Patent Publication Number: US-2010129478-A1

Title: Plant Extracts and Methods and Uses Therefore

Description:
FIELD OF INVENTION 
     The invention relates to extracts from  Terminalia  plant species that are capable of being used in methods for managing disease such as diabetes, obesity, cardiovascular diseases, and cancer, and related disease conditions. More particularly, the invention relates to certain extracts from  Terminalia arjuna , their uses as agents for the treatment of certain diseases such as diabetes, obesity, cardiovascular disease and cancer in mammals, particularly humans, processes for obtaining them and delivery formats therefore. 
     BACKGROUND OF THE INVENTION 
     Diabetes Mellitus 
     Diabetes mellitus is a common, serious disease characterized by hyperglycemia. The disease can be divided into two major subclasses: insulin-dependent diabetes mellitus (IDDM), also known as type I diabetes, and non-insulin-dependent diabetes mellitus (NIDDM), also known as type II diabetes (World Health Organization Study Group. Diabetes mellitus. WHO Tech. Rep. Ser. 727:1-113, 1985). IDDM results from insulin deficiency caused by cell-mediated autoimmune destruction of pancreatic beta cells, and generally develops in the young (Yoon J W., Insulin-dependent diabetes mellitus. In: Roitt I M 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 approximately 10-15% of the diabetic population) worldwide (World Health Organization Study Group. Diabetes mellitus. WHO Tech. Rep. Ser. 727:1-113, 1985). In contrast, NIDDM results from a variable combination of insulin resistance and insulin deficiency, and generally develops in adults (Jun H S, 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, NIDDM can also develop at a younger age, as seen in the maturity-onset diabetes of the young (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 diabetic population worldwide. Both IDDM and NIDDM can cause microvascular and macrovascular complications, resulting in increases in morbidity and mortality (Fajans S S, et al., Prediabetes, subclinical diabetes, and latent clinical diabetes: interpretation, diagnosis and treatment. In: Leibel D S, 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 currently thought to be influenced by more than a single gene or environmental factor (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 J L, et al., Genetics of diabetes mellitus. In: Rifkin H, Porte D (Eds.) Diabetes Mellitus Theory and Practice. Elsevier, N.Y., pp. 378-413, 1990). Familial aggregation and the high concordance rate for the disease (60-100%) in identical twins suggest that genetic factors play an important role in the pathogenesis of NIDDM (O&#39;Rahilly S, et al., Type 2 (noninsulin dependent) diabetes mellitus. New genetics for old nightmares. Diabetologia 31:407-414, 1988; Barnett A H, 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 also play a strong role in the development of the disease (Knowler W C, et al., Gm and type 2 diabetes mellitus: an association in American Indians with genetic admixture. Am. J. Hum. Genet. 43:520-526, 1988; Bennett P H, et al., Epidemiology and natural history of NIDDM: non-obese and obese. In: Alberti KGMM, DeFronzo R A, Keen H, Zimmett P (Eds.) International Textbook of Diabetes Mellitus. Wiley, N.Y., pp. 147-176, 1992; Helmrich S P, et al., Physical activity and reduced occurrence of NIDDM. N. Engl. J. Med 325:147-152, 1991). Although the relative contribution of genetic and environmental factors to the development of NIDDM differs among individuals, patients generally have two common metabolic abnormalities, insulin resistance and defects in glucose-stimulated insulin secretion, which lead to the disease state (Saad M F, et al., A two step model for development of non-insulin-dependent diabetes. Am. J. Med. 90:229-235, 1991; DeFronzo R A, et al., Pathogenesis of NIDDM: A balanced overview. Diabetes 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). 
     The insensitivity of the target tissue in response to insulin (insulin resistance) appears to develop first in genetically predisposed subjects in the presence of the necessary environmental factors (Jun H S, 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). To compensate for this, that is, to lower blood glucose and maintain normoglycemia, the secretion of insulin from the beta cells increases, resulting in hyperinsulinemia. Over time, the insulin resistance worsens, and the compensatory action fails, leading eventually to impaired glucose tolerance. Insulin secretion reaches a plateau, and beta cell function is impaired, resulting in insulin deficiency, and leading finally to hyperglycemic NIDDM. In addition, hyperglycemia itself leads to impaired insulin resistance and insulin secretion, exacerbating the disease. 
     The regulation of diet and exercise and/or treatment with insulin or hypoglycemia drugs has been used for the control of diabetes. Treatment with these agents is successful in some cases, but the mortality index continues to rise. Insulin treatment provides symptomatic relief rather than a cure for NIDDM. Hypoglycemic agents such as sulfonylureas and biguanides (metformin) also lower blood glucose, but again, simply provide symptomatic relief. Sulfonylureas lower the blood glucose level by stimulating the release of insulin from pancreatic beta cells. These agents directly stimulate insulin release by closing adenosyl triphosphate (ATP)-sensitive potassium channels and depolarizing the cell membrane (Aguilar-Bryan L, et al., Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. Science 268:423-426, 1995; Tan G H, et al., Pharmacologic treatment options for non-insulin-dependent diabetes mellitus. Mayo Clinic Proceedings 71:763-768, 1996; Lubbos H, et al., Oral hypoglycemic agents in type II diabetes mellitus. American Family Physician. 52:2075-2078, 1995) The side effects of sulfonylureas include hypoglycemia, renal and hepatic disease, gastrointestinal disturbances, increased cardiovascular mortality, dermatological reactions, dizziness, drowsiness and headache. Biguanides lower blood glucose levels by reducing intestinal glucose absorption and hepatic glucose, but not by stimulating insulin secretion. The major side effects of biguanidine are lactic acidosis and increased cardiovascular mortality. Alpha glucosidase inhibitors inhibit intestinal alpha glucosidases and consequently delay the digestion of sucrose and complex carbohydrates. The side effects of alpha glucosidase inhibitors include gastrointestinal side effects and hypoglycemia. Thiazolidinediones improve insulin resistance directly, enhancing the effects of circulating insulin, directly stimulate peripheral glucose uptake and inhibit glucose production in the liver. Thiazolidinediones are only effective in the presence of insulin and may cause red blood cell abnormalities, and headache. 
     Therefore, more effective drugs for the treatment of diabetes are clearly needed. 
     Obesity 
     It is generally recognized in the medical profession that obesity among the populations of the United States and some other countries has been steadily increasing in the last several decades. 
     Obesity is a condition characterized by excessive accumulation of fat on the body. Obesity can be measured by either body weight or by body mass index (BMI). By convention, obesity is said to be present when body weight exceeds by 20 percent the weight listed in typical height-weight index tables. The other measurement of obesity, BMI, is the amount of fat present in the body and is considered a reliable indication of fatness in non-athletic adults. The BMI may be calculated by using the following formula: BMI equals [body weight in kg] divided by [height in meters].sup.2. In general, a normal BMI is between the range of 20 to 25, whereas the BMI of obese individuals is greater than or equal to 30. 
     Such obesity has caused or contributed to marked increases in the occurrence of heart disease, hypertension, diabetes, osteoarthritis of the knees and hips, and increased morbidity resulting from related medical conditions. It has been reported that 50% of all American adults are overweight. 
     There are a variety of methods commonly used for treating obesity. These include dietary adjustments such as caloric restriction, exercise regimens, pharmacotherapy, surgery, and psychotherapy. However, there are problems associated with many of these treatments. 
     The use of human growth (hGH) hormone to treat obesity is described in U.S. Pat. No. 5,597,595. hGH, the most abundant hormone secreted by the pituitary gland, is the primary hormone responsible for growth in mammals, accelerating lipolysis (fat metabolism), and protein synthesis. hGH secretion is markedly decreased in obese individuals. hGH interacts with specific growth hormone receptors, which are widely distributed throughout the body. For example, fat cells (adipocytes) have hGH receptors, and when hGH binds to these receptors, it stimulates the adipocytes to breakdown triglycerides as well as suppresses their ability to take up and accumulate circulating lipids. Administration of hGH needs to be closely monitored, however, as diabetes mellitus may develop in individuals receiving hGH treatment. Diabetes may result because hGH reduces the amount of glucose uptake by cells, thus causing an increase of blood glucose concentration. Not only does this increase in blood glucose levels stimulate the .beta.-cells of the pancreas to secrete extra insulin, but hGH also directly stimulates the .beta.-cells to produce insulin. The combination of these two stimulatory effects so greatly overstimulates insulin secretion that the .beta.-cells literally “burn out.” When this occurs, diabetes mellitus may develop. Guyton and Hall; TEXTBOOK OF MEDICAL PHYSIOLOGY, 938 (1995). 
     One of the best ways to induce weight loss is through dieting or caloric restriction, i.e., establish a caloric deficit by bringing intake below output. The most common method of reducing caloric intake is by means of a low calorie diet. There are, however, a couple of common pitfalls to dieting. One is that when the dieter stops the diet and returns to the usual fare, the incentives to overeat are multiplied. Second, many obese people choose novel or bizarre diets. One example of this type of diet is a high-fat/high-protein diet. These diets are known as ketogenic diets because they contain a high level of cholesterol, which facilitates ketosis. Ketosis is associated with nausea, hypotension, and lethargy. Kaplan et al., supra, 735. 
     Pharmacotherapy may include the administration of drugs that function by increasing serontonin levels in the brain and ultimately controlling appetite. Serotonin (5-hydroxytryptamine, 5HT) is related to several general behaviors such as sleep, emotion, sex, and appetite. The drug Redux™ (dexfenfluramine), which is commonly used as a weight control medication, functions by increasing serotonin levels. Specifically, Redux™ functions by inhibiting re-uptake of serotonin and at the same time stimulating release of serotonin. Several controlled clinical trials have demonstrated that Redux™ can help individuals maintain caloric restriction and a lower body weight for a least a year. It should also be noted that Redux™ appears to reduce appetite in obese more than non-obese individuals. Katzung, BASIC &amp; CLINICAL PHARMACOLOGY 276 (1998). However, there are some adverse side-effects affiliated with Redux™ such as headache, insomnia, and pulmonary hypertension. Id. 
     Surgical methods that cause malabsorption of food or reduce gastric volume have been widely used in people who are markedly obese. Gastric bypass is a procedure used to make the stomach smaller. In particular, the stomach is reduced by stapling or transecting one of the curvatures of the stomach. The primary objective of reducing the volume of the stomach is to slow the passage of food, thereby limiting the amount of food the individual can intake. Although the results are successful, many adverse side effects result. For example, vomiting, electrolyte imbalance, and obstructions may occur. An alternate surgical procedure is lipectomy, which is the removal of fat. This technique is primarily used for cosmetic purposes, however, and has no real impact on weight loss in the long run. Kaplan et al., supra, 276. 
     Hence, for all of the reasons detailed above, a better method for facilitating weight loss in obese or overweight individuals that is readily available and cost-efficient is needed. 
     Cardiovascular Diseases 
     Cardiovascular disease (CVD) is a leading cause of mortality and is responsible for one-third of all global deaths. Nearly 85% of the global mortality and disease burden from CVD is borne by low- and middle-income countries. In India, for example, approximately 53% of CVD deaths are in people younger than 70 years of age; in China, the corresponding figure is 35%. The majority of the estimated 32 million heart attacks and strokes that occur every 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 events are preventable if meaningful action is taken against these risk factors. 
     A staggering number of people are destined to experience a cardiovascular-related disorder sometime in their lives. Almost 1 million Americans die each year as a result of cardiovascular disease, whereas 556,000 die each year from cancer. Despite these facts, people are often more afraid of cancer than they are of vascular disease. 
     Americans have become complacent about the dangers of arterial disease. One reason is that the percentage of young people dying from acute heart attack has plummeted over the past 50 years. Explanations for these reductions include lifestyle changes, greater use of dietary supplements/preventive medications, and improved cardiac medical care. 
     The question is why are so many Americans continuing to die from heart attack and stroke? The fundamental answer is that people are living longer. What has happened is that much of the human population has succeeded in delaying the development of arterial disease. So instead of suddenly dying from a heart attack at age 50, the vascular symptoms do not manifest until the 60s or 80s are reached. At this point, systemic arteriosclerosis has damaged the major organ systems, and multiple degenerative diseases result in diminished quality and quantity of life. 
     Many of the underlying causes of arterial disease have been identified in the scientific literature. Regrettably, cardiologists have only addressed a limited number of these factors, such as prescribing cholesterol-lowering drugs, controlling hypertension, etc. By ignoring the other proven causes for the epidemic of vascular-related diseases, a significant number of Americans are experiencing needless suffering and are dying prematurely. 
       Terminalia Arjuna  Plant Extracts 
       Terminalia arjuna  is a deciduous tree found throughout India growing to a height of around 60-90 feet.  Terminalia arjuna  belongs to the family Combretaceae and is called “ Arjuna ” in vernacular.  Terminalia arjuna  has been used for over 1500 years in India as a cardio tonic and has been indicated for derangement of all three humoursin, vata, pitta and kapha in Ayurveda. The bark of  Terminalia arjuna  has been widely used in Indian system of medicine for a variety of purposes. 
     U.S. Pat. No. 6,162,438 describes an edible herbal compositions, mixture of at least three, preferably at least six, herbs selected from the group consisting of  Terminalia arjuna, Cynara scolymus, Zingibar officinale, Allium sativum, Crataegus oxycantha, Curcuma longa, Boerhaavia diffusa  and  Trigonella foenumgraecum , for use as agents for the control of hypertension, hyper-cholesterolemia and hyperlipidemia in mammals. 
     Sharma V N et al. evaluated the antioxidant and hypocholesterolaemic effects of  Terminalia arjuna  tree bark (a popular cardiotonic substance in Indian pharmacopoeia) and compared it with a known antioxidant, vitamin E by a randomised controlled trial. It was concluded from this trial that,  Terminalia arjuna  tree bark powder has significant antioxidant action that is comparable to vitamin E. In addition, it also has a significant hypocholesterolaemic effect. (Antioxidant and hypocholesterolaemic effects of  Terminalia arjuna  tree-bark powder: a randomised placebo-controlled trial, J Assoc Physicians India 2001 February; 49:231-235) 
     Bioassay-guided separation methods was used to determine the cancer cell growth inhibitory constituents residing in the bark, stem and leaves of the Mauritius medicinal plant  Terminalia arjuna  (Combretaceae) by Chapuis et. al. The cancer cell line active components were found to be gallic acid, ethyl gallate, and the flavone luteolin. Only gallic acid was previously known to occur in this plant. Luteolin has a well-established record of inhibiting various cancer cell lines and may account for most of the rationale underlying the use of  T. arjuna  in traditional cancer treatments. Luteolin was also found to exhibit specific activity against the pathogenic bacterium  Neisseria gonorrhoeae . (Antineoplastic agents 338. The cancer cell growth inhibitory constituents of  Terminalia arjuna  (Combretaceae). J Ethnopharmacol. 1996 August; 53(2):57-63). 
     The bark of  Terminalia arjuna  tree has a long history of use as a cardiac tonic as well, and has been indicated in the treatment of coronary artery disease, heart failure, hypercholesterolemia and for relief of anginal pain. (Miller, A. L. Botanical Influences on cardiovascular disease. Alternative Medicine Review. December 1998, vol 3. No. 6, pages 421-431. 
     Water and methanol extracts of stem bark of  Terminalia arjuna  were found to inhibit the HIV-1 activity by more than 70% at 0.2 mg/ml. (Kandil F. E. et al. A Tannin anti-cancer promoter from  Terminalia arjuna . April 1998, Vol 47. No. 8, pages 1567-1568. 
     Adjuvant therapy with  Terminalia arjuna  bark powder in post Myocardial Infarction angina patients produced relief in angina alongwith improvement in left ventricular ejection fraction (LVEF) and left ventricular mass (LVM). (Dwivedi, S. et al. Beneficial effects of  Terminalia arjuna  in coronary Artery Disease. Indian Heart Journal. September-October 1997, vol. 49. No. 5, pages 507-510.) 
     Ethanolic extract of  Terminalia arjuna  tree bark in doses of 100 mg/kg and 500 mg/kg significantly reduced total and LDL cholesterol levels in hypercholesterolaemic rabbits. (Ram et al. Hypocholesterolaemic effects of  Terminalia arjuna  tree bark. Journal of Ethnopharmacology. Vol 55. No. 3, pages 165-169.) 
     There is no data or technical information in the prior art, which indicates the use of the  Terminalia arjuna  extracts in the treatment or prevention or management of Diabetes and Obesity. 
     It is reported that water and methanol extracts of stem of  T. - arjuna  has shown to have HIV-1 activity. But there are no other references to the use of  T. arjuna  bark extract with successive extraction solvent system, in the treatment of Cancer. 
       Terminalia arjuna  plant has been known for its cardiotonic effect. Also there are reports indicating the use of  T. arjuna  in the treatment of coronary artery disease, heart failure, hypercholesterolemia and for relief of anginal pain. But there are no clear cut evaluation of the mechanisms of action responsible for these activities. In the present invention however, we have established that cells treated with  Terminalia arjuna  extracts showed marked decrease in the amount of total lipids. Also the said extracts have shown 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibition activity. HMG-CoA is the first committed step in cholesterol biosynthetic pathway. HMG-CoA reductase the rate-limiting enzyme step in cholesterol biosynthesis has become the most successful drug treatment for lowering total plasma cholesterol, in particular LDL-cholesterol. 
     There exists a need for the development of new medicines which are effective in treating obesity, cardiovascular disease, certain cancers, and diabetes and related diseases. 
     SUMMARY OF THE INVENTION 
     Objects of the invention will become apparent from the following description and examples. 
     The present invention relates to certain extracts of plant parts from plants of the  Terminalia  species, such as  Terminalia arjuna  that are indicated as being potentially useful in the treatment of diseases such as obesity, cardiovascular disease, certain cancers, diabetes and related diseases. Furthermore, the invention relates to prophylactic compositions comprising extracts of  Terminalia  species, such as extracts from  Terminalia arjuna  species. 
     Brief Description of the Extract Nomenclature: 
     Nomenclature of Plant Extracts. 
     
       
         
         
             
             
         
       
         
         
           
             1. AV-first two letters represents Avesthagen. 
             2. Plant Name: The Plants used and in use are assigned with unique 3-digit number, 016 represents  Terminalia arjuna.    
             3. Part of the plant/Tissue: There is a two letter ID for each plant part used. For example Ba for Bark, Fr for whole Fruit. 
             4. Solvents: The solvents used for extraction are also assigned with two digit numbers 01 for Acetone, 02 for Benzene, 03 for Chloroform, 04 for Ethanol, 05 for Hexane, 06 for Methanol, 07 for Petroleum ether, 08 for water, 09 for ethyl acetate. Percentage of solvents used for extraction is given within bracket (20) for 20% of that solvent. For example if 20% of Ethanol was used for extraction, 04(20). 
             5. Method of Extraction: Successive extraction is referred to as Su whereas direct extraction is referred to as Di, temperature for extraction is written in bracket. For example, Su(65) represents successive extraction at 65° C. 
             6. Type of extract, g: gluey and ng: non-gluey. 
           
         
       
    
    
    
     
       BRIEF DESCRIPTION OF THE TABLES AND FIGURES 
       Table 1: HPLC fingerprint of the extract AV016BaDi(65)04(100). 
       Table 2: HPLC fingerprint of the extract AV016BaDi(28)04(20). 
       Table 3: HPLC fingerprint of the extract AV016BaSu(65)09(100). 
       Table 4: HPLC fingerprint of the extract AV016BaSu(65)01(100). 
       Table 5: HPLC fingerprint of the extract AV016BaSu(65)01(100)ng. 
       Table 6: HPLC fingerprint of the extract AV016BaSu(65)01(100)g. 
       Table 7: HPLC fingerprint of the extract AV016BaSu(65)04(100). 
       Table 8: HPLC fingerprint of the extract AV016BaSu(65)06(100). 
       Table 9: HPLC fingerprint of the extract AV016BaSu(105)08(100). 
       Table 10: HPLC fingerprint of the extract AV016Fr(105)08(100). 
       Table 11: LC/MS Fingerprint of extract AV016BaDi(28)04(20) (TIC Spectrum (Q1+ve mode) 
       Table 12: LC/MS Fingerprint of extract AV016BaDi(28)04(20) (TIC Spectrum (Q1 ve mode) 
       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 AVO16BaSu(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 of extract AV016BaSu(65)01(100) (TIC Spectrum (Q1−ve mode) 
       Table 18. LC/MS Fingerprint of extract AV016BaSu(65)01(100)ng (TIC Spectrum (Q1+ve mode) 
       Table 19. LC/MS Fingerprint of extract AV016BaSu(65)01(100)g (TIC Spectrum (Q1+ve mode) 
       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 inhibition activity of extracts from different  T. arjuna  plant parts 
       Table 26: The effect of insulin and  T. arjuna  extracts on glucose up-take in 3T3-L1 adipocytes. 
       Table 27: Anti-obesity potential of  Terminalia arjuna  extracts. 
       Table 28: Effect of  Terminalia arjuna  extracts on lipid levels in CHO-K1 cells. 
       Table 29: Effect of 20% ethanol extract from  T. arjuna  bark AV016BaDi(28)04(20) on HMG-CoA reductase activity. 
       Table 30: Effect of direct 100% ethanol extract from  T arjuna  bark AV016BaDi(65)04(100) on HMG-CoA reductase activity. 
       Table 31: Effect of  Terminalia arjuna  extracts on cell viability of HepG2 cells. 
         FIG. 1 : alpha-Glucosidase inhibition potential of  T. arjuna  direct ethanol extract, AV016BaDi(65)04(100) and AV016BaDi(28)04(20). 
         FIG. 2 : alpha-Glucosidase inhibition potential of  T. arjuna  fruit extracts, AV016FrDi(65)04(100) AV016FrSu(65)08(100). 
         FIG. 3 : alpha-Glucosidase inhibition potential of  T. arjuna  successive extracts, AV016BaSu(65)01(100)g, AV016BaSu(65)01(100)ng, AV016BaSu(65)04(100) 
         FIG. 4 : alpha-Glucosidase inhibition potential of  T. arjuna  successive methanol and water, AV016BaSu(65)06(100) and AV016BaSu(105)08(100) extracts 
         FIG. 5 : alpha-Glucosidase inhibition potential of  T. arjuna  successive AV016BaSu(65)09(100) extracts 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In a first aspect of the invention there is provided a method for treating a disease in a mammal, which comprises administering to the said mammal an effective non-toxic amount of at least an extract from  Terminalia arjuna  as defined herein. Preferably the mammal is a human being. The skilled addressee will appreciate that “treating a disease” in a mammal means treating, that is to say, alleviating symptoms of the disease and may also mean managing a disease in the sense of preventing such a disease state either advancing ie getting worse or becoming more invasive, or slowing down the rate of advance of a disease. 
     In a second aspect of the invention, there is a provided a prophylactic method for preventing the occurrence of a disease state in a mammal which comprises administering to the said mammal an effective non-toxic amount of an extract from  Terminalia arjuna  as defined herein in the preparation of a comestible (=foodstuff) for prophylaxis against the occurrence of a disease state selected from the group diabetes, obesity, cardiovascular disease, and cancer. Preferably the mammal is human and the said extract comprises a single extract from a plant part of  Terminalia arjuna  or a combination of extracts therefrom as detailed herein. Thus the present invention further relates to extracts which may be isolated from different parts of the  Terminalia arjuna  plant such as the bark and fruit thereof, the preparation of such extracts, medicaments comprising such extracts, and the use of these extracts and constituents for the preparation of a medicament. 
     Extracts of the present invention can be isolated from  Terminalia  tree species, such as  Terminalia arjuna , using conventional organic solvent extraction and supercritical fluid extraction technology. Generally, extracts of the invention capable of functioning in a prophylactic or therapeutic manner as outlined herein can be extracted from any  Terminalia arjuna  plant tissue, such as bark or fruit, depending on the end purpose that is required of the extract. 
     In a third aspect of the present invention there is provided a process for preparing extracts of the invention from plant parts of  Terminalia arjuna  that comprises:
         (i) Pulverizing selected plant material to a powder;   (ii) Subjecting the powdered plant material to solvent extraction;   (iii) Lyophilizing the obtained extracts.       

     The choice of selected plant material may be of any type but is preferably selected from the bark or the fruit of the  Terminalia arjuna  plant. 
     The solvent extraction process may be selected from direct or successive extraction types such as extraction from plant parts in soxhlet apparatus or in flasks at room temperature or at higher temperature with polar and/or non-polar solvent(s). Typically, the extraction process is as outlined herein. 
     It will be apparent to the skilled addressee that the selection of solvent, or mixtures of solvents for each step in the isolation of extracts of the invention showing activity can be guided by results of bioassay analysis of separate fractions, for example as indicated herein and/or as shown in the examples. 
     Also encompassed within the ambit of the invention is a pharmaceutical formulation suitable for use in the treatment of a disease selected from the group diabetes, obesity, cardiovascular disease, and cancer comprising at least one extract as isolated from a  Terminalia  species, such as  Terminalia arjuna , in admixture with a pharmaceutically acceptable carrier. Preferably, the at least one extract is selected from those listed in Tables 1-24 inclusive, depending on design and disease of interest. Preferably the at least one extract is selected from the group of extracts as defined in Tables 25-30 inclusive, again depending on end purpose. Naturally, the skilled addressee will appreciate that such compositions may comprise of two or more plant extracts of the invention in any concentration, which is capable of giving rise to a therapeutic effect. Thus, therapeutic compositions can comprise plant extracts of Terminalia substantially devoid of undesirable contaminating compounds. The plant extracts may have, for example, undergone a number of solvent extraction steps substantially to separate out undesirable components from desirable components such as those alluded to in the examples and aforementioned tables. 
     The invention thus further provides a method for the treatment of a disease selected from the group obesity, cancer, cardiovascular disease, and diabetes in mammals, including humans, which comprises the use of a clinically useful amount of an extract selected from those listed in Tables 1-24 inclusive, preferably those listed in Tables 25-30 inclusive, in a pharmaceutically useful form, once or several times a day or in any other appropriate schedule for example, orally, or intravenously or by delivery to the lungs in a dry or “wet” spray. 
     The amount of compound of extract required to be effective in the treatment of the aforementioned diseases will, of course, vary with the disease being treated and is ultimately at the discretion of the medical or veterinary practitioner. The factors to be considered include the condition being treated, the route of administration, and nature of the formulation, the mammal&#39;s body weight, surface area, age and general condition and the particular compound to be administered. A suitable effective dose of an extract of the invention generally lies in the range of about 0.01 to about 120 mg/kg bodyweight, e.g. 0.1 to about 120 mg/kg body weight, preferably in the range of about 0.1 to 50 mg/kg, for example 0.5 to 50 mg/kg. The total daily dose may be given as a single dose, multiple doses, e.g. two to six times applications per day. For example, for a 75 kg mammal (e.g. a human) the dose range would be about 8 to 9000 mg per day, and a typical dose could be about 50 mg per day. If discrete multiple doses are indicated treatment might typically be 15 mg of a compound of Formula (I) given up to 4 times per day. 
     Whilst it is possible for the active extract to be administered alone, it is preferred to present the active extract in a pharmaceutical formulation. Formulations of the present invention, for medical use, comprise an extract of the invention together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The carrier(s) should be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and substantially non-deleterious to the recipient thereof. 
     The present invention, therefore, further provides a pharmaceutical formulation comprising at least one extract selected from those listed in tables 1-24 inclusive, preferably from those mentioned in tables 25-30 inclusive together with a pharmaceutically acceptable carrier therefore. In a preferment the pharmaceutical formulation comprises at least an extract selected from those listed in tables 25-30, depending on the disease type being treated. Naturally, the skilled addressee will appreciate that when selecting more than one extract from those given in the aforementioned tables for the treatment of any single disease type, that an appropriate selection of extracts fro the disease type will be made. Thus, for example, for the treatment of diabetes, extracts appropriate for doing so will be selected from the said tables. 
     Naturally, the skilled addressee will appreciate that any pharmaceutical formulation comprising an active extract of the invention can include at least one active extract purified from an extract derived from a  Terminalia  species. Thus a pharmaceutical formulation may contain more than one active extract derived from two or more  Terminalia  species. 
     There is also provided a method for the preparation of a pharmaceutical formulation comprising bringing into association an extract of the invention, and a pharmaceutically acceptable carrier therefore. 
     Formulations according to the present invention include those suitable for oral or intravenous administration. 
     Intravenous formulations including at least one extract of the invention and may also be administered in the form of suitable liposomal or niosomal preparations or other suitable delivery vehicle. 
     Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glycerol mono-stearate and sodium laury sulphate. 
     The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active extracts(s) into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active extract(s) into association with a liquid carrier or a finely divided solid carrier or both and then, if necessary, shaping the product into desired formulations. 
     Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets, tablets, lozenges, comprising the active ingredient in a flavoured based, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier. Each formulation generally contains a predetermined amount of the active extract; as a powder or granules; or a solution or suspension in an aqueous or non-aqueous liquid such as a syrup, an elixir, an emulsion or draught and the like. 
     A tablet may be made by compression or moulding optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing an a suitable machine the active extract in a free-flowing form such as a powder or granules, optionally mixed with a binder, (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered extract moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile. 
     A syrup may be made by adding the active extract to a concentrated, aqueous solution of a sugar, for example sucrose, to which may also be added any necessary ingredients. Such accessory ingredient(s) may include flavourings, an agent to retard crystallisation of the sugar or an agent to increase the solubility of any other ingredients, such as a polyhydric alcohol for example glycerol or sorbitol. 
     In addition to the aforementioned ingredients, the formulations of this invention may further include on or more accessory ingredients) selected from diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like. 
     Alternatively, the compositions are dietary supplements, food compositions or beverage compositions suitable for human or animal consumption. 
     The invention describes the HPLC profiles and Mass spectrums of direct and successive solvent extracts of  Terminalia arjuna  plant parts thereby giving each extract an identity of itself. The various solvents used for successive extraction are in order from non-polar to polar side i.e hexane, petroleum ether, ethyl acetate, acetone, ethanol, methanol and water. In case of direct extraction alcoholic solvent alone and in combination with water was used as solvent for extraction. 
     The invention further encompasses novel extracts defined by reference to their HPLC and MS fingerprints as defined in Tables 1-24 inclusive, which are isolated from different parts of  Terminalia arjuna  plant, the preparation of such extracts, the medicaments containing said extracts, and the use of these extracts and constituents for the preparation of a medicament. 
     In a further aspect of the invention, the pharmaceutical formulations for preventing, treating, or managing diabetes and diabetes related disorders, comprise direct extracts of  T. arjuna  bark with 100% ethanol solvent [AV016BaDi(65)04(100)] and 20% ethanol solvent [AV016BaDi(28)04(20)], successive extracts of  T. arjuna  bark with ethyl acetate solvent [AV016BaSu(65)09(100)], successive extract of  T arjuna  bark with acetone solvent [AV016BaSu(65)01(100)g], successive extracts of  T. arjuna  bark with acetone solvent [AV016BaSu(65)01(100)ng], successive extracts of  T. arjuna  bark with ethanol solvent [AV016BaSu(65)04(100)], successive extracts of  T. arjuna  bark with methanol solvent [AV016BaSu(65)06(100)] and successive extracts of  T. arjuna  bark with water solvent [AV016BaSu(105)08(100)], direct extract of  T. arjuna  fruit with ethanol solvent [AV016FrDi(65)04(100)] and successive extract of  T. arjuna  fruit with water solvent [AV016FrSu(105)08(100)], alone or in combination thereof. Such pharmaceutical formulations may contain a pharmaceutically acceptable carrier, excipient, or diluent as outlined herein. 
     In a further aspect of the invention, the pharmaceutical formulations for preventing, treating, or managing obesity and obesity related disorders, comprise direct extracts of  T. arjuna  bark with 100% ethanol solvent [AV016BaDi(65)04(100)] and 20% ethanol solvent [AV016BaDi(28)04(20)], successive extracts of  T. arjuna  bark with bark ethyl acetate, gluey acetone, non-gluey acetone, ethanol and methanol extracts AV016BaSu(65)09(100), AV 016BaSu(65)01(100) g , AV016BaSu(65)01(100)ng, AV016BaSu(65)04(100), AV016BaSu(65)06(100) and AV016FrDi(65)04(100), alone or in combination thereof. The pharmaceutical formulations may contain a pharmaceutically acceptable carrier, excipient, or diluent as outlined herein. 
     In a further aspect of the invention, the pharmaceutical formulations for preventing, treating, or managing cardiovascular diseases (CVD) and CVD related disorders, comprise a direct extract of  T. arjuna  bark with 100% ethanol solvent [AV016BaDi(65)04(100)] and 20% ethanol solvent [AV016BaDi(28)04(20)], successive extract of  T. arjuna  bark with ethyl acetate [AV016BaSu(65)09(100)] and acetone [AV016BaSu(65)01(100)], alone or in combination thereof. The pharmaceutical formulations may contain a pharmaceutically acceptable carrier, excipient, or diluent as outlined herein. 
     In a further aspect of the invention, the pharmaceutical formulations for preventing, treating, or managing cancer and cancer related disorders, comprise successive extracts of  T. arjuna  bark with ethyl acetate [AV016BaSu(65)09(100)], acetone [AV016BaSu(65)01(100)], ethanol [AV016BaSu(65)04(100)], methanol [AV016BaSu(65)06(100)] and water [AV016BaSu(105)08(100)], alone or in combination thereof. The pharmaceutical formulations may contain a pharmaceutically acceptable carrier, excipient, or diluent as outlined herein. 
     In a further aspect of the invention there is provided a comestible, that is to say, a foodstuff comprising at least an extract of the invention, typically in dried form, such as in a lyophilized form selected from those listed in Tables 1-24 herein, and in particular, from those extracts selected from those mentioned in Tables 25-30. The skilled addressee will appreciate that such comestibles may contain more than one extract of the invention and may be used. Such foodstuffs may be used in a prophylactic manner and may contain further extracts having a similar function to the first added extract or further added extracts may be added that have a different prophylactic function. Thus a foodstuff could either comprise extracts that provide for a comestible having a single functional aspect, for example that of having a prophylactic effect against the occurrence of diabetes, or a comestible may have a multi-functional prophylactic effect against two or more disease types. It is thought that a similar multi-functional role could also be assigned to pharmaceutical formulations comprising two or more extracts possessing dissimilar therapeutic or prophylactic properties designed either for prophylaxis or for the treatment of more than one disease(s) in a mammal, particularly in a human. 
     The type of foodstuff or comestible to which at least an extract of the invention may be added includes any processed food such as confectionaries, baked products including breads such as loafs, and flat breads such as pitta bread, naan bread and the like, cakes, snack foods such as muesli bars, compressed dried fruit bars, biscuits, dairy products such as yoghurts, milk and milk-based products such as custards, cream, cheese, butter and crème fraiche, simulated dairy food products such as margarine, olive oil-based spreads, and low fat cream substitutes such as Elmlea products, fruit and vegetable juices, aerated drinks, such as carbonated soft drinks and non-aerated drinks such as squashes, soya milk, rice milk and coconut milk and the like, pastas, noodles, vegetable, seed and nut oils, fruited oils such as sunflower oil, rapeseed oil, olive oil, walnut, hazelnut, and sesame seed oil and the like, and frozen confections such as ice creams, iced yoghurts and the like. 
     A suitable effective dose of an extract of the invention to be included in a comestible generally lies in the range of about 0.01 to about 120 mg/kg bodyweight, e.g. 0.1 to about 120 mg/kg body weight, preferably in the range of about 0.1 to 50 mg/kg, for example 0.5 to 50 mg/kg. The total daily dose may be given as a single dose, multiple doses, e.g. two to six times applications per day. 
     In a further aspect of the invention there is provided use of an extract selected from those of Tables 1-24, and in particular those of Tables 25-30 for the preparation of a medicament for the treatment of a disease selected from the group consisting of diabetes, cancer, cardiovascular disease and obesity. 
     Thus, there is provided use of an extract selected from the group consisting of 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), AV016BaSu(105)08(100), AV016FrDi(65)04(100), and AV016FrSu(105)08(100) for the preparation of a medicament for the treatment or prophylaxis of diabetes and/or related diseases. 
     Additionally, there is provided use of an extract selected from the group consisting AV016BaDi(65)04(100), AV016BaDi(28)04(20), AV016BaSu(65)09(100), AV016BaSu(65)01(100) g , AV016BaSu(65)01(100)ng, AV 016BaSu(65)04(100) and AV016BaSu(65)06(100), and AV016FrDi(65)04(100)) for the preparation of a medicament for the treatment or prophylaxis of obesity. 
     Additionally, there is provided use of an extract selected from the group consisting of AV016BaDi(65)04(100), AV016BaDi(28)04(20), AV016BaSu(65)09(100), AV016BaSu(65)01(100), either singly or in one or more combinations thereof for the preparation of a medicament for the treatment or prophylaxis of cardiovascular disease. 
     Additionally, there is provided use of an extract selected from the group consisting of AV016BaSu(65)09(100), AV016BaSu(65)01(100), AV016BaSu(65)04(100), AV016BaSu(65)06(100) and AV016BaSu(105)08(100), either singly or in one or more combinations thereof for the preparation of a medicament for the treatment or prophylaxis of cancer. 
     The invention will now be exemplified with reference to the following Examples section and accompanying tables and Figures. It is to be understood that the examples are not to be construed as limiting the scope of the invention in any way. 
     EXAMPLES SECTION 
     Example 1 
     Extraction of  Terminalia arjuna    
     Extraction of  Terminalia arjuna  plant parts was carried out by both direct extraction as well as successive extraction method, at room temperature as well as in soxhlet apparatus and related liquid-liquid techniques followed by lyophilization. 
     I. Successive Extraction: 
     Successive extraction from bark of  Terminalia arjuna  was carried out using soxhlet extractor. The solvents used, were based on their sequential polarity starting from non-polar to polar, viz; Hexane, petroleum ether, chloroform, acetone, ethanol, methanol and water at 65° C. 
     The detailed process is given below:
         1. Weigh 100 grams of powdered plant material into the extractor (Soxhlet extractor body) with the cotton on the bottom of the soxhlet apparatus. Cover with cotton on the top. Add 1000 ml of solvent (start with Hexane) in to the round-bottomed flask and place it on the mantle and add few ceramic chips in to it. Add 100 ml of solvent over the material just wetting it.   2. Place the extractor on the flask, which is in turn connected with the condenser.   3. Let the cold water circulate continuously in the condenser from the tap. The set up fits tightly as it is fabricated as one set.   4. Switch on the mantle and set it at 65° C. The vapors of the solvent from the flask would pass through the inlet of the extractor and will get condensed. The condensed (distilled) Solvent will get collected in the Extractor (body) thus extracting the compounds from it.   5. When the plant material is completely filled with solvents, it will get drained in the flask. This process is continuous as long as there is stable heat and water circulation.   6. Continue the extraction for 8 hours, 4-5 cycles per hour.   7. The extract collected in the flask is concentrated by vacuum lyophilization.   8. Follow the same procedure as above successively for the following solvents in the same order. Petroleum Ether, Ethyl acetate, Acetone, Ethanol, Methanol and Water.       

     II. Direct Extraction 
     a. Soxhlet Based Extraction Procedure with 100% Ethanol Solvent:
         1. Weigh 100 grams of powdered plant material in the cloth bag and transfer it into the extractor (Soxhlet extractor body). Cover with cotton on the top. (Make sure the level of material is below one inch of the vapour inlet tube.)   2. Add 1 liter of solvent (start with Pet, ether) in to the round-bottomed flask and place it on the mantle and add few ceramic chips in to it. Add 100 ml of solvent over the material just wetting it.   3. Place the extractor on the flask, which is in turn connected with the condenser.   4. Let the cold water circulate continuously in the condenser from the tap. The set up fits tightly as it is fabricated as one set.   5. Switch on the mantle and set it at 65° C. The vapours of the solvent from the flask would pass through the inlet of the extractor and will get condensed. The condensed (distilled) Solvent will get collected in the Extractor (body) thus extracting the compounds from it.   6. When the plant material is completely filled with solvents, it will get drained in the flask. This process is continuous as long as there is stable heat and water circulation.   7. Continue the extraction for 8 hours, 4-5 cycles per hour.   8. The extract collected in the flask is concentrated by vacuum lyophilization.
 
b. Extraction of  T. Arjuna  Bark with 20% Ethanol Solvent at Room Temperature:
   1. Weigh known quantity (100 grams) of powdered plant material into the conical flask and cover the mouth with aluminum foil to avoid solvent evaporation.   2. Add known volume (500 ml) of 20% ethanol (100 ml ethanol+400 ml water) solvent in to the flask and place it on to the orbital shaker and set the speed at 210 rpm and room 28° C. temperature for the extraction.   3. Extract the plant material for 4 hr and drain the solvent through   4. Centrifuge the filtrate at 1000 rpm for 10 mins. Collect the supernatant and subject it, to lyophilization.   5. Re-extract with 250 ml of solvent for (2×2 hrs).   6. Centrifuge the filtrate at 1000 rpm for 10 mins.   7. Concentrate extract using lyophilizer under vacuum.       

     Example 2 
     Metabolic Fingerprinting of the  Terminalia arjuna  Extracts 
     Metabolic fingerprinting of all the direct and successive extracts from  Terminalia arjuna  plant parts is done by HPLC and LC/MS technique. 
     I. HPLC Fingerprinting: 
     The plant extracts obtained by direct/successive extraction are subjected to HPLC analysis. High Performance Liquid Chromatography (HPLC) is a technique wherein small quantity of the sample is injected into a C-18 column under high pressure and the constituents are allowed to separate based on their interaction with the column and their retention time within the column. The main purpose of HPLC analysis is to find out the total number of constituents in the plant extracts. 
     The samples are prepared for HPLC analysis by dissolving the appropriate weight of the extract in methanol. These samples are filtered and collected in the total recovery HPLC vials. These samples are subjected to separation by Waters 2695 HPLC instrument and then analyzed. 
     Run Conditions: 
     
         
         
           
             1. The software used for HPLC analysis is Waters Millennium 32 . 
             2. The HPLC column used for separation is Waters μBondpack C-18, 5μ, 4.6×150 mm. 
             3. Column temperature is maintained at 25° C. 
             4. Solvent flow rate is set at 1.0 ml per min. HPLC conditions included Gradient chromatography—solvents used are acetonitrile (solvent A), methanol (solvent B) and water (Solvent C and D). 
           
         
       
    
       Terminalia Arjuna  Extracts and HPLC Run Conditions: 
     1.  Terminalia Arjuna  Extracts: 
     
         
         
           
             1. AV016BaDi(65)04(100) 
             2. AV016BaDi(28)04(20) 
             3. AV016BaSu(65)04(100) 
             4. AV016FrDi(65)04(100) 
             5. AV016BaSu(65)06(100) 
           
         
       
    
     I. Method Set: Ethanol — 11 
     Pressure Limits:
         High Limits 4000 psi Low limits 0 psi       

     Programmed Flow:
         Pump Mode: Gradient   Accelerated to 10 ml/min in: 2.0 min (5 ml/min/min)       

     
       
         
           
               
               
               
               
               
               
               
               
             
               
                   
                   
               
               
                   
                 Time 
                 Flow 
                 % A 
                 % B 
                 % C 
                 % D 
                 Curve 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 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  Extracts 
     
         
         
           
             1. AV016BaSu(65)01(100) 
             2. AV016BaSu(65)01(100)g 
             3. AV016BaSu(65)01(100)g 
           
         
       
    
     II. Method Set: Ethyl Acetate — 10a 
     Pressure Limits:
         High Limits 4000 psi Low limits 0 psi       

     Programmed Flow:
         Pump Mode: Gradient   Accelerated to 10 ml/min in: 2.0 min (5 ml/min/min)       

     
       
         
           
               
               
               
               
               
               
               
               
             
               
                   
                   
               
               
                   
                 Time 
                 Flow 
                 % A 
                 % B 
                 % C 
                 % D 
                 Curve 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 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 
                 1 
               
               
                   
               
               
                 A: Acetonitrile, B: Methanol, C: Water 
               
            
           
         
       
     
     3.  Terminalia Arjuna  Extract: 
     1. AV016BaSu(65)09(100) 
     III. Method Set: Ethyl Acetate — 4a 
     Pressure Limits:
         High Limits 4000 psi Low limits 0 psi       

     Programmed Flow:
         Pump Mode: Gradient   Accelerated to 10 ml/min in: 2.0 min (5 ml/min/min)       

     
       
         
           
               
               
               
               
               
               
               
               
             
               
                   
                   
               
               
                   
                 Time 
                 Flow 
                 % A 
                 % B 
                 % C 
                 % D 
                 Curve 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 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 
                 1 
               
               
                   
               
               
                 A: Acetonitrile, B: Methanol, C: Water 
               
            
           
         
       
     
     4.  Terminalia Arjuna  Extract: 
     I. AV016BaSu(105)08(100) 
     2. AV016FrSu(105)08(100) 
     IV. Method Set: Gy_Water — 12 
     Pressure Limits:
         High Limits 4000 psi Low limits 0 psi       

     Programmed Flow:
         Pump Mode: Gradient   Accelerated to 10 ml/min in: 2.0 min (5 ml/min/min)       

     
       
         
           
               
               
               
               
               
               
               
               
             
               
                   
                   
               
               
                   
                 Time 
                 Flow 
                 % A 
                 % B 
                 % C 
                 % D 
                 Curve 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 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 
                 1 
               
               
                   
               
               
                 A: Acetonitrile, B: Methanol, C: Water 
               
            
           
         
       
     
     II. Liquid Chromatography Mass Spectrometry (LC/MS) Fingerprinting: 
     Mass spectroscopy, is an instrumental approach that allows for the mass measurement of molecules. The five basic components of mass spectrometer are a vacuum system, a sample introduction device, an ionization source, a mass analyzer and an ion detector. Combining these parts a mass spectrometer determines the molecular weight of chemical compounds by ionizing, separating and measuring molecular ions according to their mass-to-charge ratio (m/z). 
     Run conditions used for LC/MS fingerprinting of  Terminalia arjuna  is shown down.
         1. Q-Trap LC/MS instrument from Applied Biosystems 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 mmI.D.x 150 mm.   3. Column temperature is maintained at 25° C.   4. Solvent flow rate is set at 1.0 ml per min. HPLC conditions included Gradient chromatography—solvents used are acetonitrile (solvent C), methanol (solvent B) and water (Solvent D).       

     1.  Terminalia Arjuna  Extracts: 
     AV016BaSu(65)09(100), AV016BaSu(65)01(100), AV016BaSu(65)01(100)g, AV016BaSu(65)01(100)g, AV016BaSu(65)04(100), AV016BaSu(65)06(100), AV016BaSu(65)06(80), AV016BaDi(65)04(100), and AV016FrDi(65)04(100) 
     a. LC/MS Sample Run Conditions for all the Above-Mentioned  Terminalia Arjuna  Samples: 
     
       
         
           
               
               
             
               
                   
               
             
            
               
                 Mass Spectrometer 
                 QTrap 0 MASS SPEC 
               
               
                 Config Table Ver 
                 01 
               
               
                 Firmware Ver 
                 M401400 B4T0301 M3L1400 B3T0300 
               
               
                 Component Name 
                 Linear Ion Trap Quadrupole 
               
               
                   
                 LC/MS/MS Mass Spectrometer 
               
               
                 Component ID 
                 QTrap 
               
               
                 Manufacturer 
                 AB Sciex Instruments 
               
               
                 Model 
                 027170 - C 
               
               
                 S/N 
                 M1100301 
               
               
                 Time from start = 
                 0.0500 min 
               
               
                 Mass Spectrometer 
                 Qtrap 0 MASS SPEC 
               
               
                 Start of Run - Detailed Status 
               
               
                 Vacuum Status 
                 At Pressure 
               
               
                 Vacuum Gauge (10e−5 Torr) 
                 0.7 
               
               
                 Backing Pump 
                 Ok 
               
               
                 Dual Turbo Pump 
                 Normal 
               
               
                 Sample Introduction Status 
                 Ready 
               
               
                 Source/Ion Path Electronics 
                 On 
               
               
                 Source Type 
                 Turbo Spray 
               
               
                 Source Temperature (at setpoint) 
                 400.0 C. 
               
               
                 Source Exhaust Pump 
                 Ok 
               
               
                 Interface Heater 
                 Ready 
               
               
                 Mass Spectrometer 
                 Qtrap 0 MASS SPEC 
               
               
                 End of Run - Detailed Status 
               
               
                 Vacuum Status 
                 At Pressure 
               
               
                 Vacuum Gauge (10e−5 Torr) 
                 0.7 
               
               
                 Backing Pump 
                 Ok 
               
               
                 Dual Turbo Pump 
                 Normal 
               
               
                 Sample Introduction Status 
                 Ready 
               
               
                 Source/Ion Path Electronics 
                 On 
               
               
                 Source Type 
                 Turbo Spray 
               
               
                 Source Temperature (at setpoint) 
                 400.0 C. 
               
               
                 Source Exhaust Pump 
                 Ok 
               
               
                 Interface Heater 
                 Ready 
               
               
                 Time from start = 
                 61.4833 min 
               
               
                   
               
            
           
         
       
     
     PE LC-200 Pump Method Properties 
     PE LC-200 Quaternary Pump 
     Minimum Pressure (psi): 0.0
 
Maximum Pressure (psi): 6100.0
 
Shutdown Time (min): 999.9
 
     Step Table: 
       
     
       
         
           
               
               
               
               
               
               
               
               
               
               
             
               
                   
               
               
                   
                 Total Time 
                 Flow Rate 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Step 
                 (min) 
                 (μl/min) 
                 GradientProfile 
                 A (%) 
                 B (%) 
                 C (%) 
                 D (%) 
                 TE#1 
                 TE#2 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 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 
               
               
                   
               
            
           
         
       
     
     Quantitation Information: 
     Sample Type Unknown 
     Dilution Factor: 1.000000 
     Custom Data: 
     Quantitation Table: 
     Period 1: 
     Scans in Period: 2243 
     Relative Start Time: 0.00 msec 
     Experiments in Period: 1 
     Period 1 Experiment 1: 
     Scan Type: Q1 MS (Q1) 
     Polarity: Positive 
     Scan Mode: Profile 
     Resolution Q1: UNIT 
     Intensity Thres.: 0.00 cps 
     Settling Time: 0.0000 ms 
     MR Pause: 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  Extracts: 
     AV016BaSu(105)08(100), AV016FrSu(105)08(100) and AV016BaDi(28)04(20). 
     a. LC/MS Sample Run Conditions for all the Above-Mentioned  Terminalia Arjuna  Samples: 
     
       
         
           
               
               
             
               
                   
               
             
            
               
                 Mass Spectrometer 
                 QTrap     0     MASS 
               
               
                 SPEC 
               
               
                 Config Table Ver 
                 01 
               
               
                 Firmware Ver 
                 M401400 B4T0301 M3L1400 B3T0300 
               
               
                 Component Name 
                 Linear Ion Trap Quadrupole LC/MS/ 
               
               
                   
                 MS Mass Spectrometer 
               
               
                 Component ID 
                 QTrap 
               
               
                 Manufacturer 
                 AB Sciex Instruments 
               
               
                 Model 
                 027170-C 
               
               
                 S/N 
                 M1100301 
               
               
                 Time from start = 2.1000 min 
               
               
                 Mass Spectrometer 
                 QTrap     0     MASS 
               
               
                 SPEC 
               
               
                 Start of Run - Detailed Status 
               
               
                 Vacuum Status 
                 At Pressure 
               
               
                 Vacuum Gauge (10e−5 Torr) 
                 0.7 
               
               
                 Backing Pump 
                 Ok 
               
               
                 Dual Turbo Pump 
                 Normal 
               
               
                 Sample Introduction Status 
                 Ready 
               
               
                 Source/Ion Path Electronics 
                 On 
               
               
                 Source Type 
                 Turbo Spray 
               
               
                 Source Temperature (at setpoint) 
                 400.0 C. 
               
               
                 Source Exhaust Pump 
                 Ok 
               
               
                 Interface Heater 
                 Ready 
               
               
                 Time from start = 2.1167 min 
               
               
                 Mass Spectrometer 
                 QTrap     0     MASS 
               
               
                 SPEC 
               
               
                 End of Run - Detailed Status 
               
               
                 Vacuum Status 
                 At Pressure 
               
               
                 Vacuum Gauge (10e−5 Torr) 
                 0.7 
               
               
                 Backing Pump 
                 Ok 
               
               
                 Dual Turbo Pump 
                 Normal 
               
               
                 Sample Introduction Status 
                 Ready 
               
               
                 Source/Ion Path Electronics 
                 On 
               
               
                 Source Type 
                 Turbo Spray 
               
               
                 Source Temperature (at setpoint) 
                 400.0 C. 
               
               
                 Source Exhaust Pump 
                 Ok 
               
               
                 Interface Heater 
                 Ready 
               
               
                 Time from start = 42.9333 min 
               
               
                   
               
            
           
         
       
     
     PE LC-200 Pump Method Properties 
     PE LC-200 Quaternary Pump 
     Minimum Pressure (psi): 0.0
 
Shutdown Time (min): 999.9
 
     Step Table: 
       
     
       
         
           
               
               
               
               
               
               
               
               
               
               
             
               
                   
               
               
                   
                 Total Time 
                 Flow Rate 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Step 
                 (min) 
                 (μl/min) 
                 GradientProfile 
                 A (%) 
                 B (%) 
                 C (%) 
                 D (%) 
                 TE#1 
                 TE#2 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 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 Properties 
     Interval (sec): 0.200
 
Rate (pts/sec): 5
 
     Polarity: Bipolar 
     Channel Summary 
       
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                 Name: 
                 Interpreted Value Full Scale: 
                   
               
               
                 No. 
                 Voltage (volts): 
                 Status: 
                 Interpreted Unit: 
               
               
                   
               
             
            
               
                 1 
                 100.0 
                 % 
                 1.0 Used 
               
               
                   
               
            
           
         
       
     
     Quantitation Information: 
     Sample Type Unknown 
     Dilution Factor: 1.000000 
     Custom Data: 
     Quantitation Table: 
     Period 1: 
     Scans in Period: 1495 
     Relative Start Time: 0.00 msec 
     Experiments in Period: 1 
     Period 1 Experiment 1: 
     Scan Type: □1 MS (Q1) 
     Polarity: Positive 
     Scan Mode Profile 
     Resolution Q1: UNIT 
     Intensity Thres.: 0.00 cps 
     Settling Time: 0.0000 ms 
     MR Pause: 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 
     Determination of the Bio-Therapeutic Potential of  Terminalia Arjuna  Extracts 
     I. Anti-Diabetic Potential: 
     Acute glucose elevations after meal ingestion are associated with a variety of glucose-mediated tissue defects oxidative stress, glycation, and advanced glycation end product formation which have far reaching structural and functional consequences for virtually every human organ system. Lowering glycosylated hemoglobin to levels that prevent or delay these complications can be achieved only by reducing both postprandial and fasting plasma glucose levels. 
     The α-Glucosidase complex in the small intestine is involved in sugar absorption. Thus, inhibitors of α-Glucosidase would limit the absorption of dietary carbohydrates and in turn suppress postprandial hyperglycemia. Therefore, α-Glucosidase inhibitors are promising drug candidates in the treatment and prevention of diabetes. The α-Glucosidase inhibitors (acarbose, voglibose, miglitol) have been effective in delaying the digestion and absorption of carbohydrates, thus diminishing the postprandial surge in blood glucose levels without loss of calories. 
     1. Alpha-Glucosidase Assay 
     Alpha-Glucosidase hydrolyzes the terminal, non-reducing 1,4-linked alpha-D-glucose residues with release of alpha-D-glucose. In the assay the enzymatic activity is determined by measuring the increase in absorbance at 400 nm caused by the hydrolysis of p-nitrophenyl-α-D-glucopyranoside with the release of p-nitrophenol. 
     a. Reagents:
         Potassium phosphate buffer 0.1M, pH 6.8   PNPG 4 mM   Sodium carbonate 0.2 M   BSA 0.2% in Buffer   Enzyme 1.2 U/ml in 0.2% BSA
 
b. Procedure:
       

     
       
         
         
             
             
         
       
     
     c. Results and Discussion: 
     Successive and direct extract from  Terminalia arjuna  bark and fruit extracts assayed for their alpha-Glucosidase inhibition potential showed that  T. arjuna  extract showed dose dependent decrease in alpha-Glucosidase inhibition. IC 50  is defined as the amount of extract required for 50% inhibition in the enzyme activity. 
     It was found that all the extracts of  T. arjuna  showed higher alpha-Glucosidase inhibition potential than commercially available acarbose, as indicated by IC 50  values. (Table 25). 
     2 Glucose Uptake Assay: 
     The deoxy-glucose uptake assay by fat cells or adipoctes (3T3-L1) help to identify the extracts/bioactives that helps to increase the glucose uptake by the fat cells. Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. Green and Kehinde (1975) first demonstrated that confluent 3T3-L1 cells could be hormonally induced to differentiate into adipocytes over the course of 6-9 d. 
     The assay will thereby help in screening of phytoextracts that will help the diabetic individuals by quick removal of glucose from blood stream and simultaneously will help the fat cells to build a better storage of energy. 
     Tritiated deoxyglucose uptake by 3T3-L1 is one of the most accepted assay for the in-vitro screening of active principles with anti-diabetic activity 
     a. 3T3-L1 Cell Culture: 
     3T3-L1 fibroblasts were cultured in DME-F12 media containing 10% FCS, NEAA, glutamine, antibiotics, anti-mycotics, etc., in an atmosphere of 5% CO2 at 37° C. Fibroblasts were cultured up to confluency. Subcultures were done at three-day intervals with trypsin-EDTA solution treatment. 
     b. Maturation of the Fibroblasts to Adipocytes 
     Differentiation was induced by treating the cells with 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 h. The cells were cultured in DMEM media with standard pre-described supplements for the next 6-10 days. Cells were used only when at least 95% of the cells showed an adipocyte phenotype by accumulation of lipid droplets. These were taken up for glucose uptake studies with tritiated glucose. 
     c. Assay Procedure 
     Distribute 50,000 of differentiated 3T3-L1 adipocytes in each well of a new 96 well plate. Incubate with KRH buffer at 37° C. for 10 min. Add insulin 10 nM, 25 nM and 50 nM to the respective. To the experimental sets treatment was done with or without insulin 10 nM in presence of test compound (333.0 μg). This was followed by incubation further at 37° C. for 15 min. Wash the cells once with KRH buffer. Initiate glucose uptake reaction by adding 0.1 mM 2-deoxy glucose containing 2-deoxy[3H] glucose (final concentration 12.2 kBq/ml). Incubate for 1 hr at 37° C. Terminate assay by adding 40 μM Cytochalasin B. Wash the cells trice with ice-cold KRH buffer Solubilize cells in 20 μl 1% Triton X. Incubate for 10 min at 37° C. Count radioactivity on a micro-titre plate radioactive counter 
     d. Results and Discussion: 
     Glucose uptake activity was analyzed by measuring the uptake of 0.1 mM deoxy-glucose containing deoxy-tritiated-glucose containing 2.2 kBq/mL. 
     As seen from Table 26, direct extract of  T. arjuna  bark with 100% ethanol solvent [AV016BaDi(65)04(100)] showed stimulation index of 3.0 in absence of insulin and 2.0 in presence of 10 nM Insulin. The data thus suggest that AV016BaDi(65)04(100) has potent insulin mimetic potential. 
     2. Anti-Obesity Potential: 
     Studies have demonstrated that central obesity is more strongly related to lipid/lipoprotein abnormalities than is general obesity. Obesity may confer cardiac dysfunction due to lipid accumulation in cardiomyocytes and also induces type II diabetes. These adverse lipid/lipoprotein profiles in obese individuals are important, because they may be responsible for their increased risk for cardiovascular disease (CVD). 
     The assay is a semi quantitative method used for quantification of Adipose differentiation and hence the lipid and triglycerides contents in the cell after the stimulation of the cells for differentiation in presence and absence of the plant extracts. 
     a. Assay Principle 
     Oil Red staining is a semi quantitative method used for quantification of Adipose differentiation. Staining with oil-soluble dyes is based on the greater solubility of the dye in the lipoid substances than in the usual hydroalcoholic dye solvents. Oil Red 0 is a lipophilic red dye which stains intracellular lipid drops. The quantitation of lipid can be done following the extraction of intracellular lipid with organic solvent. Hence it is used to estimate lipid and triglycerides contents in the cell after plant extract stimulation of cells 
     b. Assay Protocol 
     Seed 3T3-L1 cells at a density of 10 5  cells/well with the differentiating medium and extracts (0.33 mg) and incubated for 24 h at 37° C. and 5% CO 2 . The medium was then removed after 24 hr and fresh differentiating medium was added and the cells were kept in the medium for another 24 hrs. Post 48 hrs from day one the medium was removed and Insulin only medium (DMEM F12+FBS+Insulin) was added and the cells were kept for 24 h. Post 24-hr incubation medium was aspirated and Oil O red staining was performed. 
     c. Oil Red O Staining: 
     Total volume in each well for reagents is 500 μl/well. Media was removed and cells were washed with PBS thrice. For fixing the cells they were incubated for 1 h with 4% Formalin. Remove the Formalin and again wash with PBS twice. For staining with Oil Red O, incubate it with the stain for 1 h. After 1 h remove the stain and wash it with PBS and dry it for 10 minutes. Add 4% NP40. Read absorbance at 520 nm in Spectrophotometer. 
     d. Results and Discussion: 
     Anti-obesity assay with 3T3-L1 cells showed that  Terminalia arjuna  extracts showed inhibition in the lipid accumulation. As seen from Table 27  Terminalia arjuna  bark direct 100% and 20% ethanol extracts, AV016BaDi(65)04(100) and AV016BaDi(28)04(20) extracts showed 76.3 and 64.3% inhibition as compared to control. 
     In case of  Terminalia arjuna  successive bark ethyl acetate, gluey acetone, non-gluey acetone, ethanol and methanol extracts AV016BaSu(65)09(100), AV016BaSu(65)01(100) g , AV016BaSu(65)01(100)ng, AV016BaSu(65)04(100) and AV016BaSu(65)06(100) gave inhibition of 61.9, 58.5, 86.8, 49.4 and 57.9% inhibition as compared to control.  Terminalia arjuna  fruit direct 100% ethanol extract AV016FrDi(65)04(100) gave inhibition of 41.9% inhibition as compared to control. 
     Assay+ve control showed 104% inhibition as compared to control. 
     3. Cardiovascular Disease: 
     Elevated cholesterol is associated with a greater-than-normal risk of atherosclerosis and cardiovascular disease. High blood triglyceride levels alone do not cause atherosclerosis. But lipoproteins that are rich in triglycerides also contain cholesterol, which causes atherosclerosis in many people with high triglycerides. So high triglycerides may be a sign of a lipoprotein problem that contributes to CHD. 
     1. Cell Based Assay for Checking Levels of Lipid Accumulation: 
     a. Assay Principle: 
     Growing the cells in presence of lipoprotein deficient serum induces the lipid biosynthesis in the cells. Assay is performed on the cells induced by growing in lipoprotein deficient serum followed by total lipid estimation using oil red staining. Oil Red O is a lipophilic red dye used to stain neutral lipids in cells. The amount of staining is directly proportional to the amount of lipid in the cell. The content of lipid was checked across the extract treated and control sets and the amount of dye and hence lipid content was measured spectrophotometrically. 
     b. Assay Procedure 
     CHO-K1 cells were seeded at density of 10 cells/well in 12 well-plate format. The cells were grown to 80% confluence in DMEM-F12 medium with 10% fetal calf serum, 2 mM glutamine, 1.5 gm/L sodium bicarbonate and anti-mycotics and penicillin and streptomycin at 37 C in 5% CO2 environment. After 2 days of incubation the medium was replaced with DMEM-F 12 medium with 10% lipoprotein deficient serum, 2 mM glutamine, 1.5 gm/L sodium bicarbonate and anti-mycotics and penicillin and streptomycin and grown for additional 24 hrs at 37 C in 5% CO2 environment. At the end of 24 hrs to the extract treated sets  Terminalia arjuna  at concentration of 0.5 mg/ml were added whereas the control sets were treated with vehicle (DMSO). The incubation was continued for additional 4 hrs after which the cells were washed with sterile PBS buffer, fixed and stained with Oil Red O. 
     c. Oil Red O Staining: 
     Total volume in each well for reagents is 500 μl/well. Media was removed and cells were washed with PBS thrice. For fixing the cells they were incubated for 1 h with 4% Formalin. Remove the Formalin and again wash with PBS twice. For staining with Oil Red O, incubate it with the stain for 1 h. After 1 h remove the stain and wash it with PBS and dry it for 10 minutes. Add 4% NP40. Read absorbance at 520 nm in Spectrophotometer. 
     d. Results and Discussion: 
     As seen from Table 28,  Terminalia arjuna  extracts showed marked decrease in the amount of total lipids in the extract treated cells.  Terminalia arjuna  bark successive extracts AV016BaSu(65)09(100) and AV016BaSu(65)01(100) showed 22% and 21% inhibition as compared to control set.  Terminalia arjuna  direct ethanol extracts AV016BaDi(65)04(100) and AV016BaDi(28)04(20) showed 31% and 28% inhibition in the levels of total lipids as compared to the control set. 
     2. HMG-CoA Reductase Assay: 
     3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is the first committed step in cholesterol biosynthetic pathway. HMG-CoA reductase the rate-limiting enzyme step in cholesterol biosynthesis has become the most successful drug treatment for lowering total plasma cholesterol, in particular LDL-cholesterol. 
     a. Assay Principle: 
     HMG-CoA reductase assay is based on the oxidation of NADPH to NADP catalyzed by a fixed concentration of HMG-CoA reductase from liver microsomal fraction. 
     
       
         
         
             
             
         
       
     
     The oxidation of NADPH to NADP +  is accompanied by a decrease in absorbance at 340 nm and is directly proportional to the HMG-CoA reductase activity in the sample. 
     b. Results and Discussion: 
     The potential of  Terminalia arjuna  bark direct 100% ethanol and 20% ethanol extracts AV016BaDi(28)04(20) and AV016BaDi(65)04(100) were checked for their HMG-CoA reductase inhibition potential. It was found that both AV016BaDi(28)04(20) and AV016BaDi(65)04(100) showed 93% inhibition (Table 29) and 63.1% (Table 30) in HMG-CoA reductase activity as compared to control set. 
     4. Anti-Cancer Assay: 
     The Anti-cancer potential of the  Terminalia arjuna  plant extracts was determined by screening the plant extracts in vitro for cytotoxicity on the human hepatic cell line HepG2. 
     a. Experimental Details: 
     The cells were seeded at a density of 2.5×10 5  cell per ml in a 24 well plate. The cells were allowed to attach for 2 hour before adding the extracts at a concentration of 10 μg/ml. 48 hour after incubation with vehicle i.e DMSO, ascorbic acid and extracts AV016BaSu(65)09(100), AV016BaSu(65)01(100), AV016BaSu(65)04(100). The cell viability was determined after 24 hours. 
     b. Assay Protocol 
     Seed HepG2 at a cell density of 2.5×10 5  cells per ml. Add of the Ascorbic acid and  Terminalia arjuna  extracts to final concentration of 10 μg/ml. Incubate in 5% CO 2  incubator at 37° C. for 48 hour. Trypsinize cell. Mix with 1 part trypan blue and 4 parts PBS and count the viable cells and the non-viable cells by counting the cells that has taken typan blue (non-viable cells) and the ones that has excluded it (viable cells). 
     c. Results and Discussions: 
     It was found that treatment of HepG2 cells with  T. arjuna  successive ethyl acetate AV016BaSu(65)09(100), acetone AV016BaSu(65)01(100), and ethanol AV016BaSu(65)04(100), extracts there was decrease in the total cell count and in the number of viable cells in case of drug treated set as compared to the untreated set or the cells with DMSO set (Table 31). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 HPLC fingerprint of extract AV016BaDi(65)04(100) 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Retention 
                   
                   
                   
               
               
                   
                 Time 
                 Area 
                 % Area 
                 Height 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 1.746 
                 83501 
                 0.03 
                 18746 
               
               
                 2 
                 1.887 
                 21939706 
                 9.17 
                 3508953 
               
               
                 3 
                 2.482 
                 424664 
                 0.18 
                 27549 
               
               
                 4 
                 3.165 
                 694426 
                 0.29 
                 38196 
               
               
                 5 
                 4.499 
                 535430 
                 0.22 
                 20883 
               
               
                 6 
                 5.860 
                 1140766 
                 0.48 
                 31447 
               
               
                 7 
                 6.488 
                 2287279 
                 0.96 
                 105829 
               
               
                 8 
                 7.680 
                 2247351 
                 0.94 
                 73739 
               
               
                 9 
                 8.259 
                 1172094 
                 0.49 
                 53148 
               
               
                 10 
                 8.702 
                 2209397 
                 0.92 
                 93831 
               
               
                 11 
                 9.425 
                 2269048 
                 0.95 
                 89553 
               
               
                 12 
                 10.177 
                 3848206 
                 1.61 
                 111334 
               
               
                 13 
                 10.485 
                 1536103 
                 0.64 
                 99241 
               
               
                 14 
                 10.730 
                 1306318 
                 0.55 
                 102448 
               
               
                 15 
                 11.155 
                 3705535 
                 1.55 
                 129030 
               
               
                 16 
                 12.263 
                 10146875 
                 4.24 
                 464227 
               
               
                 17 
                 12.335 
                 6747979 
                 2.82 
                 548102 
               
               
                 18 
                 13.000 
                 30474340 
                 12.74 
                 1778085 
               
               
                 19 
                 13.697 
                 3437393 
                 1.44 
                 233361 
               
               
                 20 
                 14.232 
                 8223596 
                 3.44 
                 277375 
               
               
                 21 
                 14.532 
                 14638851 
                 6.12 
                 819721 
               
               
                 22 
                 15.023 
                 12657916 
                 5.29 
                 590365 
               
               
                 23 
                 15.452 
                 5471266 
                 2.29 
                 306520 
               
               
                 24 
                 15.998 
                 16599596 
                 6.94 
                 1078704 
               
               
                 25 
                 16.299 
                 4397452 
                 1.84 
                 318952 
               
               
                 26 
                 16.463 
                 2424066 
                 1.01 
                 310668 
               
               
                 27 
                 16.863 
                 6414264 
                 2.68 
                 310396 
               
               
                 28 
                 17.166 
                 12652492 
                 5.29 
                 355032 
               
               
                 29 
                 17.683 
                 4940680 
                 2.07 
                 294832 
               
               
                 30 
                 18.118 
                 11457746 
                 4.79 
                 318670 
               
               
                 31 
                 18.673 
                 2951869 
                 1.23 
                 232444 
               
               
                 32 
                 18.872 
                 6643592 
                 2.78 
                 219376 
               
               
                 33 
                 19.475 
                 1933618 
                 0.81 
                 176183 
               
               
                 34 
                 19.668 
                 2454443 
                 1.03 
                 182659 
               
               
                 35 
                 19.884 
                 4858876 
                 2.03 
                 167366 
               
               
                 36 
                 20.329 
                 950214 
                 0.40 
                 139162 
               
               
                 37 
                 20.543 
                 6254360 
                 2.61 
                 134329 
               
               
                 38 
                 21.427 
                 1804592 
                 0.75 
                 107259 
               
               
                 39 
                 21.716 
                 7198552 
                 3.01 
                 109172 
               
               
                 40 
                 23.185 
                 2885830 
                 1.21 
                 72715 
               
               
                 41 
                 23.905 
                 3042057 
                 1.27 
                 54167 
               
               
                 42 
                 24.977 
                 665797 
                 0.28 
                 38055 
               
               
                 43 
                 25.258 
                 1486354 
                 0.62 
                 35170 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 HPLC fingerprint of extract AV016BaDi(28)04(20). 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Retention 
                   
                   
                   
               
               
                   
                 Time 
                 Area 
                 % Area 
                 Height 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 1.724 
                 259750 
                 0.10 
                 58489 
               
               
                 2 
                 1.897 
                 27826788 
                 10.53 
                 3799266 
               
               
                 3 
                 2.486 
                 571316 
                 0.22 
                 50306 
               
               
                 4 
                 3.045 
                 1106233 
                 0.42 
                 51910 
               
               
                 5 
                 3.732 
                 674291 
                 0.26 
                 29021 
               
               
                 6 
                 4.182 
                 1056425 
                 0.40 
                 32454 
               
               
                 7 
                 5.572 
                 2403677 
                 0.91 
                 47788 
               
               
                 8 
                 6.082 
                 3557804 
                 1.35 
                 161895 
               
               
                 9 
                 6.484 
                 757991 
                 0.29 
                 55308 
               
               
                 10 
                 7.264 
                 3549212 
                 1.34 
                 97606 
               
               
                 11 
                 7.883 
                 2512425 
                 0.95 
                 92337 
               
               
                 12 
                 8.299 
                 3676625 
                 1.39 
                 140954 
               
               
                 13 
                 9.060 
                 4300244 
                 1.63 
                 140449 
               
               
                 14 
                 9.699 
                 6186918 
                 2.34 
                 155916 
               
               
                 15 
                 10.124 
                 2085208 
                 0.79 
                 151077 
               
               
                 16 
                 10.796 
                 7036369 
                 2.66 
                 179944 
               
               
                 17 
                 11.555 
                 17109248 
                 6.48 
                 350960 
               
               
                 18 
                 12.574 
                 31568344 
                 11.95 
                 1286226 
               
               
                 19 
                 13.038 
                 2562494 
                 0.97 
                 285400 
               
               
                 20 
                 13.307 
                 4871494 
                 1.84 
                 288682 
               
               
                 21 
                 14.002 
                 26201944 
                 9.92 
                 838854 
               
               
                 22 
                 14.597 
                 14156853 
                 5.36 
                 465017 
               
               
                 23 
                 15.150 
                 4497591 
                 1.70 
                 322199 
               
               
                 24 
                 15.663 
                 16422598 
                 6.22 
                 962872 
               
               
                 25 
                 15.912 
                 3863537 
                 1.46 
                 326789 
               
               
                 26 
                 16.103 
                 6416104 
                 2.43 
                 315587 
               
               
                 27 
                 16.564 
                 4817209 
                 1.82 
                 305649 
               
               
                 28 
                 16.767 
                 8560395 
                 3.24 
                 305526 
               
               
                 29 
                 17.221 
                 2824887 
                 1.07 
                 283965 
               
               
                 30 
                 17.400 
                 2813123 
                 1.06 
                 282612 
               
               
                 31 
                 17.661 
                 16467191 
                 6.23 
                 382391 
               
               
                 32 
                 18.625 
                 6447644 
                 2.44 
                 200677 
               
               
                 33 
                 19.302 
                 3348258 
                 1.27 
                 168434 
               
               
                 34 
                 19.568 
                 11717199 
                 4.44 
                 149595 
               
               
                 35 
                 21.405 
                 4945619 
                 1.87 
                 87932 
               
               
                 36 
                 22.629 
                 4393424 
                 1.66 
                 69440 
               
               
                 37 
                 24.162 
                 2305522 
                 0.87 
                 34152 
               
               
                 38 
                 25.963 
                 255400 
                 0.10 
                 9559 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 HPLC fingerprint of extract AV016BaSu(65)09(100). 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Retention 
                   
                   
                   
               
               
                   
                 Time 
                 Area 
                 % Area 
                 Height 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 2.475 
                 25395907 
                 5.13 
                 3334075 
               
               
                 2 
                 3.244 
                 1688929 
                 0.34 
                 136464 
               
               
                 3 
                 4.656 
                 9262251 
                 1.87 
                 280029 
               
               
                 4 
                 5.254 
                 1188708 
                 0.24 
                 48754 
               
               
                 5 
                 6.603 
                 12296585 
                 2.48 
                 364675 
               
               
                 6 
                 6.899 
                 5848912 
                 1.18 
                 389909 
               
               
                 7 
                 7.233 
                 1367772 
                 0.28 
                 130776 
               
               
                 8 
                 7.428 
                 1332175 
                 0.27 
                 138611 
               
               
                 9 
                 7.896 
                 7084034 
                 1.43 
                 280115 
               
               
                 10 
                 8.260 
                 6705156 
                 1.35 
                 380995 
               
               
                 11 
                 8.576 
                 2924953 
                 0.59 
                 246136 
               
               
                 12 
                 8.782 
                 2453519 
                 0.50 
                 228061 
               
               
                 13 
                 9.184 
                 8669542 
                 1.75 
                 320819 
               
               
                 14 
                 9.521 
                 4295969 
                 0.87 
                 304404 
               
               
                 15 
                 9.816 
                 4279516 
                 0.86 
                 324956 
               
               
                 16 
                 10.243 
                 30792046 
                 6.22 
                 3248747 
               
               
                 17 
                 10.516 
                 84937402 
                 17.16 
                 4333685 
               
               
                 18 
                 11.442 
                 12774541 
                 2.58 
                 1056539 
               
               
                 19 
                 11.603 
                 33723533 
                 6.81 
                 4613519 
               
               
                 20 
                 11.705 
                 63394649 
                 12.81 
                 4142892 
               
               
                 21 
                 12.316 
                 44870739 
                 9.06 
                 3991785 
               
               
                 22 
                 12.575 
                 5106719 
                 1.03 
                 582499 
               
               
                 23 
                 12.754 
                 2812210 
                 0.57 
                 307827 
               
               
                 24 
                 12.983 
                 7556632 
                 1.53 
                 836964 
               
               
                 25 
                 13.270 
                 4609110 
                 0.93 
                 360754 
               
               
                 26 
                 13.418 
                 4222758 
                 0.85 
                 346063 
               
               
                 27 
                 13.925 
                 16253486 
                 3.28 
                 1431902 
               
               
                 28 
                 14.456 
                 7163578 
                 1.45 
                 605726 
               
               
                 29 
                 14.797 
                 1631474 
                 0.33 
                 141283 
               
               
                 30 
                 15.240 
                 3874910 
                 0.78 
                 285386 
               
               
                 31 
                 15.388 
                 3029615 
                 0.61 
                 225745 
               
               
                 32 
                 15.588 
                 2719921 
                 0.55 
                 159504 
               
               
                 33 
                 16.139 
                 1333368 
                 0.27 
                 123152 
               
               
                 34 
                 16.346 
                 3614771 
                 0.73 
                 345601 
               
               
                 35 
                 16.570 
                 1244982 
                 0.25 
                 111424 
               
               
                 36 
                 16.813 
                 1256674 
                 0.25 
                 123386 
               
               
                 37 
                 16.995 
                 1494705 
                 0.30 
                 110575 
               
               
                 38 
                 17.367 
                 1278577 
                 0.26 
                 73925 
               
               
                 39 
                 17.823 
                 2517030 
                 0.51 
                 117093 
               
               
                 40 
                 18.123 
                 2322326 
                 0.47 
                 179787 
               
               
                 41 
                 18.419 
                 841112 
                 0.17 
                 86329 
               
               
                 42 
                 18.673 
                 2150256 
                 0.43 
                 93219 
               
               
                 43 
                 18.954 
                 429553 
                 0.09 
                 73333 
               
               
                 44 
                 19.255 
                 1852803 
                 0.37 
                 78786 
               
               
                 45 
                 19.627 
                 1385755 
                 0.28 
                 84299 
               
               
                 46 
                 19.912 
                 1175927 
                 0.24 
                 106435 
               
               
                 47 
                 20.169 
                 2238619 
                 0.45 
                 198261 
               
               
                 48 
                 20.362 
                 1903752 
                 0.38 
                 136402 
               
               
                 49 
                 20.725 
                 1388092 
                 0.28 
                 86158 
               
               
                 50 
                 21.002 
                 2038182 
                 0.41 
                 112055 
               
               
                 51 
                 21.221 
                 957990 
                 0.19 
                 84615 
               
               
                 52 
                 21.507 
                 1049496 
                 0.21 
                 79603 
               
               
                 53 
                 21.799 
                 1772753 
                 0.36 
                 102185 
               
               
                 54 
                 22.203 
                 1643957 
                 0.33 
                 76435 
               
               
                 55 
                 22.543 
                 831012 
                 0.17 
                 71872 
               
               
                 56 
                 22.690 
                 572833 
                 0.12 
                 72239 
               
               
                 57 
                 22.941 
                 3535908 
                 0.71 
                 220985 
               
               
                 58 
                 23.299 
                 2133293 
                 0.43 
                 142496 
               
               
                 59 
                 23.648 
                 2144927 
                 0.43 
                 151460 
               
               
                 60 
                 23.960 
                 1493722 
                 0.30 
                 100416 
               
               
                 61 
                 24.429 
                 2297448 
                 0.46 
                 123572 
               
               
                 62 
                 24.639 
                 908084 
                 0.18 
                 85150 
               
               
                 63 
                 24.933 
                 2402551 
                 0.49 
                 104466 
               
               
                 64 
                 25.280 
                 583388 
                 0.12 
                 83878 
               
               
                 65 
                 25.564 
                 1925541 
                 0.39 
                 92355 
               
               
                 66 
                 25.800 
                 1153719 
                 0.23 
                 90624 
               
               
                 67 
                 25.974 
                 1727846 
                 0.35 
                 88759 
               
               
                 68 
                 26.404 
                 1397501 
                 0.28 
                 91036 
               
               
                 69 
                 26.591 
                 795071 
                 0.16 
                 89416 
               
               
                 70 
                 26.751 
                 1482526 
                 0.30 
                 89528 
               
               
                 71 
                 27.018 
                 763878 
                 0.15 
                 86118 
               
               
                 72 
                 27.157 
                 1700407 
                 0.34 
                 86915 
               
               
                 73 
                 27.647 
                 2637337 
                 0.53 
                 78919 
               
               
                 74 
                 28.193 
                 4360949 
                 0.88 
                 662358 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 HPLC fingerprint of extract AV016BaSu(65)01(100). 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Retention 
                   
                   
                   
               
               
                   
                 Time 
                 Area 
                 % Area 
                 Height 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 2.391 
                 6928 
                 0.00 
                 852 
               
               
                 2 
                 2.556 
                 1739539 
                 0.82 
                 304280 
               
               
                 3 
                 3.313 
                 84646 
                 0.04 
                 9680 
               
               
                 4 
                 3.686 
                 223139 
                 0.10 
                 9176 
               
               
                 5 
                 4.269 
                 63325 
                 0.03 
                 3419 
               
               
                 6 
                 5.024 
                 13893 
                 0.01 
                 769 
               
               
                 7 
                 6.157 
                 4281 
                 0.00 
                 572 
               
               
                 8 
                 6.736 
                 197041 
                 0.09 
                 6916 
               
               
                 9 
                 7.391 
                 117641 
                 0.06 
                 5898 
               
               
                 10 
                 7.878 
                 616604 
                 0.29 
                 17813 
               
               
                 11 
                 8.308 
                 278599 
                 0.13 
                 15829 
               
               
                 12 
                 8.860 
                 611104 
                 0.29 
                 22047 
               
               
                 13 
                 9.391 
                 187896 
                 0.09 
                 12237 
               
               
                 14 
                 9.564 
                 276548 
                 0.13 
                 14167 
               
               
                 15 
                 12.058 
                 8846367 
                 4.16 
                 183309 
               
               
                 16 
                 12.644 
                 751247 
                 0.35 
                 38688 
               
               
                 17 
                 13.199 
                 1719360 
                 0.81 
                 43466 
               
               
                 18 
                 13.988 
                 1735451 
                 0.82 
                 56406 
               
               
                 19 
                 14.555 
                 2169673 
                 1.02 
                 76948 
               
               
                 20 
                 14.875 
                 2254289 
                 1.06 
                 115963 
               
               
                 21 
                 15.626 
                 4155110 
                 1.95 
                 123796 
               
               
                 22 
                 15.956 
                 1080279 
                 0.51 
                 86186 
               
               
                 23 
                 16.580 
                 5912054 
                 2.78 
                 175797 
               
               
                 24 
                 17.766 
                 7200465 
                 3.38 
                 141141 
               
               
                 25 
                 18.423 
                 2986827 
                 1.40 
                 111890 
               
               
                 26 
                 19.067 
                 6971841 
                 3.28 
                 151382 
               
               
                 27 
                 19.737 
                 4017460 
                 1.89 
                 145309 
               
               
                 28 
                 20.104 
                 1488984 
                 0.70 
                 136621 
               
               
                 29 
                 20.459 
                 4173594 
                 1.96 
                 175505 
               
               
                 30 
                 20.795 
                 3235535 
                 1.52 
                 167773 
               
               
                 31 
                 21.079 
                 1788929 
                 0.84 
                 164753 
               
               
                 32 
                 21.272 
                 4796914 
                 2.25 
                 175363 
               
               
                 33 
                 21.910 
                 4124406 
                 1.94 
                 168234 
               
               
                 34 
                 22.267 
                 6071222 
                 2.85 
                 185861 
               
               
                 35 
                 22.997 
                 5930428 
                 2.79 
                 190259 
               
               
                 36 
                 23.451 
                 5868309 
                 2.76 
                 231002 
               
               
                 37 
                 23.866 
                 16287689 
                 7.65 
                 1076501 
               
               
                 38 
                 24.287 
                 4035419 
                 1.90 
                 266032 
               
               
                 39 
                 24.627 
                 3569391 
                 1.68 
                 266672 
               
               
                 40 
                 24.844 
                 42015183 
                 19.75 
                 2046204 
               
               
                 41 
                 26.107 
                 8757964 
                 4.12 
                 239370 
               
               
                 42 
                 26.900 
                 25586681 
                 12.02 
                 821474 
               
               
                 43 
                 27.926 
                 7429374 
                 3.49 
                 323025 
               
               
                 44 
                 28.156 
                 13400123 
                 6.30 
                 654328 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 HPLC fingerprint of extract AV016BaSu(65)01(100)ng. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Retention 
                   
                   
                   
               
               
                   
                 Time 
                 Area 
                 % Area 
                 Height 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 2.579 
                 1411822 
                 0.59 
                 207187 
               
               
                 2 
                 3.336 
                 80911 
                 0.03 
                 9988 
               
               
                 3 
                 3.518 
                 367100 
                 0.15 
                 14428 
               
               
                 4 
                 4.289 
                 54629 
                 0.02 
                 3130 
               
               
                 5 
                 4.766 
                 46285 
                 0.02 
                 1682 
               
               
                 6 
                 6.659 
                 58963 
                 0.02 
                 2489 
               
               
                 7 
                 7.316 
                 3635 
                 0.00 
                 454 
               
               
                 8 
                 7.852 
                 277194 
                 0.12 
                 10198 
               
               
                 9 
                 8.240 
                 150308 
                 0.06 
                 8971 
               
               
                 10 
                 8.774 
                 321493 
                 0.14 
                 13773 
               
               
                 11 
                 9.207 
                 113505 
                 0.05 
                 7075 
               
               
                 12 
                 9.458 
                 151700 
                 0.06 
                 8682 
               
               
                 13 
                 10.403 
                 841535 
                 0.35 
                 23389 
               
               
                 14 
                 11.877 
                 5609438 
                 2.36 
                 151067 
               
               
                 15 
                 12.469 
                 548895 
                 0.23 
                 28503 
               
               
                 16 
                 12.997 
                 1315579 
                 0.55 
                 34524 
               
               
                 17 
                 13.798 
                 1396765 
                 0.59 
                 46557 
               
               
                 18 
                 14.301 
                 1552115 
                 0.65 
                 56533 
               
               
                 19 
                 14.658 
                 1693118 
                 0.71 
                 88234 
               
               
                 20 
                 15.378 
                 2309177 
                 0.97 
                 76351 
               
               
                 21 
                 15.719 
                 1849902 
                 0.78 
                 89567 
               
               
                 22 
                 16.341 
                 3751422 
                 1.58 
                 131368 
               
               
                 23 
                 16.651 
                 705622 
                 0.30 
                 59259 
               
               
                 24 
                 17.537 
                 5824011 
                 2.45 
                 121156 
               
               
                 25 
                 18.170 
                 2202916 
                 0.93 
                 92276 
               
               
                 26 
                 19.153 
                 16377423 
                 6.89 
                 384476 
               
               
                 27 
                 20.514 
                 11154160 
                 4.69 
                 206032 
               
               
                 28 
                 21.248 
                 27468304 
                 11.56 
                 1141614 
               
               
                 29 
                 22.005 
                 3503203 
                 1.47 
                 178231 
               
               
                 30 
                 22.665 
                 5722669 
                 2.41 
                 184191 
               
               
                 31 
                 23.185 
                 15337448 
                 6.45 
                 535651 
               
               
                 32 
                 23.666 
                 13965998 
                 5.88 
                 926816 
               
               
                 33 
                 24.155 
                 2815614 
                 1.18 
                 263179 
               
               
                 34 
                 24.360 
                 4975496 
                 2.09 
                 287271 
               
               
                 35 
                 24.677 
                 30074237 
                 12.65 
                 1853497 
               
               
                 36 
                 25.316 
                 9050757 
                 3.81 
                 411682 
               
               
                 37 
                 25.712 
                 6189308 
                 2.60 
                 264816 
               
               
                 38 
                 26.607 
                 20990123 
                 8.83 
                 826640 
               
               
                 39 
                 26.996 
                 4842260 
                 2.04 
                 310728 
               
               
                 40 
                 27.520 
                 16829287 
                 7.08 
                 814850 
               
               
                 41 
                 27.942 
                 15732031 
                 6.62 
                 542909 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 HPLC fingerprint of extract AV016BaSu(65)01(100)g. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Retention 
                   
                   
                   
               
               
                   
                 Time 
                 Area 
                 % Area 
                 Height 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 2.384 
                 12028 
                 0.01 
                 947 
               
               
                 2 
                 2.570 
                 2633549 
                 1.23 
                 435477 
               
               
                 3 
                 3.507 
                 735108 
                 0.34 
                 33546 
               
               
                 4 
                 5.043 
                 9805 
                 0.00 
                 651 
               
               
                 5 
                 6.822 
                 78175 
                 0.04 
                 3779 
               
               
                 6 
                 7.045 
                 83478 
                 0.04 
                 5089 
               
               
                 7 
                 7.203 
                 66115 
                 0.03 
                 5355 
               
               
                 8 
                 7.820 
                 581086 
                 0.27 
                 16595 
               
               
                 9 
                 8.749 
                 775713 
                 0.36 
                 25475 
               
               
                 10 
                 9.447 
                 325440 
                 0.15 
                 11555 
               
               
                 11 
                 10.387 
                 882547 
                 0.41 
                 20400 
               
               
                 12 
                 11.017 
                 953840 
                 0.45 
                 28086 
               
               
                 13 
                 11.853 
                 2915239 
                 1.36 
                 86837 
               
               
                 14 
                 12.467 
                 565867 
                 0.26 
                 27905 
               
               
                 15 
                 13.088 
                 924183 
                 0.43 
                 31678 
               
               
                 16 
                 13.160 
                 468207 
                 0.22 
                 31979 
               
               
                 17 
                 13.766 
                 1224906 
                 0.57 
                 43299 
               
               
                 18 
                 14.633 
                 3503010 
                 1.64 
                 84712 
               
               
                 19 
                 15.471 
                 1966123 
                 0.92 
                 60085 
               
               
                 20 
                 15.728 
                 1016548 
                 0.48 
                 66817 
               
               
                 21 
                 16.317 
                 3374916 
                 1.58 
                 110591 
               
               
                 22 
                 16.627 
                 663866 
                 0.31 
                 60558 
               
               
                 23 
                 17.526 
                 4303710 
                 2.01 
                 88642 
               
               
                 24 
                 17.688 
                 741644 
                 0.35 
                 86630 
               
               
                 25 
                 18.026 
                 1554964 
                 0.73 
                 90925 
               
               
                 26 
                 18.356 
                 1800352 
                 0.84 
                 101584 
               
               
                 27 
                 18.852 
                 3360093 
                 1.57 
                 117653 
               
               
                 28 
                 19.607 
                 6271811 
                 2.93 
                 186866 
               
               
                 29 
                 19.840 
                 4324472 
                 2.02 
                 201700 
               
               
                 30 
                 20.208 
                 6132533 
                 2.87 
                 247918 
               
               
                 31 
                 20.820 
                 2351992 
                 1.10 
                 151443 
               
               
                 32 
                 21.075 
                 3520296 
                 1.65 
                 173642 
               
               
                 33 
                 23.221 
                 22630392 
                 10.58 
                 235431 
               
               
                 34 
                 23.623 
                 17865849 
                 8.35 
                 1114913 
               
               
                 35 
                 24.057 
                 4708441 
                 2.20 
                 290733 
               
               
                 36 
                 24.359 
                 4086311 
                 1.91 
                 276895 
               
               
                 37 
                 24.661 
                 35429569 
                 16.57 
                 1765464 
               
               
                 38 
                 26.639 
                 30653977 
                 14.33 
                 480095 
               
               
                 39 
                 26.977 
                 5710437 
                 2.67 
                 393204 
               
               
                 40 
                 27.595 
                 12852254 
                 6.01 
                 398867 
               
               
                 41 
                 27.911 
                 21792801 
                 10.19 
                 563202 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 HPLC fingerprint of extract AV016BaSu(65)04(100). 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Retention 
                   
                   
                   
               
               
                   
                 Time 
                 Area 
                 % Area 
                 Height 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 1.241 
                 10089 
                 0.00 
                 743 
               
               
                 2 
                 1.737 
                 139563 
                 0.04 
                 29368 
               
               
                 3 
                 1.897 
                 10508564 
                 3.02 
                 2120419 
               
               
                 4 
                 2.474 
                 168462 
                 0.05 
                 20793 
               
               
                 5 
                 3.560 
                 244245 
                 0.07 
                 13079 
               
               
                 6 
                 5.156 
                 118208 
                 0.03 
                 5005 
               
               
                 7 
                 6.455 
                 515048 
                 0.15 
                 12630 
               
               
                 8 
                 7.011 
                 397795 
                 0.11 
                 16720 
               
               
                 9 
                 7.792 
                 1203998 
                 0.35 
                 35331 
               
               
                 10 
                 8.408 
                 1782066 
                 0.51 
                 81114 
               
               
                 11 
                 9.287 
                 3153097 
                 0.90 
                 85209 
               
               
                 12 
                 10.010 
                 3363741 
                 0.97 
                 117298 
               
               
                 13 
                 10.521 
                 3211695 
                 0.92 
                 129563 
               
               
                 14 
                 10.919 
                 3272829 
                 0.94 
                 151739 
               
               
                 15 
                 11.201 
                 2910895 
                 0.84 
                 187760 
               
               
                 16 
                 11.469 
                 2011099 
                 0.58 
                 187543 
               
               
                 17 
                 12.570 
                 22668982 
                 6.51 
                 608697 
               
               
                 18 
                 13.072 
                 22315645 
                 6.40 
                 1372355 
               
               
                 19 
                 13.943 
                 15908037 
                 4.56 
                 466306 
               
               
                 20 
                 14.267 
                 13002097 
                 3.73 
                 755966 
               
               
                 21 
                 14.499 
                 14043370 
                 4.03 
                 677681 
               
               
                 22 
                 15.168 
                 18500419 
                 5.31 
                 803270 
               
               
                 23 
                 15.443 
                 8340109 
                 2.39 
                 561189 
               
               
                 24 
                 15.645 
                 7732494 
                 2.22 
                 554586 
               
               
                 25 
                 15.999 
                 15671975 
                 4.50 
                 569336 
               
               
                 26 
                 16.333 
                 14724201 
                 4.23 
                 556235 
               
               
                 27 
                 16.825 
                 11690960 
                 3.35 
                 545534 
               
               
                 28 
                 17.178 
                 11285355 
                 3.24 
                 522522 
               
               
                 29 
                 18.532 
                 79310513 
                 22.76 
                 612406 
               
               
                 30 
                 19.864 
                 57401105 
                 16.47 
                 441406 
               
               
                 31 
                 25.389 
                 2531990 
                 0.73 
                 47750 
               
               
                 32 
                 29.165 
                 4724 
                 0.00 
                 733 
               
               
                 33 
                 29.850 
                 337961 
                 0.10 
                 60806 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 HPLC fingerprint of extract AV016BaSu(65)06(100). 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Retention 
                   
                   
                   
               
               
                   
                 Time 
                 Area 
                 % Area 
                 Height 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 1.726 
                 325346 
                 0.17 
                 47632 
               
               
                 2 
                 1.893 
                 23371224 
                 12.48 
                 3640871 
               
               
                 3 
                 2.482 
                 714853 
                 0.38 
                 60900 
               
               
                 4 
                 3.177 
                 1875237 
                 1.00 
                 99118 
               
               
                 5 
                 3.690 
                 485133 
                 0.26 
                 25416 
               
               
                 6 
                 4.103 
                 679406 
                 0.36 
                 27133 
               
               
                 7 
                 4.359 
                 352324 
                 0.19 
                 24825 
               
               
                 8 
                 4.857 
                 272252 
                 0.15 
                 14769 
               
               
                 9 
                 5.596 
                 1486044 
                 0.79 
                 49063 
               
               
                 10 
                 6.072 
                 4857288 
                 2.59 
                 165611 
               
               
                 11 
                 7.074 
                 715830 
                 0.38 
                 46771 
               
               
                 12 
                 7.247 
                 928001 
                 0.50 
                 52209 
               
               
                 13 
                 7.925 
                 1065970 
                 0.57 
                 45480 
               
               
                 14 
                 8.330 
                 2132695 
                 1.14 
                 79394 
               
               
                 15 
                 9.096 
                 2308356 
                 1.23 
                 80800 
               
               
                 16 
                 9.743 
                 2396105 
                 1.28 
                 79390 
               
               
                 17 
                 11.179 
                 12808928 
                 6.84 
                 205854 
               
               
                 18 
                 12.413 
                 25125074 
                 13.42 
                 602504 
               
               
                 19 
                 13.130 
                 1899754 
                 1.01 
                 160549 
               
               
                 20 
                 14.031 
                 28439970 
                 15.19 
                 935325 
               
               
                 21 
                 14.646 
                 9859006 
                 5.27 
                 326112 
               
               
                 22 
                 15.208 
                 3351792 
                 1.79 
                 211034 
               
               
                 23 
                 15.744 
                 11230538 
                 6.00 
                 540161 
               
               
                 24 
                 16.030 
                 4547672 
                 2.43 
                 212887 
               
               
                 25 
                 16.651 
                 5194350 
                 2.77 
                 232212 
               
               
                 26 
                 16.896 
                 3162049 
                 1.69 
                 216298 
               
               
                 27 
                 17.244 
                 5839204 
                 3.12 
                 208158 
               
               
                 28 
                 17.782 
                 12488631 
                 6.67 
                 281615 
               
               
                 29 
                 18.729 
                 4904610 
                 2.62 
                 146569 
               
               
                 30 
                 19.386 
                 2196346 
                 1.17 
                 115299 
               
               
                 31 
                 19.673 
                 8273125 
                 4.42 
                 99688 
               
               
                 32 
                 21.529 
                 667601 
                 0.36 
                 50722 
               
               
                 33 
                 21.763 
                 1946885 
                 1.04 
                 46660 
               
               
                 34 
                 22.802 
                 1281629 
                 0.68 
                 26705 
               
               
                 35 
                 26.305 
                 16760 
                 0.01 
                 1806 
               
               
                 36 
                 27.757 
                 52103 
                 0.03 
                 5397 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 HPLC fingerprint of extract AV016BaSu(105)08(100). 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Retention 
                   
                   
                   
               
               
                   
                 Time 
                 Area 
                 % Area 
                 Height 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 2.531 
                 251954 
                 2.83 
                 45170 
               
               
                 2 
                 3.153 
                 91508 
                 1.03 
                 7618 
               
               
                 3 
                 3.590 
                 53827 
                 0.60 
                 2846 
               
               
                 4 
                 5.809 
                 95383 
                 1.07 
                 5176 
               
               
                 5 
                 6.237 
                 84290 
                 0.95 
                 3408 
               
               
                 6 
                 7.377 
                 237161 
                 2.66 
                 7025 
               
               
                 7 
                 7.781 
                 208394 
                 2.34 
                 12462 
               
               
                 8 
                 7.968 
                 105188 
                 1.18 
                 10471 
               
               
                 9 
                 8.523 
                 386758 
                 4.34 
                 15438 
               
               
                 10 
                 8.922 
                 255053 
                 2.86 
                 13182 
               
               
                 11 
                 9.164 
                 103465 
                 1.16 
                 11779 
               
               
                 12 
                 9.356 
                 146409 
                 1.64 
                 11519 
               
               
                 13 
                 9.690 
                 263555 
                 2.96 
                 11664 
               
               
                 14 
                 10.035 
                 197458 
                 2.22 
                 12951 
               
               
                 15 
                 10.220 
                 188840 
                 2.12 
                 14479 
               
               
                 16 
                 10.471 
                 188182 
                 2.11 
                 14258 
               
               
                 17 
                 10.814 
                 371997 
                 4.18 
                 13832 
               
               
                 18 
                 11.142 
                 112966 
                 1.27 
                 12605 
               
               
                 19 
                 11.344 
                 318863 
                 3.58 
                 13928 
               
               
                 20 
                 11.735 
                 280148 
                 3.15 
                 11849 
               
               
                 21 
                 12.451 
                 609274 
                 6.84 
                 21533 
               
               
                 22 
                 13.107 
                 585271 
                 6.57 
                 38319 
               
               
                 23 
                 13.648 
                 210651 
                 2.37 
                 8967 
               
               
                 24 
                 14.205 
                 227248 
                 2.55 
                 13908 
               
               
                 25 
                 14.513 
                 2594830 
                 29.14 
                 208042 
               
               
                 26 
                 15.951 
                 40742 
                 0.46 
                 3155 
               
               
                 27 
                 16.683 
                 52708 
                 0.59 
                 5023 
               
               
                 28 
                 17.691 
                 70180 
                 0.79 
                 6181 
               
               
                 29 
                 24.062 
                 198328 
                 2.23 
                 4386 
               
               
                 30 
                 24.791 
                 13467 
                 0.15 
                 1398 
               
               
                 31 
                 24.993 
                 12465 
                 0.14 
                 996 
               
               
                 32 
                 25.557 
                 2542 
                 0.03 
                 494 
               
               
                 33 
                 26.037 
                 62889 
                 0.71 
                 3648 
               
               
                 34 
                 26.578 
                 50279 
                 0.56 
                 5059 
               
               
                 35 
                 28.119 
                 5587 
                 0.06 
                 665 
               
               
                 36 
                 28.951 
                 42494 
                 0.48 
                 3689 
               
               
                 37 
                 29.707 
                 184276 
                 2.07 
                 17288 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 HPLC fingerprint of extract AV016Fr(105)08(100). 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Retention 
                   
                   
                   
               
               
                   
                 Time 
                 Area 
                 % Area 
                 Height 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 2.238 
                 10762330 
                 4.73 
                 1150702 
               
               
                 2 
                 2.553 
                 5628824 
                 2.47 
                 655829 
               
               
                 3 
                 2.914 
                 2203056 
                 0.97 
                 125091 
               
               
                 4 
                 3.208 
                 3512641 
                 1.54 
                 318280 
               
               
                 5 
                 3.357 
                 1070868 
                 0.47 
                 212573 
               
               
                 6 
                 3.602 
                 1899749 
                 0.84 
                 121669 
               
               
                 7 
                 4.052 
                 3407226 
                 1.50 
                 136020 
               
               
                 8 
                 4.503 
                 2919013 
                 1.28 
                 94633 
               
               
                 9 
                 5.093 
                 2190949 
                 0.96 
                 89619 
               
               
                 10 
                 5.889 
                 12371237 
                 5.44 
                 338787 
               
               
                 11 
                 6.223 
                 10361831 
                 4.55 
                 315486 
               
               
                 12 
                 7.163 
                 4782640 
                 2.10 
                 237340 
               
               
                 13 
                 7.700 
                 40604091 
                 17.85 
                 1251589 
               
               
                 14 
                 9.163 
                 11851832 
                 5.21 
                 268920 
               
               
                 15 
                 9.857 
                 3502688 
                 1.54 
                 228636 
               
               
                 16 
                 10.125 
                 4862586 
                 2.14 
                 227948 
               
               
                 17 
                 10.448 
                 2710010 
                 1.19 
                 231182 
               
               
                 18 
                 10.625 
                 2889073 
                 1.27 
                 233695 
               
               
                 19 
                 10.818 
                 1656694 
                 0.73 
                 207765 
               
               
                 20 
                 11.218 
                 23908811 
                 10.51 
                 1250520 
               
               
                 21 
                 12.067 
                 40804495 
                 17.94 
                 1657258 
               
               
                 22 
                 13.556 
                 507546 
                 0.22 
                 127495 
               
               
                 23 
                 13.790 
                 6006453 
                 2.64 
                 134107 
               
               
                 24 
                 14.940 
                 8534623 
                 3.75 
                 102587 
               
               
                 25 
                 17.771 
                 960263 
                 0.42 
                 27876 
               
               
                 26 
                 18.313 
                 820905 
                 0.36 
                 25276 
               
               
                 27 
                 20.696 
                 13722 
                 0.01 
                 1129 
               
               
                 28 
                 23.988 
                 77588 
                 0.03 
                 2021 
               
               
                 29 
                 24.355 
                 9612 
                 0.00 
                 1658 
               
               
                 30 
                 25.622 
                 204766 
                 0.09 
                 9215 
               
               
                 31 
                 25.787 
                 301167 
                 0.13 
                 11470 
               
               
                 32 
                 26.472 
                 205806 
                 0.09 
                 9613 
               
               
                 33 
                 27.231 
                 486773 
                 0.21 
                 12154 
               
               
                 34 
                 28.270 
                 5013669 
                 2.20 
                 94934 
               
               
                 35 
                 29.001 
                 2956931 
                 1.30 
                 213028 
               
               
                 36 
                 29.400 
                 7494149 
                 3.29 
                 238402 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 LC/MS Fingerprint of extract AV016BaDi(28)04(20) 
               
               
                 (TIC Spectrum (Q1 +ve mode) 
               
               
                 Peak List: 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                 Area 
                   
                 Height 
                   
                 Width 
                   
               
               
                 (min) 
                 (counts) 
                 % Area 
                 (cps) 
                 % Height 
                 (min) 
                 Baseline Type 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 2.1775 
                 1.06E+08 
                 12.3131 
                 1.97E+07 
                 17.7412 
                 0.1874 
                 Valley 
               
               
                 2.3167 
                 3.07E+08 
                 35.522 
                 3.45E+07 
                 31.1894 
                 0.2408 
                 Valley 
               
               
                 2.8588 
                 2.07E+07 
                 2.398 
                 2.52E+06 
                 2.2768 
                 0.2408 
                 Base to Base 
               
               
                 3.2879 
                 1.84E+07 
                 2.1314 
                 2.99E+06 
                 2.7 
                 0.1873 
                 Base to Base 
               
               
                 3.7661 
                 1.19E+07 
                 1.3822 
                 1.74E+06 
                 1.5668 
                 0.2408 
                 Base to Base 
               
               
                 5.0086 
                 9.58E+06 
                 1.1091 
                 1.08E+06 
                 0.9759 
                 0.2941 
                 Base to Base 
               
               
                 6.1538 
                 8.09E+06 
                 0.9365 
                 1.03E+06 
                 0.9294 
                 0.2141 
                 Base to Base 
               
               
                 6.8962 
                 6.00E+06 
                 0.6949 
                 1.39E+06 
                 1.2572 
                 0.1605 
                 Base to Base 
               
               
                 7.7876 
                 8.31E+06 
                 0.9627 
                 1.05E+06 
                 0.9452 
                 0.2676 
                 Valley 
               
               
                 7.9719 
                 1.18E+07 
                 1.3624 
                 1.89E+06 
                 1.7057 
                 0.214 
                 Valley 
               
               
                 10.735 
                 8.06E+06 
                 0.9334 
                 1.45E+06 
                 1.3135 
                 0.2408 
                 Base to Base 
               
               
                 12.0425 
                 2.48E+06 
                 0.2866 
                 6.74E+05 
                 0.6081 
                 0.107 
                 Base to Base 
               
               
                 12.3092 
                 1.54E+06 
                 0.1783 
                 8.28E+05 
                 0.7472 
                 0.0803 
                 Base to Base 
               
               
                 12.7311 
                 4.83E+06 
                 0.5597 
                 9.68E+05 
                 0.8739 
                 0.1605 
                 Base to Base 
               
               
                 14.3893 
                 3.76E+06 
                 0.4351 
                 9.54E+05 
                 0.8611 
                 0.1605 
                 Base to Base 
               
               
                 15.0886 
                 3.66E+06 
                 0.4237 
                 9.12E+05 
                 0.8235 
                 0.1338 
                 Base to Base 
               
               
                 17.5521 
                 1.82E+07 
                 2.1125 
                 2.34E+06 
                 2.1145 
                 0.3211 
                 Base to Base 
               
               
                 17.9476 
                 1.90E+07 
                 2.1971 
                 1.86E+06 
                 1.6753 
                 0.2408 
                 Base to Base 
               
               
                 19.5586 
                 1.44E+07 
                 1.6648 
                 1.53E+06 
                 1.3819 
                 0.3211 
                 Base to Base 
               
               
                 20.3886 
                 4.78E+06 
                 0.553 
                 1.22E+06 
                 1.1028 
                 0.1605 
                 Base to Base 
               
               
                 21.5431 
                 1.49E+07 
                 1.7195 
                 1.31E+06 
                 1.1854 
                 0.3746 
                 Base to Base 
               
               
                 23.6233 
                 8.28E+06 
                 0.9586 
                 1.77E+06 
                 1.5935 
                 0.1873 
                 Base to Base 
               
               
                 24.5583 
                 8.58E+06 
                 0.9939 
                 1.18E+06 
                 1.0651 
                 0.2676 
                 Base to Base 
               
               
                 25.1938 
                 9.77E+06 
                 1.131 
                 1.24E+06 
                 1.117 
                 0.3211 
                 Base to Base 
               
               
                 25.6005 
                 5.11E+07 
                 5.9143 
                 5.26E+06 
                 4.7467 
                 0.3478 
                 Base to Base 
               
               
                 25.9137 
                 1.34E+07 
                 1.5528 
                 1.76E+06 
                 1.5921 
                 0.2141 
                 Base to Base 
               
               
                 26.7469 
                 1.82E+07 
                 2.1106 
                 2.05E+06 
                 1.8533 
                 0.2943 
                 Base to Base 
               
               
                 27.3985 
                 1.09E+07 
                 1.2594 
                 1.39E+06 
                 1.2536 
                 0.2675 
                 Base to Base 
               
               
                 27.6349 
                 4.60E+06 
                 0.5329 
                 1.06E+06 
                 0.9601 
                 0.1338 
                 Base to Base 
               
               
                 28.7272 
                 1.89E+07 
                 2.1893 
                 1.77E+06 
                 1.5968 
                 0.3478 
                 Base to Base 
               
               
                 28.8934 
                 1.03E+07 
                 1.1905 
                 2.09E+06 
                 1.8883 
                 0.1338 
                 Base to Base 
               
               
                 29.1613 
                 3.01E+06 
                 0.3489 
                 9.23E+05 
                 0.8329 
                 0.1338 
                 Base to Base 
               
               
                 29.9922 
                 8.20E+07 
                 9.495 
                 5.03E+06 
                 4.5397 
                 0.6957 
                 Base to Base 
               
               
                 34.628 
                 1.16E+07 
                 1.3485 
                 1.01E+06 
                 0.9137 
                 0.3746 
                 Base to Base 
               
               
                 36.8649 
                 2.86E+06 
                 0.3315 
                 8.91E+05 
                 0.804 
                 0.107 
                 Base to Base 
               
               
                 37.1874 
                 1.77E+06 
                 0.2051 
                 6.81E+05 
                 0.6146 
                 0.0802 
                 Base to Base 
               
               
                 37.7494 
                 4.85E+06 
                 0.5616 
                 7.24E+05 
                 0.6535 
                 0.1873 
                 Base to Base 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 LC/MS Fingerprint of extract AV016BaDi(28)04(20) 
               
               
                 (TIC Spectrum (Q1 −ve mode) 
               
               
                 Peak List: 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                 Area 
                   
                 Height 
                   
                 Width 
                   
               
               
                 (min) 
                 (counts) 
                 % Area 
                 (cps) 
                 % Height 
                 (min) 
                 Baseline Type 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 2.1961 
                 2.85E+08 
                 24.4168 
                 3.04E+07 
                 25.3851 
                 0.2676 
                 Valley 
               
               
                 2.4834 
                 4.77E+08 
                 40.8241 
                 2.58E+07 
                 21.5134 
                 0.5084 
                 Valley 
               
               
                 3.4214 
                 7.47E+06 
                 0.6387 
                 1.44E+06 
                 1.2067 
                 0.1873 
                 Base to Base 
               
               
                 4.4188 
                 3.03E+06 
                 0.2592 
                 1.25E+06 
                 1.0442 
                 0.107 
                 Base to Base 
               
               
                 7.9809 
                 8.39E+06 
                 0.7181 
                 1.33E+06 
                 1.1079 
                 0.1873 
                 Base to Base 
               
               
                 9.6559 
                 5.75E+06 
                 0.4914 
                 1.14E+06 
                 0.9551 
                 0.1873 
                 Base to Base 
               
               
                 11.4246 
                 2.60E+06 
                 0.2223 
                 8.53E+05 
                 0.7128 
                 0.1338 
                 Base to Base 
               
               
                 12.387 
                 6.72E+06 
                 0.575 
                 1.29E+06 
                 1.0785 
                 0.1873 
                 Base to Base 
               
               
                 12.7886 
                 3.28E+07 
                 2.8027 
                 4.15E+06 
                 3.4643 
                 0.2408 
                 Base to Base 
               
               
                 14.0996 
                 3.48E+06 
                 0.2974 
                 1.12E+06 
                 0.9338 
                 0.107 
                 Base to Base 
               
               
                 14.8736 
                 3.87E+06 
                 0.3307 
                 5.13E+05 
                 0.4282 
                 0.1605 
                 Base to Base 
               
               
                 15.1974 
                 2.00E+06 
                 0.1714 
                 1.08E+06 
                 0.9018 
                 0.0803 
                 Base to Base 
               
               
                 16.2118 
                 1.20E+07 
                 1.0275 
                 1.28E+06 
                 1.0691 
                 0.2676 
                 Base to Base 
               
               
                 17.1494 
                 6.42E+06 
                 0.5495 
                 1.26E+06 
                 1.0499 
                 0.214 
                 Base to Base 
               
               
                 17.3337 
                 1.35E+07 
                 1.157 
                 2.57E+06 
                 2.1439 
                 0.1873 
                 Base to Base 
               
               
                 17.6906 
                 3.73E+07 
                 3.1891 
                 2.51E+06 
                 2.0939 
                 0.4013 
                 Valley 
               
               
                 17.8922 
                 9.86E+06 
                 0.8435 
                 1.99E+06 
                 1.6607 
                 0.1338 
                 Valley 
               
               
                 18.3572 
                 4.74E+06 
                 0.4051 
                 2.29E+06 
                 1.9131 
                 0.0803 
                 Base to Base 
               
               
                 18.7319 
                 9.90E+06 
                 0.8468 
                 1.37E+06 
                 1.144 
                 0.2408 
                 Base to Base 
               
               
                 19.6901 
                 6.66E+06 
                 0.5693 
                 1.68E+06 
                 1.4049 
                 0.1338 
                 Base to Base 
               
               
                 20.1151 
                 1.26E+07 
                 1.082 
                 1.77E+06 
                 1.4749 
                 0.2408 
                 Base to Base 
               
               
                 21.084 
                 5.17E+06 
                 0.4419 
                 1.45E+06 
                 1.2082 
                 0.1338 
                 Base to Base 
               
               
                 21.4301 
                 6.15E+06 
                 0.5263 
                 2.11E+06 
                 1.7665 
                 0.0803 
                 Valley 
               
               
                 21.5887 
                 1.05E+07 
                 0.8972 
                 2.53E+06 
                 2.1134 
                 0.1605 
                 Valley 
               
               
                 23.7467 
                 8.39E+06 
                 0.7173 
                 1.24E+06 
                 1.0376 
                 0.2675 
                 Base to Base 
               
               
                 23.9916 
                 4.44E+06 
                 0.3795 
                 8.19E+05 
                 0.6845 
                 0.1338 
                 Base to Base 
               
               
                 24.7774 
                 6.82E+06 
                 0.5835 
                 1.14E+06 
                 0.9516 
                 0.2943 
                 Base to Base 
               
               
                 25.5831 
                 2.43E+07 
                 2.0804 
                 2.38E+06 
                 1.9909 
                 0.3211 
                 Base to Base 
               
               
                 25.9059 
                 1.37E+07 
                 1.1725 
                 2.04E+06 
                 1.7081 
                 0.2675 
                 Base to Base 
               
               
                 26.2502 
                 1.00E+07 
                 0.8555 
                 1.08E+06 
                 0.9016 
                 0.2408 
                 Base to Base 
               
               
                 26.7168 
                 1.72E+07 
                 1.471 
                 2.26E+06 
                 1.8859 
                 0.2408 
                 Valley 
               
               
                 26.9142 
                 2.14E+07 
                 1.8288 
                 2.20E+06 
                 1.8395 
                 0.2676 
                 Valley 
               
               
                 28.3515 
                 3.10E+07 
                 2.6494 
                 3.47E+06 
                 2.8963 
                 0.4281 
                 Valley 
               
               
                 28.6747 
                 2.53E+07 
                 2.1615 
                 3.53E+06 
                 2.9518 
                 0.3478 
                 Valley 
               
               
                 28.9455 
                 4.47E+06 
                 0.3823 
                 1.21E+06 
                 1.0119 
                 0.1338 
                 Base to Base 
               
               
                 29.6212 
                 7.70E+06 
                 0.6586 
                 7.87E+05 
                 0.6574 
                 0.2408 
                 Base to Base 
               
               
                 30.0197 
                 1.10E+07 
                 0.939 
                 2.58E+06 
                 2.1565 
                 0.1873 
                 Base to Base 
               
               
                 30.4766 
                 9.79E+06 
                 0.8377 
                 1.86E+06 
                 1.552 
                 0.1605 
                 Base to Base 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 LC/MS Fingerprint of extract AV016BaDi(65)04(100) 
               
               
                 (TIC Spectrum (Q1 +ve mode) 
               
               
                 Peak List: 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                 Area 
                   
                 Height 
                   
                 Width 
                   
               
               
                 (min) 
                 (counts) 
                 % Area 
                 (cps) 
                 % Height 
                 (min) 
                 Baseline Type 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1.6819 
                 5.13E+07 
                 3.3673 
                 1.07E+07 
                 7.702 
                 0.1873 
                 Base to Base 
               
               
                 2.1223 
                 4.05E+07 
                 2.6634 
                 4.33E+06 
                 3.1211 
                 0.2408 
                 Base to Base 
               
               
                 2.4137 
                 3.19E+08 
                 20.9505 
                 4.49E+07 
                 32.3621 
                 0.3479 
                 Base to Base 
               
               
                 35.37 
                 7.19E+07 
                 4.7236 
                 3.26E+06 
                 2.3544 
                 0.6422 
                 Base to Base 
               
               
                 43.1584 
                 2.30E+08 
                 15.0758 
                 1.02E+07 
                 7.3293 
                 0.7759 
                 Base to Base 
               
               
                 46.4236 
                 1.38E+07 
                 0.9069 
                 1.66E+06 
                 1.1971 
                 0.3746 
                 Base to Base 
               
               
                 48.1523 
                 2.43E+07 
                 1.5953 
                 2.55E+06 
                 1.8369 
                 0.3211 
                 Base to Base 
               
               
                 48.5731 
                 7.28E+07 
                 4.7831 
                 6.02E+06 
                 4.3446 
                 0.3746 
                 Base to Base 
               
               
                 49.1844 
                 4.59E+07 
                 3.0145 
                 4.22E+06 
                 3.0466 
                 0.4014 
                 Base to Base 
               
               
                 49.7221 
                 1.53E+08 
                 10.0401 
                 1.05E+07 
                 7.5644 
                 0.6421 
                 Base to Base 
               
               
                 50.41 
                 1.98E+08 
                 13.0047 
                 1.31E+07 
                 9.4266 
                 0.6422 
                 Base to Base 
               
               
                 53.0591 
                 1.83E+07 
                 1.2049 
                 2.30E+06 
                 1.6573 
                 0.3478 
                 Base to Base 
               
               
                 53.7262 
                 1.26E+08 
                 8.2723 
                 8.93E+06 
                 6.4387 
                 0.5619 
                 Base to Base 
               
               
                 55.4126 
                 1.19E+08 
                 7.8049 
                 1.12E+07 
                 8.068 
                 0.4013 
                 Base to Base 
               
               
                 55.8032 
                 3.95E+07 
                 2.5926 
                 4.92E+06 
                 3.5508 
                 0.3478 
                 Base to Base 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 LC/MS Fingerprint of extract AV016BaDi(65)04(100) 
               
               
                 (TIC Spectrum (Q1 −ve mode) 
               
               
                 Peak List: 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                 Area 
                   
                 Height 
                   
                 Width 
                   
               
               
                 (min) 
                 (counts) 
                 % Area 
                 (cps) 
                 % Height 
                 (min) 
                 Baseline Type 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1.5858 
                 4.95E+08 
                 33.2934 
                 5.09E+07 
                 33.5845 
                 0.3211 
                 Valley 
               
               
                 1.6837 
                 4.68E+08 
                 31.4658 
                 4.70E+07 
                 30.9815 
                 0.2675 
                 Valley 
               
               
                 2.281 
                 2.71E+08 
                 18.2632 
                 2.23E+07 
                 14.6901 
                 0.3479 
                 Valley 
               
               
                 2.3736 
                 1.40E+08 
                 9.4462 
                 2.07E+07 
                 13.6345 
                 0.214 
                 Valley 
               
               
                 42.7019 
                 3.33E+06 
                 0.2239 
                 1.08E+06 
                 0.7156 
                 0.2141 
                 Base to Base 
               
               
                 51.6555 
                 9.78E+06 
                 0.6583 
                 1.30E+06 
                 0.8605 
                 0.2408 
                 Base to Base 
               
               
                 54.1648 
                 7.99E+07 
                 5.3782 
                 4.28E+06 
                 2.8245 
                 0.7224 
                 Base to Base 
               
               
                 55.1059 
                 3.72E+06 
                 0.2501 
                 1.06E+06 
                 0.697 
                 0.107 
                 Base to Base 
               
               
                 57.6108 
                 6.57E+06 
                 0.4422 
                 1.30E+06 
                 0.855 
                 0.214 
                 Base to Base 
               
               
                 58.0334 
                 6.71E+06 
                 0.4517 
                 9.03E+05 
                 0.5959 
                 0.2141 
                 Base to Base 
               
               
                 58.9956 
                 1.89E+06 
                 0.127 
                 8.50E+05 
                 0.5609 
                 0.0802 
                 Base to Base 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 LC/MS Fingerprint of extract AV016BaSu(65)09(100) 
               
               
                 (TIC Spectrum (Q1 +ve mode). 
               
               
                 Peak List 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                 Area 
                   
                 Height 
                   
                 Width 
                   
               
               
                 (min) 
                 (counts) 
                 % Area 
                 (cps) 
                 % Height 
                 (min) 
                 Baseline Type 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 0.6837 
                 2.30E+07 
                 1.9654 
                 2.66E+06 
                 2.8378 
                 0.3211 
                 Base to Base 
               
               
                 1.7359 
                 3.23E+08 
                 27.6122 
                 2.10E+07 
                 22.4515 
                 0.6155 
                 Base to Base 
               
               
                 2.1817 
                 6.42E+08 
                 54.9509 
                 5.24E+07 
                 55.9679 
                 0.3212 
                 Base to Base 
               
               
                 46.2873 
                 2.68E+06 
                 0.2294 
                 1.06E+06 
                 1.1283 
                 0.0802 
                 Base to Base 
               
               
                 51.3977 
                 1.34E+07 
                 1.1443 
                 1.86E+06 
                 1.987 
                 0.2408 
                 Base to Base 
               
               
                 51.7551 
                 1.14E+07 
                 0.9773 
                 2.30E+06 
                 2.4614 
                 0.1605 
                 Base to Base 
               
               
                 53.5882 
                 4.82E+07 
                 4.1246 
                 4.09E+06 
                 4.3665 
                 0.4281 
                 Base to Base 
               
               
                 56.8025 
                 3.06E+07 
                 2.6205 
                 2.29E+06 
                 2.4491 
                 0.2943 
                 Base to Base 
               
               
                 57.7937 
                 7.45E+07 
                 6.3753 
                 5.94E+06 
                 6.3504 
                 0.4549 
                 Base to Base 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 LC/MS Fingerprint of extract AV016BaSu(65)01(100) 
               
               
                 (TIC Spectrum (Q1 +ve mode) 
               
               
                 Peak List: 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                 Area 
                   
                 Height 
                   
                 Width 
                   
               
               
                 (min) 
                 (counts) 
                 % Area 
                 (cps) 
                 % Height 
                 (min) 
                 Baseline Type 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 0.9306 
                 6.79E+06 
                 0.9084 
                 1.15E+06 
                 1.8547 
                 0.1873 
                 Base to Base 
               
               
                 1.6126 
                 3.36E+07 
                 4.493 
                 1.23E+06 
                 1.9756 
                 0.2943 
                 Base to Base 
               
               
                 2.2467 
                 5.48E+08 
                 73.275 
                 2.92E+07 
                 46.9161 
                 0.5887 
                 Base to Base 
               
               
                 2.6959 
                 5.61E+06 
                 0.7509 
                 2.37E+06 
                 3.8115 
                 0.0803 
                 Base to Base 
               
               
                 8.7292 
                 4.57E+06 
                 0.6115 
                 7.87E+05 
                 1.2645 
                 0.1605 
                 Base to Base 
               
               
                 22.1814 
                 1.19E+07 
                 1.5974 
                 9.94E+05 
                 1.5977 
                 0.3478 
                 Base to Base 
               
               
                 22.538 
                 5.22E+06 
                 0.6979 
                 8.29E+05 
                 1.3331 
                 0.1873 
                 Base to Base 
               
               
                 24.7315 
                 5.89E+06 
                 0.7887 
                 1.07E+06 
                 1.7263 
                 0.1605 
                 Base to Base 
               
               
                 26.7098 
                 3.29E+06 
                 0.4408 
                 1.01E+06 
                 1.6234 
                 0.107 
                 Base to Base 
               
               
                 28.9401 
                 1.24E+07 
                 1.6572 
                 1.40E+06 
                 2.2533 
                 0.2676 
                 Base to Base 
               
               
                 29.316 
                 3.87E+06 
                 0.5185 
                 1.47E+06 
                 2.3673 
                 0.0803 
                 Base to Base 
               
               
                 33.8775 
                 4.43E+06 
                 0.5927 
                 1.17E+06 
                 1.883 
                 0.1605 
                 Base to Base 
               
               
                 35.7436 
                 4.34E+06 
                 0.5805 
                 8.92E+05 
                 1.4329 
                 0.1873 
                 Base to Base 
               
               
                 37.7239 
                 6.26E+06 
                 0.8374 
                 1.17E+06 
                 1.8777 
                 0.214 
                 Base to Base 
               
               
                 37.8343 
                 1.99E+06 
                 0.2659 
                 7.72E+05 
                 1.2402 
                 0.107 
                 Base to Base 
               
               
                 39.1183 
                 5.39E+06 
                 0.7212 
                 9.96E+05 
                 1.6006 
                 0.1606 
                 Base to Base 
               
               
                 44.5179 
                 7.33E+06 
                 0.9815 
                 9.63E+05 
                 1.5473 
                 0.2676 
                 Base to Base 
               
               
                 49.8635 
                 3.66E+06 
                 0.4897 
                 8.38E+05 
                 1.347 
                 0.1338 
                 Base to Base 
               
               
                 51.9325 
                 3.25E+06 
                 0.4342 
                 1.23E+06 
                 1.9711 
                 0.0803 
                 Base to Base 
               
               
                 52.2278 
                 3.39E+06 
                 0.4532 
                 1.25E+06 
                 2.0023 
                 0.1071 
                 Base to Base 
               
               
                 52.5806 
                 1.33E+07 
                 1.7735 
                 1.73E+06 
                 2.7832 
                 0.2676 
                 Valley 
               
               
                 52.735 
                 1.12E+07 
                 1.5033 
                 1.69E+06 
                 2.7234 
                 0.1873 
                 Valley 
               
               
                 53.8915 
                 1.27E+07 
                 1.706 
                 2.22E+06 
                 3.5732 
                 0.2141 
                 Base to Base 
               
               
                 54.4726 
                 7.86E+06 
                 1.0524 
                 1.51E+06 
                 2.4278 
                 0.1338 
                 Base to Base 
               
               
                 57.8304 
                 1.22E+07 
                 1.6307 
                 2.45E+06 
                 3.9445 
                 0.1873 
                 Base to Base 
               
               
                 59.5907 
                 5.79E+06 
                 0.7754 
                 1.11E+06 
                 1.7919 
                 0.1338 
                 Base to Base 
               
               
                 59.9224 
                 3.46E+06 
                 0.463 
                 7.03E+05 
                 1.1303 
                 0.1338 
                 Base to Base 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 LC/MS Fingerprint of extract AV016BaSu(65)01(100) 
               
               
                 (TIC Spectrum (Q1 −ve mode) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                 Area 
                   
                 Height 
                   
                 Width 
                   
               
               
                 (min) 
                 (counts) 
                 % Area 
                 (cps) 
                 % Height 
                 (min) 
                 Baseline Type 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 0.9354 
                 4.30E+06 
                 1.2293 
                 1.22E+06 
                 2.4333 
                 0.214 
                 Base to Base 
               
               
                 2.2806 
                 1.29E+08 
                 36.7178 
                 1.08E+07 
                 21.6409 
                 0.4549 
                 Base to Base 
               
               
                 2.6726 
                 4.11E+07 
                 11.7354 
                 1.62E+07 
                 32.297 
                 0.0803 
                 Base to Base 
               
               
                 2.9712 
                 3.79E+07 
                 10.8273 
                 7.74E+06 
                 15.4562 
                 0.1873 
                 Base to Base 
               
               
                 51.3175 
                 1.61E+06 
                 0.4586 
                 6.88E+05 
                 1.3736 
                 0.0803 
                 Base to Base 
               
               
                 53.8048 
                 2.24E+07 
                 6.3996 
                 1.62E+06 
                 3.2379 
                 0.3211 
                 Base to Base 
               
               
                 54.3764 
                 3.18E+07 
                 9.0759 
                 2.29E+06 
                 4.5779 
                 0.5351 
                 Base to Base 
               
               
                 56.2363 
                 1.47E+07 
                 4.2035 
                 1.55E+06 
                 3.1017 
                 0.3478 
                 Base to Base 
               
               
                 56.9396 
                 2.67E+06 
                 0.7637 
                 1.65E+06 
                 3.2962 
                 0.0535 
                 Base to Base 
               
               
                 57.4693 
                 1.54E+07 
                 4.4064 
                 2.12E+06 
                 4.2359 
                 0.2676 
                 Valley 
               
               
                 57.8919 
                 4.50E+07 
                 12.8633 
                 3.30E+06 
                 6.595 
                 0.5886 
                 Valley 
               
               
                 58.5708 
                 4.62E+06 
                 1.3192 
                 8.79E+05 
                 1.7544 
                 0.2141 
                 Base to Base 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 LC/MS Fingerprint of extract AV016BaSu(65)01(100)ng 
               
               
                 (TIC Spectrum (Q1 +ve mode) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                 Area 
                   
                 Height 
                   
                 Width 
                   
               
               
                 (min) 
                 (counts) 
                 % Area 
                 (cps) 
                 % Height 
                 (min) 
                 Baseline Type 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 0.2815 
                 2.64E+07 
                 2.56 
                 1.89E+06 
                 2.0165 
                 0.4014 
                 Base to Base 
               
               
                 0.6177 
                 2.15E+07 
                 2.0898 
                 4.89E+06 
                 5.2322 
                 0.214 
                 Base to Base 
               
               
                 1.4595 
                 6.17E+07 
                 5.9816 
                 5.53E+06 
                 5.911 
                 0.3478 
                 Valley 
               
               
                 1.6305 
                 2.63E+07 
                 2.5549 
                 3.33E+06 
                 3.5575 
                 0.1873 
                 Valley 
               
               
                 2.2215 
                 5.21E+08 
                 50.5534 
                 3.02E+07 
                 32.3009 
                 0.5352 
                 Base to Base 
               
               
                 4.4158 
                 5.86E+06 
                 0.5685 
                 1.43E+06 
                 1.5317 
                 0.1605 
                 Base to Base 
               
               
                 10.5664 
                 6.14E+06 
                 0.5957 
                 7.92E+05 
                 0.8469 
                 0.2408 
                 Base to Base 
               
               
                 23.1719 
                 7.20E+06 
                 0.6985 
                 1.10E+06 
                 1.1733 
                 0.2141 
                 Base to Base 
               
               
                 26.6471 
                 2.14E+06 
                 0.2071 
                 1.07E+06 
                 1.1409 
                 0.0803 
                 Base to Base 
               
               
                 28.4723 
                 8.22E+06 
                 0.7973 
                 9.73E+05 
                 1.04 
                 0.2676 
                 Base to Base 
               
               
                 29.1292 
                 3.99E+06 
                 0.387 
                 7.08E+05 
                 0.7574 
                 0.1873 
                 Base to Base 
               
               
                 31.5736 
                 8.16E+06 
                 0.7917 
                 8.24E+05 
                 0.8813 
                 0.2408 
                 Base to Base 
               
               
                 31.6945 
                 2.56E+06 
                 0.2481 
                 7.61E+05 
                 0.8141 
                 0.107 
                 Base to Base 
               
               
                 33.9826 
                 1.00E+07 
                 0.9715 
                 1.28E+06 
                 1.3722 
                 0.2943 
                 Base to Base 
               
               
                 36.5048 
                 4.65E+06 
                 0.4512 
                 8.70E+05 
                 0.93 
                 0.1338 
                 Base to Base 
               
               
                 37.6212 
                 3.23E+06 
                 0.3131 
                 1.19E+06 
                 1.2726 
                 0.107 
                 Base to Base 
               
               
                 39.8509 
                 3.18E+06 
                 0.3083 
                 7.69E+05 
                 0.822 
                 0.1338 
                 Base to Base 
               
               
                 42.0646 
                 4.16E+06 
                 0.4038 
                 9.70E+05 
                 1.0375 
                 0.1605 
                 Base to Base 
               
               
                 42.4115 
                 3.05E+06 
                 0.2954 
                 6.68E+05 
                 0.7142 
                 0.1338 
                 Base to Base 
               
               
                 42.5886 
                 5.36E+06 
                 0.5203 
                 1.14E+06 
                 1.2137 
                 0.1338 
                 Base to Base 
               
               
                 43.7214 
                 1.66E+06 
                 0.1606 
                 8.64E+05 
                 0.9238 
                 0.0803 
                 Base to Base 
               
               
                 49.6266 
                 3.44E+06 
                 0.3332 
                 6.23E+05 
                 0.6663 
                 0.1605 
                 Base to Base 
               
               
                 49.8477 
                 1.80E+06 
                 0.175 
                 9.30E+05 
                 0.9949 
                 0.0803 
                 Base to Base 
               
               
                 50.2725 
                 2.67E+06 
                 0.2585 
                 1.22E+06 
                 1.3044 
                 0.0803 
                 Base to Base 
               
               
                 50.4625 
                 2.57E+06 
                 0.2489 
                 1.25E+06 
                 1.3321 
                 0.0803 
                 Base to Base 
               
               
                 51.6971 
                 9.21E+06 
                 0.8936 
                 1.33E+06 
                 1.4175 
                 0.3211 
                 Base to Base 
               
               
                 52.2644 
                 3.77E+06 
                 0.3661 
                 1.08E+06 
                 1.1526 
                 0.1338 
                 Base to Base 
               
               
                 52.6743 
                 1.05E+07 
                 1.0181 
                 1.27E+06 
                 1.3617 
                 0.3478 
                 Base to Base 
               
               
                 53.3229 
                 1.80E+06 
                 0.175 
                 7.11E+05 
                 0.7607 
                 0.0803 
                 Base to Base 
               
               
                 54.0466 
                 5.16E+07 
                 5.0087 
                 5.02E+06 
                 5.3685 
                 0.4281 
                 Base to Base 
               
               
                 54.5611 
                 7.42E+06 
                 0.7194 
                 1.35E+06 
                 1.439 
                 0.1873 
                 Base to Base 
               
               
                 55.786 
                 3.29E+06 
                 0.3187 
                 1.22E+06 
                 1.304 
                 0.107 
                 Base to Base 
               
               
                 56.3205 
                 9.29E+06 
                 0.9008 
                 1.81E+06 
                 1.9397 
                 0.1605 
                 Base to Base 
               
               
                 56.982 
                 1.01E+07 
                 0.9748 
                 1.91E+06 
                 2.0474 
                 0.1873 
                 Base to Base 
               
               
                 57.5849 
                 6.16E+07 
                 5.9742 
                 5.18E+06 
                 5.5408 
                 0.4549 
                 Valley 
               
               
                 57.9722 
                 1.07E+08 
                 10.4 
                 6.43E+06 
                 6.8743 
                 0.6421 
                 Valley 
               
               
                 59.2956 
                 8.01E+06 
                 0.7772 
                 9.41E+05 
                 1.0067 
                 0.2676 
                 Base to Base 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 LC/MS Fingerprint of extract AV016BaSu(65)01(100)g 
               
               
                 (TIC Spectrum (Q1 +ve mode) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                 Area 
                   
                 Height 
                   
                 Width 
                   
               
               
                 (min) 
                 (counts) 
                 % Area 
                 (cps) 
                 % Height 
                 (min) 
                 Baseline Type 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 0.2462 
                 4.54E+06 
                 0.4516 
                 1.28E+06 
                 1.5367 
                 0.1605 
                 Base to Base 
               
               
                 0.7783 
                 2.91E+07 
                 2.896 
                 2.30E+06 
                 2.7508 
                 0.4281 
                 Base to Base 
               
               
                 1.4229 
                 1.44E+08 
                 14.3325 
                 1.54E+07 
                 18.4396 
                 0.2676 
                 Valley 
               
               
                 1.6808 
                 2.48E+08 
                 24.6093 
                 1.67E+07 
                 20.0141 
                 0.4281 
                 Valley 
               
               
                 2.2103 
                 5.05E+08 
                 50.1865 
                 3.12E+07 
                 37.4091 
                 0.5084 
                 Base to Base 
               
               
                 2.6741 
                 2.06E+07 
                 2.0484 
                 6.02E+06 
                 7.2093 
                 0.1338 
                 Base to Base 
               
               
                 3.5511 
                 4.01E+06 
                 0.3982 
                 1.49E+06 
                 1.7817 
                 0.107 
                 Base to Base 
               
               
                 4.8158 
                 1.41E+07 
                 1.4004 
                 2.22E+06 
                 2.6599 
                 0.2676 
                 Base to Base 
               
               
                 27.5026 
                 4.41E+06 
                 0.4385 
                 1.10E+06 
                 1.3192 
                 0.2141 
                 Base to Base 
               
               
                 53.9115 
                 1.52E+07 
                 1.5124 
                 1.73E+06 
                 2.0716 
                 0.3479 
                 Base to Base 
               
               
                 55.1432 
                 1.04E+07 
                 1.0317 
                 1.45E+06 
                 1.735 
                 0.3478 
                 Base to Base 
               
               
                 56.3186 
                 2.19E+06 
                 0.2172 
                 8.58E+05 
                 1.0289 
                 0.0803 
                 Base to Base 
               
               
                 57.4751 
                 1.54E+06 
                 0.153 
                 7.41E+05 
                 0.8876 
                 0.0803 
                 Base to Base 
               
               
                 57.9589 
                 3.26E+06 
                 0.3241 
                 9.65E+05 
                 1.1566 
                 0.107 
                 Base to Base 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 LC/MS Fingerprint of extract AV016BaSu(65)04(100) 
               
               
                 (TIC Spectrum (Q1 +ve mode) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                 Area 
                   
                 Height 
                   
                 Width 
                   
               
               
                 (min) 
                 (counts) 
                 % Area 
                 (cps) 
                 % Height 
                 (min) 
                 Baseline Type 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1.3402 
                 1.14E+08 
                 17.1348 
                 1.35E+07 
                 13.7133 
                 0.2676 
                 Valley 
               
               
                 1.6073 
                 1.74E+07 
                 2.6038 
                 1.93E+06 
                 1.9612 
                 0.1606 
                 Valley 
               
               
                 2.2967 
                 1.87E+08 
                 28.0404 
                 1.01E+07 
                 10.3069 
                 0.4817 
                 Base to Base 
               
               
                 4.0358 
                 4.53E+06 
                 0.6786 
                 1.01E+06 
                 1.0332 
                 0.1338 
                 Valley 
               
               
                 4.1691 
                 3.10E+06 
                 0.4647 
                 1.31E+06 
                 1.3305 
                 0.0803 
                 Valley 
               
               
                 4.385 
                 5.02E+06 
                 0.7515 
                 1.42E+06 
                 1.4467 
                 0.1338 
                 Base to Base 
               
               
                 4.9989 
                 5.94E+06 
                 0.8892 
                 1.16E+06 
                 1.1771 
                 0.1605 
                 Base to Base 
               
               
                 5.3019 
                 3.46E+06 
                 0.5183 
                 1.06E+06 
                 1.0776 
                 0.1338 
                 Base to Base 
               
               
                 5.9113 
                 2.78E+06 
                 0.4167 
                 1.30E+06 
                 1.3198 
                 0.0803 
                 Base to Base 
               
               
                 6.1778 
                 4.49E+06 
                 0.6721 
                 7.58E+05 
                 0.7719 
                 0.1873 
                 Base to Base 
               
               
                 6.3704 
                 2.71E+06 
                 0.4056 
                 7.84E+05 
                 0.7985 
                 0.107 
                 Base to Base 
               
               
                 6.8924 
                 6.19E+06 
                 0.9275 
                 7.75E+05 
                 0.7891 
                 0.214 
                 Base to Base 
               
               
                 7.2702 
                 3.77E+06 
                 0.5654 
                 1.17E+06 
                 1.1885 
                 0.107 
                 Base to Base 
               
               
                 7.4357 
                 2.04E+06 
                 0.3051 
                 9.28E+05 
                 0.945 
                 0.0803 
                 Base to Base 
               
               
                 7.9721 
                 4.93E+06 
                 0.7391 
                 9.22E+05 
                 0.939 
                 0.1873 
                 Base to Base 
               
               
                 8.8605 
                 6.09E+06 
                 0.9128 
                 7.53E+05 
                 0.7667 
                 0.2141 
                 Base to Base 
               
               
                 9.4252 
                 2.87E+06 
                 0.4292 
                 7.85E+05 
                 0.7996 
                 0.1071 
                 Base to Base 
               
               
                 9.789 
                 4.10E+06 
                 0.6138 
                 6.12E+05 
                 0.6234 
                 0.2676 
                 Base to Base 
               
               
                 10.7492 
                 1.94E+06 
                 0.2902 
                 7.58E+05 
                 0.7718 
                 0.0803 
                 Base to Base 
               
               
                 13.0858 
                 2.60E+06 
                 0.3897 
                 6.98E+05 
                 0.7105 
                 0.1094 
                 Valley 
               
               
                 13.1888 
                 3.36E+06 
                 0.5029 
                 7.11E+05 
                 0.7238 
                 0.1582 
                 Valley 
               
               
                 15.4704 
                 4.27E+06 
                 0.64 
                 1.29E+06 
                 1.3121 
                 0.1338 
                 Base to Base 
               
               
                 16.5549 
                 2.37E+06 
                 0.3545 
                 8.66E+05 
                 0.8822 
                 0.0802 
                 Base to Base 
               
               
                 17.1515 
                 4.19E+06 
                 0.6275 
                 1.24E+06 
                 1.2608 
                 0.107 
                 Valley 
               
               
                 17.2577 
                 5.17E+06 
                 0.7752 
                 9.38E+05 
                 0.9557 
                 0.1606 
                 Valley 
               
               
                 17.4256 
                 5.27E+06 
                 0.7899 
                 8.05E+05 
                 0.8196 
                 0.1605 
                 Base to Base 
               
               
                 17.7689 
                 5.45E+06 
                 0.816 
                 1.09E+06 
                 1.1149 
                 0.1338 
                 Base to Base 
               
               
                 19.3648 
                 2.95E+06 
                 0.4416 
                 8.37E+05 
                 0.8528 
                 0.1606 
                 Base to Base 
               
               
                 19.5311 
                 1.19E+06 
                 0.1786 
                 5.57E+05 
                 0.5667 
                 0.0803 
                 Base to Base 
               
               
                 20.302 
                 5.74E+06 
                 0.8605 
                 9.81E+05 
                 0.9987 
                 0.1605 
                 Base to Base 
               
               
                 23.1422 
                 4.84E+06 
                 0.7254 
                 8.61E+05 
                 0.8767 
                 0.1605 
                 Base to Base 
               
               
                 23.4055 
                 3.51E+06 
                 0.5262 
                 7.76E+05 
                 0.7901 
                 0.1345 
                 Base to Base 
               
               
                 25.5012 
                 2.20E+06 
                 0.3299 
                 1.04E+06 
                 1.0621 
                 0.0803 
                 Base to Base 
               
               
                 25.9266 
                 2.59E+06 
                 0.3882 
                 9.14E+05 
                 0.931 
                 0.107 
                 Base to Base 
               
               
                 26.7339 
                 3.31E+06 
                 0.496 
                 1.09E+06 
                 1.1142 
                 0.107 
                 Base to Base 
               
               
                 27.8535 
                 3.91E+06 
                 0.5853 
                 1.02E+06 
                 1.0382 
                 0.1873 
                 Base to Base 
               
               
                 28.7851 
                 2.65E+06 
                 0.3968 
                 9.95E+05 
                 1.0129 
                 0.0803 
                 Valley 
               
               
                 28.9276 
                 5.31E+06 
                 0.7949 
                 8.69E+05 
                 0.8849 
                 0.1873 
                 Valley 
               
               
                 29.0503 
                 4.46E+06 
                 0.6684 
                 1.13E+06 
                 1.1496 
                 0.1605 
                 Valley 
               
               
                 29.6159 
                 2.76E+06 
                 0.413 
                 1.10E+06 
                 1.1195 
                 0.107 
                 Base to Base 
               
               
                 31.7858 
                 2.66E+06 
                 0.3987 
                 7.40E+05 
                 0.7538 
                 0.1338 
                 Valley 
               
               
                 31.9243 
                 2.84E+06 
                 0.4248 
                 5.33E+05 
                 0.543 
                 0.1605 
                 Valley 
               
               
                 32.5063 
                 3.51E+06 
                 0.5259 
                 1.20E+06 
                 1.2204 
                 0.107 
                 Base to Base 
               
               
                 33.128 
                 3.73E+06 
                 0.558 
                 7.30E+05 
                 0.7436 
                 0.2141 
                 Base to Base 
               
               
                 33.3651 
                 1.37E+06 
                 0.2059 
                 5.85E+05 
                 0.596 
                 0.0803 
                 Base to Base 
               
               
                 34.7807 
                 3.81E+06 
                 0.5705 
                 9.24E+05 
                 0.9412 
                 0.1338 
                 Base to Base 
               
               
                 35.2633 
                 2.05E+06 
                 0.3075 
                 7.05E+05 
                 0.7181 
                 0.107 
                 Base to Base 
               
               
                 36.9489 
                 5.65E+06 
                 0.8458 
                 9.78E+05 
                 0.9955 
                 0.1605 
                 Base to Base 
               
               
                 38.1394 
                 2.87E+06 
                 0.4303 
                 8.43E+05 
                 0.8586 
                 0.107 
                 Base to Base 
               
               
                 38.6896 
                 6.18E+06 
                 0.9252 
                 1.03E+06 
                 1.0486 
                 0.2408 
                 Base to Base 
               
               
                 40.5898 
                 2.15E+06 
                 0.3224 
                 5.81E+05 
                 0.5917 
                 0.1338 
                 Base to Base 
               
               
                 41.5733 
                 7.00E+06 
                 1.0491 
                 9.43E+05 
                 0.96 
                 0.2408 
                 Base to Base 
               
               
                 42.0674 
                 3.46E+06 
                 0.5184 
                 8.98E+05 
                 0.9143 
                 0.1338 
                 Base to Base 
               
               
                 42.2718 
                 8.14E+06 
                 1.2191 
                 1.05E+06 
                 1.0705 
                 0.2676 
                 Base to Base 
               
               
                 42.4327 
                 2.55E+06 
                 0.3818 
                 6.87E+05 
                 0.6998 
                 0.107 
                 Base to Base 
               
               
                 44.1144 
                 7.48E+06 
                 1.1211 
                 1.08E+06 
                 1.1033 
                 0.2943 
                 Base to Base 
               
               
                 46.1964 
                 6.08E+06 
                 0.9103 
                 8.94E+05 
                 0.9104 
                 0.1873 
                 Base to Base 
               
               
                 46.8725 
                 3.21E+06 
                 0.4811 
                 7.64E+05 
                 0.7785 
                 0.1338 
                 Base to Base 
               
               
                 47.3852 
                 1.47E+06 
                 0.2207 
                 7.06E+05 
                 0.7194 
                 0.0803 
                 Base to Base 
               
               
                 50.6192 
                 1.54E+06 
                 0.2307 
                 6.74E+05 
                 0.6865 
                 0.0803 
                 Base to Base 
               
               
                 50.8303 
                 3.42E+06 
                 0.5122 
                 1.03E+06 
                 1.0527 
                 0.107 
                 Base to Base 
               
               
                 51.2141 
                 6.26E+06 
                 0.9376 
                 1.25E+06 
                 1.2757 
                 0.1873 
                 Base to Base 
               
               
                 51.531 
                 8.74E+06 
                 1.3088 
                 1.14E+06 
                 1.1594 
                 0.2408 
                 Valley 
               
               
                 51.6971 
                 7.62E+06 
                 1.1419 
                 1.49E+06 
                 1.5193 
                 0.1606 
                 Valley 
               
               
                 51.9366 
                 3.58E+06 
                 0.5358 
                 1.13E+06 
                 1.146 
                 0.1338 
                 Base to Base 
               
               
                 52.5223 
                 4.27E+06 
                 0.6399 
                 1.01E+06 
                 1.0277 
                 0.1338 
                 Base to Base 
               
               
                 53.2728 
                 4.39E+06 
                 0.6573 
                 7.78E+05 
                 0.7923 
                 0.1606 
                 Base to Base 
               
               
                 53.8949 
                 1.32E+07 
                 1.9795 
                 1.63E+06 
                 1.6568 
                 0.3211 
                 Base to Base 
               
               
                 54.4009 
                 4.66E+06 
                 0.6975 
                 1.34E+06 
                 1.3633 
                 0.107 
                 Valley 
               
               
                 54.5073 
                 1.82E+07 
                 2.7254 
                 1.63E+06 
                 1.6624 
                 0.2676 
                 Valley 
               
               
                 55.9786 
                 4.07E+06 
                 0.6101 
                 9.34E+05 
                 0.9515 
                 0.1338 
                 Base to Base 
               
               
                 56.3475 
                 1.33E+07 
                 1.9928 
                 1.13E+06 
                 1.1551 
                 0.3211 
                 Valley 
               
               
                 56.5847 
                 4.61E+06 
                 0.6907 
                 8.48E+05 
                 0.864 
                 0.1605 
                 Valley 
               
               
                 56.8275 
                 9.26E+06 
                 1.3875 
                 1.22E+06 
                 1.243 
                 0.1873 
                 Valley 
               
               
                 57.0145 
                 4.88E+06 
                 0.7309 
                 9.98E+05 
                 1.016 
                 0.1606 
                 Valley 
               
               
                 57.1188 
                 2.45E+06 
                 0.3669 
                 7.75E+05 
                 0.7891 
                 0.107 
                 Base to Base 
               
               
                 57.7155 
                 6.35E+06 
                 0.952 
                 1.11E+06 
                 1.1327 
                 0.1606 
                 Valley 
               
               
                 57.8152 
                 9.52E+06 
                 1.4268 
                 1.38E+06 
                 1.4031 
                 0.2141 
                 Valley 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 LC/MS Fingerprint of extract AV016BaSu(65)06(100) 
               
               
                 (TIC Spectrum (Q1 +ve mode) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                 Area 
                   
                 Height 
                   
                 Width 
                   
               
               
                 (min) 
                 (counts) 
                 % Area 
                 (cps) 
                 % Height 
                 (min) 
                 Baseline Type 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1.3425 
                 1.34E+08 
                 15.6624 
                 2.35E+07 
                 34.2569 
                 0.2141 
                 Base to Base 
               
               
                 2.3068 
                 6.22E+08 
                 72.7416 
                 3.24E+07 
                 47.2835 
                 0.5886 
                 Base to Base 
               
               
                 4.4357 
                 5.31E+06 
                 0.6201 
                 1.63E+06 
                 2.3849 
                 0.1605 
                 Base to Base 
               
               
                 5.2161 
                 2.96E+06 
                 0.3463 
                 1.29E+06 
                 1.8752 
                 0.107 
                 Base to Base 
               
               
                 5.6491 
                 9.55E+06 
                 1.1164 
                 1.58E+06 
                 2.3015 
                 0.1873 
                 Base to Base 
               
               
                 6.6417 
                 9.07E+06 
                 1.0594 
                 2.04E+06 
                 2.9753 
                 0.1605 
                 Base to Base 
               
               
                 17.6827 
                 1.27E+07 
                 1.4888 
                 1.34E+06 
                 1.954 
                 0.4281 
                 Base to Base 
               
               
                 54.1984 
                 5.00E+07 
                 5.8447 
                 3.32E+06 
                 4.8386 
                 0.4548 
                 Base to Base 
               
               
                 57.3586 
                 9.59E+06 
                 1.1203 
                 1.46E+06 
                 2.1301 
                 0.1873 
                 Base to Base 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 LC/MS Fingerprint of extract AV016BaSu(105)08(100) 
               
               
                 (TIC Spectrum (Q1 +ve mode) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                 Area 
                   
                 Height 
                   
                 Width 
                   
               
               
                 (min) 
                 (counts) 
                 % Area 
                 (cps) 
                 % Height 
                 (min) 
                 Baseline Type 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 2.0569 
                 2.14E+08 
                 27.1727 
                 2.15E+07 
                 36.3993 
                 0.2677 
                 Base to Base 
               
               
                 3.0165 
                 5.13E+08 
                 65.1587 
                 2.56E+07 
                 43.3466 
                 0.7224 
                 Base to Base 
               
               
                 4.4937 
                 1.06E+07 
                 1.3463 
                 2.07E+06 
                 3.4978 
                 0.1873 
                 Base to Base 
               
               
                 4.8116 
                 9.88E+06 
                 1.255 
                 1.36E+06 
                 2.304 
                 0.2141 
                 Base to Base 
               
               
                 7.1128 
                 9.55E+06 
                 1.2133 
                 1.79E+06 
                 3.0334 
                 0.1606 
                 Base to Base 
               
               
                 20.2582 
                 6.97E+06 
                 0.8856 
                 1.05E+06 
                 1.7785 
                 0.1873 
                 Base to Base 
               
               
                 21.0285 
                 2.37E+06 
                 0.3008 
                 8.07E+05 
                 1.3647 
                 0.107 
                 Base to Base 
               
               
                 21.9949 
                 5.91E+06 
                 0.751 
                 1.41E+06 
                 2.3921 
                 0.1605 
                 Base to Base 
               
               
                 26.1397 
                 4.67E+06 
                 0.5933 
                 1.60E+06 
                 2.7139 
                 0.1338 
                 Base to Base 
               
               
                 28.479 
                 1.04E+07 
                 1.3233 
                 1.87E+06 
                 3.1697 
                 0.1873 
                 Base to Base 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 LC/MS Fingerprint of extract AV016FrDi(65)04(100) 
               
               
                 (TIC Spectrum (Q1 +ve mode) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                 Area 
                   
                 Height 
                   
                 Width 
                   
               
               
                 (min) 
                 (counts) 
                 % Area 
                 (cps) 
                 % Height 
                 (min) 
                 Baseline Type 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1.6028 
                 1.52E+09 
                 29.4877 
                 6.12E+07 
                 29.3519 
                 0.6423 
                 Valley 
               
               
                 2.2961 
                 3.50E+09 
                 68.0186 
                 1.30E+08 
                 62.3426 
                 0.99 
                 Valley 
               
               
                 4.0708 
                 3.33E+06 
                 0.0647 
                 1.35E+06 
                 0.6474 
                 0.107 
                 Base to Base 
               
               
                 4.8702 
                 1.71E+07 
                 0.3327 
                 2.48E+06 
                 1.1882 
                 0.2675 
                 Base to Base 
               
               
                 5.4024 
                 9.12E+06 
                 0.1773 
                 2.48E+06 
                 1.1914 
                 0.1338 
                 Base to Base 
               
               
                 6.2587 
                 4.84E+06 
                 0.0941 
                 1.53E+06 
                 0.7348 
                 0.107 
                 Base to Base 
               
               
                 47.0653 
                 5.85E+07 
                 1.1379 
                 2.31E+06 
                 1.1097 
                 0.6689 
                 Base to Base 
               
               
                 48.8324 
                 4.52E+06 
                 0.0879 
                 1.54E+06 
                 0.7369 
                 0.1338 
                 Base to Base 
               
               
                 49.5832 
                 5.13E+06 
                 0.0998 
                 1.18E+06 
                 0.5668 
                 0.1338 
                 Base to Base 
               
               
                 51.2074 
                 3.34E+06 
                 0.065 
                 1.38E+06 
                 0.6601 
                 0.0802 
                 Base to Base 
               
               
                 51.9303 
                 6.03E+06 
                 0.1171 
                 1.52E+06 
                 0.7281 
                 0.1338 
                 Base to Base 
               
               
                 57.0683 
                 1.63E+07 
                 0.3173 
                 1.55E+06 
                 0.7421 
                 0.3211 
                 Base to Base 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 LC/MS Fingerprint of extract AV016FrSu(105)08(100) 
               
               
                 (TIC Spectrum (Q1 +ve mode) 
               
               
                 Peak List: 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Time 
                 Area 
                   
                 Height 
                   
                 Width 
                   
               
               
                 (min) 
                 (counts) 
                 % Area 
                 (cps) 
                 % Height 
                 (min) 
                 Baseline Type 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1.9597 
                 2.64E+08 
                 29.489 
                 2.96E+07 
                 45.0514 
                 0.2677 
                 Base to Base 
               
               
                 3.1068 
                 6.21E+08 
                 69.4326 
                 3.28E+07 
                 49.9315 
                 0.6689 
                 Base to Base 
               
               
                 5.6942 
                 4.34E+06 
                 0.485 
                 1.88E+06 
                 2.8549 
                 0.0802 
                 Base to Base 
               
               
                 22.1023 
                 5.31E+06 
                 0.5934 
                 1.42E+06 
                 2.1622 
                 0.1605 
                 Base to Base 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 IC 50  values of alpha-glucosidase inhibition activity 
               
               
                 of extracts from different  T. arjuna  plant parts 
               
            
           
           
               
               
               
            
               
                   
                   
                 IC 50   
               
               
                 Plant Part 
                 Extract-ID 
                 (μg/ml) 
               
               
                   
               
               
                  1. Bark 
                 AV016BaDi(65)04(100) 
                 13.2 
               
               
                  2. Bark 
                 AV016BaDi(28)04(20) 
                 14.2 
               
               
                  3. Bark 
                 AV016BaSu(65)01(100)g 
                 14.3 
               
               
                  4. Bark 
                 AV016BaSu(65)01(100)ng 
                 15.0 
               
               
                  5. Bark 
                 AV016BaSu(65)04(100) 
                 16.5 
               
               
                  6. Bark 
                 AV016BaSu(65)06(100) 
                 24.0 
               
               
                  7. Bark 
                 AV016BaSu(105)08(100) 
                 31.5 
               
               
                  8. Bark 
                 AV016BaSu(65)09(100) 
                 42.7 
               
               
                  9. Fruits 
                 AV016FrDi(65)04(100) 
                 16.3 
               
               
                 10. Fruits 
                 AV016FrSu(105)08(100) 
                 31.5 
               
               
                 11. Acarbose (Standard) 
                   
                 80.7 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 The effect of insulin and  T. arjuna  extracts on glucose up-take in 3T3-L1 adipocytes. 
               
            
           
           
               
               
               
               
            
               
                   
                 cpm 
                   
                 Stimulation 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Set 
                 Treatment 
                 Set I 
                 Set II 
                 Set III 
                 Average 
                 SD value 
                 Index 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 cells alone 
                 314.6 
                 390 
                 1200.8 
                 635.13 
                 491.33 
                   
               
               
                 2 
                 cells + Insulin (10 nM) 
                 2185.8 
                 1705.4 
                 3010.7 
                 2300.63 
                 660.18 
                 2.6 
               
               
                 3 
                 cells + Insulin (25 nM) 
                 4806.8 
                 1399.7 
                 996.7 
                 2401.07 
                 2093.15 
                 2.8 
               
               
                 4 
                 cells + Insulin (100 nM) 
                 2906.5 
                 3776.6 
                 2215.6 
                 2966.23 
                 782.21 
                 3.7 
               
               
                 5 
                 cells + AV016BaDi(65)04(100) 
                 1946.5 
                 2570.2 
                 3106.7 
                 2541.13 
                 580.65 
                 3.0 
               
               
                   
                 (Ter1) 
               
               
                 6 
                 cells + Insulin (10 nM) + 
                 2178.5 
                 1873 
                 1667.3 
                 1906.27 
                 257.22 
                 2.0 
               
               
                   
                 AV016BaDi(65)04(100) 
               
               
                   
                 (Ter1) 
               
               
                   
               
               
                 Stimulation index = {mean of sample − mean of control (cells alone)/mean of control (cells alone)} 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 Anti-obesity potential of  Terminalia arjuna  extracts. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Final Reading 
                   
               
               
                   
                 Reading 
                   
                 (Reading − 
               
               
                 Treatment 
                 OD520 nm 
                 Average 
                 Well Blank) 
                 % Inhibition 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Well Blank (−cells) 
                 0.887 
                   
                 0 
                 — 
               
               
                 Cells Alone 
                 2.883 
                 2.883 
                 1.996 
                 0.0 
               
               
                   
                 2.883 
               
               
                 Cells + DMSO 
                 2.786 
                 2.786 
                 1.899 
                 0.0 
               
               
                   
                 2.786 
               
               
                 Cells + AV016BaDi(65)04(100) 
                 1.428 
                 1.344 
                 0.457 
                 76.3 
               
               
                   
                 1.26 
               
               
                 Cells + AV016BaSu(65)09(100) 
                 2.105 
                 1.616 
                 0.729 
                 61.9 
               
               
                   
                 1.127 
               
               
                 Cells + AV016BaSu(65)01(100)ng 
                 1.161 
                 1.146 
                 0.259 
                 86.8 
               
               
                   
                 1.131 
               
               
                 Cells + AV016BaSu(65)01(100)g 
                 2.23 
                 1.6805 
                 0.7935 
                 58.5 
               
               
                   
                 1.131 
               
               
                 Cells + AV016BaSu(65)04(100) 
                 1.813 
                 1.8535 
                 0.9665 
                 49.4 
               
               
                   
                 1.894 
               
               
                 Cells + AV016BaSu(65)06(100) 
                 1.905 
                 1.693 
                 0.806 
                 57.9 
               
               
                   
                 1.481 
               
               
                 Cells + AV016BaDi(28)04(20) 
                 1.832 
                 1.6 
                 0.713 
                 64.3 
               
               
                   
                 1.368 
               
               
                 Cells + AV016FrDi(65)04(100) 
                 1.941 
                 1.9895 
                 1.1025 
                 41.9 
               
               
                   
                 2.038 
               
               
                 Cells + +ve Control 
                 0.774 
                 0.807 
                 −0.08 
                 104.0 
               
               
                   
                 0.84 
               
               
                   
               
               
                 % Inhibition = {(Mean of Control − Mean of Sample)/Mean of Control}*100 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28 
               
             
            
               
                   
               
               
                 Effect of  Terminalia arjuna  extracts 
               
               
                 on lipid levels in CHO-K1 cells. 
               
            
           
           
               
               
               
               
            
               
                 Sr. 
                   
                   
                 % Inhibition 
               
               
                 No. 
                 Treatment 
                 OD 520 nm   
                 w.r.t control 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1. 
                 Cells alone + DMSO 
                 1.206 
                 0 
               
               
                 2. 
                 Cells + AV016BaSu(65)09(100) 
                 0.944 
                 22 
               
               
                 3. 
                 Cells + AV016BaSu(65)01(100) 
                 0.964 
                 21 
               
               
                 4. 
                 Cells + AV016BaDi(65)04(100) 
                 0.844 
                 31 
               
               
                 5. 
                 Cells + AV016BaDi(28)04(20) 
                 0.873 
                 28 
               
               
                   
               
               
                 % Inhibition = {(Control − Extract treated/Control) * 100} 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 29 
               
             
            
               
                   
               
               
                 Effect of 20% ethanol extract from  T. arjuna  bark 
               
               
                 AV016BaDi(28)04(20) on HMG-CoA reductase activity. 
               
            
           
           
               
               
               
            
               
                   
                 Without Inhibitor 
                 With Inhibitor 
               
               
                 Time (mins) 
                 Reading 
                 Reading 
               
               
                   
               
            
           
           
               
               
               
            
               
                 0 
                 1.3465 
                 1.7372 
               
               
                 1 
                 1.3103 
                 1.726 
               
               
                 2 
                 1.2891 
                 1.726 
               
               
                 3 
                 1.2768 
                 1.726 
               
               
                 4 
                 1.2689 
                 1.7042 
               
               
                 5 
                 1.2534 
                 1.7042 
               
               
                 Substrate Blank 
                 0.0277 
                 0.0307 
               
               
                   
                 0 
               
               
                 Final Reading 
                 0.0654 
                 0.0046 
               
               
                 % Inhibition 
                 0 
                 93.0 
               
               
                   
               
               
                 % Inhibition = {(Control − Extract treated/Control) * 100} 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 Effect of direct 100% ethanol extract from  T. arjuna   
               
               
                 bark AV016BaDi(65)04(100) on HMG-CoA reductase activity. 
               
            
           
           
               
               
               
            
               
                   
                 Without Inhibitor 
                 With Inhibitor 
               
               
                 Time (mins) 
                 Reading 
                 Reading 
               
               
                   
               
            
           
           
               
               
               
            
               
                 0 
                 1.162 
                 1.7815 
               
               
                 1 
                 1.1125 
                 1.7637 
               
               
                 2 
                 1.1048 
                 1.7637 
               
               
                 3 
                 1.0828 
                 1.7637 
               
               
                 4 
                 1.0756 
                 1.746 
               
               
                 5 
                 1.0652 
                 1.7467 
               
               
                 Substrate Blank 
                 0.023 
                 0.0212 
               
               
                   
                 0 
               
               
                 Final Reading 
                 0.0738 
                 0.0272 
               
               
                 % Inhibition 
                 0 
                 63.1 
               
               
                   
               
               
                 % Inhibition = {(Control − Extract treated/Control) * 100} 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 31 
               
             
            
               
                   
               
               
                 Effect of  Terminalia arjuna  extracts 
               
               
                 on cell viability of HepG2 cells. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 No. of live 
                 No. of dead 
               
               
                   
                 Treatment 
                 cells 
                 cells 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Cells 
                 85 
                 20 
               
               
                   
                 Cells + DMSO 
                 82 
                 11 
               
               
                   
                 Cells + Ascorbic acid 
                 66.5 
                 18.5 
               
               
                   
                 Cells + AV016BaSu(65)09(100) 
                 27 
                 7 
               
               
                   
                 Cells + AV016BaSu(65)01(100) 
                 55 
                 2 
               
               
                   
                 Cells + AV016BaSu(65)04(100) 
                 11 
                 1