WO2008125928A2 - Standardized bioactive extracts of annona squamosa - Google Patents

Abstract

The invention relates to standardized extracts of aerial parts of plant Annona squamosa, a method of identifying, characterizing and isolating bioactive marker compounds of Formulae I and II from aerial parts of plant Annona squamosa and use thereof. The invention also relates to a method for the preparation of standardized extracts of Annona squamosa aerial parts.

Description

STANDARDIZED BIOACTIVE EXTRACTS OF ANNONA SQUAMOSA

Field of the Invention

The invention relates to standardized extracts of aerial parts of plant Annona squamosa, a method of identifying, characterizing and isolating bioactive marker compounds of Formulae I and II from aerial parts of plant Annona squamosa and use thereof. The invention also relates to a method for the preparation of standardized extracts of Annona squamosa aerial parts.

Background of the Invention Diabetes Mellitus (DM) is a metabolic disease of epidemic proportions and is increasing linearly in every part of the world, currently affecting more than 170 million people globally, whose numbers are projected to grow to about 360 million by year 2030 (WHO). Annually diabetes contributes to death of about 3.2 million worldwide, costing US alone about $132 billion a year. Diabetes is characterized primarily by hyperglycemia resulting from defects in insulin secretion or hyperinsulinimea causing insulin resistance or both. Diabetic patients are at risk of long-term damage, dysfunction, and failure of various organs, especially, retina nerves and kidney and also increased risk of cardiovascular disease. Patients may suffer either from insulin dependent type I, or non- insulin dependent type 2 diabetes. The less prominent type I diabetes is characterized by lack of insulin release, which can be corrected by administration of exogenous insulin. In contrast, about 90% of diabetics suffer from type 2 diabetes, a heterogeneous, multifactorial, and polygenic metabolic syndrome, which is caused by an inadequate secretion of insulin and its response in peripheral tissues.

Insulin is required to transfer the sugar from the blood into the cells for the production of energy. Human insulin is produced by beta cells of the islets of langerhans in pancreas. In a non-diabetic person, the beta cells secrete insulin, and when the blood sugar level drops the production of insulin stops. But in a diabetic person, the beta cells produce little or no human insulin (type 1), or cells are unable to utilize it (type 2). When this happens there is a fast elevation of blood sugar levels. This condition is called diabetes mellitus.

Apart from hyperglycemia, patients are also afflicted with other concurrent maladies like insulin resistance, obesity, hypertension, inflammation and dyslipidemia that are collectively clubbed as 'metabolic syndrome' or 'syndrome X' or 'the deadly quartet' and are associated with increased risk of cardiovascular disease. Given its prevalence and complexity, there is a growing need for novel strategies and effective therapeutic approaches for the treatment of diabetes. Insulin resistance arises from the inability of insulin to act normally in regulating nutrient metabolism in peripheral tissues. Increasing evidence from human population studies and animal research has established correlative as well as causative links between chronic inflammation and insulin resistance. High levels of glucose auto oxidize, that is, start a chain reaction that produces large amounts of free radicals and "advanced glycation products," both of which damage the body. Free radicals stimulate inflammatory responses and, in this way, people with diabetes develop high levels of inflammation.

Alternatively, people suffering from chronic inflammatory conditions are more prone to develop insulin resistance. This situation has been well documented in several studies that have found sharp elevations of pro-inflammatory markers like CRP (C- reactive protein) and interleukin-6 and reduced levels of anti-inflammatory markers such as adiponectin in people with diabetes. Because of the ability of inflammatory cytokines to stimulate one another, people with diabetes typically have a strong undercurrent of inflammation, which increases the risk of other diseases, such as heart disease. Based on pre-clinical data, several cytokines, chemokines and transcription factors have been implicated in the inflammatory process which ultimately leads to diabetes. One key transcription factor where much attention has been focused is Nuclear factor-kB (NF-kB).

AMP- activated protein kinase (AMPK) is a member of a metabolite-sensing protein kinase family that is found in all eukaryotes (Hardie et al., Annu Rev Biochem 67; 821-855 (1998)). AMPK has been proposed to act as a cellular fuel sensor, and is a potent mediator of exercise-induced glucose uptake in skeletal muscles (Hardie, Endocrinology 144; 5179-5183 (2003).

Impairment in fuel metabolism occurs in obesity and this impairment is a leading pathogenic factor in type 2 diabetes. The insulin resistance associated with T2DM is most profound at the level of skeletal muscle as this is the primary site of glucose and fatty acid utilization. Activation of AMPK, in skeletal muscle, leads to increased glucose uptake, enhanced insulin sensitivity and oxidation of fatty acids. In the liver, AMPK activation causes an increase in fatty acid oxidation and inhibition of glucose production. These effects on glucose and fat metabolism make AMPK an important pharmacological target for the treatment of type 2 diabetes. It has already been shown that two existing classes of drugs used to treat diabetes i.e. the biguanides (Metformin) and thiazolidinediones, exert some of their effects via activation of AMPK. AMPK is also required for cardiovascular beneficial effects of Metformin.

An increasing body of evidence has linked AMP- activated protein kinase (AMPK) and malonyl coenzyme A (CoA) to the regulation of energy balance. AMPK activation also increases the expression of uncoupling proteins and the transcriptional regulator peroxisome proliferator-activated receptor "? coactivator-lα (PGCIa), which could possibly increase energy expenditure.

Several findings also point to a link between AMPK and the growth and/or survival of some cancer cells. Searching for the upstream kinase(s) that regulate AMPK led to the identification of LKBl. The link between LKBl and the cancer-prone Peutz- Jeghers syndrome (PJS) (Giardiello et al., Gastroenterology 119; 1447-1453 (2000), and the identification of LKB 1 as a tumor suppressor and now as the long-sought AMPK, provide a molecular basis for the interaction between metabolism and cell proliferation. It is possible that AMPK-activating drugs could prove promising in the treatment of LKBl- deficient cancers. Furthermore LKBl now joins AMPK as an attractive target for activating drugs that would be useful in the treatment of obesity and Type 2 diabetes. Another tumor suppressor, tuberous sclerosis complex 2 (TSC2), is phosphorylated and activated by AMPK. In addition, other studies indicate that mammalian homolog of target of rapamycin (mTOR), which has been implicated in the pathogenesis of insulin resistance and many types of cancer, is inhibited by AMPK. Another potential mechanism by which AMPK might function in cancer therapy is via inhibition of FAS expression and of translation elongation factor 2 (EF2) (Horman et al.,. Curr Bio, 12; 1419-1423 (2002)).

From ancient times until the discovery of insulin in the 1920's, nutritional therapy was the only available means of treating diabetes. As early as 2500-1800 BC, there is a mention of curative properties of medicinal plants in Rigveda. Charaka Samhita and Sushruta Samhita give extensive description of various medicinal plants. It is interesting to note that till today metformin is the only drug approved for the treatment of non insulin dependent diabetes mellitus (NIDDM) patients, which is derived from a medicinal plant, the French lilac. In past there have been many medicinal plants, which have been investigated for their anti-diabetic properties but little efforts have been made to isolate new chemical entities based on defined mode of action or preparing herbal extracts in a standardized formulation form using well defined bio-markers.

It is also well known that the incidence of diabetes mellitus is highest in Asia. Different types of oral hypoglycemic agents such as biguanidines and sulphonylurea are available along with insulin for the treatment of diabetes mellitus, but have side effects associated with their uses. There is a growing interest in herbal remedies because of their effectiveness, minimal side effects in clinical experience and relatively low cost. Herbal drugs or their extracts are prescribed widely, even when their biological active compounds are unknown. Even World Health Organization (WHO) approves the use of plant drugs for different diseases, including diabetes mellitus.

Plant products and derivatives are being intensively researched nowadays for various pharmacological conditions. The synergistic components found in botanical mixtures represent a largely untapped source of new pharmaceutical products with novel and multiple mechanisms of action and targeting multiple diseases simultaneously. So, a good anti-diabetic agent might also have anti-inflammatory properties and anticancer properties. Many botanical products (herbs) are known to correct glucose metabolism, improve lipid metabolism and also exhibit antioxidant properties. A number of medicinal herbs have been found to possess hypoglycemic activities. Annona squamosa is one such plant.

The plant Annona squamosa (Annonaceae), commonly known as custard apple in English and sharifa in Hindi, is a native of West Indies and is cultivated throughout India, mainly for its edible fruit. The plant is known to possess varied medicinal properties, which include anti-fertility and anti-tumor activities in mice and rats (Pardhasaradhi BVV et al., Journal ofBioscience, 30(2); 237-244 (2005) and Rao, VSN et al., Ind J Med Res, 70; 517-520 (1979)). Several workers have investigated its use as insecticidal agent. Various photochemical, pharmacological, antibacterial and antiovulatory studies have been carried out with its seed extract. Tribal populations in parts of Northern India use the young leaves of Annona squamosa extensively for its anti-diabetic activity. The aqueous leaf extract of Annona squamosa has also been reported to ameliorate hyperthyroidism, which is often considered as one of the etiological factors for diabetes mellitus. WO 2004/060383 discloses a process of preparing an extract of Annona Squamosa for the treatment of diabetes.

As mentioned above, even though herbs are extensively used in the treatment of various diseases apart from their common use in cooking, in modern day, herbal medicines face specific challenges. US Patent No. 6,156,291 discloses such challenges, one of which is that traditional medical practitioners are concerned with the lack of both qualitative and quantitative standards for herbal medicines. Such a lack is viewed as hindering the ability to prescribe and adjust dosages of such herbal medications. Thus, it is important to employ methods which can result in the standardization of herbal compositions with respect to pharmaceutical activity of such chemical mixtures.

Summary of the Invention

In one aspect of the present invention, there is provided a bioassay guided fractionation leading to identification and characterization of compounds, 16- hydroxyoctadeca-9 (Z or E), 12 (Z or E), 14(Z or E)-trienoic acid of Formula I

and 13-hydroxy-9Z-llE-15E-octadecatrienoic acid of Formula II

from the aerial parts of the plant, Annona squamosa. In another aspect of the present invention, there is provided a method for the isolation of compounds of Formulae I or II from the aerial parts of the plant, Annona squamosa.

In another aspect, the present invention provides a method for the preparation of extracts from the aerial parts of the plant Annona squamosa enriched with bioactive marker compounds.

In another aspect of the present invention, there is provided a method for the standardization of the extracts of Annona squamosa aerial parts. In another aspect of the present invention, there are provided standardized extracts of Annona squamosa aerial parts.

Another aspect of the invention is to provide a method for treating, preventing, inhibiting or suppressing diseases mediated through PTP-IB, for example, a disease caused due to insulin resistance, for example, diabetes and related complications including hyperglycemia, impaired glucose tolerance, insulin resistance syndrome, and other disorders in a mammal, comprising administering a therapeutically effective amount of a compound of Formula I, Formula II, or a standardized extract of Annona squamosa aerial parts. Another aspect of the invention is to provide a method for treating, preventing, inhibiting or suppressing diseases mediated through NFKB and cPLA2, for example, inflammatory conditions, for example, AIDS, asthma, arthritis, bronchitis, chronic obstructive pulmonary disease (COPD), psoriasis, allergic rhinitis, shock, atopic dermatitis, Crohn's disease, adult respiratory distress syndrome (ARDS), eosinophilic granuloma, allergic conjunctivitis, osteoarthritis or ulcerative colitis, or some cancers in a mammal, comprising administering a therapeutically effective amount of a compound of Formula I, Formula II, or a standardized extract of Annona squamosa aerial parts.

Another aspect of the invention is to provide a method for treating, preventing, inhibiting or suppressing diseases having their iteology through AMP kinase, for example, metabolic syndrome or cancer in a mammal, comprising administering a therapeutically effective amount of a compound of Formula I, Formula II, or a standardized extract of Annona squamosa aerial parts.

Another aspect of the invention is to provide a method for treating, preventing, inhibiting or suppressing diabetes in a mammal, comprising administering a therapeutically effective amount of a compound of Formula I, Formula II, or a standardized extract of Annona squamosa aerial parts.

Another aspect of this invention is to provide a pharmaceutical composition comprising of a standardized extract of Annona squamosa aerial parts along with one or more of pharmaceutically acceptable carriers, excipients or diluents. Another aspect of this invention is to provide a pharmaceutical composition comprising of a therapeutically effective amount of a compound of Formula I along with one or more of pharmaceutically acceptable carriers, excipients or diluents. Another aspect of this invention is to provide a pharmaceutical composition comprising of a therapeutically effective amount of a compound of Formula II along with one or more of pharmaceutically acceptable carriers, excipients or diluents.

Another aspect of this invention is to provide a combination comprising bioactive marker compounds of Formula I, Formula II, or a standardized extract of Annona squamosa aerial parts, and one or more of extracts of plants known to have antidiabetic use.

The details of one or more embodiments of the inventions are set forth in the description below. Other features, objects, and advantages of the inventions will be apparent from the description.

Detailed Description of the Invention

The present invention provides a bioassay guided fractionation of the aerial parts of the plant, Annona squamosa, leading to the identification and characterization of bioactive marker compounds of the plant. The process includes preparing different extracts of Annona squamosa, subjecting the extracts to the primary screening for bioactivity using glucose uptake assay, and further, evaluating the most active extract against secondary target assays, such as, IR phosphorylation, IRS phosphorylation, PD kinase mRNA expression, glut 4 mRNA expression, PTP Ib inhibition, NFKB inhibition, cPLA2 inhibition and AMP kinase activation. The active extracts are subjected to fractionation by one or more of solvents and each fraction is evaluated for the primary bioactivity assay. The active fraction is evaluated against secondary bioactivity assays and subjected to column chromatography, fractions are isolated from active fraction and evaluated for the bioactivity using primary assay, the active fractions obtained are screened against secondary assays and the most active compounds isolated from these active fractions are characterized as compounds of Formulae I and II using spectroscopy. A new series of extracts enriched with bioactive marker compounds is prepared, the enriched extracts are evaluated for the bioactivity using primary and secondary target assays and the most active extracts are evaluated for in vivo activity.

The solvents for preparing different extracts of Annona squamosa may be water, alcohols, for example, methanol or ethanol, and mixture(s) thereof. The solvents for fractionating the active extracts may be hexane, petroleum ether, toluene, diethyl ether, dichloromethane, dichloroethane, chloroform, carbon tetrachloride, ethyl acetate, butanol and mixture(s) thereof.

The present invention provides a method for the isolation of compounds of Formula I or Formula II from the aerial parts of plant Annona Squamosa. The process comprises extracting aerial parts of Annona Squamosa in one or more of solvents, concentrating the extracts, suspending the extracts in water, partitioning in one or more of solvents and isolating the compounds of Formulae I and II.

Powdered Annona squamosa aerial parts are extracted with a solvent selected from methanol, water: methanol (1:1) and water at room temperature and the combined extract is concentrated under reduced pressure. The concentrated extract is suspended in water and partitioning is carried out with a solvent selected from hexane, petroleum ether, diethyl ether, toluene, dichloromethane, dichloroethane, chloroform, carbon tetrachloride, ethyl acetate, butanol and mixture(s) thereof. The organic layers are combined and dried. The extract thus obtained is purified by column chromatography and eluted with hexane, ethyl acetate, methanol and mixture(s) thereof. Different fractions are collected and each fraction is observed by thin layer chromatography. Detection is done by observation under UV and with spraying reagents. Fraction containing compounds of Formulae I and II are collected and purified further by prep HPLC. Sample preparation is done by sonicating the said fraction in an organic solvent selected from acetonitrile and methanol followed by the addition of a buffer selected from formic acid, trifluoro acetic acid, orΛo-phosphoric acid, ammonium acetate, sodium perchlorate, potassium dihydrogen orthophosphate, dipotassium hydrogen orthophosphate, sodium dihydrogen orthophosphate, disodium hydrogen orthophosphate, diammonium hydrogen orthophosphate, ammonium dihydrogen orthophosphate, ammonium formate, tetramethyl ammonium hydroxide, tetrabutyl ammonium hydroxide and tetrabutyl ammonium hydrogen sulphate in one fourth volume of organic solvent. The mixture is subsequently filtered and subjected to preparative HPLC for isolation of compounds of Formulae I or II at room temperature by using gradient method.

Compounds of Formulae I and II were obtained by evaporating the solvent from pure fractions. Working example 1 describes the process for the isolation of compounds of Formulae I and II. The compounds were characterised on the basis of spectroscopic data. Characterization of compound of Formula I

Compound of Formula I was isolated as a colourless oil from the methanolic extract by silica gel column chromatography followed by Prep-HPLC. The LC-MS data revealed the M⁺ ion at m/z = 294 corresponding to the molecular formula C₁₈H₃₀O₃. The ¹H NMR (Table 1) suggested an unsaturated fatty acid residue with signals typical for six olefinic protons (three double bond equivalents), a one proton multiplet for an oxymethine group at δ 4.01, seven methylenes as a broad singlet at δ 1.42 two downfield methylene groups at δ 2.20 and 2.54 and finally a methyl group at δ 0.88 as a triplet. The ¹³C NMR spectrum displayed signals for eighteen carbons, comprising of nine methylenes, one carbinol carbon, one methyl carbon, an acid carbonyl and six sp² carbons. The assignments were confirmed using 2D-NMR experiments (HSQC, COSY and HMBC). For instance, the methine resonating at δ 5.25 m, H-9 showing strong COSY cross peaks with the H- 10 olefin (δ 5.50 m) which was further coupled to a down- field methylene, H₂- 11. This methylene was coupled to a third olefinic proton that appeared as a multiplet at δ 5.35 and displayed a cross peak to H-13 methine. In the HMBC spectrum, the H-14 methine (δ 6.46, m) showed two / correlations with the olefinic carbon at δ 134.6 and the carbinol carbon at δ 73.2, which by HMBC experiment could be placed at C- 16. On the basis of the above spectral evidence, Formula I was identified as 16-hydroxyoctadeca-9 (Z or E), 12 (Z or E), 14(Z or E)-trienoic acid.

Table 1: ¹H (400 MHz), ¹³C NMR (100 MHz), COSY (correlated spectroscopy) and HMBC (Heteronuclear Multiple Bond Coherence) spectral data for the compound of Formula I

Position ¹H (Hz) ¹³C COSY HMBC

CO 178.1

2 2.20 t, (6.5) 32.4 H-2 CO

3 1.42 br s 25.2 C-I

4 1.42 br s 30.1

5 1.42 br s 30.1

6 1.42 br s 30.1

7 1.42 br s 30.1 C-9

8 2.01 m 33.1 H-8, H- 10

9 5.25 m 128.6^a H- 10 C-9, C-11

10 5.50 m 126.7 H-I l

11 2.54 m 36.1 H-13

12 5.35 m 128.5^a H- 14 C-12, C-14

13 6.05 m 131.3^b H-15

14 6.46 m 132.6^b H-16 C-16

15 5.60 m 134.6 H- 17

16 4.01 m 73.2

17 1.32 m 28.3

18-CH₃ 0.88 t, (6.5) 8.1 H-18 C-18

All spectra were recorded in CDCI₃. ^{a b}Signals within a column are interchangeable.

Coupling constants in Hz.

Characterization of compound of Formula II

Compound of Formula II was isolated as a colourless oil from the methanolic extract by silica gel column chromatography followed by Prep-HPLC. The LC-MS data revealed the M⁺ ion at m/z = 294 corresponding to the molecular formula C₁₈H₃₀O₃. The ¹H and ¹³C NMR data (Table 2) were very similar to that of Formula I indicating the presence of six olefinic protons (three double bond equivalents), an oxymethine group, a carbonyl carbon, seven methylenes, two downfield methylene groups and finally a methyl group.

The HMBC spectrum showed a / correlation between a downfield methylene (5_H 2.28, m, H₂-2) and the carbonyl carbon (δ_c: 178.4, C-I) of the carboxyl acid group. An unresolved multiplet for a second downfield methylene (δπ 2.17) displayed two COSY cross peaks, first with an olefinic proton, H-15 (5_H 5.44, q, 15.6), which further was coupled to H-16 proton (δπ 5.44, q, 15.6), showing trans- geometry, while the second cross peak was with an oxymethine proton, placing this oxymethine at C- 13 (δπ 4.20, q, 6.5). This oxymethine proton in turn showed a COSY coupling to an olefinic proton (δπ 5.64, dd, 15.1, 6.5) assignable to H-12. Furthermore, the H-12 methine was coupled to the olefinic proton, H-I l (δπ 6.50 dd, / = 15.1, 11.0 Hz) and the large coupling constant shown between H-12 and H-I l (/ = 15.1 Hz) indicated that these protons were trans- orientated, while H-I l also showed vicinal coupling (J=I LO Hz) with another olefinic methine at H-10 (δ_H 5.95, t, /=11.0) further coupled to H-9 (δ_H 5.35, q, /=11.0) showing cis- orientation (/=11.0). Hence it was clear that C-9 and C-Il were in a diene configuration with 9Z and 1 IE. The H-9 olefinic methine showed a / (HMBC) correlation with a methylene carbon at C-8 (δc: 28.6), indicating the beginning of the alkyl chain. The alkyl chain was found to consist of 7 methylene groups and a terminal carboxylic acid group. From the above spectral data, Formula II was characterized as 13- hydroxy-9Z,l lE,15E-octadecatrienoic acid.

Table 2: ¹H (400 MHz), ¹³C NMR (100 MHz), COSY (correlated spectroscopy) and HMBC (Heteronuclear Multiple Bond Coherence) spectral data for the compound of Formula II

Position ¹H (Hz) ¹³C COSY HMBC

CO 178.4

2 2.28 m 36.2 H-2 CO

3 1.60 m 26.3 C-I

4 1.38 br s 30.3

5 1.38 br s 30.3

6 1.38 br s 30.3

7 1.38 br s 30.3 H-8 C-8

8 2.17 m 28.6

9 5.35 q, (11.0) 125.6^a H-8, H- 10

10 5.95 t, (11.0) 129.3 H-Il C-9, C-Il

11 6.50 dd, (15.1, 126.6^a H- 12

11.0)

12 5.64 dd, (15.1, 136.7 H-13

6.5)

13 4.20 q, (6.5) 73.2 H-14 C-12, C-14

14 2.17 m 28.6 H-15

15 5.44 q, (15.6) 134.6^b H-16 C-16

16 5.44 q, (15.6) 133.2^b H- 17

17 2.04 m 21.7

18-CH₃ 0.94 t, (7.5) 14.5 H- 17 C- 17

All spectra were recorded in CDCI₃. ^a' Signals within a column are interchangeable. Coupling constants in Hz.

In compounds of Formulae I and II, Z (German word 'Zusammen' (together)) and E (German word 'Entgegen' (opposite)) are stereodescriptors, which indicate the configuration at a double bond. The present invention also provides a method for the preparation of extracts of the aerial parts of Annona squamosa enriched with bioactive marker compounds, which method includes, extracting Annona squamosa aerial parts with a solvent in the extractor, concentrating the combined extracts under reduced pressure at low temperature, pouring the extract into stainless steel trays and drying in high vacuum oven. On the basis of bioassay guided fractionation approach and identification of bioactive marker compounds of Formulae I and II, extraction can be carried out in solvents selected from non polar to polar solvents, for example, petroleum fractions (such as hexane, petroleum ether, heptane, cyclohexane or toluene), chloroform, acetone, methanol, water: methanol (1:1), chloroform: methanol (1:1) and water, for the preparation of extracts. Examples 2-8 are directed towards preparation of extracts of Annona squamosa enriched with bioactive marker compounds.

Various extracts prepared are subjected to primary bioactivity assay and the bioactive extracts are further evaluated for the secondary bioactivity assays. Among various evaluated extracts, petroleum fraction extracts displayed highest bioactivity as well as a high content of the two bioactive marker compounds of Formulae I and II.

The present invention provides standardized extracts of the aerial parts of Annona squamosa and a method for standardization of these extracts with bioactive marker compounds of Formulae I and II by HPLC method. The method includes diluting the extracts in one or more of organic solvents, sonicating the solution, filtering the supernatant liquid to form test solution, injecting the test solution in chromatographic column, running test chromatogram using a mobile phase, scanning, detecting bioactive marker compounds of Formulae I and II in the extracts by matching retention times of these bioactive marker compounds in the test chromatogram with that of standard chromatogram and quantifying them.

The extracts can be diluted with one or more of organic solvents, for example, alcohols (such as methanol, isopropyl alcohol or ethanol), nitriles (such as acetonitrile) and mixture(s) thereof.

The test chromatogram can be run in a mobile phase comprising one or more of solvents, for example, alcohols (such as methanol, isopropyl alcohol or ethanol), nitriles (such as acetonitrile), water and mixture(s) thereof, and one or more of buffers (such as formic acid, trifluoro acetic acid, orΛo-phosphoric acid, ammonium acetate, sodium perchlorate, potassium dihydrogen orthophosphate, dipotassium hydrogen orthophosphate, sodium dihydrogen orthophosphate, disodium hydrogen orthophosphate, diammonium hydrogen orthophosphate, ammonium dihydrogen orthophosphate, ammonium formate, tetramethyl ammonium hydroxide, tetrabutyl ammonium hydroxide, tetrabutyl ammonium hydrogen sulphate and mixture(s) thereof.

Each of the standard chromatograms is obtained by injecting the standard bioactive marker solutions separately in chromatographic column and running standard chromatogram using a mobile phase and scanning.

The preparation of standard bioactive marker solutions can be carried out by dissolving compounds of Formulae I and II separately in one or more of organic solvents, for example, alcohols (such as methanol, isopropyl alcohol or ethanol), nitriles (such as acetonitrile) and mixture(s) thereof. The solution can be sonicated and then made up to a desired fixed volume using the same solvent.

The standard chromatogram can be run in a mobile phase comprising one or more of solvents, for example, alcohols (such as methanol, isopropyl alcohol or ethanol), nitriles (such as acetonitrile), water and mixture(s) thereof and one or more of buffers (such as formic acid, trifluoro acetic acid, orΛo-phosphoric acid, ammonium acetate, sodium perchlorate, potassium dihydrogen orthophosphate, dipotassium hydrogen orthophosphate, sodium dihydrogen orthophosphate, disodium hydrogen orthophosphate, diammonium hydrogen orthophosphate, ammonium dihydrogen orthophosphate, ammonium formate, tetramethyl ammonium hydroxide, tetrabutyl ammonium hydroxide, tetrabutyl ammonium hydrogen sulphate and mixture(s) thereof.

The scanning is done at a wavelength of about 235 nm.

HPLC system used is a gradient system attached with PDA detector. Column used is Ci₈, 150X4.6 mm 5μ. (Purospher¹¹ Star) or equivalent.

The percentage content of bioactive marker compounds of Formula I or Formula II in the test sample may be calculated as follows:

= x x x p x 100

where,

A_SPL - Average peak area corresponding to compounds of Formula I or Formula II from the sample chromatogram A_STD - Average peak area corresponding to compounds of Formula I or Formula II from the standard chromatogram D_SPL - Dilution of test solution D_STD - Dilution of reference standard solution W_STD - wt. of reference standard taken in mg W_SPL - wt. of test sample taken in mg P - Purity of the reference standard

The HPLC method is provided for the detection and quantification of bioactive marker compounds of Formulae I and II in extracts of Annona squamosa. a. Preparation of reference standard solutions

(i) Compound of Formula I Compound of Formula I was weighed in a 10 ml volumetric flask. Methanol (5.0 ml) was added, sonication was done in an ultrasonic water bath to dissolve and the volume was made up with methanol. The resulting solution was used as reference standard solution for compound of Formula I.

(ii) Compound of Formula II Compound of Formula II was weighed in a 10 ml volumetric flask. Methanol (5.0 ml) was added, sonication was done in an ultrasonic water bath to dissolve and the volume was made up with methanol. The resulting solution was used as reference standard solution for compound of Formula II. b. Preparation of test solutions

Extracts (examples 2 to 8) were weighed separately, in volumetric flasks (10 mL). Methanol (5 mL) was added to the extracts and sonication was done in an ultrasonic water bath for about 15 minutes. Filtration was done through 0.45 μ membrane filter and the resulting solutions were used as test solutions. c. Identification and quantification of the compound of Formula I Standard solutions and test solutions were injected twice separately and the chromatograms were obtained. HPLC conditions Instrument: A Gradient High Performance Liquid Chromatographic System attached with PDA detector (Waters with class EMPOWER software) Mobile Phase: 0.1 % formic acid : acetonitrile Column: Ci_8, 150 mm X 4.6 mm, 5μ (Purospher^R Star) or equivalent

Detector: PDA Detector

Wavelength For Recording The Chromatogram: 235 nm

Flow Rate: l.O mL/min. Injection Volume: 20μL

Run Time: 50 minutes

Time Flow rate 0.1 % Formic acid Acetonitrile

0 lml/min 90 10

35 lml/min 25 75

40 lml/min 25 75

41 lml/min 90 10

50 lml/min 90 10 d. Identification and quantification of the compound of Formula II

Standard solutions (μL) and test solutions (μL) were injected twice separately and the chromatograms were obtained. HPLC conditions

Instrument: A Gradient High Performance Liquid Chromatographic System attached with PDA detector (Waters with class EMPOWER software) Mobile Phase: 0.1 % formic acid : acetonitrile

Column: Ci_8, 150 mm X 4.6 mm, 5μ (Purospher^R Star) or equivalent

Detector: PDA Detector Wavelength For Recording The Chromatogram: 235 nm Flow Rate: 1.0 mL/min.

Injection Volume: 20μL

Run Time: 50 minutes

Time Flow rate 0.1 % Formic acid Acetonitrile

0 lml/min 90 10 35 lml/min 25 75

40 lml/min 25 75

41 lml/min 90 10

50 lml/min 90 10

Calculations:

The percentage content of compounds of Formula I or Formula II in the test sample was calculated as follows:

= x x x p x 100

where,

A_SPL - Average peak area corresponding to compounds of Formula I or Formula II from the sample chromatogram

A_STD - Average peak area corresponding to compounds of Formula I or Formula II from the standard chromatogram

D_SPL - Dilution of test solution

D_STD- Dilution of reference standard solution W_STD - wt. of reference standard taken in mg

W_SPL - wt. of test sample taken in mg

P - Purity of the reference standard

Percentage content of the compounds of Formulae I and II in different extracts was found to be in the range of 0.0001% to 5%. The present invention also provides a pharmaceutical composition comprising of a standardized extract of Annona squamosa aerial parts along with one or more of pharmaceutically acceptable carriers, excipients or diluents.

The present invention also provides a pharmaceutical composition comprising of a therapeutically effective amount of a compound of Formula I along with one or more of pharmaceutically acceptable carriers, excipients or diluents.

The present invention also provides a pharmaceutical composition comprising of a therapeutically effective amount of a compound of Formula II along with one or more of pharmaceutically acceptable carriers, excipients or diluents.

The invention also provides a combination comprising bioactive marker compounds of Formula I, Formula II, or a standardized extract of Annona squamosa aerial parts and one or more of extracts of plants known to have antidiabetic use, for example, Cinnamomum cassia, Capparis moonii, Azadirachta indica, Salacia chinensis, Delphinium denudatum, Chicorium intybus, Enicostemma littorale, Bauhinia variegata, Smilax china, Alstonia scholaris , Swertia chirayta, Coptis teeta, Caesalpinia bonduc, Cissampelos parreira, Citrullus colocynthis, Saraca asoca, Sphaeranthus indicus, Tecomella undulata, Trichosanthes dioica, Prunus cerasoides , Polyalthia longifolia, Ailanthus excelsa, Helicteres isora or Stereospermum suaveolens.

"Aerial parts of Annona squamosa" include stems, bark, flowers, fruits, seeds and leaves of Annona squamosa. A standardized extract of Annona squamosa aerial parts refers to an extract of

Annona squamosa aerial parts, wherein the compounds of Formulae I and II are detected and quantified. The extracts of the present invention, for example, hexane extract, chloroform extract, acetone extract, chloroform: methanol extract, water: methanol extract, methanol extract or aqueous extracts are obtained by extraction with such solvents and the solvents are removed to a level acceptable in accordance with FDA and ICH guidelines.

The pharmaceutical compositions comprising of a compound of Formula I, Formula II, or a standardized extract of Annona squamosa aerial parts, along with one or more of pharmaceutically acceptable carriers, excipients or diluents may be administered to a mammal for treatment, prevention, inhibition or suppression of diabetes, diseases mediated through PTP-IB, NFKB and cPLA2 or diseases having their iteology through

AMP kinase in a mammal, by any route, which effectively transports the active compound to the appropriate or desired site of action such as oral, nasal, pulmonary, transdermal or parenteral (rectal, subcutaneous, intravenous, intraurethral, intramuscular or intranasal). The choice of pharmaceutical carrier, excipient or diluent can be made with regard to the intended route of administration and standard pharmaceutical practice.

Brief Description of the Figures Figure 1: Dissection of the Insulin Signaling pathway

Figure 2: Flow-chart showing bio-assay guided fractionation of the plant Annona squamosa Figure 3: 2-Deoxy Glucose uptake by compound of Formula I

Figure 4: 2-Deoxy Glucose uptake by compound of Formula II Figure 5: PTPlB enzyme inhibition by compound of Formula I Figure 6: PTPlB enzyme inhibition by compound of Formula II

Figure 7: % Inhibition of NF-kB survival pathway by compounds of Formulae I and II

Figure 8: cPLA2 activity with compounds of Formulae I and II Figure 9: 2-Deoxy Glucose uptake by hexane extract in L6 myotubes

Figure 10: Effect of hexane extract on GLUT4 mRNA expression at 18 hours Figure 11 : Effect of hexane extract on PI3 Kinase mRNA expression at 18 hours Figure 12: Effect of hexane extract on fasting plasma glucose of diabetic (Ob/Ob) animals after 21 days of treatment Figure 13a: Effect of hexane extract on fasting plasma glucose curve during OGTT

(oral glucose tolerance test) of diabetic (ob/bb) animals after 21 days of treatment

Figure 13b: Effect of hexane extract on fasting plasma glucose AUCo-₁₂0 during

OGTT (oral glucose tolerance test) of diabetic (ob/ob) animals after 21 days of treatment

Figure 14: Effect of hexane extract on plasma triglycerides of diabetic (ob/ob) animals after 21 days of treatment

Figure 15: Effect of hexane extract on body weight of diabetic (ob/ob) animals after

21 days of treatment

Biological Assay Insulin resistance in patients with type 2 diabetes is attributable mostly to insulin- stimulated glucose uptake into skeletal muscle, which is a major mass peripheral tissue that accounts for -40% of the total body mass and is a major player in energy balance. It accounts for >30% of energy expenditure and is the primary tissue for insulin stimulated glucose uptake, the rate-limiting step in glucose metabolism. The L6 cell line has been widely used to study the insulin- stimulated glucose transport. Glucose uptake in L6 myoblasts may be used as the primary assay to test the anti-diabetic activity of extracts or fractions for a bioassay-guided identification of bio-actives in Annona squamosa. Glucose uptake is mediated by specific glucose transporters of the plasma membrane. In normal muscle cells and adipocytes, the glucose transporter isoform is GLUT-4, a 12-transmembrane domain protein that mediates transport of glucose in the direction of glucose gradient. Insulin promotes GLUT-4 incorporation into plasma membrane, and this translocation from intracellular compartments appears to fail in the insulin resistance present in some form of diabetes. As glucose transport is primarily mediated by Glut 4 transporter in the skeletal muscle, changes in mRNA expression of Glut 4 is also carried out by RT-PCR, along with PD kinase which is one of the key downstream players in the insulin signaling pathway. Insulin receptor tyrosine kinase catalyses the phosphorylation of IRS proteins that recruit and activate PI3K (PI3 Kinase) to form phosphatidylinositol (3, 4, 5) triphosphate, which leads to the activation of PDK-I. The protein kinase PDKl can phosphorylate multiple downstream protein kinases, such as Akt/PKB and protein kinase C (PKC) resulting in the translocation of Glut4 to the plasma membrane and thereby facilitating glucose uptake into cells. Wortmannin is a specific inhibitor of PI3K and hence insulin- stimulated glucose transport into cells. PTPlB (a protein tyrosine phosphatase) acts as a negative regulator of the insulin signaling pathway and acts by dephosphorylation of important tyrosine residues on the IR to reduce its activity (Rondinone, C. M., Endocrinology (2006) 147(6); 2650-2656). Induction of diabetes in rabbits and rats

The experimental animals used were rabbit and Wistar rats. Alloxan was used for inducing diabetes in rabbits and streptozotocin in rats. (Alloxan and streptozotocin were purchased from Aldrich Chem. Co. USA). Intravenous injections of alloxan in rabbits and intraperitoneal injections of streptozotocin in rats were administered at a dose of 80 mg/kg and 50 mg/kg of body weight respectively to overnight starved animals. Fasting blood glucose (FBG) levels were estimated by commercial kits based on glucose oxidase method. Diabetes was confirmed by testing FBG and postprandial blood glucose levels. Depending on their glucose levels the animals were divided into three groups, namely sub, mild and severe diabetic. All animals were provided free access to standard chow diet and tap water ad libitum, during the period of treatment. Measurement of 2-deoxy-d-H-3Hl glucose

L6 myoblast cells grown in 24-well plate (Corning, NY) were subjected to glucose uptake as reported (Yonemitsu S. et al Diabetes (2001) 50; 1093-1101). Differentiated myotubes were serum starved for 5 hours and were incubated with the compounds of Formula I, Formula II or extracts of Annona squamosa aerial parts for 24 hours (both in presence and absence of 100 nM Insulin). After experimental incubation, cells were rinsed once with HEPES -buffered Krebs Ringer phosphate solution (118mM NaCl, 5mM KCl, 1.3mM CaC12, 1.2mM MgSO4, 1.2mM KH2PO4 and 3OmM HEPES— pH 7.4) and were subsequently incubated for 15 min. in HEPES -buffered solution containing 0.5μCi/ml 2- deoxy-D-[l-³H] glucose. The uptake was terminated by aspiration of media. Cells were washed thrice with ice cold HEPES buffer solution and lysed in 0.1 N NaOH. An aliquot was used to measure the cell-associated radioactivity by liquid scintillation counting. Glucose uptake values were corrected for non-specific uptake in the presence of 10 μM cytochalasin B (5-10% of total uptake). All the assays were performed in triplicate and repeated thrice for concordancy. Measurement of Glut4 and PI3 Kinase mRNA expression by RT-PCR RT-PCR was carried out as described previously (Hall et al BioTechniques (1998)

24(4); 652-658). L6 myotubes after experimental incubation with compounds of Formula I, Formula II or the extracts of Annona squamosa aerial parts were lysed in total RNA isolation reagent Trizol. Proteins were extracted with chloroform and total RNA was precipitated with isopropanol. The RNA precipitate was washed with 70% ethanol and resuspended in 50 μl of DEPC-treated water. Reverse transcription was carried out to obtain cDNA using 200 units of avian reverse transcriptase and 200 ng/μl oligo d [T] 18. The primers used were as follows: Glut-4 sense, 5'-CGG GAC GTG GAG CTG GCC GAG GAG-3'; anti-sense 5'-CCC CCT CAG CAG CGA GTG A-3' (318-bp) and; PI3 kinase sense, 5'-TGA CGC TTT CAA ACG CTA TC-3'; anti-sense, 5'-CAG AGA GTA CTC TTG CAT TC-3' (248-bp) and GAPDH Sense, 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3'; Anti-sense, 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3' (588-bp). For PCR reaction, 1 μl of the cDNA mixture prepared as described earlier was added to a PCR reaction mix consisting of 1OxPCR buffer, 2 mM dNTP, 10 pM of paired primers, 2 units of Taq polymerase and distilled water in a total volume of 50 ml. The reaction mixture was overlaid with mineral oil and placed in a PCR thermal cycler for 35 cyclic reactions. PCR products were run on 1.5% agarose gels, stained with ethidium bromide and photographed. IR and IRSl tyrosine phosphorylation by Immunoprecipitation

L6 cells were seeded in 12-well plates and allowed to differentiate for 4 days in medium containing 2% serum. Cells were treated with the compounds of Formula I,

Formula II or extracts of Annona squamosa aerial parts for 24 hours and Insulin (100 nM) for 15 minutes. Thereafter, these were washed once with ice-cold PBS (phosphate buffered saline) and lysed in 1 ml of lysis buffer (50 mM Hepes, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, ImM Sodium orthovanadate, 50 mM NaF, 10 μg/ml Aprotinin, 10 μg/ml leupeptin, 1% Triton X-100 pH 7.4). The lysates were centrifuged, and the supernatants incubated with 50 μl of protein A-Sepharose beads that had been coated with monoclonal anti-IR / IRSl antibody. The immunoprecipitates were washed three times with 500 μl lysis buffer and then analyzed by SDS-polyacrylamide gel electrophoresis and Immunoblotting. Western Blotting

Samples were boiled in Laemmli SDS sample buffer, resolved by SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked in TBST (25 mM Tris-HCl, pH 8.0, 125 mm NaCl, 0.1% Tween 20) containing 5% skimmed milk for 1 hour and then incubated with anti-phosphotyrosine (primary) antibody for 1 hour at room temperature. The blot was washed extensively with TBST and further incubated with secondary antibody conjugated to HRP. After further washing with TBST, the blots were developed using enhanced chemiluminescence (ECL kit).

Anti-diabetic Activity of compounds of Formulae I and II

Compound of Formula I showed significant dose-dependent glucose uptake (Figure 3).

• EC50 = 8.17 ng/ml

• EC50 (with Insulin) = 1.176 ng/ml Compound of Formula II showed significant dose-dependent glucose uptake (Figure 4).

• EC50 = 2 ng/ml

• EC50 with Insulin = 0.8 ng/ml PTPl-B Enzyme Inhibition

The enzymatic assay was carried out in sodium acetate buffer containing ImM DTT, ImM EDTA and 0.5% Igepal (pH 5.5), in a 96 well format. The initial rate of

PTP IB -catalyzed hydrolysis of pNPP was measured by following the absorbance change at 405 nm. PTPlB enzyme used in the assay was purified recombinant human PTPlB from Biomol. IC50 values were determined at fixed enzyme (25 ng/well) and substrate (5 mM) concentration with varying concentrations of the extracts of Annona squamosa aerial parts or compounds of Formula I or Formula II. The enzyme reaction was carried out at

30⁰C for 20 minutes in dark and arrested by IN NaOH. RK682 from Biomol was used as a positive control for PTPlB enzyme inhibition (IC50=100 μM). The compounds of Formulae I and II showed potent inhibition of PTPlB enzyme. (Figure 5 = PTP-IB dose response curve for Formula I, Figure 6= PTP-IB dose response curve for Formula II and IC50 for Formula I =1 μg/ml and for Formula II, IC50=0.25 μg/ml). Protein tyrosine phosphatases, most significantly PTPlB, have emerged as a promising drug target for diabetes and obesity. These have been implicated in the negative regulation of insulin action through dephosphorylation of the IR. Blocking or inhibiting the PTPlB enzyme would result in increased signaling through the IR and IRS-I thereby decreasing insulin resistance. NFKB Enzyme Inhibition J774A.1 cell lines (ATCC) were maintained in RPMI- 1640 supplemented with

10% FBS (Fetal bovine serum), 2mM L-Glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin and cultured at 37 ⁰C in 5% CO₂ incubator. J774A.1 cells were seeded in a 96 well plate at a density of 0.2 million cells/well (in 180 μl of RPMI medium with FBS). The dilutions of standard compound (BAY- 11 -7082), the extracts of Annona squamosa aerial parts and compounds of Formula I or Formula II were made in dimethylsulphoxide and RPMI- 1640 medium. 20 μl of each dilution was added to the cells. The effect of the compounds of Formula I, Formula II or extracts of Annona squamosa aerial parts on the death of J774A.1 was measured after 18 hours of treatment. The cell viability was measured by MTT assay which relies on the fact that viable cells converts the water soluble MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) to an insoluble formazan salt. The formazan was then solubilized and the concentration was determined by optical density at 540 nm. Without discarding the media, 10 MTT solution (5mg/ml in RPMI medium) was added to each well and the cells were kept for 4 h at 37 ⁰C in 5% CO₂ incubator. Finally supernatant was discarded, pellet was dissolved in dimethylsulfoxide (DMSO) and absorbance of the converted dye was measured at a wavelength of 540 nm. Cell survival was estimated as a percentage of the value of untreated controls. Figure 7 shows % inhibition of NFKB survival pathway by compounds of Formulae I and II. Percentage inhibition = (Mean OD_untreated ceil control - Mean OD_compound_S treated)/ Mean OD untreated ceil control x 100 , wherein OD=optical density. cPLA2 Enzyme Inhibition

Mast cells release arachidonic acid (AA) in response to stimulation with antigen and the release is thought to be responsible in part of mast cell-mediated inflammatory reactions. RLB 2H3 cells contain PLA2 that cleaves AA from membrane phospholipids. Method

Cells were seeded into 24 well plate at a density of 1 x 10⁵/well and incubated overnight with [¹⁴C] - arachidonic acid (0.1 μCi) and the extracts of Annona squamosa aerial parts or compounds of Formula I or Formula II at 37°C. Supernatant was removed to measure the amount of [¹⁴C] - arachidonic acid incorporated into the cells. Cells were resuspended in PBS (phosphate buffered saline) (pH 7.4) with calcium and exposed to a Ca²⁺ ionophore, A23817 (5 μM) for 20 min. Supernatant was collected to count the radioactivity released into the media. The radioactivity released into the medium reflects cPLA2 activity and was measured with liquid scintillation counter. The cPLA2 activity was expressed as percentage of the total radioactivity incorporated into the cells. Figure 8 shows cPLA2 activity with compounds of Formulae I and II. Anti-diabetic Activity of hexane extract

Hexane extract (example 2) showed 2-Deooxy Glucose uptake by hexane extract in L6 myotubes as shown in Figure 9.

• EC50 = 39 ng/ml • EC50 (with Insulin) = 40 ng/ml

The effect of hexane extract (example 2) on Glut 4 mRNA expression at 18 hrs is shown in Figure 10, wherein M- 50bp marker 1- Control 2- Insulin (Positive control)

3- Rosiglitazone (Positive control) 4 - Hexane extract (1 ng/ml) 5- PCR Negative control

The effect of hexane extract (example 2) on PI3kinase mRNA expression at 18 hrs is shown in Figure 11, wherein M- 50bp marker 1- Control 2- Insulin (Positive control)

3- Rosiglitazone (Positive control) 4 - Hexane extract (lng/ml)

5- PCR Negative control AMPK activity assay

AMPK activity was measured by monitoring phosphorylation of the SAMS peptide substrate (Davies et. al. Eur. J. Biochem. (1989) 186; 123-128) in a 96-well format. Each well carried a reaction volume of 30 ml containing 10 mg partially purified AMPK enzyme, 100 mM SAMS peptide and the extracts of Annona squamosa aerial parts or compounds of Formula I a or Formula II in a buffer containing Hepes (sodium salt) buffer (40 mM, pH 7.0), NaCl (80 mM), glycerol (8% by volume), EDTA (0.8 mM), MgCl₂ (5mM) and [g-33P] ATP (200 mM, 200-500 cpm/pmol). After incubation for 10 minutes at 30⁰ C, the reaction was stopped using 10% phosphoric acid and 15 ml of reaction mix was transferred to unifilter plates. Wells were washed 8-10 times with 1% phosphoric acid, followed by two washes with acetone, before air drying, adding scintillant and measuring cpm (counts per minute) in a Microbeta Wallac counter. Results were expressed as fold activation compared to untreated cells. At concentrations above 1 μM, hexane extract (example 2) showed fold activation in the range of 1-1.3, with maximal activation, 1.3 at 10 μM concentration. Aminoimidazole-4-carboxamide ribonucleoside (AICAR), a pharmacological activator of AMPK, was used as positive control and gave a fold activation of 1.3 at 1 mM concentration. Antidiabetic effect of hexane extract in ob/ob mouse

The ob/ob homozygous mouse is leptin deficient, hyper-insulinemic and is susceptible to extreme obesity. The obese (ob/ob) mouse is a commonly used animal model of genetic non-insulin-dependent diabetes mellitus. Methods and study design

The study was conducted in ob/ob mice, weighing 35-60 g, age 8-10 weeks obtained from the Experimental Animal Facility, Ranbaxy Research Laboratories Ltd. All the animals were housed in polypropylene cages and maintained at a temperature of 24 ± 2°C with controlled illumination to provide a light dark cycle of 12 hrs, till the end of experimentation. Diabetic animals of both sexes were divided into three groups (4 male + 4 female in each group) on the basis of random plasma glucose level. First group was orally given vehicle (0.25% CMC), once daily, while second group was given Rosiglitazone (10mg/kg, once daily), a PPAR-γ agonist, used as a standard compound (table 3). Hexane extract was freshly prepared with 1% Tween 80 and was administered twice daily for 21 days (table 3). Control animals were dosed with vehicle once only. During study period, body weights of animals were measured at 7, 14 and 21 days. On day 21, blood samples were collected after 1 hour of drug administration to measure blood glucose, triglyceride and total cholesterol levels. Blood samples were collected by orbital sinus bleeding under light ether anesthesia. On day 22, oral glucose tolerance test was performed. Following oral glucose loading (2g/kg), blood samples were collected at 15, 30, 60 and 120 min. Samples were analyzed for plasma glucose levels. Area under the curve (AUC) of plasma glucose level was calculated using graph pad prism software.

Plasma samples were analyzed for triglyceride, glucose and total cholesterol using commercial diagnostic kits and automated biochemical Auto analyzer (Dade Behring, USA). Data was analyzed using student t test and considered significant for P values less than 0.05.

Table 3: Study design and treatment protocol of diabetic animals

Oral administration of hexane extract for 21 days caused a significant reduction in random glucose (27.7 %; p<0.01), comparable to rosiglitazone treatment (32.3%; Figure 12). Oral administration of hexane extract for 21 days caused a significant glucose lowering following OGTT (Figure 13a). During OGTT (AUCo-₁₂0 _mm), hexane extract exhibited significant decrease (27.6%; p<0.01) in AUCo-₁₂0 _mm following oral glucose loading (Figure 13b). Hexane extract (500mg/kg) showed significant reduction in plasma triglyceride level (30.5%) comparable to that of Rosiglitazone at 10 mg/kg (34.8%) (Figure 14). Hexane extract did not alter the body weight of diabetic animals as compared to a significant (p<0.05) increase of body weight by rosiglitazone after 21 days of treatment (Figure 15).

Table 4: Anti-diabetic activity of hexane extract and bioactive marker compounds of Formulae I and II

Abbreviation: IR-P Insulin Receptor phosphorylation; IRSl-P Insulin Receptor Substratel phosphorylation; PTPlB Protein tyrosine phosphataselB; ff Significant up-regulation/expression; + moderate activity; + + Significant activity; — No effect; ND - not done

Hexane extract (example 2) showed a significant antidiabetic and hypo- triglyceridemic activity and this effect could be partly explained by its PTPIb inhibitory property and significant effect on insulin signalling events, ob/ob mouse is an established animal model to study insulin resistance hence the anti diabetic effect seen in ob/ob mice with hexane extract after 21 days of treatment pointed towards the insulin sensitizing effect (PTPIb inhibition) and insulin mimetic effect. Moreover, non-significant effect on body weight compared to controls suggested that hexane extract was devoid of an undesirable adverse effect on body weight, which is the case with PPAR-γ agonists (rosiglitazone). This also explains that the extract may be acting through pathway(s) independent of PPAR-γ.

Examples The following examples and the preparations describe the manner of making and using the invention and are illustrative rather than limiting. Example 1 : Isolation of the compounds of Formulae I and II

5.0 kg of Annona squamosa powdered leaves were extracted with methanol (40 liters) at room temperature five times for 15 hours each and the combined organic extract was concentrated under reduced pressure at room temperature. Dry methanolic extract was suspended in water (1 liter) and partitioning was carried out with chloroform (500 ml) four times. The chloroform fractions were combined and dried over anhydrous sodium sulphate. The organic extract was purified by column chromatography and eluted with hexane, hexane: ethyl acetate in the ratio of 90: 10, 80:20, 70:30, 65:35, 60:40 and finally with ethyl acetate. Different fractions were collected and each fraction was observed by thin layer chromatography using mobile phase (ethyl acetate in hexane, methanol in ethyl acetate and methanol in chloroform). Detection was done by observation under 254, 366 nm of UV and with anisaldehyde sulphuric acid. Fraction containing the compounds of Formulae I and II was collected and purified further by prep HPLC. Sample preparation was done with sonication by taking the said fraction in acetonitrile followed by the addition of 0.1% formic acid in water in one fourth volume of organic solvent. The reaction mixture was sonicated and was subsequently filtered. The mixture was subjected to preparative HPLC for isolation of the compounds of Formulae I and II at room temperature. The mixture was eluted by using gradient method. Prep HPLC Column: YMC Pack ODS-AM (250mmx50mm)

Buffer: 0.1% formic acid or 15mM Ammonium acetate filtered through 0.45μ filter. Organic phase: Acetonitrile Flow: 20ml/min Detector: UV@235nm Gradient: Time Buffer Acetonitrile

0 60 40

20 50 50

40 45 55

60 30 70 80 15 85

100 60 40

120 60 40

The eluted fractions were collected and analyzed on analytical HPLC to get their purity. Following chromatographic conditions were used to analyze the purity of fractions: HPLC Column: Kromasil (100mmx4.6mm)

Buffer: 0.1% formic acid filtered through 0.45μ filter. Organic phase: Acetonitrile Flow: lml/min

Detector: UV@235nm

Isocratic elution: Buffer: Acetonitrile: 60:40

Pure fractions containing the compounds of Formulae I and II were worked up by evaporating the solvent in rotavapor under vacuum at room temperature.

Yield of compound of Formula I = 0.0006 %

Yield of compound of Formula II = 0.0006%

Example 2: Preparation of hexane extract of Annona squamosa

Powdered Annona squamosa leaves (5.0 kg) were macerated three times with hexane (20 lit X 3) for 16 hours each in the extractor. The hexane extracts were combined and concentrated to one fifth under reduced pressure at low temperature. The extract was poured into stainless steel trays and dried in high vacuum oven at room temperature for about 16 - 18 hours.

Yield ~ 4.25 % Example 3: Preparation of chloroform extract of Annona squamosa

Powdered Annona squamosa leaves (5.0 kg) were macerated three times with chloroform (20 lit X 3) for 16 hours each in the extractor. The chloroform extracts were combined and concentrated to one fifth under reduced pressure at low temperature. The extract was poured into stainless steel trays and dried in high vacuum oven at room temperature for about 16 - 18 hours.

Yield - 6.10 %

Example 4: Preparation of acetone extract from Annona squamosa

Powdered Annona squamosa leaves (5.0 kg) were macerated three times with acetone (20 lit X 3) for 16 hours each in the extractor. The acetone extracts were combined and concentrated to one fifth under reduced pressure at low temperature. The extract was poured into stainless steel trays and dried in high vacuum oven at room temperature for about 16 - 18 hours.

Yield - 5.70 %

Example 5: Preparation of chloroform : methanol :: 1:1 extract from Annona squamosa Powdered Annona squamosa leaves (5.0 kg) were macerated three times with chloroform: methanol (20: 20 lit X 3) for 16 hours each in the extractor. The chloroform: methanol extracts were combined and concentrated to one fifth under reduced pressure at low temperature. The extract was poured into stainless steel trays and dried in high vacuum oven at room temperature for about 16 - 18 hours.

Yield -16.2 %

Example 6: Preparation of water: methanol: 1:1 extract from Annona squamosa Powdered Annona squamosa leaves (5.0 kg) were macerated three times with methanol: water (20: 20 lit X 3) for 16 hours each in the extractor. The methanol: water extracts were combined and concentrated to one fifth under reduced pressure at low temperature. The extract was poured into stainless steel trays and dried in high vacuum oven at room temperature for about 16 - 18 hours. Yield ~ 20.8 %

Example 7: Preparation of methanol extract of Annona squamosa

Powdered Annona squamosa leaves (5.0 kg) were macerated three times with methanol (20 lit X 3) for 16 hours each in the extractor. The methanol extracts were combined and concentrated to one fifth under reduced pressure at low temperature. The extract was poured into stainless steel trays and dried in high vacuum oven at room temperature for about 16 - 18 hours.

Yield ~ 13.70%

Example 8: Preparation of aqueous extract from Annona squamosa

Powdered Annona squamosa leaves (5.0 kg) were macerated three times with water (20 lit X 3) for 16 hours each in the extractor. The water extracts were combined and concentrated to one fifth under reduced pressure at low temperature. The extract was poured into stainless steel trays and dried in high vacuum oven at room temperature for about 16 - 18 hours.

Yield ~ 25%

Claims

We Claim: 1. A standardized extract of Annona squamosa aerial parts. 2. The standardized extract of claim 1, comprising bioactive marker compounds, 16- hydroxyoctadeca-9 (Z or E), 12 (Z or E), 14 (Z or E)-trienoic acid of Formula I

and 13-hydroxy-9Z-l lE-15E-octadecatrienoic acid of Formula II,

wherein the percentage content compounds of Formula I and Formula II is 0.0001% to 5% each.

A pharmaceutical composition comprising the standardized extract of claim 1 or 2 along with one or more of pharmaceutically acceptable carriers, excipients or diluents.

A pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I along with one or more of pharmaceutically acceptable carriers, excipients or diluents.

A pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula II along with one or more of pharmaceutically acceptable carriers, excipients or diluents.

A method for the isolation of 16-hydroxyoctadeca-9 (Z or E), 12 (Z or E), 14 (Z or E)-trienoic acid of Formula I, 13-hydroxy-9Z-l lE-15E-octadecatrienoic acid of Formula II or mixtures thereof from Annona squamosa aerial parts, the method comprising, a extracting Annona squamosa aerial parts in a solvent, b. concentrating the extract, c. suspending the extract in water, d. partitioning the extract in a solvent, e. isolating the compounds of Formulae I, II, or mixtures thereof.

7. The method of claim 6, wherein the extraction of Annona squamosa aerial parts is carried out with a solvent selected from the group consisting of alcohol, water and mixture(s) thereof.

8. The method of claim 7, wherein the alcohol is methanol.

9. The method of claim 6, wherein the partitioning of the extract is carried out in a solvent selected from the group consisting of hexane, petroleum ether, diethyl ether, toluene, dichloromethane, dichloroethane, chloroform, carbon tetrachloride, ethyl acetate, butanol and mixture(s) thereof.

10. A method for the preparation of extracts of Annona squamosa aerial parts enriched with bioactive marker compounds, the method comprising, a. extracting Annona squamosa aerial parts with a solvent selected from the group consisting of petroleum fraction, chloroform, acetone, methanol, water and mixture(s) thereof, and b. drying the extract.

11. A method for the standardization of extracts of Annona squamosa aerial parts, the method comprising detecting and quantifying bioactive marker compounds of Formulae I and II.

12. A standardized extract of Annona squamosa aerial parts prepared by a method comprising a extracting Annona squamosa aerial parts with a solvent, b. drying the extract and c. standardizing the extract by using compounds of Formulae I and II as bioactive marker compounds.

13. The method of claim 12, wherein the solvent is selected from a group consisting of petroleum fraction, chloroform, acetone, methanol, water and mixture(s) thereof.

14. A method for treating, preventing, inhibiting or suppressing diseases mediated through PTP-IB in a mammal comprising administering a therapeutically effective amount of a compound of Formula I, Formula II, a standardized extract of Annona squamosa aerial parts, or mixtures thereof.

15. A method for treating, preventing, inhibiting or suppressing diseases mediated through NFKB and cPLA2, in a mammal, comprising administering a therapeutically effective amount of a compound of Formula I, Formula II, a standardized extract of Annona squamosa aerial parts, or mixtures thereof.

16. A method for treating, preventing, inhibiting or suppressing diseases having their iteology through AMP kinase in a mammal, comprising administering a therapeutically effective amount of a compound of Formula I, Formula II, a standardized extract of Annona squamosa aerial parts, or mixtures thereof.

17. A method for treating, preventing, inhibiting, or suppressing diabetes in a mammal comprising administering a therapeutically effective amount of a compound of Formula I, Formula II, a standardized extract of Annona squamosa aerial parts, or mixtures thereof.

18. A combination comprising bioactive marker compounds of Formula I, Formula II, a standardized extract of Annona squamosa aerial parts, or mixtures thereof and one or more of extracts of plants known to have antidiabetic use.

19. The combination of claim 18, wherein the plants are selected from the group consisting of Cinnamomum cassia, Capparis moonii, Azadirachta indica, Salacia chinensis, Delphinium denudatum, Chicorium intybus, Enicostemma littorale, Bauhinia variegata, Smilax china, Alstonia scholaris , Swertia chirayta, Coptis teeta, Caesalpinia bonduc, Cissampelos parreira, Citrullus colocynthis, Saraca asoca, Sphaeranthus indicus, Tecomella undulata, Trichosanthes dioica, Prunus cerasoides , Polyalthia longifolia, Ailanthus excelsa, Helicteres isora and Stereospermum suaveolens.