The anti-infl ammatory activity of standard aqueous stem bark extract of Mangifera indica L. as evident in inhibition of Group IA sPLA2

The standard aqueous stem bark extract is consumed as herbal drink and used in the pharmaceutical formulations to treat patients suffering from various disease conditions in Cuba. This study was carried out to evaluate the modulatory effect of standard aqueous bark extract of M. indica on Group IA sPLA2. M. indica extract, dose dependently inhibited the GIA sPLA2 (NN-XIa-PLA2) activity with an IC50 value 8.1 μg/ml. M. indica extract effectively inhibited the indirect hemolytic activity up to 98% at ~40 μg/ ml concentration and at various concentrations (0-50 μg/ml), it dose dependently inhibited the edema formation. When examined as a function of increased substrate and calcium concentration, there was no relieve of inhibitory effect on the GIA sPLA2. Furthermore, the inhibition was irreversible as evidenced from binding studies. It is observed that the aqueous extract of M. indica effectively inhibits sPLA2 and it is associated infl ammatory activities, which substantiate their anti-infl ammatory properties. The mode of inhibition could be due to direct interaction of components present in the extract, with sPLA2 enzyme. Further studies on understanding the principal constituents, responsible for the anti-infl ammatory activity would be interesting to develop this into potent anti-infl ammatory agent.


INTRODUCTION
Phospholipases A 2 (PLA 2 ) (PLA 2 , EC 3.1.1.1.4)are a family of key enzymes, that cleaves fatty acids at the sn-2 position of glycerol phospholipids to liberate free fatty acid and lysophospholipid (Nanda et al. 2007, Burke andDennis 2009).The released free fatty acid i.e., arachidonic acid, is known to liberate potent and short-lived pro-inflammatory mediators such as prostaglandins, thromboxanes, prostacyclins and leukotrienes; by the action of cyclooxygenase (COX) and lipooxygenase (LOX) (Nanda et al. 2007, Vadas and Pruzanski 1984, Diaz and Arm 2003).The other sPLA 2 action product -lysophospholipid that is liberated has also been demostrated to serve as a substrate for the synthesis of pro-inflammatory platelet activating factors (PAF) (Burke andDennis 2009, Diaz andArm 2003).Thus, it can be concluded that the release of archidonic acid BHADRAPURA LAKKAPPA DHANANJAYA and SUDHARSHAN SHIVALINGAIAH from membrane lipids is the fi rst regulatory and obligatory step in the synthesis of pro-infl ammatory mediators in inflammatory.This arachidonic acid metabolic pathway is known to be regulated mainly by secretory phospholipase A 2 , suggesting the importance of enzyme in infl ammation related processes (Nanda et al. 2007).In snake venoms, only two groups of sPLA 2 s (GI and GII) have been identified.Group I (GIA) includes the svPLA 2 s from Elapinae and Hydrophiinae venoms with 115-120 amino acid residues and these svPLA 2 s are homologous to mammalian pancreatic GIB sPLA 2 .Group II (GIIA and GIIB) comprises the svPLA 2 s from Crotalinae and Viperinae venoms with 120-125 amino acid residues and homologous to mammalian non-pancreatic Group II-A sPLA 2 (Burke and Dennis 2009).Group II PLA 2 s are in turn divided into different subgroups on the basis of amino acid residue in the 49 th position: catalytically active D49 enzymes, catalytically inactive or with low activity K49, S49, N49 or R49 forms (Nevalainen et al. 2012, Lomonte et al. 2009).The subgroups described above exhibit a wide variety of physiological and pathological effects including the inflammatory one (Doley et al. 2010, Kini 2003).
An increased level of sPLA 2 are found in inflammatory exudates such as synovial fluid, pleural fl uid, ascitis fl uid (Bomalaski and Clark 1993) and also in plasma after an infl ammatory challenge (Wright et al. 1990).In addition, an excess level of sPLA 2 s is found to be associated with many physiopathological processes such as in cerebral illnesses, cardiovascular disorders, cancers, asthma, respiratory distress syndrome, and progression of tonsillitis (Mallat et al. 2010, de Luca et al. 2011, Chalbot et al. 2011, Farooqui et al. 1999).Furthermore, an increased level of sPLA 2 activity is also observed in some brain tumors, in chronic neurological disorders associated with neurodegenerative diseases such as neural trauma, Alzheimer's, and Parkinson's diseases (Farooqui et al. 1999, Sun et al. 2004).Several studies have demonstrated that the sPLA 2 inhibitors suppress inflammatory processes, which provides a strong evidence for their role in pathogenesis in infl ammation (Nanda et al. 2007, Moses et al. 2006, Balsinde et al. 1999, Granata et al. 2003, Kim et al. 2004, Rosenson et al. 2010).It has been observed that effective inhibitors of sPLA 2 are known to suppress the infl ammation and its associated aspects (Nanda et al. 2007, Meyer et al. 2005, Narendra Sharath Chandra et al. 2007).Considering the role of sPLA 2 s in the infl ammatory process, there is considerable pharmacological interest in fi nding potent and specifi c sPLA 2 inhibitors from diverse sources (Nanda et al. 2007, Narendra Sharath Chandra et al. 2007).In addition, considering the drawbacks and severe side effects exhibited by the current anti-infl ammatory therapies (non-steroidal anti-inflammatory drugs) targets either LOX or COX-1/2 enzymes (Vane and Botting 1998), Therefore, it appears rational to develop or fi nd effective inhibitors of sPLA 2 which could deplete the downstream pro-infl ammatory metabolites of arachidonic acid as well as PAF.Many plant extracts and its constituents are reported for their antiinfl ammatory activity through inhibition of sPLA 2 s (Nanda et al. 2007, Narendra Sharath Chandra et al. 2007, Springer 2001); effective and specific inhibitors of sPLA 2 are not available to date.Many studies have shown that sPLA 2 s (of Group I) have a very defi nite role in pathogenesis of infl ammation (Granata et al. 2003, Kim et al. 2004, Rosenson et al. 2010), therefore, it would be benefi cial to fi nd plant extract/compounds exhibiting inhibitory activity against group I sPLA 2 , this would be of potential therapeutic relevance in many inflammatory diseased state (Scott et al. 2010, Dessì et al. 2013, Magrioti and Kokotos 2013, Slesser et al. 2013).
Mangifera indica L. (Anacardiaceae), also known as mango, aam, grows in the tropical and sub tropical regions and is one of the most popular edible fruit yielding trees in the world.M. indica has been traditionally used to treat various diseases (Chopra et al. 1956, Coe and Anderson 1996, Shah et al. 2010, Dhananjaya et al. 2011).The aqueous stem bark extract of M. indica obtained by decoction has been traditionally used for the treatment of menorrhagia, scabies, diarrhea, syphilis, deabetes, cutaneous infections and anemia, as reported in the Naprolert database (Coe andAnderson 1996, Shah et al. 2010).This standard aqueous stem bark extract has been used in pharmaceutical formulations in Cuba under the brand name VIMANG ® , to treat patients suffering from increased stress (Guevara et al. 1998).Pharmacological studies have indicated that VIMANG ® has immunomodulatory, analgestic, antinociceptive, antioxidant and antiinfl ammatory effects (Makare et al. 2001, Garrido et al. 2004).Previously, the standard aqueous extract was demonstrated to inhibit the edema induced by carrageen and formalin in mice, rats and guinea-pigs (Garrido et al. 2001, Ojewole 2005).Even though, M. indica extract is known to possess anti-inflammatory activities, no study has been conducted with regard to sPLA 2 enzyme (main regulatory enzyme in arachidonic acid pathway) inhibition in support of its anti-inflammatory effects.Therefore, in this study, for the fi rst time we report the investigations carried out to evaluate the modulatory effect of standard aqueous extract of M. indica on Group IA sPLA 2 (i.e., purifi ed NN-XIa-PLA 2 phospholipase A 2 enzyme from Naja naja venom), to substantiate their anti-inflammatory properties.Furthermore, this study provides an insight on the possible biochemical interaction of extract/components to bring about inhibition of sPLA 2 s and its infl ammatory process.

MATERIALS AND METHODS
Venom of Naja naja (Indian Cobra) was purchased from Irula Co-operative Society Ltd., Chennai, India.All other reagents and chemicals used were of all analytical grades purchased from Sisco Research Laboratories (SRL), Bangalore, India.

PREPARATION OF EXTRACT
Mangifera indica stem bark collected in the campus of the university, which was authenticated at the university herbarium centre, Department of Botany, University of Mysore, Mysore, India, where a voucher specimen (UOM/DOSB/PL/82) was deposited.The stem bark extract of M. indica was prepared by decoction for 1 h in accordance to the method of Garrido et al. (2004)  sPLA 2 belonging to the group IA-NN-XIa-PLA 2 from the venom of Naja naja (Southern region) was purifi ed up to homogeneity as described previously by the method of Rudrammaji and Gowda (1998).This protein was further used for evaluating the anti-infl ammatory potential of aqueous extract of M. indica.The protein concentration was estimated according to the method of Lowry et al. (1951) using BSA as protein standard.

INHIBITION OF PHOSPHOLIPASE A 2 ACTIVITY
The Phospholipase A 2 assay was carried out according to the method described by Bhat and Gowda (1989).Phosphatidyl choline (PC) was diluted with petroleum ether (60 -80 °C) to a BHADRAPURA LAKKAPPA DHANANJAYA and SUDHARSHAN SHIVALINGAIAH concentration of 1000 nmoles/50 ml.The reaction mixture containing NN-XIa-PLA 2 (5 μg) was made up to 680 ml with water.To the reaction mixture, 200 μl of ether, 100 μl of Tris -HCl buffer (0.05M, pH 7.5), and 20 μl of CaCl 2 (500 mM) was added.The total reaction mixture was incubated at 37 °C for 60 min.After incubation, 0.5 ml of Doles mixture (Isopropanol: Pet ether: 1NH 2 SO 4 , 40:10:1) was added, mixed and centrifuged at 1000 rpm for 3 min.To the organic phase 0.5 ml of CHCl 3 : Pet ether (1:5) was added, mixed and centrifuged at 1000 rpm for 3 min.To the upper phase cobalt reagent [1.35 vol. of Triethanolamine made up to 10 ml with solution A (6 g of CO(NO 3 )2.-6H 2 O + 0.8 ml glacial acetic acid) and 7 ml of solution B (Saturated Na 2 SO 4 )] was added, mixed and centrifuged at 1000 rpm for 3 min.The upper organic phase was carefully transferred and 0.75 ml of a-nitroso-b-naphthol reagent (0.4% a-nitroso-b-naphthol in 96% ethanol) was added.The intensity of the orange colour is directly proportional to the amount of cobalt present.After 30 min 2 ml of ethanol was added to dilute the contents and absorbance was read at 540 nm.The amount of free fatty acid released was estimated using the standard linolenic acid curve.Enzyme activity was expressed as nmol of fatty acid released/min/mg of protein.
For inhibition studies, NN-XIa-PLA 2 (5 μg) was preincubated with or without different concentrations of aqueous extract of M. indica (0 -20 μg/ml) at 37 °C for 15 minutes.Appropriate controls were carried out and further experiments were conducted as described above.The inhibition is expressed as percentage (%) taking activity of venom alone as 100%.IC 50 values were calculated using Graphpad version 5.0.

EFFECT OF SUBSTRATE AND CALCIUM CONCENTRATION
Effects of substrate and calcium concentrations on the inhibition of NN-XIa-PLA 2 (5 μg) at IC 50 concentration of extract were determined.In general, the reaction mixture containing NN-XIa-PLA 2 alone and/or with the IC 50 concentration of aqueous extract of M. indica, in 0.05M Tris-HCl buffer, pH 7.5, and 400 mM calcium was used for the PLA 2 assay.In the substrate-dependent assay, substrate concentration in the range of 20 to 120 nM was used as in the fi nal reaction mixture.The calcium-dependent assay was carried out with concentrations ranging from 0 to 15 mM in the fi nal reaction mixture.After the reaction time, PLA 2 assay was carried out as described above.

DETERMINATION OF BINDING CHARACTERISTICS AND REVERSIBILITY OF INHIBITION BY M. Indica
The reaction mixture containing NN-XIa-PLA 2 (5 μg) with the IC 50 concentration of aqueous extract of M. indica in 0.05M Tris-HCl buffer, pH 7.5, and 40mM calcium were pre-incubated for 15 min.Following the reaction mixture was dialyzed against 1000 ml of 100mM Tris-HCl buffer, pH 7.5, containing 0.2 mM Ca at 4 °C in dialysis tubing (with a molecular weight cut off of 3000-6000) for 24 h with three buffer changes.The PLA 2 activity was assayed before and after dialysis as described above.

NEUTRALIZATION OF INDIRECT HEMOLYTIC ACTIVITY
Indirect hemolytic activity was assayed as described by Boman and Kaletta (1957).The substrate for the indirect hemolytic assay was prepared by suspending 1 ml of packed fresh human red blood cells and 1 ml fresh hen's egg yolk in 8 ml of phosphate buffered saline (PBS).Aqueous extract of M. indica (0 -40 μg/ml) was pre-incubated with or without NN-XIa-PLA 2 (4 μg), which showed 100% hemolytic activity, for 30 min at 37 °C.To the pre-incubated sample, 1 ml of substrate was added and allowed to react for 45 min at 37 °C.The reaction was stopped by adding 9 ml of ice-cold PBS.The suspension was mixed and centrifuged at 1500×g for 20 min.The released hemoglobin was read at 530 nm.A sample with venom alone served as positive control.The hydrolysis of erythrocyte caused by the addition of 9 ml of distilled water is taken as 100% hydrolysis.Appropriate controls were carried out and the inhibition is expressed as percentage (%).Values are presented as the mean of 4 independent determinations.

NEUTRALIZATION OF EDEMA INDUCING ACTIVITY
The procedure of Yamakawa and Hokama (1976) as modified by Vishwanath et al. (1987) was followed.NN-XIa-PLA 2 (6 μg) was preincubated without and/or with different concentration of aqueous extract of M. indica (0 -50 μg/ml) in a total volume of 20 μl saline.The reaction mixture was injected into intra plantar surface of the right hind footpad of mice weighing 20-25 g.The left footpad that received 20 μl of saline served as control.After 45 min the mice were sacrificed giving anaesthesia (Pentobarbitone, 30 mg/kg, i.p.) and both hind limbs were removed at the ankle joint and weighed individually.The increase in weight due to edema is expressed as the ratio of the weight of oedematous limb to the weight of normal (sham injected) limb x 100.Minimum edema dose is defi ned as the microgram of protein causing an edema ratio of 120%.Injecting a fixed dose of protein into footpads of mice and sacrifi cing them at regular periods of time, provided time-course curve of edema inducing activity.Edema ratio was calculated and expressed as %.

STATISTICAL ANALYSES
The IC 50 values were calculated using Graph Pad version 5.0.Inhibition percentages were calculated from the difference between inhibitor-treated group and control animals, which received the vehicle.Student's t-test for comparisons of unpaired data was used for statistical evaluation.

RESULTS AND DISCUSSION
sPLA 2 are groups of enzymes that are the main regulating enzymes in arachidonic acid pathwayderived pro-inflammatory mediators release, and thus play an important role in infl ammatory processes (Nanda et al. 2007, Vadas andPruzanski 1984).Considering the drawbacks and the severe side effects exhibited by current anti-infl ammatory therapies that include the non-steroidal antiinfl ammatory drugs that inhibit either of the LOX or COX-1/2 enzymes (Vane and Botting 1998).It appears rational to develop effective inhibitors of sPLA 2 that could deplete the downstream proinfl ammatory metabolites of arachidonic acid as well as PAF (Nanda et al. 2007).It has been observed that effective inhibitors of sPLA 2 are known to suppress the infl ammation and its associated process (Nanda et al. 2007), thus there is renound interest among the researchers around the world for the search of new potent and specifi c sPLA 2 inhibitors from different sources.Many sPLA 2 inhibitors have been isolated from various medicinal plants (Nanda et al. 2007, Narendra Sharath Chandra et al. 2007, Springer 2001), however, effective and specifi c inhibitors of sPLA 2 are still not available.In these line of studies, the aqueous steam bark extract of M. indica is evaluated for its antiinfl ammatory potential by inhibiting infl ammatory phospholipase A 2 (PLA 2 ) belonging to group IA i.e., NN-XIa-PLA 2 , which is isolated from Naja naja venom as per the previously described method of Rudrammaji and Gowda (1989).The sPLA 2 belonging to group IA i.e., NN-XIa-PLA 2 gave a specifi c activity of around 183.6±4.6, when measured using PC as substrate (Table I).When preincubated with different concentrations of extract it was observed that the aqueous extract of M. indica, inhibited the enzymatic activity in a concentration dependent manner as shown in Fig. 1.The result shows that the extent of inhibition was >95% at 40 μg/ml of extract used.The IC 50 values BHADRAPURA LAKKAPPA DHANANJAYA and SUDHARSHAN SHIVALINGAIAH 120 nM (Fig. 2).This suggests that the inhibition is independent of substrate concentration.Furthermore, in the calcium dependent activity test, it was observed that an increase in calcium concentration from 2.5 to 15 mM, increased the NN-XIa-PLA 2 enzymatic activity in a dose dependent manner.However, while when IC 50 concentration of M. indica extract was preincubated and then used along with varying concentrations of calcium, there was no relieve of inhibition (Fig. 3), suggesting that the inhibition by M. indica extract is independent on calcium concentration.All these studies show that inhibition by aqueous extract of M. indica is independent on substrate and calcium concentration.Furthermore, it is reported that some of the sPLA 2 inhibitors have been known to mediate displacement of catalytically essential calcium from the enzyme and consequently inhibition of enzymatic activity (Pruzanski et al. 1992).In the calcium binding experiments, it was found that the PLA 2 enzyme activity, both before and after the dialysis of the enzyme inhibitor mixture, was unaltered i.e., the % of inhibitory activity of M. indica extract was not decreased upon extensive dialysis, suggesting that the inhibition  NN-XIa-PLA 2 183.6±4.6 8.1 μg/ml a nmol of fatty acid released/mg of protein/min at 37 °C.b IC 50 value is defi ned as the amount of extract (μg/ml) required to inhibit 50% of enzyme activity in the given reaction mixture.calculated was 8.1 μg/ml (Table I).Most of the sPLA 2 inhibitors are known to inhibit the activity either by binding to substrate or by chelating calcium, which is required for activity (Nanda et al. 2007).Furthermore, it was observed that the sPLA 2 inhibitors affect the "Quality of interface" by modifying the phospholipids bilayer properties which render the phospholipids inaccessible to the enzyme.The steroid inducible inhibitors of sPLA 2 like lipocartin I and II are shown to inhibit by nonspecifi c binding to the membrane phospholipids.It is also observed that their inhibition is relieved by increasing the substrate concentration (Davidson et al. 1987).In our experiments, we observed that, when examined as a function of substrate concentration, there was no relieve of inhibition of the extract preincubated, and also when the substrate concentration was increased from 20 to is irreversible, supporting the observation that inhibition by M. indica extract is independent on substrate and calcium concentration.These studies indicate that the inhibition could be due to direct interaction of components/molecules present in M. indica extract at active site residues of the sPLA 2 enzyme.
NN-XIa-PLA 2 enzyme exhibited indirect hemolytic activity, which is an indirect way of measuring PLA 2 activity using egg yolk and washed erythrocytes which is used as substrates.When the effect of aqueous extract of M. indica at different concentrations (0-40 μg/ml) was tested, it was found that the extract in general effectively inhibited indirect hemolytic activity up to 98% at ~40 μg/ml concentration (Fig. 4).This in situ inhibition activity is well correlated with the inhibitory activity of the in vitro PLA 2 enzyme.Therefore, the inhibition of NN-XIa-PLA 2 activity by molecules in M. indica extract could be attributed to the modulation of the catalytic activity of PLA 2 at the interface itself, i.e., beyond the initial steps of enzyme adsorption and activation, probably through modifi cations of the intermolecular organization of the membrane components.It is well known that sPLA 2 s cause cell membrane asymmetry by degradation of glycerol phospholipids of the membranes (Granata et al. 2003).
Many of the inflammatory sPLA 2 s induce edema (inflammatory reactions) when injected into mouse footpad as demonstrated before  Mangifera indica ANTI-INFLAMMATORY ACTIVITY 205 (Vishwanath et al. 1988).Several sPLA 2 inhibitors are demonstrated to exhibit concomitant inhibition of enzyme activity and edema-inducing activity (Nanda et al. 2007, Mohamed et al. 2010).Since in our study, the aqueous extract of M. indica effectively inhibited the in vitro sPLA 2 activity and in situ sPLA 2 activity, the inhibitory potential of in vivo edema inducing activity of NN-XIa-PLA 2 was tested.The edema inducing effect of NN-XIa-PLA 2 (6 μg) was more than 175%, when compared to the saline injected mice.Figure 5 shows that aqueous extract of M. indica at different concentrations (0-50 μg/ml), dose dependently inhibited the edema formation, when co-injected with enzyme.In addition, M. indica extract at the tested dose alone did not cause edema when injected into footpads of mice.The neutralization of edema inducing activity is known to be well correlated with the in vitro enzymatic activity inhibition.It has been demonstrated that the standard extract of M. indica, administered orally (50-200 mg/Kg body wt.) reduced ear edema induced by arachidonic acid (AA) and phorbol myristate acetate (PMA) in mice (Garrido et al. 2006).In addition, the extract was demonstrated to inhibit the edema induced by carrageen and formalin in mice, rats and guinea-pigs (Garrido et al. 2001, Magrioti andKokotos 2013).Furthermore, the extract has been shown to reduce the tumor necrosis factor alpha (TNF alpha) serum levels in both arachidonic acid (AA) and phorbol myristate acetate (PMA) induced models of infl ammation in mice (Garrido et al. 2004).It was demonstrated that the extract inhibited the induction of PGE 2 and LTB 4 , when it was stimulated with pro-infl ammatory stimuli lipopolysaccharide-interferon gamma (LPS-IFNg) or calcium ionophore A23187 in J774 macrophage cell lines (Garrido et al. 2006).Recently, it has been shown that the aqueous stem bark extract of M. indica administration reduced TBARS levels and iNOS, COX-2, TNF-α and TNF R-2 expression in colonic tissue.A decrease in IL-6 and TNF-α serum levels was also observed (Márquez et al. 2010).
Although the mechanism of action of the extract is unclear, the finding that no visible change was detected in electrophoretic pattern of NN-XIa-PLA 2 when incubated with extracts (data not shown), excludes the proteolytic degradation as a potential mechanism (Borges et al. 2000).Furthermore, considering the results of binding  studies, the inhibition is known to be of irreversible nature.In addition, inhibiton is observed to be of independent of substrate and calcium concentration.The most likely mechanism for anti-infl ammatory activities by this extract is probably due to the direct binding of the constituents of the extract with sPLA 2 s active site.The extract is known to contain a well-defi ned and standardized mixture of components such as polyphenols, terpenoids, steroids, fatty acids and microelements (Nunez-Selles et al. 2002, Shah et al. 2010), and mangiferin (20%) being the predominant one.As with other polyphenols, the phenolic constituents of the extract like phenolic acids (Gallic acid, 3, 4 dihydroxy benzoic acid, benzoic acid) and phenolic esters (Gallic acidmethyl ester, gallic acid propylester, benzoic acid propyl ester), could also be involved in binding with sPLA 2 s, thus bringing about inhibition (Nunez-Selles et al. 2002).In addition, the active constituents of this extract like mangiferin, amento fl avone, friedelin, daucosterol and betasistosterol (Sanchez et al. 2000) seems to exhibit anti-infl ammatory effect through quenching of free radicals, as they are involved in the mechanism of infl ammatory process (Nanda et al. 2007, Garrido et al. 2004, 2009, Márquez et al. 2012, Vyas et al. 2012).Previously, it was demonstrated and concluded that the standardized aqueous extract of M. indica (VIMANG ® ) can be classifi ed as a dual inhibitor, as it inhibits both COX and LOX pathways of arachidonate metabolism (Garrido et al. 2004).This anti-infl ammatory activity of the extract was believed to be due to the powerful antioxidant activities exhibited by the constituents like phenolic compounds including mangiferin.It has been demonstrated that VIMANG ® and mangiferin, exhibit inhibitory activity against synovial fl uid PLA 2 activities (Garrido et al. 2004).
The aqueous stem bark extract of M. indica inhibiting both in vitro sPLA 2 enzymatic activity and in vivo edema inducing activity of NN-XIa-PLA 2 , suggests a strong correlation between lipolytic activity and pro-inflammatory activity inhibition.It is to be noted that this standard aqueous stem bark extract of M. indica has been tested in a broad set of toxicological studies with satisfactory results, including acute and subchronic toxicity, genotoxicity, and irritability and is classifi ed as a non-toxic product (Garrido et al. 2009, Márquez et al. 2012, González et al. 2007).Thus, M. indica bark extract can be developed for topical application, as it is non-toxic and an effective anti-inflammatory formula contains potent antiinfl ammatory molecules.

CONCLUSIONS
The aqueous extract of M. indica effectively inhibited sPLA 2 and its associated infl ammatory activities.The inhibition is irreversible and also independent on substrate and calcium concentration, suggesting that the constituents of the extract might possibly directly interact to bring about inhibition.In addition, it was found that there is a strong correlation between lipolytic activity and pro-infl ammatory activity inhibition.Therefore, the study suggests that the extract possesses potent antiinfl ammatory agents, which could be developed as a potential therapeutic agent against infl ammatory related diseases.This study also substantiates their anti-infl ammatory properties.Further studies on compounds responsible for the anti-infl ammatory activity would be interesting, as these are highly attractive candidates for clinical development as a new class of anti-infl ammatory agents.

ACKNOWLEDGMENTS
DBL thanks Jain University for the constant encouragement to progress in research.DBL acknowledge DST, Govt of India for providing the grant INT/SLP/P-007/2012, dated 15 th May, 2014.SS and DBL acknowledge the Adichunchanagiri Mahasamstana Mutt and Shikshana Trust for providing facilities in the Adichunchanagiri Biotechnology and Cancer Research Institute (ABCRI).We thank Balagangadharanatha Swamiji Institute for Technology (BGS-IT) and Sri Adichunchangiri College of Pharmacy for the support to carry out the research.

Figure 1 -
Figure 1 -Dose dependent inhibition of in vitro PLA 2 (NN-XIa-PLA 2 ) activity by aqueous stem bark extract of M. indica.Briefl y, Phosphatidyl choline (PC)corresponding to 1000 nmol/ml was made up to 680 μl with NN-XIa-PLA 2 (5 μg), with or without aqueous stem bark extract of M. indica at various concentrations (0-20 μg/ml) and was incubated with other reaction mixture at 37 °C for 60 min and color developed was read at 540 nm.The results show ± S.E.M. for n=3.

Figure 2 -Figure 4 -
Figure 2 -Dose dependent inhibition of indirect hemolytic activity of NN-XIa-PLA 2 by aqueous stem bark extract of M. indica.NN-XIa-PLA 2 (4 μg) in 100 μl of phosphate-buffered Saline (PBS) was preincubated with or without aqueous stem bark extract of M. indica at various concentrations (0-40 μg/ml).The reaction was started by adding erythrocytes, egg yolk and PBS (1:1:8 v/v) incubated for 10 min at 37 °C.The released hemoglobin in the supernatant was measured by taking absorbance at 540 nm.The results show ± S.E.M. for n=3.

Figure 5 -
Figure 5 -Effect of calcium concentration on inhibition of NN-XIa-PLA 2 by aqueous stem bark extract of M. indica.The reaction mixture contained NN-XIa-PLA 2 enzyme in 0.05 M Tri-HCl buffer pH 7.5, and 0-15mM CaCl 2 in fi nal volume.The reaction was initiated by adding substrate.The reaction was carried out in the absence (rhombus) and presence of IC 50 concentration of extract (open square) with increasing concentration of calcium.The fi gure inset represents percentage inhibition in the presence of the IC 50 concentration.Data represents ± S.E.M for n=3.