FIELD OF THE INVENTION
The invention relates to a process of preparing an extract ofAnnona squamosa. The extracts ol Annona Squamosa are useful for the treatment of cancer, diabetes and related complications including inflammatory conditions such as 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.
BACKGROUND OF THE INVENTION
Diabetes Mellitus (DM) is a metabolic disease of epidemic proportions and increasing linearly ir every parts 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 diabetei 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 secretior or hyperinsulinimea causing insulin resistance or both. Diabetic patients are at risk of long-terrr damage, dysfunction and failure of various organs, especially retina, kidney nerves and alsc 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 characterizec by lack of insulin release, which can be corrected by administration of exogenous insulin. Ir contrast, about 90% of diabetics suffer from type 2 diabetes, a heterogeneous, multifactorial anc polygenic metabolic syndrome, which is caused by an inadequate secretion of insulin and it! response in peripheral tissues.
Insulin is required to transfer the sugar from the blood into the cells for the production o 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 when blood sugar rises, and when the blood suga level drops the production of insulin stops. But in a diabetic person, the beta cells produce littl< or no human insulin (type 1) or cells are unable to utilize it (type 2), when this happens there is i fast elevation of blood sugar levels. This condition is called diabetes mellitus.
Apart from hyperglycemia, patients are also afflicted with other concurrent maladies like insulii resistance, obesity, hypertension, inflammation and dyslipidemia that are collectively clubbed a: 'metabolic syndrome' or 'syndrome X' or 'the deadly quartet' and are associated with increase< risk of cardiovascular disease. Given its prevalence and complexity, there is a growing need fo

novel strategies and effective therapeutic approaches treatment of diabetes that is central to the metabolic syndrome.
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)
Recent developments reveal a broader involvement of NF-kB in various auto-immune diseases including rheumatoid arthritis (RA) pathology, including development of T helper 1 responses, activation, abnormal apoptosis and proliferation of RA fibroblast-like synovial cells, and differentiation and activation of bone resorbing activity of osteoclasts. In agreement with this, studies in animal models of RA have demonstrated the high therapeutic efficacy of specific inhibitors of NF-kB pathway, indicating the feasibility of anti-NF-kB therapy for human disease. Chemical entities down-regulating the expression of cell adhesion molecules are also effective in controlling various inflammatory diseases. With these aspects in mind NF-kB has also been shown to be essential for the expression of cell adhesion molecules and thus an important target for various anti-inflammatory diseases.
Cytosolic phospholipase A2 (cPLA2) plays a key role in inflammation by its ability to release arachidonic acid from cell membrane glycerophospholipids. The oxidative metabolism of released arachidonic acid by 5-lipoxygenase and cyclooxygenase (COX) results in the generation of pro-inflammatory leukotrienes and prostaglandins. These pro-inflammatory mediators possess a wide spectrum of biological activities and alone, or in combination, are

responsible for variety of inflammatory conditions. cPLAi also affect the inflammatory response by regulating the secretion of many pro-inflammatory cytokines viz., TNFa, IL-6, IL-1 and IL-8. Hence inhibiting the activity of cPLA2 could potentially regulate the disease progression.
Treatment Of Diabetes
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 ethical drug approved for the treatment of 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 and anti-cancer properties. Many botanical products 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 reputed to possess varied medicinal properties, which include anti-fertility and anti-tumor activities in mice and rats. Several workers have investigated its use as insecticidal agent. Various photochemical, pharmacological, antibacterial and antiovulatory studies have been carried out with 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. The nonaqueous extracts of plant annona squamosa of the present invention or the purified components, however, have not been evaluated till date for its antidiabetic activity or inflammatory activity.
WO04/060383 discloses a process for preparing an extract of Annona Squamosa for the treatment of diabetes and related complications. Annona squamosa with its anti-diabetic as well as anti-inflammatory properties promises to reduce the risk of both conditions
SUMMARY OF THE INVENTION
According to this invention there is provided a process of extracting 16-Hydroxyoctadeca-9 (Z or E),12 (Z or E),14(Z or E)-trienoic acid of Formula I OH
(Formula Remove)

and 13-Hydroxy-9Z-l lE-15E-octadecatrienoic acid of Formula II
from the plant Annona squamosa wherein the process comprises collecting leaves of Annona squamosa, drying the said leaves, preparing an aqueous extract of the leaves. Suspending dry methanolic extract in water (1 litre) and carry out partitioning with chloroform.
An object of this invention is to prepare an extract of Annona squamosa.
Further object of this invention is to screen extracts of Annona Squamosa as anti-diabetic agents.

Other object of this invention is to screen extracts of Annona Squamosa as anti-inflammatory agents.
Other object of this invention is to screen extracts of Annona Squamosa as anti-cancer agents.
DETAILED DESCRIPTION OF THE INVENTION
Powdered Annona squamosa Leaves were extracted with methanol at room temperature and the combined methanolic extract was concentrated under reduced pressure at room temperature. Dry organic extract was suspended in water and partitioning was carried out with organic solvents selected from the group consisting of hexane, petroleum ether, diethyl ether, toluene, xylene, benzene, dichloromethane, dichloroethane, chloroform, carbon tetrachloride, dioxane or ethyl acetate. The organic fractions were combined and dried over anhydrous sodium sulphate. The mixture was filtered and concentrated under reduced pressure. The organic extract was purified by column chromatography and eluted with hexane, ethyl acetate in hexane, ethyl acetate and rnethanol in ethylacetate. Different fractions were collected and observed each fraction by thin layer chromatography using mobile phase ethyl acetate in hexane, ethyl acetate in methanol, dichloromethane in methanol and methanol in chloroform. Detection was done by observation under UV and with spraying reagents. Fraction containing the compounds of Formula I and II above were collected. Sample preparation was done with sonication by taking the said fraction in organic solvents selected from the group consisting of acetonitrile or methanol followed by the addition of buffer selected from the group consisting of formic acid, trifluoro acetic acid, ort/zo-phosphoric acid, ammonium acetate, sodium perchlorate, potasium dihydrogen orthophosphate, dipotasium 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 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 compounds of Formula I and II at room temperature. The mixture was eluted by using gradient method.
Pure fractions containing compound of Formula I and II were worked up by evaporating the solvent in rotavapor under vacuum at room temperature to yield compounds of Formula I and Formula II.

Reference is now made to a working example for the process for preparation of the extract of Annona squamosa.
Example 1; Powdered 3.2 kg Annona squamosa leaves were extracted with methanol (20 liters) at room temperature three times successively 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 methanolic fractions were combined and dried over anhydrous sodium sulphate. The mixture was filtered and concentrated under reduced pressure. The organic extract was purified by column chromatography and eluted with hexane, ethyl acetate in hexane in ratio of 60:40, 65:35, 70:30, 80:20, 90:10 and finally with ethyl acetate. Different fractions were collected and observed each fraction by thin layer chromatography using mobile phase ethyl acetate in hexane, ethyl acetate in methanol, dichloromethane in methanol 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 Formula I and II above were collected. Sample preparation was done with sonication by taking the said fraction in acetonitrile followed by the addition of Buffer 0.1% Formic acid in one fourth volume of acetonitrile. The reaction mixture was sonicated and was subsequently filtered. The mixture was subjected to preparative HPLC for isolation of compounds of Formula I and II at room temperature. The mixture was eluted by using following gradient method:
Prep HPLC Column: YMC Pack ODS-AM (250mmx50mm)
Buffer: 0.1% formic acid or 15mM Ammonium acetate filtered through 0.45u filter.
Organic phase: Acetonitrile Flow: 20ml/min Detector: (Table Remove)

The eluted fractions were collected and analyzed on analytical HPLC to get their purity. Following chromatographic conditions was used to analyze the purity of fractions:
HPLC Column: Kromasil (100mmx4.6mm)
Buffer: 0.1% formic acid filtered through 0.45^i filter.
Organic phase: Acetonitrile
Flow: Iml/min
Detector: UV@235nm
Isocratic elution: Buffer: Acetonitrile: 60:40
Pure fractions containing compound of Formula I and II were worked up by evaporating the solvent in rotavapor under vacuum at room temperature to yield compounds of Formula I and Formula II.
Yield of compound of Formula I = 0.0006 % Yield of compound of Formula II = 0.0006%
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: Dissection of the Insulin Signaling pathway
Figure 2: Depicts flow-chart showing bio-assay guided fractionation of the plant Annona squamosa, based on glucose uptake activity in L6 cells.
Figure 3: 2-Deoxy Glucose uptake by compound of Formula I Figure 4: 2-Deoxy Glucose uptake by compound of Formula II Figure 5: FTP IB enzyme inhibition by compound of Formula I. IC50 DiDg/ml Figure 6: FTP IB enzyme inhibition by compound of Formula II. IC50 = 0.25 Dg/ml Figure 7: Table summarizing the anti-diabetic activity of Formula I and II Figure 8: % Inhibition of NF-kB survival pathway by compounds of Formula I and II. Figure 9: cPLA2 activity with compounds of Formula I and II BIOLOGICAL ASSAY METHOD
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 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. We used glucose uptake in L6 myoblasts as the primary assay to test the anti-diabetic activity of extracts and/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 rnRNA expression of Glut 4 was also carried out by RT-PCR, along with PI3 kinase which is one of the key downstream players in the insulin signaling pathway.
Figure 1: Dissection of the Insulin Signaling pathway
(Figure Remove)
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-1. The protein kinase PDK1 can phosphorylate multiple downstream protein kinases, such as Akt/PKB and protein kinase C £PKCQ; 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. PTP1B (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, Endocrinology (2006) 147(6);2650-2656.
Induction of diabetes in rabbits and rats;
The experimental animals used are rabbit and Wistar rats. Alloxan is used for inducing diabetes in rabbits and streptozotocin in rats. (Alloxan and streptozotocin are purchased from Aldrich Chem. Co. USA). Intravenous injections of alloxan in rabbits and intraperitoneal injections of streptozotocin in rats are 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 are estimated by commercial kits based on glucose oxidase method (Ranbaxy Laboratories, Delhi). Diabetes was confirmed by testing FBG and postprandial blood glucose levels, depending on their glucose levels the animals are divided into three groups, namely sub, mild and severe diabetic. All animals had free access to standard chow diet and tap water ad libitum, during the period of treatment.
Measurement of 2-deoxy-d-[l-3H] glucose
L6 myoblast cells grown in 24-well plate (Corning, NY) were subjected to glucose uptake as
reported (Yonemitsu et al., 2001). Differentiated myotubes were serum starved for 5 hours and were incubated with the plant extracts 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 KC1, 1.3mM CaC12, 1.2mM MgSO4, 1.2mM KH2PO4 and 30mM HEPES—pH 7.4) and were subsequently incubated for 15 min. in HEPES-buffered solution containing 0.5|j,Ci/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 uM 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., 1998). In brief, L6 myotubes after experimental incubation with the plant extracts 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 ul of DEPC-treated water. Reverse transcription was carried out to obtain cDNA using 200 units of avian reverse transcriptase and 200ng/ul 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 AGO TCC ACC-3' (588-bp). For PCR reaction, 1 ul of the cDNA mixture prepared as described earlier was added to a PCR reaction mix consisting of IQxPCR buffer, 2mM 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.
1R and IRS1 tyrosine phosphorvlation 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 compound for 24 hours and Insulin (lOOnM) for 15 minutes. Thereafter, they were washed once with ice-cold PBS 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 Dg/ml Aprotinin, 10 Dg/ml leupeptin, 1% Triton X-100 pH 7.4). The lysates were centrifuged, and the supernatants incubated with 50 ul of protein A-Sepharose beads that had been coated with monoclonal anti-IR / IRS1 antibody. The immunoprecipitates were washed three times with 500 ul 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 I 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).
Figure II
(Figure Remove)
Anti-diabetic Activity of compound of Formula I and Formula II:
Figure 3: 2-Deooxy Glucose uptake by compound of Formula I
(Figure Remove)



• Compound of Formula I showed significant dose-dependent glucose uptake
• EC50 = 8.17ng/ml
• EC50 (with Insulin) - 1.176 ng/ml
Figure 4; 2-Deoxy Glucose uptake by compound of Formula II
Glucose Uptake by Formula II

(Figure Remove)
Concentration (pg to ng)
• Compound of Formula II showed significant dose-dependent glucose uptake
• 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 FTP IB-catalyzed
hydrolysis of pNPP was measured by following the absorbance change at 405 nm. PTP1B enzyme used in the assay was purified recombinant human FTP IB from Biomol. IC50 values were determined at fixed enzyme (25 ng/well) and substrate (5 mM) concentration with varying concentrations of drug molecule. The enzyme reaction was carried out at 30°C for 20 minutes in dark and arrested by IN NaOH. RK682 from Biomol was used as a positive control for PTP1B enzyme inhibition (IC50-100 DM).
Figure 5: PTP1B enzyme inhibition by compound of Formula I. IC50 = 1 ug/ml
PTPlbDRC for Formula 1
(Figure Remove)

Figure 6; PTP1B enzyme inhibition by compound of Formula II. IC50 = 0.25 ug/ml
PTPIbDRC for Formula II
(Figure Remove)

Our studies showed that Annona squamosa extracts / fractions increased glucose uptake in an efficient manner, both in the presence and absence of Insulin. The enhanced uptake of glucose would lead to increased utilization of glucose from the blood. Potency in glucose uptake assays is a good indication of the compound's insulin sensitizing and therefore, anti-diabetic properties.
The compounds also showed potent inhibition of FTP IB enzyme. Protein tyrosine phosphatases, most significantly PTP1B, have emerged as a promising drug target for diabetes and obesity. They have been implicated in the negative regulation of insulin action through dephosphorylation of the IR. Blocking or inhibiting the FTP IB enzyme would result in increased signaling through the IR and IRS-1 thereby decreasing insulin resistance.
Figure 7; Table summarizing the anti-diabetic activity of Formula I and II
(Table Remove)
AbbreviationsIR-P Insulin receptor phosphorylation; IRS 1-P Insulin receptor substrate 1 phosphorylation; PTP1B Protein tyrosine phosphatase IB;
Dl- Methanolic extract; D2 - hydroalcoholic extract; D3 - Aqueous extract; Fl & F2 Chloroform fraction; F3 - Butanol fraction. 11 significant up-regulation/expression + + Significant activity — no effect
NFicB Enzyme Inhibition:
The J774A.1 cell lines (ATCC) were maintained in RPMI-1640 supplemented with 10% FBS, 2mM L-Glutamine, 100 units/ml penicillin and 100 ug/ml streptomycin and cultured at 37 °C 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 Dl of RPMI medium with PCS). The dilution of standard compound (BAY-11-7082) and compounds to be tested were made in dimethylsulphoxide and RPMI-1640 medium. 20 Dl of each dilution was added to the cells. The effect of compounds on the death of J774 was measured 18 hours post treatment of compounds. 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, 10D1 of MTT solution (5mg/ml in RPMI medium) was added to each well and the cells were kept for 4h at 37 °C 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 540nm. Cell survival was estimated as a percentage of the value of untreated controls. (Formula Remove)
PLA2 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. In order to determine whether our compound inhibits PLA2 activity, cells were loaded with [14C]- arachidonic acid. The A23187-stimulated release of incorporated [14C]-arachidonic acid reflects the PLA2 activity.
Method
Cells were seeded into 24 well plate at a density of 1 x 105/well and incubated overnight with [I4C]- arachidonic acid (0.1 u€i) and test compound at 37°C. Supernatant was removed to measure the amount of [14C]- arachidonic acid incorporated into the cells. Cells were resuspended in PBS (pH 7.4) with calcium and exposed to A23817 (5 uM) for 20 min. Supernatant was collected to count the radioactivity released into the media. The radioactivity released into the medium reflects PLA2 activity and was measured with liquid scintillation counter. The PLA2 activity was expressed as percentage of the total radioactivity incorporated into the cells.
Figure 9: cPLA2 activity with compounds of Formula I and II
(Formula Remove)

WE CLAIM:
1. A process of extracting the 16-Hydroxyoctadeca-9 (Z or E), 12 (Z or E), 14(Z or E)-trienoic
(Formula Remove)
from the plant Annona squamosa wherein the method comprises:
(Formula Remove)
a. Collecting leaves of Annona squamosa;
b. Drying the said leaves,
c. Preparing an organic extract of the leaves,
d. Concentrating the organic extract under reduced pressure at room temperature,
e. Suspending the organic extract in water and partitioning with organic solvent,.
f. Drying the organic fractions over anhydrous sodium sulphate,
g. Filtering the mixture and concentrating under reduced pressure,
h. Subjecting the concentrated extract to the step of column chromatography;
i. Eluting the extract with hexane, ethyl acetate in hexane, ethyl acetate and methanol in ethylacetate.
j. Pooling up the fractions enriched with compounds of Formula I and II.
k. Preparing sample by taking enriched fractions in organic solvent followed by sonication for 15min. and subsequent addition of Buffer, which is further sonicated for lOmin.

1. filtering the mixture with 0.45micron polytetrafluoroethylene. m. Purifying the enriched fractions by prep HPLC.
2. The process as claimed in claim 1, wherein the leaves are air-dried upto 5 to 6 days in absence
of sunlight on blotting paper.
3. The process as claimed in claim 1, wherein the organic extract was prepared using organic
solvent selected from the group consisting of methanol, chloroform, dichloromethane, ethyl
acetate, acetone and acetonitrile.
4. The process as claimed in claim 3, wherein the organic solvent is selected as methanol.
5. The process as claimed in claim 1, wherein partitioning was carried out in an organic solvent
is selected from the group consisting of hexane, petroleum ether, diethyl ether, toluene, xylene,
benzene, dichloromethane, dichloroethane, chloroform, carbon tetrachloride, dioxane or ethyl
acetate.
6. The process as claimed in claim 5, wherein the organic solvent is selected as chloroform.

7. The process as claimed in claim 1, wherein the said column chromatography is performed
with ethyl acetate in hexane in a ratio of 60:40, 65:35, 70:30, 80:20, 90:10.
8. The process as claimed in claim 1, wherein the said thin layer chromatography is performed
with ethyl acetate.

10. The process as claimed in claim 1, wherein sample preparation was carried out in organic
solvent selected from acetonitrile or methanol.
11. The process as claimed in claim 10, wherein sample preparation was carried out in organic
solvent selected as acetonitrile.
12. The process as claimed in claim 1, wherein the buffer used is formic acid, trifluoro acetic
acid, ort/zo-phosphoric acid, ammonium acetate, sodium perchlorate, potasium dihydrogen
orthophosphate, dipotasium 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.

13. The process as claimed in claim 12, wherein the buffer used is 0.1% formic acid in water.
14. Use of compounds as defined in claim 1, in the manufacture of a medicament for treatment
or prophylaxis of an animal or a human suffering from disease or disorder mediated through
PTB-1B.
15. Use of compounds as defined in claim 1, in the manufacture of a medicament for treatment
or prophylaxis of an animal or a human suffering from disease or disorder mediated through
NFKB and PLA2.
16. The use according to claim 14, wherein the disease or disorder is diabetes mellitus and
related complications including hyperglycemia, impaired glucose tolerance, insulin resistance
syndrome, and other disorders wherein insulin resistance is a component.
17. The use according to claim 15, wherein the disease or disorder is inflammation.
18. The use according to claim 15, wherein the disease or disorder is 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.
19. The use according to claim 15, wherein the disease or disorder is cancer.