In Vitro and In Vivo Evaluation of Polyherbal Formulation against Russell's Viper and Cobra Venom and Screening of Bioactive Components by Docking Studies

The present study emphasizes to reveal the antivenom activity of Aristolochia bracteolata Lam., Tylophora indica (Burm.f.) Merrill, and Leucas aspera S. which were evaluated against venoms of Daboia russelli russelli (Russell's viper) and Naja naja (Indian cobra). The aqueous extracts of leaves and roots of the above-mentioned plants and their polyherbal (1 : 1 : 1) formulation at a dose of 200 mg/kg showed protection against envenomed mice with LD50 doses of 0.44 mg/kg and 0.28 mg/kg against Russell's viper and cobra venom, respectively. In in vitro antioxidant activities sample extracts showed free radical scavenging effects in dose dependent manner. Computational drug design and docking studies were carried out to predict the neutralizing principles of type I phospholipase A2 (PLA2) from Indian common krait venom. This confirmed that aristolochic acid and leucasin can neutralize type I PLA2 enzyme. Results suggest that these plants could serve as a source of natural antioxidants and common antidote for snake bite. However, further studies are needed to identify the lead molecule responsible for antidote activity.


Introduction
Every year snake bites cause innumerable mortalities in India. Recent annual report estimates every year nearly 94,000 deaths globally and 15,000 deaths in India due to snake bites [1]. Snake venoms are heterogeneous mixture of organic and inorganic substances which is mainly used by the snake to immobilizae and to digest the prey. It is also used as a defense mechanism against their natural predators [2]. There are 3000 species of snakes known to science; in this 30% of snakes are venomous and considered dangerous to human [3]. Elapidae and Viperidae are two families of species which cause human mortality worldwide [4]. The Elapidae family contains the cobras and their relative species. All types of vipers come under the family Viperidae. Snake envenoming causes severe local tissue damage in the victim, such as hemorrhage and myonecrosis [2].
Daboia russelli russelli (Indian subspecies of Russell's viper) appears to be the commonest cause of fatal snake bite in Southern India [5]. Russell's viper venoms act predominantly on the haemostatic system, particularly on capillary endothelium which causes swelling within minutes of the bite [4]. In cobra bite local necrosis is the most common consequence and approximately half of the victims suffer local tissue necrosis, which is difficult to treat [6]. Phospholipase A 2 (PLA 2 ), neurotoxins, and cardiotoxins are the major classes of cobra venom polypeptides involved in the toxicity and pharmacology of bite [7]. PLA 2 can be divided into three types based on its chemical structures. Elapidae and sea snakes from the genus Hydrohis venom contain type I PLA 2 . The venom of Viperidae and Crotalidae families contain type II PLA 2 . Venom of bees (Apis mellifera) and Gila monsters (Heloderma) contain type III PLA 2 [8]. Many extensive studies have been done on this enzyme due to its pharmacological and physiopathological effects in living organisms. PLA 2 activity upon cell membranes of specific tissues suggests an important role of these enzymes in venoms toxicity [9]. Antiserum, the only remedy for envenomation, may be associated with various reactions such as early anaphylactoid reaction, pyrogenic and late serum sickness, and several other manifestations [10]. In many cases the mortality of victim happens due to the wrong choice of antiserum because of misidentified snake species by the treating physicians which results in severe life-threatening envenoming to the victim [11]. In addition antiserum development in animals (like horse, goat, sheep, etc.) is time consuming, expensive, and requires ideal storage conditions [12]. In recent years, because of the costs as well as serious side effects of a number of modern drugs, attention has turned back to medicinal plants as drugs. It has been reported that about 64% of the total world population is using traditional medicine to satisfy their healthcare needs [13]. The neutralizing activity of plants against snake venom has long been recognized, but scientist paid attention to these medicinal plants only from the last 20 years [14]. Many Indian medicinal plants have been recommended by the traditional physicians for the treatment of snak ebite [15].
Aristolochia bracteolata Lam. (AB), Tylophora indica (Burm.f.) Merrill. (TI), and Leucas aspera (S.) Labiate (LA) are the major medicinal plants commonly found in India. AB belongs to the family Aristolochiaceae. Its roots and leaves are used as anthelminthic [16]. AB is used in traditional medicine as a gastric stimulant and in the treatment of cancer, lung inflammation, and dysentery [17]. TI commonly known as "Antmool" is an important medicinal plant traditionally used as a folk remedy in treatment of bronchial asthma, bronchitis, rheumatism, allergies, and inflammation. The roots and leaves contain 0.2 to 0.46% of therapeutically important alkaloids tylophorine which act as immunosuppressive, antiinflammatory, antitumor stimulant of adrenal cortex and antiamoebic properties [18]. Leaves of LA are used for the treatment of respiratory tract disorders, edema, gastrointestinal disorders, general pain, and also as an antidote to poison. It plays a major role in Indian traditional medicine to treat psoriasis, chronic skin eruption, and chronic rheumatism [19]. The present study aims to examine the potential of polyherbal formulation of plant extracts to act as an antidote to neutralize snake venoms and screening of potent active herbal compounds by docking study.

Snakes Venom.
The Indiancobra and Russell's viper venom was obtained from Irula Snake Catchers I.C.S. Ltd., Vadanemmeli village, Kancheepuram District, Tamilnadu, India and was preserved at 4 ∘ C. Before use, the venom was dissolved in normal physiological saline and centrifuged at 200 ×g for 10 min and the supernatant was used for anitvenom studies. Venom concentration was expressed in terms of dry weight. College, Nagercoil, Kanyakumari District, Tamilnadu, India. Plants were collected from the foothill of Marunthuvazh malai (terminal of western gates) Nagercoil, Kanyakumari District, Tamilnadu and the details where noted in the field notebook according to field and herbarium techniques. The leaves and roots of the plants AB, TI, and LA were washed and homogenized. The shade-dried plant parts were brought to Pharmacology laboratory, IBSD, Imphal. The dried plants parts were powdered and subjected to soxhlet extraction (70± 10 ∘ C temp.) with the purified water as solvent in 1 : 4 (w/v) ratios for 18 hrs. The extracts were concentrated using rotary vacuum evaporator (40 ∘ C; pressure 70 ± 5 psi); the thick brown masses obtained were kept in vacuum desiccators for complete drying. The dried extracts were stored in air-tight container and kept in refrigerator (<10 ∘ C). The extracts were suspended in distilled water and used for pharmacological experiments.

Phytochemical
Screening. Preliminary phytochemical screening was carried out according to the procedure given by Harborne [20], which revealed the presence of lipids, carbohydrates, flavonoids, and alkaloids in all the plant extracts.

Animals for Experimentations.
Wistar albino rats (150-200 g) and Swiss albino mice (20-30 g) of either sex were provided by the institutional central animal house (IBSD, Imphal west, Manipur, India). The animals were acclimatized for one week under laboratory conditions. These were maintained under standard environmental condition (room temperature 27 ± 3 ∘ C, relative humidity 65 ± 10%, and 12hour light/dark cycle). All the animals were fed with standard diet and water ad libitum. The litter in the cages was removed daily to ensure hygienic condition and maximum comfort for animals. Experiments were performed in accordance with the current guidelines of CPCSEA India. Ethical clearance for handling and experimentation on the animals was obtained from the Institutional Animals Ethical Committee (IAEC), IBSD, Imphal (Approval no. IBSD/IAEC/Inst./Ph.cology/3) prior to the commencement of the experimental works.

2.5.
In Vitro Antisnake Venom Activity 2.5.1. PLA 2 Inhibition Activity. A mixture A mixture containing 1.2% egg yolk (dissolved in diethyl ether) as a source of lecithin, different concentration of aqueous extracts of AB, TI and LA (10 to 500 g) in 1 mL, 1 mL venom (200 g/mL) and 0.1 mL 5% CaCl 2 in respective test tubes and the reaction tubes were spin until a homogeneous mixture was obtained. The reaction was allowed to stand under room temperature for 4 hrs. At the end 25 mL ethanol and 0.3 mL of cresol red (HiMedia, India) were added and titrated with ice cold 0.02 N methanolic NaOH. A blank was prepared by addition of ethanol, venom, and CaCl 2 to the ether in same order and was titrated immediately. The hydrolysis capacity of PLA 2 exhibited by reacting 200 g/mL of venom with lecithin, was considered as 100% phospholipase activity, and served as control [21].
Evidence-Based Complementary and Alternative Medicine 3

Inhibition of Adenosine-Diphosphate-(ADP-)Induced
Platelet Aggregation. Platelet-rich plasma from healthy albino rats ( = 3) was obtained by centrifugation of their citrated blood at 190 ×g for 15 min. The reaction mixture contains 0.5 mL different concentration of aqueous extracts of AB, TI, and LA (10 to 500 g/mL), venom solution 0.5 mL (200 g/mL), and platelet-rich plasma 0.5 mL. Theis reaction mixture was maintained at 37 ∘ C and kept for 2 min with constant stirring; after this 0.5 mL of ADP solution (Sigmaaldrich) was added and incubated for 4 min in the dark and the absorbance was measured at 414 nm with Thermo Multiskan Spectrum [21]. The results were expressed as IC 50 or inhibitory concentration 50 value: where OD1 = ADP+platelet, OD2 = ADP+venom+platelet, and OD3 = ADP + venom + platelet + plant extract.

Inhibition of Venom-Induced Hemolysis Assay.
The hemolytic activity of aqueous extract of AB, TI, and LA was determined using the method of Oguiura et al. [22] and modified in our laboratory condition. Briefly, ethylene diamine tetra-acetic acid (EDTA) anticoagulated blood was collected from healthy Swiss albino mice, centrifuged at 300 ×g for 15 min, washed twice with PBS (pH 6.8), and diluted to 0.5% in phosphate buffer saline (PBS) with 10% fetal bovine serum, followed by dispensing 100 L into micro centrifuge tubes, to that different concentration of cobra and Russell's viper venom (10 L) was separately added in triplicate and incubated at 37 ∘ C for 2 hrs. All the tubes were centrifuged at 800 ×g for 10 min; the supernatants were transferred to 96-well plates and absorbance was measured at 405 nm for released hemoglobin. Control tubes 0 and 100% hemolysis consisting of red blood corpuscle (RBC) suspended in PBS and 1% Triton X-100, respectively. Inhibitor tubes consisting of 100 L of RBC suspended in PBS, 100 L of different concentration of test samples (aqueous extracts of AB, TI, and LA), and 10 L of venom at a concentration of 40 g. The results were expressed as IC 50 or inhibitory concentration 50 value, that is, concentration of samples exhibited 50% inhibition of venom-induced hemolysis.

Determination of Acute Toxicity (LD 50 ) of Plant Extracts.
The acute toxicity of aqueous extracts of AB, TI, and LA was determined in male albino mice maintained under standard conditions. The animals were fasted overnight prior to the experiment. Acute oral toxicity-Acute toxic class method (OCED Guideline no. 423, Annexure-2d) adopted by CPCSEA, Government of India was followed for acute toxicity studies. The behavioral abnormality, normal food and water intake, and mortality were observed after single oral administration of 2000 mg/kg b.w. of each test samples to their respective groups ( = 3) of animals.

Neutralization of Hemorrhagic Activity of Snake Venoms.
The minimum hemorrhagic dose (MHD) was defined as the least dose of venom ( g/b.w) which induces a hemorrhagic lesion of 10 mm in diameter when injected into rats intradermally after 24 hrs. Aliquots of venoms (100 L) in physiological saline or saline alone (control) were injected into the shaved dorsal skin of male Wister rats (150-200 g). In a single group five rats were used for each venom study. Aqueous extracts of AB, TI, and LA and polyherbal formulation (AB : TI : LA = 1 : 1 : 1) at 200 mg/kg p.o. were administered to their respective groups of animals just after 5 min of venom challenged [25].

DPPH Free Radical Scavenging
Assay. The free radical scavenging activity of aqueous extracts of AB, TI, and LA was measured in terms of hydrogen donating or radical scavenging ability using the stable radical 2,2-diphenyl-1picrylhydrazyl (DPPH) using the method of Jain et al. [26]. Solution of 0.1 mM DPPH (HiMedia, India) in ethanol was prepared and 1.0 mL of this solution was mixed with 3.0 mL of extract solution in water at different concentrations (10-100 g/mL). Thirty minutes later the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. Rutin (Ozone, India) was used as a standard. The results were expressed as IC 50 or inhibitory concentration 50 value, that is, Figure 1: Structure of (a) leucasin isolated from LA, (b) aristolochic acids from AB.
concentration of samples exhibited 50% inhibition of DPPH free radicals:

Superoxide Radical Scavenging Activity.
The assay was based on the capacity of the aqueous extracts of AB, TI, and LA to inhibit blue formazon formation of scavenging the superoxide radicals generated in riboflavin-light-NBT (nitroblue tetrazolium) system. 100 L riboflavin solution, 200 L EDTA solution, 200 L ethanol, and 100 L NBT solution were mixed in a test tube and the reaction mixture was diluted up to 3 mL with phosphate buffer (pH 7.2). The absorbance of solution was measured at 560 nm using phosphate buffer as blank after illumination for 15 min. This was taken as control reading. Different concentration (10-100 g) of test samples in 100 L was mixed with 100 L riboflavin, 200 L EDTA, 200 L ethanol and 100 L NBT solution in the test tubes, and then the reaction mixtures were diluted to 3 mL with phosphate buffer. The absorbance of solution was measured after illumination for 15 min at 560 nm [27]. L-ascorbic acid was used as a reference control. Decrease in absorbance of the reaction mixture indicated increased superoxide anion scavenging activity. Percentage inhibition was calculated and this activity was expressed as an inhibition concentration 50 (IC 50 ):

Reducing Power
Method. Different doses of aqueous extracts of AB, TI, and LA were mixed in 1 mL of distilled water so as to get 10 g, 20 g, 40 g, 80 g, and 100 g concentrations in separate test tubes and were mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide (2.5 mL, 1%). The mixture was incubated at 50 ∘ C for 20 min. A portion (2.5 mL) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 300 ×g for 10 min. The upper layer of the solution (2.5 mL) was mixed with distilled water (2.5 mL) and ferric chloride (0.5 mL, 0.1%), and the absorbance (OD) was measured at 700 nm. Rutin (Ozone, India) was used as a reference control. Increased absorbance of the reaction mixture indicates increase in reducing power. The % reducing power was calculated by using the formula [28]: 2.9. Protein Active Site Prediction. The potential binding sites (PBS) of proteins are those residues or atoms which bind to ligand directly on protein surface and were identified using MetaPocket 2.0 online server. In this, pocket sites are represented using the standard PDB file format. MetaPocket uses LIGSITE, PASS, Q-site finder, and SURFNET methods to identify pocket sites. All identified pockets are represented as a single probe with ranking score. To make the ranking scores comparable a Z-score is calculated separately for each site in different methods. Only the top most three pocket sites in each method are taken into further consideration. Probes within a certain distance threshold are grouped together as a cluster. Each cluster is ranked by a Z-scores, which is the sum of the Z-score of the pocket sites in a cluster [31].

Molecular Docking Protocols.
Virtual screening and molecular docking studies are a series method in drug discovery. Most of the molecular docking algorithms assume the protein as rigid object which leads to poor correlation of the docking scores. There is no single docking algorithm or scoring function that can correctly predict the binding affinities of ligand in molecular interaction [32]. For these reason, in this study two docking programs such as LigandFit method in Discovery studio package and Glide were investigated to identify the interaction of ligand molecule in the active region of the protein and to predict the binding affinity between the ligand and the receptor protein molecule [33].  [34]. Throughout the docking process the top ten conformations were generated for each ligand after the energy minimization method based on steepest descent method and conjugate gradient method. Each of the saved conformation was evaluated and then ranked using the scoring function including Ligscore1, Ligscore2, PLP1, PLP2, JAIN, PMF, and LUDI. Visual inspections of receptor-ligand interaction were selected based on the Jain score for further evaluation [35].
The ligand which showed good hydrophobic and hydrogen bond interaction with active site I region of the receptor protein showed the possible character for neutralizing the activity of PLA 2 from Bungarus caeruleus venom.

Glide.
In the case of Glide v4.5, protein coordinates were preprocessed for docking using the Protein Preparation Wizard provided in the Schrodinger Maestro environment. Hydrogen atoms were added, and the bond orders were assigned after deleting the monomer B as well as water molecules. Assignment of protonation states was carried out followed by hydrogen bond optimization for hydroxyl group residues. The hydrogen atoms were then minimized with the OPLS2005 force field. Grid calculations were performed for the protein residues of the active sites coordinates. Initially Glide performs a complete systematic search of the conformational, orientational, and positional space of the docked ligand, eliminating unwanted conformations using scoring and followed by energy optimization. Finally the conformations are further refined via a Monte Carlo sampling of pose conformation. Predicting the binding affinity and rank-ordering ligands in database screens was implemented by modified and expanded version of the ChemScore18 scoring function. For our studies, three-dimensional crystal structure of a novel phospholipase A 2 from Indian common krait was used (PDB:1DPY) with a resolution of 2.45Å. Solvent molecules were deleted and bond order for crystal ligand and protein were adjusted and minimized up to 0.30Å RMSD. Using standard precision (SP) mode of Glide software, docking studies were performed [34,35].  (Tables 2 and 3). Venom-induced hemorrhagic activity was significantly inhibited by administration of the plant extracts (Table 4).

In Vitro Antioxidant
Assay. The reducing power activity, superoxide ion, and DPPH * free radical scavenging capacity of aqueous extracts of AB, LA, and TI were compared to standard Rutin (Tables 5, 6, and 7; Figure 2). For the measurements of the reducing power ability, we investigated the Fe 3+ -Fe 2+ transformation in the presence of different concentration of AB, TI, and LA, where a concentration dependent-reduction of ferric ions to ferrous ions was observed and their calculated EC 50 values as 69.34 g, 69.65 g and 69.95 g which were found almost equal.   All values are expressed as a mean ± SEM, = 6. One way analysis of variance (ANOVA) followed by multiple comparison Dunnett's test. The minimum value of < 0.05 was considered significant. * < 0.05, * * < 0.01, compared to control group. The results were expressed asIC 50 (inhibitory concentration 50) value, that is, concentration of samples exhibited 50% inhibition of DPPH radicals.
In case of the nitroblue tetrazolium (NBT) superoxide radical system test samples AB, TI, and LA possesses concentration dependent inhibition of superoxide anions. The IC 50 values of Rutin, AB, TI, and LA was calculated as 69.8 g, 74.05 g, 82.59 g and 72.5 g, respectively.
DPPH in presence of ethanol produces stable free radical and accepts hydrogen radical to become a stable diamagnetic The results were expressed as EC 50 (effective concentration 50) value, that is, concentration of samples exhibited 50% reducing power activity.    Tables 9 and 11). Glide v4.5 predicted two modes of interactions between ligand and PLA2 by means of hydrogen bond (Figures 7 and 8; Tables 10 and 12).

Methodology of Ligand-Protein
Interaction. aristolochic acid shows interaction with Active site I region of PLA 2 from Bungarus caeruleus by means of hydrogen bond. DS package and Glide were used to study the way of ligandprotein interaction. By our computational study we found that H33 of the Aristolochic acid form hydrogen bond with the receptor protein at ASP49 and another hydrogen bond occur between ASP49 with CYS45. The interaction between Leucasin and Active site I region of PLA 2 happens by means 8  1  10  20  30  40  50   60  70  80  90  100  60  70  80  90  100   110  118  110 118 Figure 3: Geneious Basic annotation of the protein structure. Table 9: Discovery Studio docking score of aristolochic acids-PLA 2 binding. Docking process top ten conformations were generated for each ligand after the energy minimization method based on steepest descent method and conjugate gradient method. Each of the saved conformation was evaluated and then ranked using the dock score. Ligand pose 1 ranged as best pose with dock score and Jain value.  of H-bond between ligand atom O18, O17 with the Lys6 of PLA 2 by Ligandfit method in DS package (Figures 5 and  6). Glide result shown the hydrogen bond interaction occur between the H33 of the Aristolochic Acids and Lys31 from PLA 2 and O7, H3 of Leucasin with Asp49, Gly32 ( Figures  7 and 8). From these we conclude the mode of interaction of ligand molecule with the receptor protein by forming hydrogen bond which clearly showed the possibility that drug can neutralize common krait venom.   aimed to study the antidote activity of AB, TI, and LA against snake venom.

Discussion
From the present study, the aqueous extracts of AB, TI, and LA were found to be neutralize the venom of Russell's viper and Cobra. Aqueous extract of AB showed 80% inhibition of lethality effect caused by Russell's viper and cobra venom. LA showed 60% inhibition of venom lethality effect of cobra and 100% in case of viper venom during 48 hours of observation. For cobra venom aqueous extracts of TI showed 60% inhibition of venom lethality up to 6 hours and 60% against Russell's viper venom up to 12 hrs. From the above results it is clearly understood that TI acts as a first aid in slowing down the time interval for venom spreading in the body. The polyherbal formulation of AB, TI, and LA (1 : 1 : 1)  shows promising venom-neutralizing activity against cobra and viper venom. PLA 2 is commonly found in the venom of Viperidae, Hydrophiidae, and Elapidae family [8]. Neutralizing effect of the PLA 2 activity is the common character of herbal drug which can act as the antidote for most types of snake venom. PLA 2 type present in viper and cobra venom might release a large amount of platelet-activating factor in addition to histamine and other anaphylactic mediators from mast cells [24]. In the present study the protection of the mast cells against the degranulation effect of cobra and viper venom by the aqueous extract of AB, TI, and LA was studied. In this TI showed high phospholipase inhibition activity on cobra and viper venom by preventing the release of platelet-activating factor and histamine from mast cells followed by LA and AB.
Local necrosis and hemorrhagic lesions were shown in some snake envenomation [25]. Hemorrhaging damages the vascular endothelium which leads to bleeding from vital organs and the victim will die due to excessive blood loss [36]. The aqueous extract of AB, TI, and LA shows significant reduction in viper and cobra venom-induced hemorrhagic damage. This inhibition activity of PLA 2 , ADP-induced platelet aggregation shows promising antivenom character of the polyherbal drugs since WHO report states that antivenom Table 11: Discovery Studio docking score of Leucasin-PLA 2 binding. Docking process top ten conformations were generated for each ligand after the energy minimization method based on steepest descent method and conjugate gradient method. Each of the saved conformation was evaluated and then ranked using the dock score. Ligand pose 1 ranged as best pose with dock score and jain value.  drugs should have capacity to neutralize venom lethality and haemorrhagic effects [24].
In vitro antioxidant DPPH free radical scavenging, superoxide radical scavenging, and reducing power activity of AB, TI, and LA were studied. Antioxidant properties of the plants play important role in neutralizing the snake venom [15,21]. It was evident from our study that aqueous extract of AB, TI and LA showed dose dependent antioxidant activities. This result suggests the possible snake venom neutralizing activity due to free radical scavenging and antioxidant activity which may be due to the presence of phytoflavonoids in the plants which boost the antidote properties against snake venom. It was argued that disturbances in the second messenger and regulatory activity of free radicals may also contribute significantly to the various inflammatory processes.
It is evident from our study that aqueous extract of AB, TI, and LA showed dose-dependent antioxidant activities. This result suggests the possible snake venom neutralizing activity due to free radical scavenging, and antioxidant activity which may be due to the presence of phytoflavonoids in the plants which boost the antidote properties against snake venom.
In vitro studies showed that AB, TI and LA can induce PLA 2 of cobra and Russell's viper venom. In order to know the neutralizing effect of the plants against PLA 2 from Bungarus caeruleus venom computational docking methods were followed to screen the potent compound which may be the source of venom lethality and make a conclusion on the mode of interaction of herbal drug. The active site region for the enzyme PLA 2 type I from Bungarus caeruleus venom (PDB:1DPY) is identified based on the bound inhibitor pocket region in the crystal structure. DS package and Glide docking score along with the binding orientation and hydrogen bond interaction were considered as the primary components to choose the best ligand-receptor interaction. In our study we focus on screening the possible potent compounds present in our plants against PLA 2 from the venom of Bungarus caeruleus by computational docking study. Initially, based on several reviews we identified four different secondary metabolites from AB, TI, and LA which were designed using MedChem designer v1.0. In these four compounds Lecuasin from LA and Aristolochic acid from AB showed positive docking score for type I PLA 2 of Bungarus caeruleus venom. Characterization study of the Bungarus caeruleus venom PLA 2 structure showed that different amino acids play significant role in the formation of functionally important structure. Secondary structure of PLA 2 contains 44.4% helix, 9.4% sheet, 5.1% turn, 33.3% coil, and 7.7% 3-10 helix region. The ligand active targets were present mostly in the coil region of PLA 2 such as Arg49, His48 and Lys6. The potential ligand binding sites were examined for PLA 2 and generated the binding sites I, II and III. H33 from aristolochic acid and Lecuasin O17, O18 showed the interaction with the Asp49 and Lys6 in binding site region I of PLA 2 by means of forming hydrogen bond using Ligandfit method in DS package with the docking score 48.109 and 50.76. Glide docking parameter showed H33, O2 of aristolochic acid and H15, O4 of Lecuasin interacts with Lys31, His48, and Asp49, Gly32 in active region I of the PLA 2 with the docking score −7.051, −6.585 and −6.852, −7.966. It is reported that type I PLA 2 from snake venom catalyzes the hydrolysis of 2acyl ester bond of 3-sn-phospholipids producing fatty acids and lysophospholipids. The ca 2+ ion, an essential cofactor, and Asp49 are required for catalysis activity. These catalytic activities upon cell membrane of specific tissues suggest an important role of PLA 2 in venom toxicity and cell death [9]. Our study suggests the neutralizing activity of aristolochic acid by means of forming H-bond interaction with the Asp49 which results in the alternation of the function, leading to blocking the catalytic activity of Ca 2+ cofactor in PLA 2 and prevent the cell death. In Glide docking study showed the Hbond interaction of O20 from aristolochic acid with the His48 of PLA 2 which results in the reduction of His48 enzymatic and toxic activity. Hence His48 involved with the catalysis through its N-1 group oriented towards the solvent and made toxic effect [9]. His48 is supported by hydrogen bonds from Tyr52 to the side chain of opposite helix (Asp99) with close coupling of Asp49 and defines the active site geometry of type I PLA 2 enzyme. There is an other statement as aristolochic acid failed to neutralize the toxic properties of type II PLA 2 from russell's viper venom but in circular dichroism studies showed that binding of aristolochic acid alters the secondary structure of type II PLA 2 from russell's viper; such a structural change may lead to lesser degrees of hemolysis and edemainducing activity of this enzyme. In addition aristolochic acid is a competitive inhibitor of type I PLA 2 and showed noncompetitive inhibition in the case of type II PLA 2 from russell's viper venom [37,38]. Type II PLA 2 venom enzyme differs from type I PLA 2 by having extended C-terminal tail [39]. This leads to differance in their pharmacological sites of the enzyme.
In case of leucasin, it directly binds with PLA 2 by means of forming H-bond between O18 and O17 of leucasin with Lys6 residues of PLA 2 . Lys6 residues are found in PLA 2 of all Elapidae venom. It is reported that Lys6 plays an indirect role in the Ca 2+ cofactor for catalyst activity of the PLA 2 in snake venom by means of intermolecular H-bond with Ca 2+ catalytic active site residues [39]. Modification in the Lys6 might distort the binding ability of PLA 2 for substrate and cause a drastic loss in enzymatic activity [40]. Leucasin atom O18 and atom O17 formed H-bond with the Lys6 which block the Ca 2+ catalytic activity of His48 and Asp49, Since there is intermolecular H-bond between the Lys6, His48, Asp49, Lys2, Trp19 and Tyr22 which connects the active site residues to the N-terminal -helix segment which promotes the Ca 2+ catalytic activity. Our computational study shows that the screened potent secondary metabolites can also neutralize type I PLA 2 enzyme from Bungarus caeruleus venom and these finding suggest that our selected plant can act as the source of common antidote for snake venom.

Conclusion
Present crisis of dependable and safe antivenom supply in the developing countries reflects a global loss of momentum in anti-venom research, development, and financing. But the outcome of the present study is encouraging for research on antivenom from natural resources, where aqueous extracts of Aristolochia bracteolata, Tylophora indica, and Leucas aspera exhibited neutralizing effects against Russell's viper and Cobra venom. Further study on isolation of active phytoconstituents and their antisnake venom property could lead to the development of a new natural antidote for snake envenoming.