Group IIA secreted phospholipase A2 inhibition by elemolic acid as a function of anti-inflammatory activity

Human group IIA secreted phospholipase A2 (GIIA) is a key enzyme in inflammatory reactions, worsening the condition of several chronic inflammatory diseases. The natural inhibitors of GIIA potentially block the production of inflammatory mediators. In the present study, elemolic acid, a triterpenoid from Boswellia serrata inhibited the GIIA enzyme in a concentration-dependent manner with IC50 value of 5.70 ± 0.02 µM. The mode of GIIA inhibition was studied by increasing the concentration of the substrate from 30 to 120 nM, and calcium from 2.5 to 15 mM, the level of inhibition was not changed. The inhibitor-enzyme interaction was examined by fluorimetry and Circular Dichroism (CD) studies; elemolic acid altered intrinsic fluorescence intensity and shifted far UV- CD spectra of GIIA enzyme, suggesting the direct interaction with GIIA. Elemolic acid neutralized the GIIA mediated indirect hemolytic activity from 94.5 to 9.8% and reduced GIIA induced mouse paw edema from 171.75 to 113.68%. Elemolic acid also reduced the hemorrhagic effect of GIIA along with Vipera russelii neurotoxic non-enzymatic peptide -VNTx-II (VR-HC-I). Thus, the elemolic acid has been proven as a potent inhibitor of GIIA enzyme and modulated the GIIA induced inflammatory response by in situ and in vivo methods.

Purification of GIIA. The GIIA enzyme was purified from Vipera russelii venom as described by Kasturi et al. 38 . Homogeneity of GIIA was checked by SDS-PAGE 39 . The phospholipase A 2 (GIIA) of Vipera russelii venom belongs to group IIA generally used to reveal the mode of action of human inflammatory GIIA and inhibition studies because of its availability, simple purification procedures, high degree of structural similarities, and catalytic activity to human sPLA2 40 . Molecular docking. Autodock vina 1.1.2 in PyRx 0.8 was used to conduct a molecular docking study 41 .
The crystal structure of secreted human inflammatory phospholipase A 2 (GIIA) was taken from the protein data bank (PDB id: 1POE), and the structure of phytoconstituents in 3D SDF format was retrieved from PubChem structure database 42 . GIIA and phytochemicals structures were developed and docked on a grid, and the center value was 28.3252 X 3.8846 X 69.9194. In the docking study, the drug molecule was flexible, and GIIA was inflexible. For the optimal conformation, the molecule with the lowest binding score with the highest binding affinity was selected 43 . PoseView (version 1.1.2) was used to examine the interactions of different GIIA residues with inhibitors via hydrogen bonds, hydrophobic interactions, and electrostatic interactions. www.nature.com/scientificreports/ Estimation of in vitro antioxidant activities. The in vitro antioxidant activity of elemolic acid was estimated as per the method of Blois 44 . Briefly, 25 μM of elemolic acid was added to methanolic DPPH solution (0.01 mM) and incubated for 20 min in the dark condition. Then optical density was measured at 517 nm. DPPH alone served as a positive control, and ascorbic acid was the standard. Anti-lipid peroxidation assay was performed as per the method of Gutteridge 45 . Briefly, the lipid peroxidation was induced by adding FeCl 3 (7 mM) to the solution containing egg homogenate (10%) with 25 μM elemolic acid and incubated for 30 min. The reaction was ended by adding 1 mL of TBA (0.8%) and TCA (20%). The reaction without elemolic acid served as a positive control, and alpha-lipoic acid was taken as standard. Lipid hydroperoxides released were extracted with butanol and estimated by reading optical density at 530 nm. The antioxidant activities of the above methods are expressed using the following formula, The reducing power assay was carried out as per the method of Oyaizu 46 . 25 μM of elemolic acid was mixed with 490 μL of phosphate buffer of 0.2 M (pH 6.6) and 500 μL of potassium ferricyanide (1%), which was incubated for 20 min at 50° C. The reaction ended by adding 0.5 mL of TCA (10%), and the reaction mixture was centrifuged at 3500×g for 10 min. 1 mL of supernatant was collected and mixed with 1 mL of distilled water, added 0.1 mL of 0.1% FeCl 3 to develop a colored complex which was read at 700 nm. An increase in absorbance compared to control indicated the ferric reducing antioxidant power. The reaction mixture without standard or test served as a blank. We calculated the percent increase in reducing power using the following equation.
Secreted phospholipase A 2 assay (GIIA). The GIIA enzyme activity was estimated by using autoclaved E. coli labeled with 14 C-oleic acid according to Patriarca et al. 47 and the modified method of Vishwanath et al. 48 . Briefly, a 350 µL reaction mixture consisting of 3.18 × 10 9 autoclaved E. coli cells (corresponds to 10,000 cpm and 60 nmol lipid phosphorus), 5 mM calcium (CaCl 2 ), and 100 mM Tris-HCl buffer pH 7.4 were mixed in the following order, buffer, calcium, enzyme (20 µg), water. Finally, added E. coli substrate (30 µL) and incubated at 37 °C for 60 min. Adding 100 µL of 2 N HCl and 100 μL fatty acid free BSA (10%) to terminate the reaction, vortexed the reaction mixture and centrifuged at 20,000×g for 5 min. 140 µL supernatant (containing 14 C-oleic acid) was carefully collected, and added scintillation cocktail and measured the radiation of 14 C using a Quantulus 1220 liquid scintillation spectrometer (Perkin Elmer, USA). GIIA activity was expressed as nmol of free fatty acid ( 14 C-oleate) released/min/mg of protein under standard conditions. Inhibition of GIIA activity. The 10 mg elemolic acid was dissolved in 1 mL DMSO and made up to the appropriate concentration with the Tris-HCl buffer. GIIA inhibition was carried out with indicated concentrations of elemolic acid in the range of 2 to 16 µM. The previous report showed that genistein is a promising inhibitor of GIIA proven as an anti-inflammatory molecule by in vitro, in situ, and in vivo (reduced the mouse paw edema) experiments, used as a positive control. The highest concentration of DMSO used was 0.022 percent. The GraphPad Prism Version 5.0, USA software was used to calculate IC 50 value.
Effect of calcium and substrate concentration on GIIA inhibition. The effect of calcium and substrate concentrations on GIIA inhibition was studied. In separate assays, GIIA activity was measured by increasing the concentration of calcium from 2.5 to 15 mM, and substrate from 30 to 120 nmol in the presence and absence of IC 50 concentration of elemolic acid (5.70 µM), and the assay was carried out as stated above.
Determination of binding characteristics and reversibility of GIIA inhibition. In this study, GIIA enzyme was pre-incubated with IC 50 concentration of elemolic acid (5.70 µM) in a 350 µL reaction mixture and dialyzed (MW cut off-3,000-6,000) for twenty-four hours with two buffer changes. GIIA activity was measured before and after the dialysis.
Intrinsic fluorescence interaction study. The intrinsic fluorescence intensity of GIIA enzyme with and without elemolic acid was measured in Horiba JobinYvonFluorolog-3 spectrofluorometer (Centre of excellence and Nano Science (CeNS, Bangalore, India). The 2.0 mL reaction mixture in quartz cuvette of 1 cm path length consists, GIIA (20 µg/mL), 100 mM Tris-HCl buffer (pH 7.4), 5 mM calcium and increasing concentrations of elemolic acid (0.02 to 0.10 µM). The fluorescence spectra was measured between 300 and 370 nm after the excitation at 280 nm. Due to the internal absorption and filtration, the elemolic acid caused the quenching of spectra non-specifically. The tryptophan standard was used to correct it empirically 49 . The spectra for blank containing 100 mM Tris-HCl buffer (pH 7.4), 5 mM calcium, and 0.02% DMSO were substracted from spectra of GIIA and GIIA with different concentrations of elemolic acid. Circular dichroism study. Far UV-CD spectrum was recorded for GIIA enzyme (30 µg/mL) with or without elemolic acid (IC 50 concentration, 5.70 µM) in a standard reaction mixture using Jasco J-810 spectropolarimeter at the Centre of excellence and Nano science (CeNS), Bangalore, India. The spectra was obtained using a quartz cuvette with a path length of 1 cm between 200 and 240 nm at room temperature. The response time was 2 s and www.nature.com/scientificreports/ the bandwidth was 1 nm. A total of ten scans were used to get the final spectra. Spectrum of blank contained Tris-HCl buffer (100 mM), 5 mM calcium, and DMSO (0.022%) and was subtracted to correct the protein spectra. K2D3 software was used to calculate the secondary structure of GIIA using CD spectral data.

Neutralization of GIIA induced indirect hemolytic activity.
The assay was carried out as per the method of Boman and Kaletta 50 . The substrate was prepared by mixing freshly packed human RBC (1 mL) and egg yolk (1 mL) in 8 mL of PBS. GIIA (30 µg) was pre-incubated with elemolic acid in the range of 2 to 16 µM at 37 °C for 30 min. Then, 1 mL of the substrate was added to this pre-incubated reaction mixture and incubated at 37 °C for 45 min. Halted the reaction by adding 9 mL of ice-cold PBS and centrifuged it for 20 min at 1500×g. The hemolytic activity in terms of released hemoglobin was measured at 530 nm. GIIA enzyme without corosolic acid in the sample served as a positive control.
Neutralization of edema inducing activity. The method of Yamakawa et al. 51 , adapted by Vishwanath et al. 52 was followed. The GIIA (5 µg) alone or with different concentrations of elemolic acid (3 to18 µM) in a total volume of 20 µL was injected into the intraplantar surface of the right hind footpad of mice weighing 20 to 25 g. 20 µL saline was injected into the respective left footpad for control. The animals were euthanized after 45 min by administering anesthesia (30 mg/kg of pentobarbital i.p.), and both the hind limbs were cut at the ankle joint and weighed separately. The percentage of edema was calculated by the following formula, Neutralization of hemorrhagic activity of GIIA was estimation. The method of Kondo et al. 53 , modified by Venkatesh et al. 54 was used to determine hemorrhagic activity. Briefly, mice were injected 10 μg of hemorrhagic complex containing 5:2 ratio of GIIA enzyme and non-enzymatic peptide (Vipera neurotoxin-II VNTx-II) subcutaneously (s.c). For the inhibition study, the hemorrhagic complex was pre-incubated with indicated concentrations of elemolic acid (5 µM, 10 µM, and 15 µM) for 30 min. Saline alone served as a negative control. After three hours, the mice were euthanized by administering pentobarbital (30 mg/kg, i.p.) and sacrificed by cervical dislocation. The skin was removed and hemorrhagic spots on the dorsal surface of the skin were measured using graph sheet. The results were expressed in mm 2 of hemorrhagic spots.

Statistical analysis.
The experimental results were reported as the mean ± SD of three determinations.
Graph Pad prism version 5.0 was used to calculate the IC 50 values and dissociation constant (KD) (La Jolla, USA) and calculated the percentage of inhibition from the difference between animals of inhibitor-treated and control that received the vehicle.

Ethics approval.
All experiments were performed in accordance with AVMA guidelines 55

Result and discussion
The previous studies reported that the methanolic extract of B. serrata contains the bioactive molecules such as monoterpenoids (phellendrene, cadinene, limonene, p-cymene), diterpenoid (serratol) and triterpenoids (lupeolic acid, elemolic acid, α Boswellic acid, acetyl-α-boswellic acid) exhibited several pharmacological activities [56][57][58] (Fig. 2). These molecules were subjected to in silico docking study for identification of potent GIIA inhibitor/s. The docking study with respect to enzyme-inhibitor binding affinity was exploited and expressed as energy-value (E-value  (Table 1). Additionally, elemolic acid was reported for inhibitory activity against 12-O-tetradecanoyl phorbol-13acetate-induced inflammation in mice 57 . If the elemolic acid inhibits inflammatory GIIA, that can also suppress inflammation by regulating the MAPK pathway 59 is an added benefit. The GIIA enzyme consists of active site His-47/Asp-48 (1POE) diad (active site sequence Asp-Xxx-Cys-Cys-Xxx-Xxx-His-Asp), and calcium binding loop (loop sequence Xxx-Cys-Gly-Xxx-Gly-Gly) are important for the activity 66 . Most of the GIIA inhibitors such as aristolochic acid, ascorbic acid, palmitate, and p-BPB interfere with the catalytic site by binding with His-47/Asp-48 and weakening the Ca 2+ coordination that lowers the catalytic activity of enzyme 67,68 . Many GIIA inhibitors, such as gallic acid, vannilic acid, syringic acid, and protocatechuic acid, interact with substrate binding pockets and avoid enzyme-substrate interaction by forming van der Waals contacts with amino acids Phe-23, Phe-5, Leu-31, and Leu-2 69 . Considering the above aspects, elemolic acid was docked against the GIIA enzyme. The GIIA containing three major alpha-helices located at amino acid sequences 0-17, 38-53, and 80-100 play a vital role in catalysis. The elemolic acid interacted with N terminus helices (almost), mainly to active site containing Gly-29, His-47, and Asp-48 through a hydrogen bond (H bond), as well as hydrophobic interaction with amino acids such as Leu-2, Gly-22, and Tyr-51 (Fig. 3a,b) www.nature.com/scientificreports/ space-filling model confirmed the coverage of elemolic acid in the catalytic site of GIIA (Fig. 3c, d). Thus, elemolic acid established the interaction with both the catalytic site and substrate-binding pocket of the GIIA enzyme.
There is a high degree of similarity between snake venom PLA 2 and human GIIA, which share similar biological functions like acute muscle damage, pain, edema development, and leukocyte influx into tissues 70 . The structural homology and binding pattern of vannilic acid with human GIIA and Bothrops jararacussu Toxin II (BthTX-II), were very close to hydrogen bond energies, interaction energies and the score function 71 . Bothrops jararacussu and Vipera russelii are belongs to Viperidae family; their secreted enzymes, BthTX-II and V. russelii PLA 2 (GIIA) are basic phospholipases. Hence, it is suggested that the use of V. russelii PLA 2 (GIIA) as a tool for studying the mechanism of action and development of new inhibitors for the human GIIA enzyme 71 . Also, sequencing alignment of human GIIA (1POE), with V. russelii GIIA (3H1X) and B. jararacussu GIIA (3JR8)  www.nature.com/scientificreports/ showed 97.6% and 98.4% homology, respectively, and 100% homology in active site residues (Fig. 4). Hence, in the present study, V. russelii PLA 2 (GIIA) was considered for evaluating human GIIA inhibitor/s. Further, elemolic acid was subjected to inhibit GIIA enzyme, which showed concentration-dependent inhibition (Fig. 5). The extent of GIIA inhibition was 96% at 16 µM concentration with F-statistic value of 0.0035 and p-value 0.9965 (F static value is quantified additional errors of variances of the experimental data, this can be converted to probability value (p-value), and which is a statistical measure describe the probability of obtaining the observed results). Both the F-stat and p-values of GIIA inhibition by elemolic acid following the null hypothesis. The IC 50 value of elemolic acid was 5.70 ± 0.02 µM, whereas the IC 50 value of the positive control genistein was 11.92 ± 1.45 μM (Table 2) 65 .
Because some of the inhibitors limit the activity of GIIA by either chelating metal ion calcium or some of the steroid inducible inhibitors (lipocortin I and II) non-specifically binding to GIIA impact the 'quality of interface of phospholipids 72 . Hence, we investigated the effect of calcium and substrate concentrations on GIIA inhibition by elemolic acid. GIIA activity was measured in the presence and absence of IC 50 concentration of elemolic acid (5.70 ± 0.02) by increasing the calcium concentration from 2.5 to 15 mM. The activity was increased linearly with constant inhibition, i.e., 49.2 ± 1.46% in all over the ranges of calcium concentrations (Fig. 6). Similarly, GIIA activity was measured by increasing the concentration of substrate from 30 to 120 nmoles, in the presence of IC 50 concentration of elemolic acid; the activity was increased linearly and maintained constant inhibition i.e., 48.27 ± 1.38% in all over the ranges of substrate concentrations (Fig. 7). These results suggest that GIIA inhibition by elemolic acid is independent of calcium and substrate concentrations and does not alter enzyme activity by either binding to the substrate or chelating calcium ions.
Further, the intrinsic fluorescence of GIIA was measured to determine the changes in the structure of the enzyme in the presence of elemolic acid. The altered intrinsic fluorescence indicates the structural changes in the enzyme due to the interaction with the inhibitor. The interaction of many GIIA inhibitors with enzymes resulted in fluorescence quenching 73 . Generally, aromatic amino acids of proteins (tryptophan, tyrosine, and phenylalanine) contribute to the intrinsic fluorescence. The intensity, quantum yield, and wavelength of maximum fluorescence emission of these amino acids depend on the microenvironment of the amino acid molecules. The fluorescence spectrum shifts to a shorter wavelength, and the intensity of the fluorescence increases as the polarity of the solvent surrounding the aromatic amino acid residue decreases 74,75 . Elemolic acid alters the relative intrinsic fluorescence of GIIA in concentration dependent manner. The maximum intensity of GIIA was recorded at 338 nm and shifted towards a lower wavelength of 322 nm at 0.1 μM concentration of elemolic acid (Fig. 8I,II). GIIA contains aromatic amino acids such as Tryptophan 30; Tyrosine 21, 24, 27, 51, 64, 66, 103, 107, 110; Phenylalanine 45, 113 might be responsible for increased intrinsic fluorescence. Either elemolic acid or DMSO does not alter intrinsic fluorescence. Altered intrinsic fluorescence on the addition of elemolic acid indicates that the inhibitor interacts with the GIIA enzyme directly. Further, the dissociation constant (KD) of the elemolic acid was 6.805 ± 0.06 μM.
A circular Dichroism experiment is commonly performed to analyze the structural changes in enzymes due to enzyme-inhibitor complex formation 76 . Generally, in CD analysis, the α-helix gives negative bands at 222 and 208 nm, β-sheet structures give a negative band at 210-220 nm, and the random coil has a characteristic negative band at 200 nm 77 . The far UV-CD spectrum of GIIA exhibited two distinct negative bands at 210 nm and 222 nm. The maximum absorbance of negative bands of GIIA was substantially reduced in the presence of elemolic acid at its IC 50 concentration. The peak at 210 nm shifted abruptly towards a higher wavelength and formed a peak at 215 nm, and the peak at 222 nm shifted towards a lower wavelength and formed a peak at 220 nm (Fig. 9). The change in the secondary structure of GIIA upon the interaction of elemolic acid (IC 50 concentration) was calculated using K2D3 software ( Table 3). The changes in the CD spectrum of GIIA enzyme substantiate the findings of fluorimetric studies.
Further, the reversibility of GIIA inhibition was examined by subjecting the reaction mixture to dialysis. GIIA enzyme was pre-incubated with IC 50 concentration of elemolic acid, and activity was checked before and after www.nature.com/scientificreports/ dialysis. The percentage inhibition before and after the dialysis was 50.4 ± 1.6 and 48.6 ± 1.7, respectively. This indicates that the elemolic acid binds to the GIIA irreversibly ( Table 4). The indirect hemolytic activity is an indirect approach to determine GIIA activity by using different substrates, i.e., egg yolk phospholipid and cleansed erythrocyte 78 . Elemolic acid (2 to 16 μM) was employed to neutralize the indirect hemolytic activity of GIIA, which neutralized the indirect hemolytic activity of GIIA in a concentrationdependent way. The GIIA (30 μg) alone caused the erythrocyte lysis to 94.5% ± 2.19 and which is reduced to 9.8% ± 2.39 at 16 μM elemolic acid (Fig. 10). . Briefly, 350µL reaction mixture consists of 3.18 × 10 9 autoclaved E. coli cells, 5 mM calcium, and 100 mM Tris-HCl buffer pH 7.4, with GIIA and indicated concentration of inhibitors, incubated at 37 °C for 60 min. GIIA activity was measured by the radiation of 14 C using Quantulus 1220 liquid scintillation spectrometer (Perkin Elmer, USA). GIIA inhibition was noted as a percentage of control. The data are represents mean ± SD (n = 3). www.nature.com/scientificreports/   www.nature.com/scientificreports/ The p-Bromophenacylbromide (p-BPB) neutralizes the phospholipase A 2 (V. russelii) enzyme induced mouse paw edema 79 by alkylating histidine-48, which is a highly conserved residue at the active site of V. russelii PLA 2 (GIIA) 80,81 and suggests that catalytic activity of GIIA is necessary to induce edema. Triterpenoids inhibitors of GIIA, such as celastrol, ursolic acid, oleanolic acid, neutralized GIIA induced mouse paw edema by binding to the catalytic domain of the enzyme. Hence, elemolic acid was tested for neutralizing GIIA induced edema. The different doses of elemolic acid were pre-incubated with GIIA and injected into the right hind paw of mice, and the left hind paw received saline as a negative control. Elemolic acid reduced the edema in a dose-dependent pattern and the edema ratio was reduced from 171.75% ± 2.39 (edematous leg) to 113.68% ± 2.74 at 18 μM concentration (Fig. 11). The apparent IC 50 value of elemolic acid for reducing edema was found to be 7.98 μM. The edema ratio of the standard was 119% ± 2.20.
In the living system, protein-protein interaction leads to protein complexes and is crucial for almost all aspects of cellular dynamics 82 , which enhances the pharmacological injuries by their synergistic action. Many human diseases are the result of abnormal protein-protein interactions 83 . For instance, the binding of human vimentin (an intracellular protein externalized during platelet activation) to GIIA enhances its catalytic activity 84 . It suggested that interaction GIIA-vimentin causes a more deleterious effect during inflammation. The addition of LY311727 (GIIA inhibitor which binds to the active site of enzyme) causes substantial structural changes in Table 3. Effect of elemolic acid on the secondary structure of GIIA.  Table 4. Inhibition of GIIA enzyme activity by elemolic acid before and after dialysis. The data are expressed in mean ± standard deviation (n = 3).
Reaction mixture % of inhibition (Before dialysis)

% of inhibition (After dialysis)
GIIA + IC 50 of elemolic acid 50.4 ± 1.6 48.6 ± 1.7 Figure 10. Neutralization of indirect hemolytic activity of GIIA by elemolic acid. The GIIA enzyme was pre-incubated with indicated concentrations of elemolic acid for 15 min. The reaction was initiated by adding 1 mL of the substrate (erythrocytes, egg yolk, and PBS -1: 1: 8 V/V) and incubated for 30 min at 37 °C. The hemoglobin released due to hemolysis was measured at 540 nm. The reaction mixture without enzymes served as a positive control. The data represents mean ± SD (n = 3). www.nature.com/scientificreports/ the amino terminus of the GIIA 85 . The structural displacement around the active site of GIIA by inhibitor is enough to reduce its interaction with vimentin. In snake bites, the synergistic interaction between GIIA and non-enzymatic peptides leads to increased hemorrhage 54 . In the current study, GIIA and V. russelii neurotoxic non-enzymatic peptide (VNTx-II) were used to form a protein-protein complex (5:2 molar ratio) called V. russelii Hemorrhagic Complex-I (VR-HC-I) 54 . Administration of the VR-HC-I complex into mice causes a synergistic hemorrhage at the injection site (Fig. 12c). On the other hand, either the GIIA or VNTx-II separately did not cause a hemorrhagic effect (Fig. 12a,b). Further, VR-HC-I was pre-incubated with different concentrations of elemolic acid (5 µM, 10 µM, and 15 μM) and administered to mice, resulting in the reduced hemorrhagic potential of VR-HC-I (Fig. 12a1, b1 and c1 respectively). After 30 min, mice were sacrificed, the skin was removed, the hemorrhagic spots on the dorsal surface were measured using a graph sheet, and the results were expressed in mm 2 . Elemolic acid significantly neutralized the hemorrhagic activity at 15 μM concentration.
In addition, the formation of oxidants to a higher extent signifies outrage of inflammatory response. Natural compounds that neutralize or reduce the generation of oxidants are known to have anti-inflammatory activity 86 . Hence, elemolic acid has been examined for its antioxidant activity in terms of its capacity to scavenge free radicals. At 25 µM concentration, elemolic acid efficiently scavenged the DPPH free radicals to 86.9% ± 2.3 compared to ascorbic acid, 98.5% ± 1.7. Elemolic acid showed a reducing power activity to 43.56% ± 1.97 compared to standard quercetin, which was 56.25% ± 2.5. The percentage of anti-lipid peroxidation activity of elemolic acid was 82% ± 2.2, whereas standard α-lipoic acid exhibited 89.6% ± 1.87 (Table 5). Thus, we can conclude that elemolic acid effectively scavenged the free radicals in all the three experimental methods.

Conclusion
The above data suggested that elemolic acid irreversibly binds to the GIIA and inhibits its activity proven by in vitro, in situ, in vivo studies. The studies on the mode of inhibition interpret that the inhibition of GIIA is not dependent on the concentration of metal ions or substrate. Altered intrinsic fluorescence and substantially reduced negative bands of CD spectrum by elemolic acid, indicating that the inhibitor interacted with GIIA enzyme directly. Also, elemolic acid neutralized the GIIA induced indirect hemolytic activity, mouse paw edema, and synergistic hemorrhagic effect (VNTx-II). Therefore, the elemolic acid is a candidate for drug development for both the inflammatory pathologies and snakebite envenomation. Further, necessary experiments are needed to prove elemolic acid as an anti-inflammatory drug. Figure 11. Neutralization of edema inducing activity of GIIA by elemolic acid. GIIA (5 µg) was pre-incubated with indicated concentration of elemolic acid (3 to 18 µM) for 30 min and injected into the right footpad of the hind limb of mice and the respective left footpad received vehicle (saline). After 45 min, mice were euthanized, and their legs were removed at the ankle joints and weighed separately. The edema ratio was calculated. The data are expressed in mean ± standard deviation (n = 3).

Data availability
We declare that 'The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request' .