) diabetes mellitus (NIDDM)-Genetic predisposition and metabolic abnormalities.Advanced Drug Delivery Reviews 35: 157-177, 1999; DeFronzo R A., The triumvirate: .beta.-cell, muscle, liver: a collusion responsible for NIDDM.Diabetes 37: 667-687, 1988). However, it can also occur at a young age, as seen in childhood adult developmental diabetes (Pirart J., Diabetes mellitus and its degenerative complications: a prospective study of 4400 patients observed between 1947 and 1973. Diabetes Care 1: 168-188, 1978 ). NIDDM accounts for over 85% of the world's diabetic population. Both IDDM and NIDDM can cause microvascular and capillary complications, resulting in increased morbidity and mortality (Fajans SS, et al., Prediabetes, subclinical diabetes, and latent clinical diabetes: interpretation, diagnosis and treatment. In: Leibel DS, Wrenshall G S. (Eds.) On the Nature and Treatment of Diabetes.Excerpta Medica, Amsterdam, pp. 641-656, 1965).

NIDDM is a complex disease that is considered to be affected by more single genes or environmental factors (Ghosh S, et al., Genetic analysis of NIDDM. Diabetes 45: 1-14. 1995; Kobberling J. Studies on the genetic heterogeneity of diabetes mellitus.Diabetologia 7: 46-49, 1971; Rotter JL, et al., Genetics of diabetes mellitus.In: Rifkin H, Porte D (Eds.) Diabetes Mellitus Theory and Practice.Elsevier, NY, pp. 378- 413, 1990). Family integration and high agreement (60-100%) of disease in identical twins suggest that genetic factors play an important role in the development of NIDDM (O'Rahilly S, et al., Type 2 (noninsulin dependent). diabets mellitus.New genetics for old nightmares.Diabetologia 31: 407-414, 1988; Barnett AH, et al., Diabetes in identical twins.A study of 200 pairs.Diabetologia 20: 87-93, 1981). In addition, environmental factors such as obesity, physical activity and diet play a very important role in the development of the disease (Knowler WC, et al., Gm and type 2 diabetes mellitus: an association in American Indians with genetic admixture. Hum. Genet. 43: 520-526, 1988; Bennett PH, et al., Epidemiology and natural history of NIDDM: non-obese and obese.In: Alberti KGMM, DeFromzo RA, Keen H, Zimmett P (Eds.) International Textbook of Diabetes Mellitus.Wieley, NY, pp. 147-176, 1992; Helmrich SP, et al., Physical activity and reduced occurrence of NIDDM.N. Engl. J. Med 325: 147-152, 1991). The relative contributions of genetic and environmental factors to the development of NIDDM may vary from person to person, but in general, patients have two common metabolic abnormalities: deficiency in insulin and glucose-stimulated insulin secretion leading to disease states. Saad MF et al., A two step model for development of non-insulin-dependent diabetes.Am J. Med. 90: 229-235, 1991; DeFronzo RA, et al., Pathogenesis of NIDDM: A balanced overview. Care 15: 318-368, 1992; Lillioja S, et al., Insulin resistance and insulin secretory dysfunction as precursors of non-insulin-dependent diabetes mellitus.N. Engl. J. Med. 329: 1988-1992, 1993).

Insensitivity of target tissues to insulin appears to first appear in genetic predisposition in the presence of essential environmental factors (Jun Hs, et al., Pathogenesis of non-insulin-dependent (Type II) diabetes mellitus (NIDDM)-). -Genetic predisposition and metabolic abnormalities.Advenced Drug Delivery reviews 35: 157-177, 1999). To compensate for this, that is, to lower blood sugar and maintain normal blood sugar, insulin secretion from beta cells is increased to result in hyperinsulinemia. Over time, insulin resistance deteriorates, compensatory action weakens, and eventually impairs glucose tolerance. Insulin secretion reaches a plateau, and beta cell function deteriorates, leading to hyperglycemic NIDDM. In addition, hyperglycemia itself induces impaired insulin resistance and insulin secretion, exacerbating the disease.

Diet control and exercise and / or treatment with insulin or hypoglycemic agents have been used for the control of diabetes. Treatment with these agents has been successful in some cases, but mortality continues to increase. Insulin therapy is intended to alleviate symptoms rather than to treat NIDDM. Hypoglycemic agents, such as sulfonylureas and biguanides (metformin), can lower blood sugar levels, but can also ease symptoms. Sulfonylurea lowers blood sugar levels by stimulating the release of insulin from pancreatic beta cells. These agents directly stimulate insulin secretion by closing adenosyl triphosphate (ATP) -sensitive potassium channels and depolarizing cell membranes (Aguilar-Bryan, et al., Cloning of the bete cell high-affinity sulfonylures receptor: a regulator of Insulin secretion.Science 268: 423-426. 1995; Tan GH, et al., Pharmacologic treatment option for non-insulin-dependent diabetes mellitus.Mao Clinic Proceedings 71: 763-768, 1996; Lubbos H, et al., Oral hypoglycemic agents in type II diabetes mellitus.American Famiy Physician. 52: 2075-2078, 1995). Side effects of sulfonylureas include hypoglycemia, kidney and liver diseases, gastrointestinal disorders, cardiovascular necrosis, skin reactions, dizziness, drowsiness and headache. Biguanides do not lower insulin levels by stimulating insulin secretion, but by lowering glucose intake and hepatic glucose. The major side effects of biguanides are lactic acidosis and cardiovascular necrosis. Alpha glucosidase inhibitors inhibit intestinal alpha glucosidase and delay the digestion of sucrose and complex carbohydrates. Side effects of alpha glucosidase inhibitors are side effects of gastrointestinal disorders and hypoglycemia. Chiazolidinedione directly improves insulin resistance, thereby improving insulin circulating effects, directly stimulating peripheral glucose uptake, and inhibiting glucose production in the liver. Chiazolidinedione is effective only in the presence of insulin and can cause red blood cell abnormalities and headaches.

Thus, there is a clear need for more effective agents for the treatment of diabetes.

obesity

In general, specialists recognize that the obesity population has grown steadily in the United States and in some countries since recent decades.

Obesity is a phenomenon caused by excess accumulation of fat in the body. Obesity can be measured by weight or body mass index (BMI). By convention, obesity is when the body weight exceeds 20% of the body weight indicated on a typical kidney-weight index. Another measure of obesity, BMI, is the amount of fat in the body and a reliable indicator of obesity in adults, not athletes. BMI can be calculated using the following equation: BMI = weight kg / (m tall) ² . In general, normal BMI is between 20 and 25, while obese people have a BMI of 30 or more.

This obesity causes or contributes to a significant onset of heart disease, hypertension, diabetes, arthritis of the knee and hip joints, and causes an increase in mortality from these symptoms. Fifty percent of American adults are reported to be overweight.

Various methods are used for the treatment of obesity. These methods include diet such as caloric restriction, exercise therapy, drug therapy, surgery and psychotherapy. However, there are problems associated with these methods.

The use of human growth hormone (hGH) for the treatment of obesity is described in US Pat. No. 5,597,595. HGH, the most abundant hormone secreted by the pituitary gland, is the primary hormone important for mammalian growth and promotes lipolysis (fat metabolism) and protein synthesis. In obese people, hGH secretion is significantly reduced. hGH interacts with specific growth hormone receptors that are widely distributed in the body. For example, adipocytes have hGH receptors and when hGH binds to these receptors, they inhibit their ability to stimulate adipocytes, destroy triglycerides, and absorb and accumulate circulating lipids. However, the administration of hGH needs to be carefully managed because diabetes can be invented in people receiving hGH treatment. hGH can be a cause of diabetes by decreasing the amount of sugar absorbed by the cells to increase blood glucose levels. This increase in blood glucose stimulates the beta cells of the pancreas to allow excess insulin to be secreted, as well as hGH directly stimulates beta cells that produce insulin. The combination of these two stimulating effects greatly stimulates insulin secretion, causing the beta cells to be 'burned out'. Diabetes can develop when this happens. Guyton and Hall; See TEXTBOOK OF MEDICAL PHYSIOLOGY, 938 (1995).

One of the best ways to induce weight loss is diet or calorie restriction, i.e. calorie intake is less than the amount of divergence, so that weight loss is achieved through calorie restriction that causes calorie deficiency. The most common way to reduce calorie intake is by a low calorie diet. However, there are generally two pitfalls in diet. One is that the need for overeating multiplies when the person who implements the diet stops the diet and returns to the daily regimen. Second, many obese people choose novel or unusual diets. One example of this type of diet is a high fat / high protein diet. These diets are known as ketone diets because they envelop high levels of cholesterol and encourage ketoneosis. Ketonosis is associated with vomiting, hypotension and lethargy. See Kaplan et al. 735, above.

Drug therapy involves the administration of drugs that increase the level of serotonin produced in the brain, thereby controlling appetite. Serotonin (5-hydroxytryptamine, 5HT) is involved in many common behaviors such as sleep, emotions, libido and appetite. The drug Redux.RTM. (Dexfenfluramine), commonly used as a weight control agent, exerts its function by increasing serotonin levels. In particular, Redux.RTM. Inhibits serotonin reuptake and simultaneously stimulates serotonin secretion. Several clinical trials have demonstrated that Redux.RTM. Will help maintain calorie restriction and lower body weight for at least one year. It should also be noted that Redux.RTM. Reduces appetite for non-obese people. See Katzung, BASIC & CLINICAL PHARMACOLOGY 276 (1998). However, Redux.RTM. Has side effects such as headache, insomnia and pulmonary hypertension.

Surgical procedures to reduce food absorption or reduce gastric capacity have been widely used in obese people. Gastrointestinal fusion is a method of reducing the size of the stomach. In particular, the stomach is incised to partly shrink. The main purpose of reducing the size of the stomach is to limit the amount of food a person consumes by allowing food to pass slowly. Although the results were successful, there are many side effects. For example, disorders such as vomiting and electrolyte imbalance can occur. Another surgical procedure is liposuction to remove fat. However, this technique is mainly used for cosmetic purposes, and there is no substantial effect of weight loss over an organ. See Kaplan et al., 276 above.

Thus, for all the reasons mentioned above, there is a need for a best method that is readily available and cost effective for weight loss of obese or overweight people.

Cardiovascular disease

Cardiovascular disease (CVD) is the leading cause of death, accounting for one third of all global deaths. Nearly 85% of deaths and diseases on Earth due to CVD are found in low and middle income countries. For example, in India, about 53% of CVD deaths are under 70 and 35% are equivalent in China. Most of the 32 million heart attacks and heart attacks that occur each year are caused by one or more cardiovascular risk factors: hypertension, diabetes, smoking, high levels of blood lipids and physical inactivity, and most of these CVD factors contribute to these risks. This can be avoided by carrying out specific material actions that address the factors.

Many people will experience cardiovascular disorders sometime in their lifetimes. Nearly one million Americans die each year from these cardiovascular diseases, while 556,000 people die from cancer. Despite this fact, people fear cancer more than vascular diseases.

Americans have become indifferent to the risk of arterial disease. One reason is that in the last 50 years, the mortality rate of acute heart attacks in young people has dropped dramatically. This reduction in mortality can be explained by lifestyle changes, the use of more dietary supplements / preventive drugs, and improved heart health care.

The question is why so many Americans die from heart attacks and heart attacks. The basic answer is that people live longer. Many people have succeeded in delaying the development of arterial disease. As such, there is no symptomatic vascular symptom from the age of 50 to 60 or 80 years of age instead of a sudden death due to a heart attack. At this point, aortic sclerosis has compromised key mechanisms, and many degenerative diseases have resulted in reduced quality and quantity of life.

The basic causes of many arterial diseases have been identified in scientific papers. However, cardiologists deal with only a limited number of these factors, such as the prescription of cholesterol lowering drugs and the control of hypertension. By ignoring other proven causes of the prevalence of vascular disease, many Americans suffer from useless pain and die early.

Terminalia Arjuna  Plant extracts

Terminalia arjuna is a deciduous tree that can be found throughout India and grows to about 60 to 90 feet. Terminalia Arjuna belongs to the Combretaceae and is named "Arjuna". In India, Terminalia Arjuna has been used as a cardiovascular drug for more than 1500 years and is based on Ayurveda medicine, all three humoursin, namely vata, pitta and kapha. It has been used to treat confusion of constitution. The bark of Terminalia Arjuna is widely used as a versatile medicine in India.

U. S. Patent No. 6,162, 438 discloses a terminalia arjuna , thistle ( Cynara ) for use as a medicament for the control of mammalian hypertension, hypercholesterolemia and hyperlipidemia. Scolymus ), ginger ( Zingiber officinale ), garlic ( Allium sativum ), wild lion ( Crataegus) Oxycantha , Curcuma longa ), Pork ( Boerhaavia A edible herb composition is described which is a mixture of at least three, preferably at least six, herbs selected from the group consisting of diffusa ) and fenugreek ( Trigonella foenumgraecum ).

Sharma VN et al. Evaluated the antioxidant and hypocholesterolemia effects of the bark of the Terminia Arjuna tree (which is a folk cardiac drug in India) and compared it with known antioxidant vitamin E in a randomized clinical trial. From these experiments, it was concluded that the bark powder of the termini arjuna tree has a significant antioxidant activity compared to vitamin E. In addition, it has a significant hypocholesterolemia effect (Antioxidant and hypocholesterolaemic effects of Terminalia arjuna tree-bark powder: a randomised placebo-controlled trials, J Assoc Physician India 2002 Feb; 49: 231-235).

A bioactive tracking separation method was used by Chapuis et al. To determine the growth inhibitory components of cancer cells in the bark, stem and leaves of Termini Arjuna (Combretoaceae), a medicinal plant from Mauritius. The cancer cell-based active ingredients were found to be gallic acid, ethyl gallate and flavone luteolin. Only gallic acid is already known to be included in these plants. It is well known that luteolin inhibits several cancer cell systems, and this could be the most rationale for using Terminalia Arjuna in traditional cancer therapy. Luteolin has also been shown to have special activity against the pathogenic bacterium Neisseria gonorrhoeae (Antineoplastic agents 338. The cancer cell growth inhibitory constituents of Terminalia arjuna (Combretaceae). J Ethnopharmacol. 1996 Aug; 53 (2): 57-63).

 The bark of the Terminia Arjuna tree has a long history of cardiovascular use and has been used to treat coronary heart disease, heart failure and hypercholesterolemia and to relieve angina pain (Miller, AL Botanical Influences on cardiovascular disease.Alternative Medicine Review. Dec 1998, vol 3. No. 6, pages 421-431.

 Water and methanol extracts of the bark of Terminalia Arjuna were found to inhibit HIV-1 activity by more than 70% at 0.2 mg / ml (Kandil FE et al., A Tannin anti-cancer promoter from Terminalia arjuna. April 1998, Vol 47. No. 8, pages 1567-1568).

Candidate therapy of the Terminalia Arjuna bark powder for patients with myocardial infarction improved the left ventricular blood count (LVEF) and left ventricular mass (LVM) with relief of angina (Dwivedi, S. et al., Beneficial effects of Terminalia arjuna). in coronary Artery Disease.Indian Heart Journal.Spet-Oct 1997, vol. 49. No. 5, pages 507-510).

Total cholesterol and LDL cholesterol levels were markedly reduced when the ethanol extracts of the termini arjuna bark were administered at doses of 100 mg / kg and 500 mg / kg in hypercholesterolemia rabbits (Ram et al., Hypocholesterolaemic effects of Terminalia arjuna tree bark.Journal of Ethnopharmacology.Vol 55. No. 3. pages 165-169).

In the prior art, there is no data or technical information showing the use of the termini arjuna extract in the treatment or prevention or management of diabetes and obesity.

It has been reported that the water and methanol extracts of the termini arjuna stems appear to have HIV-1 activity. However, there are no other references showing the use of Terminalia Arjuna bark extract with a continuous extraction solvent system for the treatment of cancer.

The Terminalia Arjuna plant is known for its cardiovascular effect. There is also a report showing the use of Terminalia Arjuna for the treatment of coronary artery disease, heart failure, hypercholesterolemia and alleviation of angina pain. However, there is no clear evaluation of the mechanism of action for these activities. However, in the present invention, we demonstrated that the cells treated with the Terminalia Arjuna extract showed a significant decrease in total lipid content. The extract showed 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitory activity. HMG-CoA is the first dedicated step in the cholesterol biosynthesis pathway. At the rate of cholesterol biosynthesis, HMG-CoA reductase has become the most successful drug for lowering total plasma cholesterol, especially LDL-cholesterol.

There is a need to develop new drugs effective for the treatment of obesity, cardiovascular disease, some cancers, diabetes and related diseases.

The object of the present invention will become apparent from the following description and examples.

The present invention relates to extracts of plant parts from the terminus plant species, such as the terminus arjuna, which are useful for the treatment of diseases such as obesity, cardiovascular disease, some cancers, diabetes and related diseases. Furthermore, the present invention relates to a prophylactic composition consisting of extracts of the terminus plant species, such as extracts from the terminus arjuna plant species.

Brief description of extract nomenclature:

Nomenclature of plant extracts.

1. AV -The first two letters represent Avesthagen .

2. Plant Name: The used or used plant is designated by three digits. 016 represents the Terminalia Arjuna .

3. Plant Part / Organization: The two letter ID represents the part of each plant used. For example, Ba stands for Bark and Fr stands for whole. Fruit ), ie whole fruit.

4. Solvent: The solvent used for extraction was specified in two digits. 01 is acetone , 02 is benzene , 03 is chloroform , 04 is ethanol , 05 is hexane , 06 is methanol , 07 is petroleum ether , 08 is water and 09 is ethyl acetate . The proportion of solvent used for extraction is shown in parentheses . For example, (20) represents 20% of the solvent. If 20% ethanol is used for extraction, label it 04 (20).

5. Extraction Method: Continuous extraction (successive extraction) is referred to Su, and was referred to a direct extraction is Di (direct extraction), the extraction temperature was shown in parentheses. For example, Su 65 represents continuous extraction at 65 ° C.

6. Extract form, g: gluey, ng: non-gluey

Short description of the table

Table 1: HPLC fingerprint of extract AV016BaDi (65) 04 (100).

Table 2: HPLC fingerprint of extract AV016BaDi (28) 04 (20).

Table 3: HPLC fingerprint of extract AV016BaSu (65) 09 (100).

Table 4: HPLC fingerprint of extract AV016BaSu (65) 01 (100).

Table 5: HPLC fingerprint of extract AV016BaSu (65) 01 (100) ng.

Table 6: HPLC fingerprint of extract AV016BaSu (65) 01 (100) g.

Table 7: HPLC fingerprint of extract AV016BaSu (65) 04 (100).

Table 8: HPLC fingerprint of extract AV016BaSu (65) 06 (100).

Table 9: HPLC fingerprint of extract AV016BaSu (105) 08 (100).

Table 10: HPLC fingerprint of extract AV016Fr (105) 08 (100).

Table 11: LC / MS fingerprint of the extract AV016BaDi (28) 04 (20) (TIC spectrum (Q1 + ve mode).

Table 12: LC / MS fingerprint (TIC spectrum (Q1-ve mode) of extract AV016BaDi (28) 04 (20).

Table 13: LC / MS fingerprint of extract AV016BaDi (65) 04 (100) (TIC spectrum (Q1 + ve mode).

Table 14: LC / MS fingerprint of extract AV016BaDi (65) 04 (100) (TIC spectrum (Q1-ve mode).

Table 15: LC / MS fingerprint of extract AV016BaSu (65) 09 (100) (TIC spectrum (Q1 + ve mode).

Table 16: LC / MS fingerprint of extract AV016BaSu (65) 01 (100) (TIC spectrum (Q1 + ve mode).

Table 17: LC / MS fingerprint (TIC spectrum (Q1-ve mode) of extract AV016BaSu (65) 01 (100).

Table 18: LC / MS fingerprint of extract AV016BaSu (65) 01 (100) ng (TIC spectrum (Q1-ve mode).

Table 19: LC / MS fingerprint (TIC spectrum (Q1 + ve mode) of extract AV016BaSu (65) 01 (100) g.

Table 20: LC / MS fingerprint of extract AV016BaSu (65) 04 (100) (TIC spectrum (Q1 + ve mode).

Table 21: LC / MS fingerprint of extract AV016BaSu (65) 06 (100) (TIC spectrum (Q1 + ve mode).

Table 22: LC / MS fingerprint of extract AV016BaSu (105) 08 (100) (TIC spectrum (Q1 + ve mode).

Table 23: LC / MS fingerprint of extract AV016FrDi (65) 04 (100) (TIC spectrum (Q1 + ve mode).

Table 24: LC / MS fingerprint of extract AV016FrSu (105) 08 (100) (TIC spectrum (Q1 + ve mode).

Table 25: IC ₅₀ values of alpha glucosidase inhibitory activity of extracts extracted from different parts of the Terminalia Arjuna plant.

Table 26: Effect of insulin and terminaria arjuna extract on glucose uptake in 3T3-L1 adipocytes.

Table 27: Antiobesity capacity of the Terminalia Arjuna extract.

Table 28: Effect of the Terminalia Arjuna extract on lipid levels of CHO-K1 cells.

Table 29: Effect of 20% ethanol extract AV016BaDi (28) 04 (20) from the termini arjuna bark on HMG-CoA reductase activity.

Table 30: Effect of 100% ethanol extract AV016BaDi (65) 04 (100) directly from the termini arjuna bark on HMG-CoA reductase activity.

Table 31: Effect of the termini arjuna extract on cell viability of HepG2 cells.

Brief description of the drawings

1 is a graph showing the alpha-glucosidase inhibitory ability of the terminus arjuna direct ethanol extract, AV016BaDi (65) 04 (100) and AV016BaDi (28) 04 (20).

Figure 2 is a graph showing the alpha-glucosidase inhibitory ability of the terminus arjuna confection extract, AV016FrDi (65) 04 (100) and AV016FrSu (65) 08 (100).

FIG. 3 is a graph showing the alpha-glucosidase inhibitory ability of the terminus arjuna continuous extract, AV016BaSu (65) 01 (100) g, AV016BaSu (65) 01 (100) ng, AV016BaSu (65) 04 (100).

Figure 4 is a graph showing the alpha-glucosidase inhibitory ability of the termini arjuna continuous ethanol and water extracts, AV016BaSu (65) 06 (100) and AV016BaSu (105) 08 (100).

Figure 5 is a graph showing the alpha-glucosidase inhibitory ability of the termini arjuna continuous extract, AV016BaSu (65) 09 (100).

In a first aspect of the present invention, the present invention provides a method for treating a disease in a mammal, the method comprising: Terminalia arjuna as defined in the text. arjuna ) comprising administering at least an effective amount of a nontoxic extract to said mammal. Experts say that "treatment of a disease" in mammals means treatment, that is, alleviation of the symptoms of the disease, and also the prevention of a disease state, such as the progression of the disease and the worse or more invasive, It will be understood that it refers to the management of diseases such as alleviation.

In a second aspect of the present invention, the present invention provides a prophylactic method for preventing a disease in a mammal, the method comprising: a food for preventing the occurrence of a disease state selected from the group of diabetes mellitus, obesity, cardiovascular disease and cancer A food preparation of the form (= food) comprising administering to said mammal at least an effective amount of a non-toxic extract from the terminus arjuna as defined herein. Here, the mammal is a human and the extract consists of a single extract from some part of the plant terminary arjuna or a combination of extracts from them as detailed in the text. The invention also relates to extracts which can be separated from various parts of the terminus arjuna plant, such as bark and fruit, preparations of such extracts, medicaments consisting of these extracts, and the use of these extracts and components for the manufacture of a medicament. will be.

The extract of the present invention can be isolated from a terminus species such as the terminus arjuna using conventional organic solvent extraction and supercritical fluid extraction techniques. In general, extracts of the present invention that can exert prophylactic or therapeutic functions as briefly described in the text can be extracted from the terminus arjuna plant tissue, such as bark or fruit, depending on the final purpose required for the extract.

In a third aspect of the present invention, the present invention provides a method for preparing an extract of the present invention from a part of a termini arjuna plant, which method comprises the following steps.

(i) milling the selected plant material into a powder.

(ii) solvent extraction of the powdered plant material.

(iii) the extract thus obtained is lyophilized.

The choice of plant material may be any choice, but may be chosen from the bark or fruit of the terminus arjuna plant.

The solvent extraction process may be selected from direct or continuous extraction processes such as extraction from parts of the plant in a Soxhlet apparatus or flask at room temperature or above using a polar solvent and / or a nonpolar solvent. Typically the extraction process is as briefly described in the text.

For each step of separating an extract of the present invention that is active, the selection of a solvent or a mixture of solvents may be performed by the expert on the results of the biological quantitation of the separate fractions, for example as indicated in the text or shown in the Examples. It will be appreciated that it may be guided by.

It can also be suitably used for the treatment of diseases selected from the group of diabetes, obesity, cardiovascular disease and cancer, at least isolated from the terminus plant species, such as the terminus arjuna, used in admixture with a pharmaceutically acceptable carrier. Pharmaceutical formulations consisting of one extract are included within the scope of the present invention. Preferably at least one extract is selected from those listed in Tables 1-24, depending on the scheme or target disease. Also preferably at least one extract is selected from the group of extracts defined in Tables 25-30 according to the end purpose. Of course, those skilled in the art will understand that such compositions may consist of two or more plant extracts of the present invention in concentrations that may have a therapeutic effect. As such, the therapeutic composition may consist of a plant extract of terminaria that is substantially free of undesirable contaminant compounds. For example, the plant extract will be subjected to a number of solvent extraction steps that enable the separation of undesirable components from the preferred components as mentioned in the Examples and the above tables.

The present invention also provides a method for the treatment of a disease selected from the group of obesity, cancer, cardiovascular disease and diabetes in a mammal, including human, in a pharmaceutically useful form, for example oral administration, Or a clinical amount of the extracts listed in Tables 1 to 24, preferably in Tables 25 to 1, once or several times per day or other suitable dosing regimen via intravenous administration or lung lungs of dry or "wet" sprays. It consists of using the extracts listed in Table 30.

Of course, the amount of extract compound required to be effective in the treatment of the above-mentioned diseases may vary depending on the disease being treated and eventually may be at the discretion of the physician or veterinarian. Factors to be considered include the condition to be treated, the route of administration, the nature of the formulation, the body weight, surface area, age, general conditions of the mammal, and the particular compound to be administered. Generally, a suitable and effective dosage of the extract of the present invention is 0.01-120 mg / kg body weight, for example 0.1-120 mg / kg body weight, preferably 0.1-50 mg / kg body weight, for example 0.5-50 mg / The weight is in the range of kilograms. The total daily dose may be given in a single dose, for example, in multiple doses, such as two to six doses per day. For example, for a 75 kg mammal (eg, a human), the daily dosage may be about 8-9000 mg, a typical dosage may be about 50 mg per day. If a plurality of discontinuous doses are administered, the dosage of the therapeutic agent will be 15 mg of the compound of formula (I) given up to four times daily.

Only active extracts may be administered, but such active extracts are preferably provided in the form of pharmaceutical preparations. As a medicament, the pharmaceutical preparation of the present invention consists of the extract of the present invention together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The carrier must be pharmaceutically acceptable to be compatible with the other ingredients of the pharmaceutical formulation and must be substantially harmless to the recipient.

Accordingly, the present invention provides a pharmaceutical formulation consisting of at least one extract selected from those listed in Tables 1 to 24, preferably Tables 25 to 30, together with a pharmaceutically acceptable carrier. In a preferred embodiment, the pharmaceutical formulation consists of at least one extract selected from those listed in Tables 25-30 according to the type of disease to be treated. Those skilled in the art will appreciate that when selecting one or more extracts from those listed in the aforementioned tables to treat any single form of disease, an appropriate selection of extracts for the disease form may be made. As such, extracts suitable for such treatment, for example for the treatment of diabetes, will be selected from the table above.

Naturally, those skilled in the art will understand that pharmaceutical preparations consisting of the active extracts of the present invention may include at least one active extract purified from extracts derived from the Terminalia plant species. Thus, the pharmaceutical preparations may comprise one or more extracts derived from two or more termini plant species.

Also provided is a method of preparing a pharmaceutical formulation comprising the step of combining the extract of the present invention and a pharmaceutically acceptable carrier therefor.

Formulations according to the invention include those suitable for oral or intravenous administration.

Intravenous formulations may contain at least one extract of the invention and may also be administered in the form of suitable liposomes or niosomal preparations or by other suitable carriers.

Suitable emulsifiers and emulsion stabilizers for use in the formulations of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glycerol mono-stearate and sodium lauryl sulfate.

Such formulations are conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. All methods include the step of bringing into association the active extract with a carrier consisting of one or more accessory ingredients. In general, formulations are prepared by uniformly and intimately binding the active extract to a liquid carrier or a micropowdered solid carrier, or both, and, if necessary, to shape the product into the required formulation. Formulations of the present invention suitable for oral administration typically consist of individual formulations such as capsules, sachets, tablets, lozenges, gelatin, and the like, consisting of sucrose and an active ingredient in a flavor base such as acacia or tragacanth rubber. Pastilles consisting of the active ingredient of an inert base such as glycerin or sucrose and acacia, and mouth-washes consisting of the active ingredient in a suitable liquid carrier may be provided. Generally each formulation comprises a predetermined amount of the active extract, powder or granules, or a solution or suspension in an aqueous or non-aqueous liquid such as syrup, elixir, emulsion or draft.

Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may optionally contain a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g. sodium starch glycolate, cross-linked povidone, cross-linking). Combined carboxymethyl sodium cellulose), free-flowing active extracts such as powders or granules mixed with surfactants or dispersants may be prepared by compressing with a suitable compressor. Molded tablets may be prepared by molding a mixture of powdered extracts moistened with an inert liquid diluent with a suitable molding machine. Tablets may be coated or divided and may be formulated to allow sustained or controlled release of the active ingredient, for example, using hydroxypropylmethyl cellulose in a proportion that provides the required release profile.

Syrups can be prepared by, for example, adding active extracts to concentrated aqueous solutions of saccharides such as sucrose, and other necessary ingredients can be added thereto. Such auxiliary ingredients may include flavoring agents, anti-crystallization agents to prevent crystallization of sugars or solubility-increasing agents to increase the solubility of other components, such as polyhydric alcohols such as glycerol or sorbitol, for example.

In addition to the components mentioned above, the formulations of the present invention may comprise one or more accessory ingredients selected from diluents, buffers, flavors, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants) and the like.

The composition is also a dietary supplement, food composition or beverage composition suitable for human or animal consumption.

The present invention describes the HPLC profiles and mass spectra of the direct and continuous solvent extracts of the termini arjuna plant parts to give each extract the same identity. Several solvents used for continuous extraction are hexane, petroleum ether, ethyl acetate, acetone, ethanol, methanol and water, from nonpolar to polar. In the case of direct extraction, only alcohol solvent was used in combination with water as a solvent for extraction.

The invention also relates to new extracts, preparations of such extracts, pharmaceuticals comprising the extracts, which are isolated from various parts of the terminus arjuna plant and defined with reference to HPLC and MS fingerprints of the extracts as defined in Tables 1-24. And the use of these extracts and components for the preparation of a medicament.

In another aspect of the present invention, a pharmaceutical preparation for preventing, treating or managing diabetes mellitus and diabetes-related diseases, alone or in combination, may be used as a direct extract of T. arjuna bark with 100% ethanol solvent [ AV016BaDi (65) 04 (100)] and Direct Extraction of Terminalia Arjuna Bark with 20% Ethanol Solvent [AV016BaDi (28) 04 (20)], Continuous Extraction of Terminalia Arjuna Bark with Ethyl Acetate Solvent (65) 09 (100)], continuous extract of the termini arjuna bark with acetone solvent [AV016BaSu (65) 01 (100) g], continuous extract of the terminus arjuna bark with acetone solvent [AV016BaSu (65) 01 (100) ng], Continuous Extraction of Terminalia Arjuna Bark with Ethanol Solvent [AVOl6BaSu (65) 04 (l00)], Continuous Extraction of Terminalia Arjuna Bark with Methanol Solvent [AVO16BaSu (65) 06 (100 )] And te by water solvent Continuous Extract of Luminalia Arjuna Bark [AV016BaSu (105) 08 (100)], Direct Extract of Terminalia Arjuna Fruits by Ethanol Solvent [AV016FrDi (65) 04 (100)] and Terminalia Arjuna by Water Solvent Continuous extract of fruit [AVO 16FrSu (105) 08 (100)]. Such pharmaceutical preparations may include pharmaceutically acceptable carriers, excipients or diluents as mentioned herein.

In another aspect of the present invention, a pharmaceutical preparation for preventing, treating or managing obesity and obesity-related diseases, either alone or in combination thereof, is a direct extract of T. arjuna bark with 100% ethanol solvent [ AV016BaDi (65) 04 (100)] and direct extracts of the termini arjuna bark with 20% ethanol solvent [AV016BaDi (28) 04 (20)], ethyl acetate, colloidal acetone, comparative acetone, ethanol and methanol solvents AVO16BaSu (65) 09 (100), AVO16BaSu (65) 01 (100) g, AVO16BaSu (65) 01 (100) ng, AV016BaSu (65) 04 (100), AV016BaSu (65) 06 (100) and AV016FrDi (65) 04 (100). Such pharmaceutical preparations may include pharmaceutically acceptable carriers, excipients or diluents as mentioned herein.

In another aspect of the present invention, a pharmaceutical preparation for preventing, treating, or managing cardiovascular disease (CVD) and CVD-related diseases, alone or in combination thereof, is made of T. arjuna bark with 100% ethanol solvent. Direct Extract of [AV016BaDi (65) 04 (100)] and Terminalia Arjuna Bark with 20% Ethanol Solvent Direct Extract of [AV016BaDi (28) 04 (20)], Terminalia Arjuna Bark with Ethyl Acetate Solvent A continuous extract [AV016BaSu (65) 09 (100)] and a continuous extract [AV016BaSu (65) 01 (100)] of the termini arjuna bark with an acetone solvent. Such pharmaceutical preparations may include pharmaceutically acceptable carriers, excipients or diluents as mentioned herein.

In another aspect of the present invention, a pharmaceutical preparation for preventing, treating or managing cardiovascular diseases (CVD) and CVD-related diseases, alone or in combination thereof, is a continuous extract of Terminalia arjuna bark with ethyl acetate solvent [AV016BaSu ( 65) 09 (100)], continuous extracts of the termini arjuna bark with acetone solvent [AV016BaSu (65) 01 (100)], continuous extracts of the termini arjuna bark with ethanol solvent [AVOl6BaSu (65) 04 ( 00)], continuous extracts of the terminus arjuna bark with methanol solvent [AVO16BaSu (65) 06 (100)] and continuous extracts of the terminus arjuna bark with water solvent [AV016BaSu (105) 08 (100)]. It is composed. Such pharmaceutical preparations may include pharmaceutically acceptable carriers, excipients or diluents as mentioned herein.

In another aspect of the invention, at least the extract of the invention in a typical dry form such as a freeze-dried form selected from extracts selected from those listed in Tables 1 to 24, in particular extracts selected from those listed in Tables 25 to 30 Foodstuffs consisting of, or food preparations are provided. Those skilled in the art will appreciate that such food preparations may comprise and be used with one or more extracts of the invention. Such foods may be used for prophylaxis and may include other extracts with functions similar to those originally added, or other additives with different preventive functions may be added. As such, the food is composed of extracts which are provided for food preparations having a multifunctional preventive effect against two or more diseases or food preparations having a single function, for example, preventing the onset of diabetes. It is contemplated that similar multifunctional roles may be defined in pharmaceutical formulations consisting of two or more extracts with dissimilar therapeutic or prophylactic properties for the prevention or treatment of one or more diseases of a mammal, in particular a human.

Foodstuffs or food preparations in the form of foods or foodstuffs to which the extract of the present invention is added at least include snacks such as sweets, flat breads such as breads, pita bread, egg bread, snacks such as cakes, muesli bars, compressed fruit bars and biscuits. Foods, milk base products such as yogurt, milk, custard, dairy products such as cream, cheese, butter and krem fresh, imitation milk products such as margarine, olive oil based spreads, low fat cream substitutes such as Elmlea products, fruits And carbonated beverages such as vegetable juices, carbonated soft drinks, non-carbonated beverages such as squash, soy milk, rice milk and coconut milk, pasta, noodles, vegetables, seed oils and nut oils, sunflower oil, rapeseed oil, olive oil, walnuts, hazelnuts And fruit fruits such as sesame oil, ice cream, ice yogurt and the like.

Suitable effective dosages of the extracts of the invention to be included in the food preparations are generally about 0.01-120 mg / kg body weight, for example 0.1-120 mg / kg body weight, preferably about 0.1-50 mg / kg body weight, for example For example, it ranges from 0.5 to 50 mg / kg body weight. The total daily dose may be given in a single dose, for example, in multiple doses, such as two to six doses per day.

In another aspect of the present invention, the present invention provides the use of an extract selected from Tables 1 to 24, in particular Tables 25 to 30, for the preparation of a therapeutic agent for a disease selected from the group consisting of diabetes, cancer, cardiovascular disease and obesity. to provide.

Thus, in order to prepare a drug for the treatment or prevention of diabetes and related diseases, AV016BaDi (65) 04 (100), AV016BaDi (28) 04 (20), AV016BaSu (65) 09 (100), AVO16BaSu (65) ) 01 (100) g, AVO16BaSu (65) 01 (100) ng, AVO16BaSu (65) 04 (100), AV016BaSu (65) 06 (100), AV016BaSu (105) 08 (100), AV016FrDi (65) 04 ( 100) and the use of extracts selected from the group consisting of AV016FrSu 105 (08) (100).

In addition, for the preparation of a medicament for the treatment or prevention of obesity, AVO16BaDi (65) 04 (l00), AVO16BaDi (28) 04 (20), AVO16BaSu (65) 09 (l00), AV016BaSu (65) 01 (100) g, AV016BaSu (65) 01 (100) ng, AV016BaSu (65) 04 (100), AV016BaSu (65) 06 (100) and AV016FrDi (65) 04 (100) provide the use of an extract selected from the group consisting of do.

In addition, for the preparation of a medicament for the treatment or prevention of cardiovascular diseases, alone or in combination with one or more of AV016BaDi (65) 04 (100), AV016BaDi (28) 04 (20), AV016BaSu (65) 09 (100) and , AV016BaSu (65) 01 (100) provides the use of an extract selected from the group consisting of.

In addition, for the preparation of a medicament for the treatment or prevention of cancer, AVO16BaSu (65) 09 (100), AVO16BaSu (65) 01 (100), AVO16BaSu (65) 04 (100), AV016BaSu (65) 06 (100) and AV016BaSu (105) 08 (100).

The invention will be described in detail with reference to the following examples and the accompanying tables and figures. It is to be understood that the examples do not limit the scope of the invention.

Example  part

Example 1 Extraction of Terminalia arjuna

The extraction of the termini arjuna plant parts was carried out by direct extraction and continuous extraction at room temperature with the relevant liquid-liquid technique in a soxhlet apparatus followed by a lyophilization process.

I. Continuous Extraction

Continuous extraction from the bark of the termini arjuna was performed using a Soxhlet extractor. Solvents used are hexane, petroleum ether, chloroform, acetone, ethanol, methane and water, based on sequential polarity from nonpolar to polar, and the temperature is 65 ° C.

The detailed process is as follows:

1. Weigh 100 grams of powdered plant material into a cotton extractor (soxlet extractor body) on the bottom of a soxhlet device. Cover the upper side with cotton. 1000 ml of solvent (starting from hexane) are placed in a round bottom flask and placed on a mantle, with some ceramic chips. Add 100 ml of solvent to the material soaked with solvent.

2. Place the extractor on the flask connected to the condenser.

3. Allow coolant to circulate continuously from the tap into the condenser. When assembled in one set, firmly assemble.

4. Power on the mantle and set its temperature to 65 ° C. Vapor of solvent from the flask will condense through the inlet side of the extractor. The condensed (distilled) solvent will be collected in the extractor (body) to extract the compound from it.

5. When plant material is completely filled with solvent, allow it to drain into the flask. This process continues as long as stable heat and water circulation occurs.

6. Continue brewing for 8 hours at 4-5 times per hour.

7. The extracts collected in the flask are concentrated by vacuum freeze drying.

8. Perform the same procedure as described above in succession with the following solvents. Petroleum ether, ethyl acetate, acetone, ethanol, methanol and water.

II . Direct extraction

a. With 100% ethanol solvent Soxhlet  Based Extraction Process:

1. Weigh 100 grams of powdered plant material into the sachet and transfer it into the extractor (soxlet extractor body). Cover the upper side with cotton. (Make sure the level of material is less than 1 inch of the steam inlet tube)

2. Add 1 liter of solvent (starting with petroleum ether) to the round bottom flask, place it on the mantle and add some ceramic chips. Add 100 ml of solvent to the material soaked with solvent.

3. Place the extractor on the flask connected to the condenser.

4. Allow coolant to circulate continuously from the tap into the condenser. When assembled in one set, firmly assemble.

5. Power on the mantle and set its temperature to 65 ° C. Vapor of solvent from the flask will condense through the inlet side of the extractor. The condensed (distilled) solvent will be collected in the extractor (body) to extract the compound from it.

6. When plant material is completely filled with solvent, allow it to drain into the flask. This process continues as long as stable heat and water circulation occurs.

7. Continue extraction for 8 hours at 4-5 times per hour.

8. The extract collected in the flask is concentrated by vacuum freeze drying.

b. With 20% ethanol solvent at room temperature Terminalia Arjuna  Extraction of Bark:

1.Measure known amounts (100 grams) of powdered plant material into the conical flask and cover the inlet with aluminum foil to prevent evaporation of the solvent.

2. Add a known amount (500 ml) of 20% ethanol (100 ml ethanol + 400 ml water) solvent to the flask and place it on an orbital stirrer, set the speed to 210 rpm for extraction and set the temperature to 28 ° C. I set it.

3. Extract the plant material for 4 hours and then drain the solvent.

4. Centrifuge the filtrate at 1000 rpm for 10 minutes. Supernatant is collected and lyophilized.

5. Reextract for 2 x 2 hrs with 250 ml of solvent.

6. Centrifuge the filtrate at 1000 rpm for 10 minutes.

7. Concentrate the extract using a lyophilizer under vacuum.

Example  2: Terminalia Arjuna  Metabolic Fingerprint Test of Extracts

Metabolic fingerprinting of all direct and continuous extracts from the Terminalia Arjuna plant parts is performed by HPLC and LC / MS techniques.

I. HPLC  Fingerprint test:

Plant extracts obtained by direct / continuous extraction are analyzed by HPLC. High performance liquid chromatography (HPLC) is a technique in which a small amount of sample is introduced into a C-18 column under high pressure and the components are separated based on interaction with the column and retention time in the column. The main purpose of HPLC analysis is to find the total number of components in plant extracts.

Samples are prepared by dissolving an appropriate amount of extract in methanol for HPLC analysis. These samples are filtered and collected in total recovery HPLC vials. These samples are separated and analyzed by Waters 2695 HPLC instrument.

Operation condition:

1. The software used for HPLC analysis is Waters Millennium ³² .

2. The HPLC column used for separation is Waters μBondpack C-18, 5 μ, 4,6 × 150 mm.

3. The column temperature is maintained at 25 ° C.

4. The solvent flow rate is set at 1.0 ml / min. HPLC Conditions Contained in Gradient Chromatography—The solvents used were acetonitrile (solvent A), methanol (solvent B) and water (solvents C and D).

Terminalia Arjuna  Extracts HPLC  Operation condition:

One. Terminalia Arjuna  extract:

   AV016BaDi (65) 04 (100)

   2.AV016BaDi (28) 04 (20)

   AV016BaSu (65) 04 (100)

   4.AV016FrDi (65) 04 (100)

   5.AV016BaSu (65) 06 (100)

I. Method Set: Ethanol_11

Pressure limit:

         Upper limit 4000 psi Lower limit 0 psi

Programmed Flow Rate:

         Pump Mode: Gradient

         Accelerated to 10 ml / min for 2.0 minutes (5 ml / min / min)

time Flow % A % B % C % D Curb One 0.01 1.00 10.0 0.0 0.0 90.0 6 2 1.00 1.00 10.0 0.0 0.0 90.0 6 3 15.00 1.00 30.0 0.0 0.0 70.0 6 4 30.00 1.00 40.0 0.0 0.0 60.0 6

A: Acetonitrile , B: methanol, C: water

2. Terminalia Arjuna  extract:

   AV016BaSu (65) 01 (100)

   2.AV016BaSu (65) 01 (100) g

   AV016BaSu (65) 01 (100) g

II . Method set: ethyl acetate_10a

Pressure limit:

         Upper limit 4000 psi Lower limit 0 psi

Programmed Flow Rate:

         Pump Mode: Gradient

         Accelerated to 10 ml / min for 2.0 minutes (5 ml / min / min)

time Flow % A % B % C % D Curb One 0.01 0.75 5.0 2.5 92.5 0.0 6 2 1.00 0.75 5.0 2.5 92.5 0.0 6 3 25.00 0.75 25.0 2.5 72.5 0.0 6 4 30.00 0.75 5.0 2.5 92.5 0.0 One

A: acetonitrile, B: methanol, C: water

3. Terminalia Arjuna  extract:

   AV016BaSu (65) 09 (100)

III . Method set: ethyl Acetate _4a

Pressure limit:

         Upper limit 4000 psi Lower limit 0 psi

Programmed Flow Rate:

         Pump Mode: Gradient

         Accelerated to 10 ml / min for 2.0 minutes (5 ml / min / min)

time Flow % A % B % C % D Curb One 0.01 0.75 0.0 5.0 95.0 0.0 6 2 1.00 0.75 0.0 5.0 95.0 0.0 6 3 15.00 0.75 0.0 20.0 80.0 0.0 6 4 25.00 0.75 0.0 50.0 50.0 0.0 6 5 30.00 0.75 0.0 5.0 95.0 0.0 One

A: acetonitrile, B: methanol, C: water

4. Terminalia Arjuna  extract:

   AV016BaSu (105) 08 (100)

   AV016FrSu (105) 08 (100)

IV . Method set: Gy _Water_12

Pressure limit:

         Upper limit 4000 psi Lower limit 0 psi

Programmed Flow Rate:

         Pump Mode: Gradient

         Accelerated to 10 ml / min for 2.0 minutes (5 ml / min / min)

time Flow % A % B % C % D Curb One 0.01 0.75 5.0 5.0 90.0 0.0 6 2 1.00 0.75 5.0 5.0 90.0 0.0 6 3 20.00 0.75 25.0 15.0 60.0 0.0 4 4 26.00 0.75 70.0 5.0 25.0 0.0 4 5 30.00 0.75 5.0 5.0 90.0 0.0 One

A: acetonitrile, B: methanol, C: water

II . Liquid Chromatography Mass Spectrometry LC Of MS Fingerprint test:

Mass spectrometry is an instrumental approach that allows mass measurement of molecules. The five basic components of a mass spectrometer are a vacuum system, a sample injection device, an ionization source, a mass spectrometer, and an ion detector. By combining these configurations, mass spectrometers ionize, separate, and measure molecular ions according to their mass-to-charge ratios (m / z) to determine the molecular weight of chemical compounds.

The operating conditions used for LC / MS fingerprinting of Terminalia Arjuna are as follows.

1. Applied Biosystems' Q-Trap LC / MS instrument was used. The software used for LC / MS analysis is Analyst.

2. The HPLC column used for separation is COSMOSIL ^® 5C ₁₈ -MS-II Packed Column C-18, 5 μm, 4.6 mm I.Dx 150 mm.

3. The column temperature is maintained at 25 ° C.

4. The solvent flow rate was set at 1.0 ml / min. HPLC conditions included in the gradient chromatography—The solvents used were acetonitrile (solvent C), methanol (solvent B) and water (solvent D).

One. Terminalia Arjuna  extract:

AVO16BaSu (65) 09 (100), AVO16BaSu (65) 01 (100), AVO16BaSu (65) 01 (100) g, AVO16BaSu (65) 01 (100) g, AVO16BaSu (65) 04 (100), AVO16BaSu (65 ) 06 (l00), AV016BaSu (65) 06 (80), AV016BaDi (65) 04 (100), AV016FrDi (65) 04 (100)

a. All mentioned above Terminalia Arjuna  For samples LC Of MS  Sample operation condition:

Mass spectrometer QTrap 0 MASS

SPEC

Configuration Table Version 01

Firmware Version M401400 B4T0301 M3L1400 B3T0300

Part Name Linear Ion Trap Quadrupole LC / MS / MS Mass Spectrometer

Part ID QTrap

Manufacturer AB Sciex Instruments

Model 027170-C

S / N M1100301

Time from start = 0.0500 minutes

Mass spectrometer QTrap 0 MASS

SPEC

Start of driving-detailed status

With vacuum pressure

Vacuum gauge (10e-5 Torr) 0.7

Auxiliary Pump Ok

Dual turbo pump tops

Sample injection state preparation

Source / Ion Path Electronics On

Source form turbo spray

Source temperature (set value) 400 ℃

Source discharge pump Ok

Interface heater preparation

Mass spectrometer QTrap 0 MASS

SPEC

End of driving-detailed state

With vacuum pressure

Vacuum gauge (10e-5 Torr) 0.7

Auxiliary Pump Ok

Dual turbo pump tops

Sample injection state preparation

Source / Ion Path Electronics On

Source form turbo spray

Source temperature (set value) 400 ℃

Source discharge pump Ok

Interface heater preparation

Time from start = 61.4833 min

PE LC -200 pump method characteristics

PE LC-200 Quaternary Pump

Pressure (psi): 0.0

Pressure (psi): 6100.0

Downtime (minutes): 999.9

Step table :

Stage Total Time Flow Gradient Form A (%) B (%) C (%) D (%) TE # 1 TE # 2

       (Min) (μl / min)

0 0.5 1000.00 1.0 0.0 0.0 10.0 90.0 Open Open

1 1.0 1000.00 1.0 0.0 0.0 10.0 90.0 Open Open

2 15.0 1000.00 1.0 0.0 0.0 15.0 85.0 Open Open

3 40.0 1000.00 1.0 0.0 0.0 25.0 75.0 Open Open

4 50.0 1000.00 1.0 0.0 0.0 35.0 65.0 Open Open

5 60.0 1000.00 1.0 0.0 0.0 50.0 50.0 Open Open

Quantitative Information:

Sample Type: Unknown

Dilution factor: 1.000000

Custom data:

Quantitative table:

Period 1:

-------

Scans within period: 2243

Relative start time: 0.00 msec

Experiment in period: 1

Period 1 Experiment 1:

--------------

Scan Type: Q1 MS (Q1)

Polarity: Positive

Scan Mode: Profile

Analysis Q1: UNIT

Intensity Thres .: 0.00 cps

Settling time: 0.0000 ms

MR downtime: 5.0070 ms

MCA: No

Center / Width: No

Step Size: 0.10 amu

Start (amu) Stop (amu) Time (sec) Param Start Stop

50.00 1700.00 1.60 CEP 6.47 66.65

Parameter  Table (period 1 experiment 1)

CUR: 20.00

TEM: 400.00

GS1: 20.00

GS2: 50.00

ihe: ON

IS: 4500.00

DP: 90.00

EP: 10.00

2. Terminalia Arjuna  extract:

AVO16BaSu (105) 08 (100), AVO16FrSu (105) 08 (100), and AVO16BaDi (28) 04 (20).

a. All mentioned above Terminalia Arjuna  For samples LC Of MS  Sample operation condition:

Mass spectrometer QTrap 0 MASS

SPEC

Configuration Table Version 01

Firmware Version M401400 B4T0301 M3L1400 B3T0300

Part Name Linear Ion Trap Quadrupole LC / MS / MS Mass Spectrometer

Part ID QTrap

Manufacturer AB Sciex Instruments

Model 027170-C

S / N M1100301

Time from start = 2.1000 minutes

Mass spectrometer QTrap 0 MASS

SPEC

Start of driving-detailed status

With vacuum pressure

Vacuum gauge (10e-5 Torr) 0.7

Auxiliary Pump Ok

Dual turbo pump tops

Sample injection state preparation

Source / Ion Path Electronics On

Source form turbo spray

Source temperature (set value) 400 ℃

Source discharge pump Ok

Interface heater preparation

Time from start = 2.1167 min

Mass spectrometer QTrap 0 MASS

SPEC

End of driving-detailed state

With vacuum pressure

Vacuum gauge (10e-5 Torr) 0.7

Auxiliary Pump Ok

Dual turbo pump tops

Sample injection state preparation

Source / Ion Path Electronics On

Source form turbo spray

Source temperature (set value) 400 ℃

Source discharge pump Ok

Interface heater preparation

Time from start = 42.9.333 min

PE LC -200 pump method characteristics

PE LC-200 Quaternary Pump

Pressure (psi): 0.0

Pressure (psi): 6100.0

Downtime (minutes): 999.9

Step table :

Stage Total Time Flow Gradient Form A (%) B (%) C (%) D (%) TE # 1 TE # 2

       (Min) (μl / min)

0 0.5 750.00 1.0 0.0 5.0 5.0 90.0 Open Open

1 1.0 750.00 1.0 0.0 5.0 5.0 90.0 Open Open

2 20.0 750.00 1.0 0.0 15.0 25.0 60.0 Open Open

3 26.0 750.00 1.0 0.0 5.0 70.0 25.0 Open Open

4 30.0 750.00 1.0 0.0 5.0 5.0 90.0 Open Open

5 40.0 750.00 1.0 0.0 5.0 5.0 90.0 Open Open

Analog / Digital Converter Characteristics

Time interval (sec): 0.200

Rate (pts / sec): 5

Polarity: bipolar

Channel Summary

No. Name: Analysis Value Real Size: Analysis Unit:

Voltage (volts): State:

1 100.00% 1.0 Used

Quantitative Information:

Sample Type: Unknown

Dilution factor: 1.000000

Custom data:

Quantitative table:

Period 1:

-------

Scan in Period: 1495

Relative start time: 0.00 msec

Experiment in period: 1

Period 1 Experiment 1:

Scan Type: Q1 MS (Q1)

Polarity: Positive

Scan Mode: Profile

Analysis Q1: UNIT

Intensity Thres .: 0.00 cps

Settling time: 0.0000 ms

MR downtime: 5.0070 ms

MCA: No

Center / Width: No

Step Size: 0.10 amu

Start (amu) Stop (amu) Time (sec) Param Start Stop

50.00 1700.00 1.60 CEP 6.47 66.65

Parameter  Table (period 1 experiment 1)

CUR: 20.00

TEM: 400.00

GS1: 20.00

GS2: 50.00

ihe: ON

IS: 4500.00

DP: 90.00

EP: 10.00

Example  3: Terminalia Arjuna  Determination of Biotherapeutic Capacity of Extracts:

I. Diabetes Treatment Ability

Sudden rise in glucose after food intake is associated with defects in various glucose-mediated tissues, oxidative stress, glycation, and the formation of many glycemic end products that have a significant structural and functional impact on virtually all organ systems. Lowering glycated hemoglobin to levels that can prevent or delay these complications can be achieved by lowering both postprandial and fasted plasma glucose levels.

In the small intestine, α-glucosidase complex is involved in glucose uptake. As such, inhibitors of α-glucosidase will inhibit postprandial hyperglycemia by limiting absorption of dietary carbohydrates. Thus, α-glucosidase inhibitors are useful drug candidates for the treatment and prevention of diabetes. α-glucosidase inhibitors (acarbose, bolibos, miglitol) effectively delay carbohydrate digestion and absorption, reducing postprandial spikes in blood sugar levels without losing calories.

Alpha Glucosidase  analysis

Alpha-glucosidase hydrolyzes the final non-reducing 1,4-binding alpha-D-glucose residues with the release of alpha-D-glucose. In the assay, enzymatic activity is determined by measuring the increase in absorbance 400 nm by hydrolysis of p-nitrophenyl-α-D-glucopyranoside with the release of p-nitrophenol.

a. reagent

인산칼륨 버퍼 0.1M, pH 6.8

Potassium Phosphate Buffer 0.1M, pH 6.8

PNPG 4 mM

PNPG 4 mM

탄산나트륨 0.2M

Sodium Carbonate 0.2M

버퍼중 BSA 0.2%

0.2% BSA in buffer

0.2% BSA 중에 효소 1.2U/ml

1.2U / ml Enzyme in 0.2% BSA

b. step

c. Results and review

Continuous and direct extracts from the terminus arjuna bark and fruit analyzed for the ability of the terminus arjuna extract to alpha-glucosidase inhibition showed that the termini arjuna extract showed a decreased dose-dependentness of alpha-glucosidase inhibition. Seems to be. IC ₅₀ is defined as the amount of extract required for 50% inhibition in enzymatic activity.

All extracts of Terminalia Arjuna are IC ₅₀ As shown by the value, it was found to show high alpha-glucosidase inhibitory ability as compared to commercially available acarbose (Table 25).

2. Glucose absorption analysis

Deoxy-glucose uptake analysis by adipocytes (3T3-L1) helps to identify extract / bioactivity that helps to increase glucose uptake by adipocytes. Differentiation of 3T3-L1 cells shows a marked increase in insulin-stimulated glucose transport rate during their conversion from proliferative fibroblasts to non-proliferative adipocytes. Initially, Green and Kehinde (1975) demonstrated that fusion 3T3-L1 cells could be induced by hormones to differentiate into adipocytes in the course of 6-9d.

This analysis will help to remove glucose from the blood stream and test plant extracts that will help people with diabetes and at the same time help fat cells store better energy.

Triple deoxyglucose uptake assay by 3T3-L1 is one of the best assays allowed for in vitro testing of active ingredients having a therapeutic effect on diabetes.

a. 3 T3 - L1  Cell culture

3T3-L1 fibroblasts were cultured in DME-F12 medium containing 10% FCS, NEAA, glutamine, antibiotics, antifungal agents and the like at 37 ° C. under atmospheric pressure of 5% CO ₂ . Fibroblasts were cultured to confluency. Secondary cultures were performed with trypsin-EDTA solution at 3 day intervals.

b. Fibroblast maturation into adipocytes

DMEM hi glucose (4.5 gm / litre) containing 0.5 mmol / l 3-isobutyl-1-methylxanthine, 4 mg / ml dexamethasone, 1 mg / ml insulin and 10% FCS for 48 hours. Differentiation was induced by treating the cells. Cells were incubated in DMEM medium with the standard supplement described above for the next 6-10 days. Cells are used only when at least 95% of the cells show adipocyte standard form by accumulation of fat droplets. These were adopted for the study of glucose absorption using triple glucose.

c. Analysis procedure

50,000 differentiated 3T3-L1 adipocytes are dispensed into each well of a new 96 well plate. Incubate with KRH buffer for 10 minutes at 37 ° C. Add 10 nM, 25 nM and 50 nM of insulin, respectively. Treatment was performed with or without insulin 10 nM in the presence of test compound (333.0 μg) for the experimental set. Thereafter, further incubation took place at 37 ° C. for 15 minutes. Wash cells once with KRH buffer. The glucose absorption reaction is started by adding 0.1 mM 2-deoxy glucose containing 2-deoxy [3H] glucose (final concentration 12.2 kBq / ml). Incubate at 37 ° C. for 1 hour. The assay is terminated by the addition of 40 μM cytocalinine D. The cells are washed three times with cold KRH buffer. Cells are solubilized in 20 μl of 1% Triton X. Incubate at 37 ° C. for 10 minutes. Calculate the radioactivity on the micro-titer plate radioactivity counter.

d. Results and review

Glucose absorbing activity was analyzed by measuring the uptake of 0.1 mM deoxy-glucose containing 2.2 kBq / mL of deoxy-triple-glucose.

As shown in Table 26, the direct extract of the terminus arjuna bark [AV016BaDi (65) 04 (100)] using 100% ethanol solvent had a stimulation index of 3.0 without insulin and a stimulation index in the presence of 10 nM insulin. Showed that it is 2.0. As such, these data suggest that AV016BaDi (65) 04 (100) has a strong insulin mimetic capacity.

2. Anti-obesity ability

Studies have shown that abdominal obesity is more strongly related to lipid / lipoprotein abnormalities than normal obesity. Obesity causes heart failure due to lipid accumulation in cardiomyocytes or type 2 diabetes. These lipid / lipoproteins are important for obese people because these poor lipid / lipoproteins are at risk of increasing cardiovascular disease (CVD).

The assay is a semiquantitative method used for lipogenesis and quantification of lipid and triglyceride content in cells following stimulation of cells for differentiation in the presence and absence of plant extracts.

a. Analysis principle

Oil Red staining is a semiquantitative method used for the quantification of fat differentiation. Dyeing with oil-soluble dyes is based on greater solubility of the dyes in oleaginous materials as compared to conventional aqueous alcoholic dye solvents. Oil Red O is a lipophilic red dye that stains droplets of hepatocytes. Quantification of lipids can be performed after extraction of hepatocyte lipids with an organic solvent. As such it is used to assess lipid and triglyceride content in cells after stimulation of plant extracts of the cells.

b. Analysis protocol

Inject wells with differentiation medium and extract (0.33mg) into 3T3-L1 cells at a density of 10 ⁵ cells / well and incubate for 24 hours at 37 ° C and 5% CO ₂ . The medium was then removed after 24 hours and replaced with fresh differentiation medium and cells were preserved in this medium for an additional 24 hours. After 48 hours, the medium was removed and insulin (DMEM F12 + FGS + Insulin) was added to the medium and cells were preserved for 24 hours. After 24 hours, the culture medium was aspirated to perform Oil O red staining.

c. Oil Red  O dyeing

The total volume of each well for reagent reaction is 500 μl / well. The medium was removed and the cells washed three times with PBS. To settle the cells, they were incubated with 4% formalin for 1 hour. Remove formalin and wash twice with PBS again. To dye with Oil Red O, it is incubated with the dye for 1 hour. After 1 hour, the dye is removed, washed with PBS and dried for 10 minutes. 4% NP40 is added. Read the absorbance at 520 nm through the spectrometer.

d. Results and review

Anti-obesity analysis of 3T3-L1 cells showed that Terminalia Arjuna extract inhibited fat accumulation. As shown in Table 27, 100% and 20% ethanol direct extracts AV016BaDi (65) 04 (100) and AV016BaDi (28) 04 (20) of the termini arjuna bark showed 76.3 and 64.3% inhibition rate compared to the control. .

Ethyl acetate, colloidal acetone, comparative acetone, ethanol and methanol consecutive extracts of the Terminalia Arjuna bark AV016BaSu (65) 09 (100), AV016BaSu (65) 01 (100) g, AV016BaSu (65) 01 (100) ng, AV016BaSu (65) 04 (100) and AV016BaSu (65) 06 (100) showed inhibition rates of 61.9, 58.5, 86.8, 49.4 and 57.9% compared to the control. The 100% ethanol direct extract AV016FrDi (65) 04 (100) of the Terminalia arjuna fruit showed 41.9% inhibition rate compared to the control.

Analysis of the + ve control showed a 104% inhibition rate compared to the control.

3. Cardiovascular Disease

Elevated cholesterol is associated with atherosclerosis and cardiovascular disease at greater than normal risk. High blood triglyceride levels alone do not cause atherosclerosis. Lipoproteins rich in triglycerides also contain cholesterol, which causes atherosclerosis in many people with high triglycerides. Such high triglycerides will be a sign of lipoprotein problems that cause CHD.

1. Geological accumulation level Check  Cell-based analysis for

a. Analysis principle

Growth of cells in the presence of lipoprotein deficient serum leads to lipid biosynthesis in cells. The assay is performed on cells induced by growth in lipoprotein deficient serum followed by a total lipid assessment using oil red staining. Oil Red O is a lipophilic red dye used to color the cells' neutral lipids. The amount of stain is directly proportional to the amount of lipid in the cell. Lipid content was checked through the extract treatment and control, and dye amount and lipid content were measured using a spectrophotometer.

b. Analysis procedure

CHO-K1 cells were injected at a density of 10 ⁵ cells / well into wells in a 12 well-plate format. The cells were treated with DMEM-F12 medium containing 10% fetal calf serum, 2 mM glutamine, 1.5 gm / L sodium bicarbonate and antifungal agents and penicillin and streptomycin at 37 ° C and 5% CO ₂ atmosphere. Has grown to 80% confluence. After 2 days of culture, the medium was replaced with DMEM-F12 medium containing 10% lipoprotein deficient serum, 2 mM glutamine, 1.5 gm / L sodium bicarbonate and antifungal and penicillin and streptomycin, and 5% temperature at 37 ° C. It was grown for an additional 24 hours in an atmosphere of CO ₂ . At the end of 24 hours the treatment was added a Terminalia Arjuna extract at a concentration of 0.5 mg / ml, while the control was treated with DMSO solution. Incubation continued for an additional 4 hours, after which it was washed with sterile PBS buffer, settled and stained with Oil Red O.

c. Oil Red  O dyeing

The total volume of each well for reagent reaction is 500 μl / well. The medium was removed and the cells washed three times with PBS. To settle the cells, they were incubated with 4% formalin for 1 hour. Remove formalin and wash twice with PBS again. To dye with Oil Red O, it is incubated with the dye for 1 hour. After 1 hour, the dye is removed, washed with PBS and dried for 10 minutes. 4% NP40 is added. Read the absorbance at 520 nm through the spectrometer.

d. Results and review

As shown in Table 28, the termini arjuna extract showed a significant decrease in total lipid content in the extract treated cells. Continuous extracts AV016BaSu (65) 09 (100) and AV016BaSu (65) 01 (100) of the termini arjuna bark showed inhibition rates of 22% and 21% compared to the control. Ethanol direct extracts AV016BaDi (65) 04 (100) and AV016BaDi (28) 04 (20) of the termini arjuna showed 31% and 28% inhibition rates as compared to the control.

2. HMG - CoA  Reductase Assay:

3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a given first step in the cholesterol biosynthetic pathway. HMG-CoA reductase, a rate limiting step in cholesterol biosynthesis, has become the most successful drug therapy for lowering total plasma cholesterol, especially LDL-cholesterol.

a. Analysis principle

The HMG-CoA reductase assay is based on the oxidation of NADPH to NADP catalyzed by a fixed concentration of HMG-CoA reductase obtained from liver microsome fragments.

Oxidation from NADPH to NADP ⁺ was performed with a decrease in absorbance at 340 nm and is directly proportional to the HMG-CoA reductase activity of the sample.

d. Results and review

The effects of 100% ethanol and 20% ethanol direct extracts AV016BaDi (28) 04 (20) and AV016BaDi (65) 04 (100) of the termini arjuna bark were checked for their ability to inhibit HMG-CoA reductase. These extracts AV016BaDi (28) 04 (20) and AV016BaDi (65) 04 (100) showed 93% inhibition rate (Table 29) and 63.1% inhibition rate (Table 30) in HMG-CoA reductase activity compared to the control. .

4. Anticancer Analysis

The anticancer capacity of the terminus arjuna plant extract was measured by screening the plant extract in vitro for cytotoxicity in the hepatocyte cell line HepG2 in humans.

a. Experiment Contents:

Cells were injected at a density of 10 ⁵ cells / ml in 24 well-plates. These cells were incubated with DMSO solution, ascorbic acid and extracts AV016BaSu (65) 09 (100), AV016BaSu (65) 01 (100), AV016BaSu (65) 04 (100) for 48 hours and then extracted at a concentration of 10 µg / ml. It was attached for 2 hours before adding. Cell viability was measured after 24 hours.

b. Analysis protocol

Inject HepG2 at a cell density of 2.5x10 ⁵ cells / ml. Add ascorbic acid and the termini arjuna extract to a final concentration of 10 μg / ml. Incubate for 24 hours at a temperature of 37 ° C. in a 5% CO ₂ incubator. Trypsinize the cells. Mixed cells with 1 part trypan blue and 4 parts PBS are used to calculate cells that have been stained with trypan blue (non-surviving cells) and cells that have not been stained (surviving cells) to obtain viable and non-surviving cells.

c. Results and review

HepG2 cells treated with ethyl acetate continuous extract AV016BaSu (65) 09 (100), acetone continuous extract AV016BaSu (65) 01 (100) and ethanol continuous extract AV016BaSu (65) 04 (100) of the termini arjuna were untreated In other words, it was confirmed that the total number of cells and the number of viable cells were decreased in the drug-treated set compared to the cells treated with DMSO.

Table 1: HPLC fingerprint of extract AV016BaDi (65) 04 (100)

Table 2: HPLC fingerprint of extract AV016BaDi (28) 04 (20)

Table 3: HPLC fingerprint of extract AV016BaSu (65) 09 (100)

Table 4: HPLC fingerprint of extract AV016BaSu (65) 01 (100)

Table 5: HPLC Fingerprint of Extract AV016BaSu (65) 01 (100) ng

Table 6: HPLC fingerprint of extract AV016BaSu (65) 01 (100) g

Table 7: HPLC fingerprint of extract AV016BaSu (65) 04 (100)

Table 8: HPLC fingerprint of extract AV016BaSu (65) 06 (100)

Table 9: HPLC fingerprint of extract AV016BaSu (105) 08 (100)

Table 10: HPLC fingerprint of extract AV016Fr (105) 08 (100)

Table 11: LC / MS Fingerprint (TIC Spectrum (Q1 + ve Mode) of Extract AV016BaDi (28) 04 (20)

Peak list:

Table 12: LC / MS Fingerprints of Extract AV016BaDi (28) 04 (20) (TIC Spectrum (Q1-ve Mode)

Peak list:

Table 13: LC / MS Fingerprints of Extract AV016BaDi (65) 04 (100) (TIC Spectrum (Q1 + ve Mode)

Peak list:

Table 14: LC / MS Fingerprints of Extract AV016BaDi (65) 04 (100) (TIC Spectrum (Q1-ve Mode)

Peak list:

Table 15: LC / MS Fingerprint of Extract AV016BaSu (65) 09 (100) (TIC Spectrum (Q1 + ve Mode)

Peak list:

Table 16: LC / MS fingerprint (TIC spectrum (Q1 + ve mode) of extract AV016BaSu (65) 01 (100)

Peak list:

Table 17: LC / MS Fingerprints of Extract AV016BaSu (65) 01 (100) (TIC Spectrum (Q1-ve Mode)

Peak list:

Table 18: LC / MS fingerprint (TIC spectrum (Q1-ve mode) of extract AV016BaSu (65) 01 (100) ng)

Table 19: LC / MS fingerprint (TIC spectrum (Q1 + ve mode)) of extract AV016BaSu (65) 01 (100) g

Table 20: LC / MS fingerprint (TIC spectrum (Q1 + ve mode) of extract AV016BaSu (65) 04 (100))

Table 21: LC / MS Fingerprints of Extract AV016BaSu (65) 06 (100) (TIC Spectrum (Q1 + ve Mode)

Table 22: LC / MS Fingerprint (TIC Spectrum (Q1 + ve Mode) of Extract AV016BaSu (105) 08 (100)

Table 23: LC / MS fingerprint (TIC spectrum (Q1 + ve mode)) of extract AV016FrDi (65) 04 (100)

Table 24: LC / MS fingerprint (TIC spectrum (Q1 + ve mode) of extract AV016FrSu (105) 08 (100)

Peak list:

Table 25: IC ₅₀ Values of Alpha Glucosidase Inhibitory Activities of Extracts Extracted from Other Parts of Terminalia Arjuna Plants

Table 26: Effects of Insulin and Terminalia Arjuna Extracts on Glucose Uptake in 3T3-L1 Adipocytes

 set  process cpm  Average  SD value  Stimulus index Set I Set II Set III One Cell alone 314.6 390 1200.8 635.13 491.33 2 Cell + insulin (10 nM) 2185.8 1705.4 3010.7 2300.63 660.18 2.6 3 Cell + insulin (25 nM) 4806.8 1399.7 996.7 2401.07 2093.15 2.8 4 Cell + insulin (100 nM) 2906.5 3776.6 2215.6 2966.23 782.21 3.7 5 Cell + AV016BaDi (65) 04 (100) (Ter1)   1946.5   2570.2   3106.7   2541.13   580.65   3.0 6 Cell + insulin (10nM) AV016BaDi (65) 04 (100) (Ter1)   2178.5   1873   1667.3   1906.27   257.22   2.0

 Stimulus index = {sample mean-control mean (cell only) / control mean (cell only)}

Table 27: Antiobesity capacity of the Terminalia Arjuna extract.

      Inhibition rate% = {(average of control-sample) / average of control} * 100

Table 28: Effect of Terminalia Arjuna Extract on Lipid Levels of CHO-K1 Cells

Table 29: Effect of 20% ethanol extract AV016BaDi (28) 04 (20) from the termini arjuna bark on HMG-CoA reductase activity

 Minutes Reading without inhibitor Readings with inhibitor 0 1.3465 1.7372 One 1.3103 1.726 2 1.2891 1.726 3 1.2768 1.726 4 1.2689 1.7042 5 1.2534 1.7042 Temperament Blank 0.0277 0.0307 0 Final reading 0.0654 0.0046 Inhibition% 0 93.0

% Inhibition = {(control-treated extract / control) * 100}

Table 30: Effect of 100% ethanol extract AV016BaDi (65) 04 (100) directly from the termini arjuna bark on HMG-CoA reductase activity

 Minutes Reading without inhibitor Readings with inhibitor 0 1.162 1.7815 One 1.1125 1.7637 2 1.1048 1.7637 3 1.0828 1.7637 4 1.0756 1.746 5 1.0652 1.7467 Temperament Blank 0.023 0.0212 0 Final reading 0.0738 0.0272 Inhibition% 0 63.1

% Inhibition = {(control-treated extract / control) * 100}

Table 31: Effect of Terminalia Arjuna Extract on Cell Viability of HepG2 Cells

process Number of viable cells Number of non-viable cells cell 85 20 Cell + DMSO 82 11 Cell + Ascorbic Acid 66.5 18.5 Cell + AV016BaSu (65) 09 (100) 27 7 Cell + AV016BaSu (65) 01 (100) 55 2 Cell + AV016BaSu (65) 04 (100) 11 One

Claims (28)

A method for the treatment of a disease for treating a disease selected from the group consisting of obesity, cancer, cardiovascular disease and diabetes in a mammal, the method comprising at least from a Terminalia arjuna selected from those as defined in Tables 1-24. A method of treating a disease, characterized by administering to said mammal an effective amount of a nontoxic extract. The method of claim 1 wherein the disease is obesity and the extract is AVOl6BaDi (65) 04 (100), AVO16BaDi (28) 04 (20), AVO16BaSu (65) 09 (l00), AVO16BaSu (65) 01 (100) g, AVO16BaSu (65) 01 (100) ng, AVO16BaSu (65) 04 (l00), AV016BaSu (65) 06 (100) and AV016FrDi (65) 04 (100) or combinations of two or more thereof Way. The method of claim 1, wherein the disease is cancer and the extract is selected from the group consisting of AV016BaSu (65) 09 (100), AV016BaSu (65) 01 (100) and AVOl6BaSu (65) 04 (100) or a combination of two or more thereof. How to feature. The method of claim 1, wherein the disease is a cardiovascular disease and the extract is AV016BaDi (65) 04 (100), AV016BaDi (28) 04 (20), AV016BaSu (65) 09 (100) and AV016BaSu (65) 01 (100) or their Selected from the group consisting of two or more combinations of; The method of claim 1, wherein the disease is obesity and the extract is AV016BaDi (65) 04 (100), AVO 16BaDi (28) 04 (20), AVO16BaSu (65) 09 (100), AVO 16BaSu (65) 01 (100) g , AVOl6BaSu (65) 01 (100) ng, AVOl6BaSu (65) 04 (l00), AVO16BaSu (65) 06 (100), AVO16BaSu (105) 08 (100), AVO16FrDi (65) 04 (100) and AVO16FrSu (105 08 (100) or a combination of two or more thereof. The method of claim 1, wherein the disease is diabetes, obesity and cardiovascular disease and the extract is selected from the group consisting of AV016BaDi (65) 04 (100) and AV016BaDi (28) 04 (20) or a combination of two or more thereof. . The method of any one of claims 1 to 6, wherein the therapeutic agent for the treatment of the disease is a prophylactic agent. For pharmaceutical preparations for use in the treatment of diabetes, obesity, cardiovascular diseases and diseases selected from the group of cancers, the pharmaceutical preparations are isolated from pharmaceutically acceptable and terminally aryona junona selected from those listed in Tables 1-24. Pharmaceutical formulation, characterized in that consisting of at least one extract used in combination with a possible carrier. The pharmaceutical preparation according to claim 8, wherein the pharmaceutical preparation is a pharmaceutical preparation for use in the treatment of diabetes mellitus, AV016BaDi (65) 04 (100), AV016BaDi (28) 04 (20), AV016BaSu (65) 09 (100), AVO16BaSu ( 65) 01 (100) g, AVO16BaSu (65) 01 (100) ng, AVO16BaSu (65) 04 (100), AVO16BaSu (65) 06 (100), AV016BaSu (105) 08 (100), AV016FrDi (65) 04 (100) and AVO16FrSu (105) 08 (100) or at least one extract selected from the group consisting of two or more combinations thereof. The pharmaceutical formulation according to claim 8, wherein the pharmaceutical formulation is a pharmaceutical formulation for use in the treatment of cancer, and AV016BaSu (65) 09 (100), AV016BaSu (65) 01 (100) and AVOl6BaSu (65) 04 (100) or their Pharmaceutical formulation, characterized in that consisting of at least one extract selected from the group consisting of two or more combinations. 9. The pharmaceutical preparation according to claim 8, wherein the pharmaceutical preparation is a pharmaceutical preparation for use in the treatment of cardiovascular diseases, AV016BaDi (65) 04 (100), AV016BaDi (28) 04 (20), AV016BaSu (65) 09 (100) and AVO16BaSu. (65) 01 (100) or a pharmaceutical formulation, characterized in that it consists of at least one extract selected from the group consisting of two or more combinations thereof. According to claim 8, wherein the pharmaceutical preparation is a pharmaceutical preparation for use in the treatment of cardiovascular diseases, AVOl6BaDi (65) 04 (100), AVO16BaDi (28) 04 (20), AVO16BaSu (65) 09 (100), AV0l6BaSu (65) 01 (100) g, AV016BaSu (65) 01 (100) ng, AV016BaSu (65) 04 (100), AV016BaSu (65) 06 (100) and AV016FrDi (65) 04 (100) or two or more thereof Pharmaceutical formulation, characterized in that consisting of at least one extract selected from the group consisting of a combination. According to claim 8, wherein the pharmaceutical preparation is a pharmaceutical preparation for use in the treatment of diabetes, obesity and cardiovascular disease, AV016BaDi (65) 04 (100) and AV016BaDi (28) 04 (20) or a combination thereof Pharmaceutical formulation, characterized in that consisting of at least one extract selected from the group consisting of. The pharmaceutical preparation according to any one of claims 8 to 13, wherein the pharmaceutical preparation is a prophylactic pharmaceutical preparation. A method of preparing a pharmaceutical preparation, wherein the method comprises the step of combining the extract of the present invention with a pharmaceutically acceptable carrier therefor. Extract from Terminalia Arjuna selected from the group consisting of extracts with HPLC and / or MS properties shown in Tables 1-24. A food preparation comprising an extract from Terminalia Arjuna selected from the group consisting of extracts having HPLC and / or MS properties shown in Tables 1-24. 18. A food preparation according to claim 17, consisting of at least one extract for use in the prevention of cancer. 18. A food preparation according to claim 17, comprising at least one extract for use in the prevention of cardiovascular disease. 18. A food preparation according to claim 17, consisting of at least one extract for use in the prevention of diabetes. 18. A food preparation according to claim 17, consisting of at least one extract for use in the prevention of obesity. 18. The method of claim 17, wherein at least one extract selected alone or in combination of one or more of AV016BaDi (65) 04 (100) and AVO16BaDi (28) 04 (20) for use in the prevention of diabetes, obesity and cardiovascular disease Foodstuff agent, characterized in that consisting of. Use of an extract selected from the group consisting of extracts having the HPLC and / or MS properties shown in Tables 1-24 for the manufacture of a medicament for the treatment of a disease selected from the group consisting of diabetes, cancer, cardiovascular disease and obesity. 24. The method of claim 23 for the manufacture of a medicament for the treatment or prevention of diabetes and / or related diseases, wherein AVO16BaDi (65) 04 (100), AVO16BaDi (28) 04 (20), AVO16BaSu (65) 09 (l00). ), AVO16BaSu (65) 01 (100) g, AVO16BaSu (65) 01 (100) ng, AVO16BaSu (65) 04 (100), AVO16BaSu (65) 06 (100), AV016BaSu (105) 08 (100), AV016FrDi (65) 04 (100) and use of an extract selected from the group consisting of AV016FrSu (105) 08 (100). 24. The method of claim 23, for the manufacture of a medicament for the treatment or prevention of obesity, AV016BaDi (65) 04 (100), AV016BaDi (28) 04 (20), AV016BaSu (65) 09 (100), AV016BaSu (65) ) 01 (100) g, AV016BaSu (65) 01 (100) ng, AV016BaSu (65) 04 (100) AV016BaSu (65) 06 (100) and AV016FrDi (65) 04 (100) . 24. The method of claim 23, for the manufacture of a medicament for the treatment or prevention of obesity, AV016BaDi (65) 04 (100), AV016BaDi (28) 04 (20), AV016BaSu (65) 09 (100) and AV016BaSu (65). Use of an extract selected from the group consisting of 01 (100) alone or in combination of one or more thereof. 24. The method according to claim 23, for the manufacture of a medicament for the treatment or prevention of obesity, alone or in AV016BaSu (65) 09 (100), AV016BaSu (65) 01 (100) and AV016BaSu (65) 04 (100). Use of an extract selected from the group consisting of one or more combinations thereof. 24. The method of claim 23, for the manufacture of a medicament for the treatment or prevention of diabetes, obesity and cardiovascular disease, the extract of AV016BaDi (65) 04 (100) and AV016BaDi (28) 04 (20) alone or one or more thereof Use of extracts in combinations.

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