Quercetin-3-O-rhamnoside from Euphorbia hirta protects against snake
Venom induced toxicity

Kadiyala Gopi, K. Anbarasu, Kadali Renu, S. Jayanthi, B.S. Vishwanath,

Gurunathan Jayaraman

PII: S0304-4165(16)30086-1
DOI: doi: 10.1016/j.bbagen.2016.03.031
Reference: BBAGEN 28434

To appear in: BBA - General Subjects

Received date: 28 November 2015

Revised date: 14 March 2016
Accepted date: 23 March 2016

Please cite this article as: Kadiyala Gopi, K. Anbarasu, Kadali Renu, S. Jayanthi,
B.S. Vishwanath, Gurunathan Jayaraman, Quercetin-3-O-rhamnoside from Euphorbia
hirta protects against snake Venom induced toxicity, BBA - General Subjects (2016), doi:
10.1016/j.bbagen.2016.03.031

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 ACCEPTED MANUSCRIPT

Quercetin-3-O-rhamnoside from Euphorbia hirta Protects against Snake Venom Induced

Toxicity

Kadiyala Gopi1, Anbarasu K1, Kadali Renu1, Jayanthi S1, Vishwanath B.S2 and Gurunathan
Jayaraman1*

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1
School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu 632 014, India

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2
Department of Biochemistry, University of Mysore, Mysuru, Karnataka, India

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Address for Correspondence

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Dr. G. Jayaraman MA
Professor,

School of Bio Sciences and Technology,

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VIT University, Vellore, Tamil Nadu-632014, India.

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Email: gjayaraman@vit.ac.in
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Phone: + 91 416 2202573

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Abstract

Background: The plant Euphorbia hirta is widely used against snake envenomations in rural
areas and it was proved to be effective in animal models. Therefore, the scientific validation of
its phytoconstituents for their antiophidian activity is aimed in the present study.

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Methods: E. hirta extract was subjected to bioactivity guided fractionation and the fractions that

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inhibited different enzyme activities of Naja naja venom in vitro was structurally characterized

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using UV, FT-IR, LC-MS and NMR spectroscopy. Edema, hemorrhage and lethality inhibition
activity of the compound was studied in mice model. In addition, molecular docking and
molecular dynamic simulations were also performed in silico.

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Results: The bioactive fraction was identified as Quercetin-3-O--rhamnoside (QR, 448.38 Da).
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In vitro experiments indicated that protease, phospholipase-A2, hemolytic activity and
hemorrhage inducing activity of the venom were inhibited completely at a ratio of 1:20 (venom:
QR) w/w. At the same concentration, the edema ratio was drastically reduced from 187% to
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107%. Significant inhibition (93%) of hyaluronidase activity was also observed at a slightly
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higher concentration of QR (1:50). Further, in in vivo analysis, QR significantly prolonged the

survival time of mice injected with snake venom.
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Conclusion: For the first time Quercetin-3-O--rhamnoside, isolated from Euphorbia hirta, has
been shown to exhibit anti-snake venom activity against Naja naja venom induced toxicity.
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General Significance: Exploring such multifunctional lead molecules with anti-venom activity
would help in developing complementary medicine for snakebite treatments especially in rural
areas where anti-snake venom is not readily available.

Keywords: Euphorbia hirta, Snake venom, Naja naja, Quercetin-3-O-rhamnoside, PLA2

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1. Introduction
Snake envenomation is an imperative public health problem in many rural areas of tropical and
sub-tropical Asian countries. Globally, it is estimated that 1.84 million snake envenomations

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occur every year of which 94,000 lead to death of the victim [1]. Unfortunately, India records the
highest number of deaths (35,000-50,000) due to snakebite [2, 3]. The major causative species

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are from Elapidae and Viperidae families [4]. Of these, cobras and kraits of Elapidae family
cause maximum envenomations [5]. Envenomation by these snakes is associated with severe

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tissue damage, local necrosis, hemorrhage and edema at the bite site [6].

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Pathology induced by envenomation is due to the collective effect of myotoxic phospholipases
A2, hemorrhagic metalloproteinases, neurotoxins, hyaluronidases and cytotoxins [7]. Although,
snake anti-venom (IgG) is generally accepted for effective treatment of snakebites, they are
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inaccessible to millions of rural people in developing countries [8], moderately effective in
neutralizing local tissue damage [9] and also induce undesirable effects like pyrogenic or
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anaphylactic reactions [10]. This intricate scenario demands extensive search for alternative
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medicines of either natural or synthetic origin that could inhibit the action of snake venom
enzymes [11, 12].
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In general, medicinal plants are the ample source of therapeutically active molecules and are
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proven to be effective against several pathophysiological disorders including snakebite.

Phytochemicals isolated from different plant species are reported to reduce edema, inflammation,
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myotoxicity, neurotoxicity and hemorrhage induced by snake venoms [13, 14]. Mors et al.,[15]
reported that the plant secondary metabolites such as flavonoids, terpenoids, steroids, saponins,
phenolics, vegetable tannins, pterocarpans, coumastans and polysaccharides possess anti-venom
activity. Although, many plant species or phytochemicals have been pharmacologically
investigated for anti-venom activity, a large number of traditionally used medicinal plants in
snakebite treatment are yet to be evaluated [16].

E. hirta belongs to the category of plants that is being used traditionally to treat various disorders
[17, 18 & 19]. In the present study, a survey in the rural places of Vellore district, Tamil Nadu,
India revealed that E. hirta is one of the most widely used herbs to treat snake envenomations.
The decoction of the whole plant extract is administered orally and applied topically to treat
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poisonous snakebites [20, 21]. In our laboratory we evaluated anti-venom activity of E. hirta, in
vitro as well as in vivo. The results strongly supported its usage in snakebite treatment [22].
Therefore in order to obtain further insight into the anti-ophidian activity of E. hirta extract,
bioactivity guided fractionation was performed to identify the phytochemical(s) which has the

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potential to inhibit/decrease the snake venom induced toxicity.

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2. Materials and Methods

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2.1. Venom and chemicals

Lyophilized snake venom (N. naja) was purchased from Irula snake catchers Industrial

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Cooperative Society Limited, Chennai (India). Venom was dissolved in 10 mM PBS and
centrifuged at 2500 rpm for 10 min and supernatant was used for the study. Hyaluronic acid was
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purchased from Sigma Aldrich (USA). All other reagents and solvents were of high quality
analytical grade and were purchased from SD Fine chemicals/Sisco Research Laboratory
Pvt Ltd/ HiMedia.
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2.2.Animals

Male Swiss-albino mice, weighing 25-30 g were used in the study. All animals were maintained
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at the University animal house, under standard conditions and diet. Experiments were
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carried out with prior permission from Institutional Animal Ethical Committee, VIT University,
Vellore (Ethical clearance no.VIT-IAEC-V-5).
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2.3.Plant collection

The plant was collected from Irrularpatti village, Gudiyatham taluk of Vellore district, Tamil
Nadu, India and authenticated by Dr. K. Madhava Chetty, (Department of Botany, Sri
Venkateswara University, Tirupati, Andhra Pradesh, India) and the voucher specimen was
deposited in the Herbarium of the same department under the number SVUTY-EU/1067.

2.4. Preparation of plant extract

Five hundred grams of shade dried and powdered whole plant material was immersed in
methanol at a ratio of 1:2 (w/v) for 24 h with continuous stirring (at room temperature). The

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extraction was repeated for 3 days by changing the solvent every 24 h and the filtrate was
concentrated using rotary vacuum evaporator under reduced pressure. The dried extract was
stored at 4 C.

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2.5. Isolation and purification of bioactive compound

2 g of air dried methanol extract of E. hirta was dissolved in 2 mL of distilled water and

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centrifuged at 6000 rpm for 10 minutes. The supernatant was loaded on to the Sephadex LH-20

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column (column height 50 cm, bed height 45 cm, column inner dia 1 cm, bed volume 35 mL)
and sequentially eluted (flow rate is 1 mL/min) with milli-Q water and methanol (50, 70 and

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100%). 5 mL fractions were collected and the homogeneity of the fractions was analyzed by
Thin layer chromatography (TLC) on Silica gel 60 F254 plates using butanol: acetic acid: water
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(70:15:15 v/v) as the developing solvent. Fractions were screened for inhibition activity of Naja
naja venom enzymes. Fractions that inhibited venom protease and phospholipase were further
fractionated using Sephadex LH-20 column, as mentioned previously. Subsequently, semi-
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preparative thin layer chromatography was performed (with 4:1 chloroform/methanol as mobile
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phase) to obtain pure compound that exhibited enzyme inhibition. In order to infer the class of
molecule that inhibited specific venom enzymes, phytochemical assay was performed using
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appropriate spray reagents [23].

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2.6. LC-MS, UV and FT-IR analysis

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The phytochemical that inhibited the enzyme activity was mixed with KBr (FT-IR grade) in a
ratio of 5:95 and FT-IR analysis was performed in the mid-IR region (4004000 cm1)
with a scan speed of 16 (cm-1/min) using Thermo Nicolet AVATAR 330 spectrometer.. UV-Vis
spectrum of the compound was recorded using Hitachi U-2800 spectrophotometer. 20 L of
compound (in methanol) was injected into the LC. A mobile phase of methanol: water (40:60), at
a flow rate of 0.2 mL min1 for 60 min, was used for the separation on a BDS HYPERSIL C-18
(4.6 250 mm, 50 m) HPLC column. Mass spectra were obtained using ESI-Mass
Spectrometer equipped with an electrospray ionization source, in positive ion mode under the
flow of Helium gas at 1 mL min1(approx).

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2.7. Structural characterization using NMR spectroscopy

One-dimensional (1H, 13
C, DEPT 13
C) and two-dimensional (COSY, TOCSY, HMBC and
HSQC) NMR spectroscopy experiments were performed for the phytochemical (20 mg in 600
L CD3OD) that inhibited selected snake venom enzymes, using a 5 mm inverse probe. Spectra

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were acquired on Bruker ASCEND 400-MHz spectrometer at 25C and were referenced with

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respect to sodium-3-(trimethylsilyl) tetradeuterio propionate (TSP). Spectra were processed and
analyzed using TOPSPIN and ACDLABS 12.0. COSY spectrum was acquired by collecting

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(1024, 128) data points and 4 transients with a sweep width of (3286.26 Hz, 3286.26 Hz) and
acquisition time of (0.31 s, 0.03 s) along t1 and t2 dimensions. TOCSY spectrum was acquired by

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collecting (1024, 256) data points with a sweep width of (3286.26 Hz, 3286.26 Hz), 8 transients
and acquisition time of (0.31 s, 0.07 s) along t1 and t2 dimensions. HMBC spectrum was acquired
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with (1024, 128) data points, acquisition time of (0.31 s, 0.01 s), sweep width of (3245.17 Hz,
22333.15 Hz) and 12 transients, whereas HSQC spectrum was acquired with (512, 256) data
points, (0.16 s, 0.02 s) acquisition time, (3286.26 Hz, 16656.80 Hz) sweep width and 8
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transients.
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2.8. Caseinolytic inhibition by SDS-PAGE

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2 g of N. naja venom was pre-incubated with various concentrations of the phytochemical for 1
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h at 37C. These samples were further incubated with 10 l of 1% casein (in 0.1M Tris HCl
(w/v) buffer, pH 8.0, 37C). After 30 minutes, the reaction was stopped by adding SDS loading
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buffer (containing 50 mM Tris HCl pH 6.8, 10% v/v glycerol, 2% w/v SDS, 100 mM -
mercaptoethanol and 0.1% w/v bromophenol blue). SDS-PAGE was performed according to
Laemmli, [24] using 15% acrylamide gel. In order to quantitate the venom caseinolytic activity,
the gel was post analyzed using myImage analysis software (Thermoscientific;
http://www.piercenet.com/product/myimageanalysis-software). The pixels of the casein band in
the absence of venom, served as control.

2.9. Phospholipase (PLA2) inhibition

2 g of N. naja venom was pre-incubated with various concentrations of the phytochemical for 1
h at 37C. PLA2 activity was then evaluated using egg yolk as substrate in 1% agarose plates

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[25]. The pre-incubated samples were then loaded into 3 mm diameter wells of 1% agarose
plates containing 0.6% egg yolk and 5 mM CaCl2 followed by overnight incubation at 37C. The
PLA2 inhibition was calculated by measuring the zone of clearance in the presence and absence
of the active molecule. PLA2 activity of venom in the absence of the active molecule served as

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control.

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2.10. Hemolytic inhibition

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2 g of N. naja venom was pre-incubated with various concentrations of the active molecule for
1 h at 37C.Hemolytic inhibition was determined as per the method of Boman and Kaletta [26],

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with slight modifications. Briefly, 100 l of human erythrocyte suspension (in 10 mM PBS, pH
7.4) was added to pre-incubated venom samples for 2 h at 37C. The reaction was stopped by
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adding 100 l of ice cold PBS and centrifuged at 2500 rpm (10 min at 4C). The amount of
hemoglobin released was estimated spectrophotometrically at 490 nm using microtitre plate
reader.
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2.11. Hyaluronidase inhibition

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The quantitative estimation of hyaluronidase activity was performed using ELISA-like assay as
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described by Tanaka et al., [27], with slight modifications. Briefly, samples of N. naja venom (2
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g) were pre-incubated with various concentrations of the phytochemical (total volume of 100
L) for 1 h at 37C, followed by addition of 10L of the hyaluronic acid (1 mg/mL in 0.1 M
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sodium acetate buffer containing 0.15 M NaCl, pH 5.8) and incubated for 30 min at 37C. The
reaction was then stopped by the addition of 2.5% cetyl trimethyl ammonium bromide (100 L)
in 2% NaOH and the absorbance was measured at 630 nm using a microtitre plate reader.

2.12. Neutralization of snake venom lethality

In order to evaluate the anti-snake venom activity of the active compound, 28 mice of male
gender were divided into VII groups of 4 in each. All the mice in group-I were injected (i.p) with
2 LD50 of venom in a constant volume of 0.2 ml saline. To determine the venom neutralization
effect, different concentrations of active compound was pre-incubated with twice the LD50 of
venom (w/w) for 1 h at 37C (in a constant volume of 0.2 ml saline) and then the mixture was
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injected intra-peritoneally (i.p) to mice of different groups. Group of mice injected with only
saline or phytochemical served as controls. Mice of all the groups were constantly monitored for
toxic symptoms, signs of neurotoxicity, behavioural changes and the survival time of all the
animals were recorded for 24 h.

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2.13. Histopathological analysis

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The major vital organs such as heart, liver, kidney and lungs were excised out from the animals

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of all the groups. All the organs were then stored in 10% formalin at room temperature before
processing the tissue. All the sections were stained with haematoxylin and eosin. The slides were

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then observed for pathological changes using high power light microscope (Olympus, Tokyo,
Japan). MA
2.14. Inhibition of edema inducing activity

Studies on edema inducing activity were carried out according to the method of Vishwanath et al
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[28]. Briefly, groups of three mice were injected (20 l) with different concentrations of venom
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in the right footpads. The left footpads that were injected with PBS served as control. After 45
min, both the legs were excised at ankle joint and the increase in weight was used to calculate the
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edema ratio using the formula: (Weight of edematous leg/ Weight of control leg) 100. For
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inhibition studies, 5 g of venom was pre-incubated with different concentrations (w/w) of the
active molecule (total volume of 20 L) for 1 h at 37C and injected in right footpads for
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determining edema inducing activity of venom in presence and absence of plant extract. Mice
injected with the plant isolate and venom alone was served as controls.

2.15. Inhibition of hemorrhagic activity

Hemorrhagic activity was assayed according to the method described by Nikai et al., [29] with
slight modifications. Swiss albino mice were injected intradermal, in the back, with different
concentrations of venom in 50 L of PBS. After 3 h, mice were sacrificed and the inner surface
of the skin was examined for hemorrhage. Venom samples pre-incubated with different
concentrations of the active molecule (50 l of PBS for 1h at 37C) were used to determine the
inhibition of hemorrhage. The mice injected with PBS served as control.

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2.16. Molecular Docking

The software Vina [30] in PyMOL plugin was used to study the molecular interactions between
phospholipase A2 [PDB code: 1A3F] enzyme of Naja naja venom and Quercetin-3-O-

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rhamnoside (QR) or Quercetin (Qn). Both protein and ligands were saved in PDB format. The
volume of the box was fixed to 27000 . The grid used for molecular docking of centre was x =

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49.68, y =10.82, z =13.3, size 60 x 60 x 60 and spacing 0.375 to target the active binding
cavity of the protein. The parameter exhaustiveness was set to 8 and docking run was set to 100

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conformations. The scoring function was represented by binding affinity in kcal/mol. PyMOL
was used to visualize the docking interactions and Ligplot+ [31] was also used to evaluate both

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hydrogen bond and hydrophobic interactions.
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2.17. Molecular dynamics (MD) simulations

MD simulations (Gromacs version 4.5.4) were performed [32] to understand the dynamic
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behavior of Naja naja venom phospholipase A2 in the ligand (QR/Qn) bound state. GROMOS96
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43a1 force field [33] was used in energy minimization of a protein model with steepest-descent
algorithm. After topology generation, the QR/Qn-PLA2 complexes were solvated in cubic box
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(size of 1.0 nm) with SPC216 water model under periodic boundary conditions. Neutralization of
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the system was achieved by adding six chlorine ions. Van der Waals interactions were calculated
with 14 cut-off and electrostatic interactions were calculated with 12 cut-off. Energy
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minimization of the complex was performed and possible steric clashes were removed with two
phases namely NVT (constant composition, volume, and temperature) and NPT (constant
composition, pressure, and temperature). Each complex was minimized with a constant
temperature of 300 K with a coupling constant of 0.1 ps for duration of 100 ps using NVT
ensemble. After NVT, minimization with constant pressure of 1 bar was employed with a
coupling constant of 5 ps for duration of 100 ps using NPT ensemble. Berendsen coupling
scheme was applied for equilibration ensembles. After equilibration of the system with desired
temperature and pressure, the final production MD run of 25 ns was performed. MD simulations
with additional step of ligand topology of compound were generated using PRODRG server [34].
The trajectory files of both protein and protein/ligand complexes were stored at one pico second

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interval and analyzed using Gromacs utilities. g_rms was used for analyzing the structural
deviation through RMSD plot. g_hbond was used for evaluating the inter-hydrogen bond
interactions between two groups by NH plot.

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2.18. Statistical analysis

All the experiments were carried out in duplicates with proper controls as specified. The values

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are expressed as mean S.D. The data was plotted using the GraphPad Prism 5.0 and the

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analysis was performed with one way analysis of variance (ANOVA). Results were considered
statistically significant if P<0.05.

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3. Results and Discussion MA
For the first time, in our laboratory we evaluated anti-venom activity of E. hirta, in vitro as well
as in vivo and results strongly supported its usage in snakebite treatment [22]. Consequently, the
present study was aimed at identifying the phytochemical(s) from E. hirta L. that could inhibit
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Naja naja venom induced toxicity.

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3.1. Purification of active constituent

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The methanol extract of E. hirta was subjected to successive bioassay-guided fractionation and
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each fraction was assayed for protease and phospholipase inhibition activity. Semi quantitative
thin layer chromatography of the active fractions indicated the presence of three different
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fractions (F11C-1 to F11C-3). The major fraction F11C-2 (27 mg), colored yellow, showed
substantial inhibition of these venom proteins and HPLC analysis revealed that the fraction
contained only one phytochemical. Phytochemical analysis revealed that this compound could be
a flavonoid derivative. Flavonoids are widely used in therapeutics for their antioxidant, anti-
inflammatory, wound healing and anti-fungal effects.

3.2.Structural characterization of the isolated flavonoid

Information on identity of the molecule was obtained from LC-MS, UV and FT-IR spectroscopic
techniques. The LC-MS chromatogram revealed a single peak with a retention time of 10.39 min
[Figure 1A]. Mass spectrum presented a major peak at m/z 471.26, consistent with the theoretical
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molecular mass of the sodium ion adduct [C21H20O11Na+] of the compound [M+Na]+ [Figure
1B]. The UV spectrum indicated three peaks at 352, 263 and 213 nm which are characteristic
[35] of B, C and A rings of the flavones [Figure 1C]. Presence of carboxyl pyrone ring and
phenolic OH groups were characteristic of the vibrational frequencies at 1651 and 3441 cm

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respectively in the FT-IR spectrum [Figure 1D]. Further, the asymmetric C=CH stretch and
C=CC of the flavonoid was evident from by the transmittance at 3238 and 1402 cm1,

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respectively. Peaks between 2850 and 2950 cm1 were attributed to the CH stretching of the

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sugar while peaks at 1089 and 1060 cm1 indicated the glycosidic CO stretching [36].

The isolated flavonoid was further characterized by suitable NMR experiments. The 1D-1H

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spectrum presented signals in the region between 3.0 4.0 ppm and 6.0 - 7.5 ppm, suggesting the
presence of sugar and aromatic residues, respectively (Supplementary Figure S1). The 1H peaks
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in the aromatic region at 6.19 ppm (J=1.38 Hz) and 6.35 ppm (J=1.63 Hz) indicated the presence
of meta coupled protons of the aromatic ring. Moreover, the presence of aromatic peaks at 7.35
ppm (J=1.76 Hz), 6.93 ppm (J=8.28 Hz), 7.32 ppm (J=8.28, 1.88 Hz) indicated the presence of
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another aromatic ring with substitutions at the 1, 3, 4 positions. The observed resonances were
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consistent with the characteristics of a flavone moiety, quercetin [37]. Further, the spectrum
showed a doublet at 5.3 ppm indicative of an anomeric proton with a coupling constant of J= 1.2
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Hz suggesting -orientation of the proton in sugar moiety. A doublet signal at 0.96 ppm (d,
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J=6.15 Hz) suggested the presence of methyl group. The chemical shifts along with the J-
coupling values were consistent with that of -rhamnose [38]. The DEPT-135 analysis presented
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10 peaks of CH groups and one CH3 (17.67 ppm) group in the spectrum. Resonance signal at
179.46 ppm in the 13C spectrum indicated the presence of C=O group. The presence of quercetin
and rhamnose ring was confirmed by the observed spin systems in the homonuclear 2D-COSY
[Figure 1E] and TOCSY [Figure 1F]. The carbon resonances were identified and confirmed
using the HSQC spectrum [Figure 1G]. Confirmation on the glycosidic linkage was derived from
the presence of cross peak between the C1 protons of the rhamnose ring (5.36 ppm) with the C3
carbon of the flavone (136.08 ppm) in HMBC spectrum [Figure 1H]. The chemical shifts of 1H
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and C derived from the homonuclear as well as heteronuclear NMR experiments are given in
Table 1. Based on the above spectral evidences, the isolated active flavonoid glycoside was
identified to be quercetin-3-O--rhamnoside (C21H20O11) [Figure 2].
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3.3. Protease inhibition activity

Snake venom contains serine proteases as well as metallo proteases. These enzymes affect the
hemostatic system apart from degrading the extra cellular matrix [39]. These proteases could

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either prolong (anticoagulants) or shorten (pro-coagulants) the blood clotting process upon
envenomation [40]. They are mostly found in Viperidae and Elapidae snake families. Therefore,

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in order to estimate the inhibitory effect of the Quercetin-3-O-rhamnoside (QR), the venom
caseinolytic activity was assayed using SDS-PAGE and the band intensities were quantified by

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densitometric analysis [Figure 3A]. In the presence of venom, degradation of casein was evident
from the decrease in the native band intensity by 63%. However, in the presence of QR, the

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changes in the band intensities were dose dependent. Even at 1:5 ratio (venom: QR) the
proteolytic activity was found to be substantially inhibited (28%) and complete inhibition of
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casein hydrolysis was observed at a ratio of 1:20. Snake venom metalloproteases act upon a
variety of substrates such as plasma proteins, membrane proteins, endothelial cells, proteins
involved in platelet aggregation, inflammatory response cells and causes severe hemorrhage at
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the site of envenomation [41]. Therefore, in order to obtain further confirmation on the
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proteolytic inhibition, changes in the hemorrhage caused by the venom were assessed in the
presence and absence of QR (Figure 3B). The minimum amount of venom that produced
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hemorrhage in mice was found to be 10 g (Gopi and Jayaraman, unpublished results). Co-
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injection of venom along with QR significantly reduced the hemorrhage suggesting the inhibition
of venom proteases by QR. Though significant inhibition was observed at 1:5, complete
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inhibition was seen at higher concentrations of QR (1:20). Both SDS-PAGE and hemorrhage
analysis, revealed that QR was able to inhibit the proteolytic activity in vitro Phospholipase
(PLA2) inhibition

Phospholipase A2 is one of the major enzymes present in snake venom that hydrolyses
membrane phospholipids at sn-2-position [42], releasing lysophospholipds and arachidonic acid
which lead to production of various pro-inflammatory mediators [43]. PLA2 induces
cardiotoxicity, myotoxicity, pre or post synaptic neurotoxicity, edema, inflammation, hemolysis
and hypotension [44, 45]. Thus, inhibition of PLA2 is a key point in the treatment of snake
envenomations. Lattig et al., [46] reported decreased eicosanoid levels and reduced inflammatory

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response upon PLA2 inhibition. In the present study, it could be observed from Fig. 3C that at a
ratio of 1:05, 1:10 and 1:15 w/w (venom: QR), 18, 32 and 40% of PLA2 activity was inhibited
while complete inhibition was achieved at 1:20 ratio [Figure 3C]. Similar inhibition was reported
by Cotrim et al., [47], wherein PLA2 activity of Crotalus durissus venom was inhibited by

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quercetin. Interestingly, the authors report that the inhibition did not reduce the inflammatory
activity of the enzyme. Recent study by Toyama et al., [48] demonstrated that quercetin

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rhamnoside was able to inhibit biochemical and pharmacological activities of PLA2 from

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Crotalus durissus venom more efficiently in comparison to quercetin alone. Molecular docking
studies suggested that quercetin glycoside interacts with Gly30, Gly32, His48 and Asp49

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residues present at the substrate binding site of PLA2, thus leading to complete inhibition of the
enzyme activity [48]. In the present study QR significantly reduced the edema inducing activity
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of PLA2 [Figure 3D]. 2 g of venom produced edema ratio of 184%. At a ratio of 1:05 and 1:10
of venom: QR (w/w), the edema ratio decreased to 161 and 137% (greater than the accepted
level of 120%), respectively. Further increase in QR concentration (1:20 ratio), reduced the
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edema to 107%, suggesting the inhibition of inflammatory activity caused by the snake venom
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enzymes. Analysis of the three dimensional structure of different PLA2s (PDB ID: 1A3D and
3MLM) revealed variations in the amino acid residues present at the substrate binding site of an
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enzyme (Gopi and Jayaraman, unpublished results). Such differences in the amino acids would
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be critical in deciding the multi-inhibition effects of the inhibitor molecules. Additionally,

several studies revealed that the rhamnose at C3 position of quercetin, enhances the
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bioavailability of the inhibitor and therefore is considered to be more effective compared to

quercetin alone [49, 50].

3.4. Hyaluronidase inhibition activity

Hyaluronidases play a crucial role in the diffusion of systemic toxins into circulation from the
site of envenomation by fragmenting the hyaluronic acid present in extracellular matrix and
create severe morbidity [51]. They degrade connective tissues surrounding the blood vessel,
capillaries, and smooth muscles resulting in loss of structural integrity and enhance the tissue
permeability [52]. Therefore, phytochemicals with hyaluronidase inhibitory activity are deemed
to be important in the treatment of snake envenomations. In the present study, QR inhibited the

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hyaluronidase activity of Naja naja venom significantly. At a ratio of 1:05 and 1:10 w/w, the
enzyme activity was reduced by 43 and 55%, respectively. Further increase in QR concentration
decreased the hyaluroidase activity in a dose dependent manner with maximum inhibition (93%)
observed at a ratio of 1:50 (Figure 3E). Similar results were reported by Girish and Kemparaju

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[53], using quercetin against hyaluronidase of Naja naja venom. Several other studies
demonstrated the hyaluronidase inhibition by proteins, polyphenols, terpenoids, flavonoids [for

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review see, Santhosh et al., [13]. However, for the first time the present study reports inhibition

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of hyaluronidase activity by QR where glycosidic compounds have greater therapeutic
importance over aglycon compounds that might be due to their higher solubility in aqueous

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media.

3.5. Inhibition of hemolytic activity MA

Apart from metalloproteases and phospholipases, Elapidae snake venoms are rich sources of
cardiotoxins, cytotoxins, hemotoxins, myotoxins that are mostly involved in hemolysis and
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cytolysis [54]. Hemolysis is a distinct feature of cobra venoms induced by multiple components.
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Previous reports revealed that, phospholipases hydrolyze the intact phospholipids present on
erythrocyte membrane and causes hemolysis [55]. In the present study, at a ratio of 1:2 (venom:
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QR) the hemolytic activity was reduced by 56% and complete reduction was observed at a ratio
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of 1:20 thus representing the strong potential of QR against venom toxicity [Figure 3F]. It is
interesting to observe that a single phytochemical was able to inhibit multiple enzymes of the
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snake venom. However, literature indicates that there exist phytochemicals (Supplementary
Table 1) that could elicit multiple inhibition effects on venom induced toxicity [56-61].

3.6. Quercetin-3-O-rhamnoside prolongs the survival time

The anti-venom potential of QR against Naja naja snake venom induced lethality was evaluated
in mice model. Twice the LD50 of N. naja venom was taken as the challenging dose to evaluate
the anti-venom activity of QR. In the present study, QR significantly prolonged the survival time
of mice in a dose dependent manner [Figure 4A]. However, the compound could not completely
inhibit the venom induced lethality. Mice injected with venom alone was able to survive for only
2.20 h, while mice injected with venom in presence of QR the survival time was slightly

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increased by 17 and 95 minutes at 1:10 and 1:20 (venom: QR w/w) respectively. Further increase
in QR concentration resulted with significant increase in mice survival time by 170 and 302
minutes at 1:40 and 1:80 ratios respectively.

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Though QR can inhibit multiple enzymes of venom in vitro, at any tested concentration, it could
not save the mice from venom induced lethality, rather it facilitated to prolong the survival time

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of mice injected with lethal dose of N. naja venom. It is possible that QR could bind with venom
enzymes but upon injection of this mixture to mice, the enzyme-ligand binding might have been

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reversible thereby enzymes become active resulting in prolonged death time of mice. It is also
possible that the concentration of QR may not be sufficient to completely in vivo conditions.

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Snake venom lethality is majorly caused by cumulative action of several toxins or enzymes
present in crude venom. Cobra venom is rich in phospholipase-A2 and neurotoxins that cause
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neurotoxicity, haemorrhage and myotoxicity. It is expected that, though the plant compound had
shown significant inhibition of major enzymes activity of snake venom in vitro, one single
compound might not have been sufficient to neutralize venom toxicity in in vivo as evidenced by
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survival time of each mice. Apparently, it is understood that crude plant extracts would have
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more number of bioactive components which strongly bind to different toxins present in snake
venom thereby effectively neutralize venom induced toxicity and lethality in vivo which was
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previously evidenced by complete inhibition of venom induced lethality by phenolic rich E. hirta
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crude extract [22], unlike QR. The toxic symptoms and behavioral changes in mice observed
during the experimental period were recorded and tabulated [Table 2]. In the mice injected with
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only venom, apathy, limb paresis and eye closure was primarily observed followed by thoracic
breathing just before animal died. From this it could be understood that animal death is majorly
due to respiratory arrest caused by venom components. Similar symptoms were previously
observed in mice injected intra-peritonially with weak neurotoxin of Naja kouthia and the mice
hid in the corner and became tousled. In the present study, though similar toxic signs were
observed in the mice co-injected with venom and quercetin-3-O-rhamnoside, the symptoms were
observed to be delayed due to partial inhibition of venom components in vivo by the plant
isolate.

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3.7. Histopathological observations

PLA2 of Bothrops insularis venom significantly damaged the renal tubules and glomeruli in the
kidney. These structural and morphological changes could be because of cytotoxic potential of

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PLA2. Intra aurticular injection of PLA2 isolated from Naja naja atra, resulted in acute
inflammation in synovial tissue of rats which was evidenced by hyperplasia, cellular infiltration

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[62]. In the present study, histopathological analysis of the vital organs such as heart, kidney,
liver and lungs of mice injected with N. naja venom in the presence and absence of the QR was

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performed. Findings showed severe damage in all the vital organs of mice injected with only
venom while mice injected with venom in presence of QR at different concentrations [Figure 4B]

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showed remarkable reduction in venom induced systemic toxicity. PLA2 being the major
multifunctional component of snake venoms, the severity and structural damages identified in
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mice vital organs could have been majorly contributed by this enzyme. A summary of tissue
specific histopathological observations are presented in Table 3.
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3.8. Inhibition of PLA2 activity by Quercetin/Quercetin-3-O-rhamnoside

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QR had shown dose dependent inhibition on venom phospaholipase-A2. At a concentration of

200 M it was able to reduce the enzyme activity upto 96%. However, at the same concentration
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Qn was able to reduce the enzyme activity only to 42% [Figure 5B]. Similar results were
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obtained by Cotrim et al [47] where QR was more potent than Qn to inhibit PLA2 activity of
Crotalus durissus terrificus venom. Such increased potential of quercetin glycoside against PLA2
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activity could be because of the sugar moiety. Thus, the role of sugar moiety in molecular
interactions between the QR/Qn- and enzyme were also investigated by in silico analysis.

3.9. Molecular docking

Molecular docking was performed to evaluate the inhibition mechanism of Quercetin (Qn) and
Quercetin-3-O-rhamnoside (QR) against Naja naja venom PLA2. Forming two hydrogen bonds
with His47 residue present in the catalytic site of an enzyme, Qn-PLA2 complex showed least
binding energy of -7.9 kcal/mol. In case of QR-PLA2 complex the least binding energy was
found to be -8.2 kcal/mol. This shift in binding energy could be due to increase in hydrogen bond
strength between QR and PLA2. The QR-PLA2 complex was found with four hydrogen bonds
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involving Tyr63, Cys44, Tyr27 and Asp48 residues in the enzyme [Table 4]. Interestingly, all
these residues interacted specifically with the rhamnose sugar moiety present in the QR. In vitro
assay revealed that rhamnose per se cannot inhibit the activity of PLA2 (data not shown). This
was further validated through molecular docking analysis. In the absence of the aromatic moiety

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(quercetin), the sugar interacts only with the surface amino acid residues of PLA2 and therefore it
might be easily displaced even in the presence of lower concentrations of the substrate.

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[Supplementary Figure S3]. In case of Qn-PLA2 complex, among the top twenty clusters, the

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binding pattern with His47 hydrogen bond was observed in ten clusters. Totally, ten residues
(Trp18, Ala22, Phe21, Phe100, His47, Cys44, Tyr51, Asp48, Gly29, Tyr63) were involved in

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hydrophobic interactions [Figure 5E]. In case of QR-PLA2 complex, four H-bond interactions
were observed with residues Tyr27, Cys44, Asp48 and Tyr63 [Figure 5F].
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3.5 Molecular dynamics simulations

In addition to molecular docking, molecular dynamics simulations studies were carried out for
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apo PLA2 and PLA2-ligand complexes to refine the binding affinity and stability in dynamic
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system. MD simulation is a more effective method near to experimental methods that provide
more structural insights on protein-ligand complex. MD simulation studies include critical
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concept of the receptor flexibility which is not implemented in docking module. After the
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production MD runs of 25 ns simulations, the docked complex of two ligands (QR, Qn) were
analyzed using RMSD and inter-hydrogen bond interactions.
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From RMSD plot based on protein backbone atoms, the apo PLA2 showed proper convergence
till 25ns and RMSD average near to 0.15 nm showed the stable conformation of protein. In case
of PLA2/quercetin complex, RMSD plot showed proper convergence pattern near to apo PLA2
with less deviation with average near to 0.18 nm. In case of PLA2/quercetin-3-O-rhamnoside
complex, RMSD plot showed better convergence pattern near to apo PLA2 till 13 ns and after
that 14-22 ns deviation was observed due to the ligand displacement during the binding, RMSD
average near to 25 nm [Figure 5G]. Comparison of RMSD indicated the less deviation in the
protein even after ligand binding.

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The inter-hydrogen bond interactions between protein and ligand are important in the simulations
study as evaluated by NH analysis. NH plot of PLA2/quercetin complex revealed one to two
hydrogen bond interactions throughout 25 ns simulation [Figure 6A]. However, the interaction
pattern was stable with two hydrogen bonds. Moreover, NH analysis confirmed the strong

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binding of PLA2 to quercetin even in the dynamic system which corroborates docking results. In
case of PLA2/quercetin-3-O-rhamnoside complex three to four hydrogen bonds were found

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throughout the 25 ns simulation [Figure 6B]. The inter-hydrogen bond interactions pattern

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suggested the plausible mode of strong binding of ligands with PLA2 that favored the inhibition.

4. Conclusion

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For the first time, the present study demonstrated that Quercetin-3-O-rhamnoside isolated from
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methanolic extract of E. hirta inhibited protease, PLA2, hyaluronidase and hemolytic activity of
Naja naja venom. Increase in survival time of mice injected with snake venom was also
observed in presence of QR. Further, in silico analysis revealed that QR was able to interact
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more efficiently than Qn through hydrogen bonds. Presence of sugar moiety in QR enhanced the
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enzyme-ligand interactions in contrast to Qn. Molecular dynamic simulations were also

supported the in vitro findings of increased potential of QR over Qn. Hence, the study also
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provides the scientific basis for the use of E. hirta extract in the treatment of snake bites. Also,
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exploring such multifunctional lead molecules with anti-venom activity would help in
developing complementary medicine for snakebite treatments especially in rural areas where
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anti-snake venom is not readily available.

Acknowledgement

The author Mr. K. Gopi, would like to thank the Indian Council of Medical Research (ICMR),
India for providing ICMR-SRF fellowship for this research work [45/33/2013/BMS/TRM]. The
authors would also like to thank the management of VIT University, Vellore, for providing
necessary support for carrying out this research work.

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Figure Legends

Figure 1: Spectroscopic characterization and identification of plant isolate. (A) HPLC analysis
of plant isolate, showing high purity with a single peak at 10.39 retention time. (B) Mass
spectrum analysis of the isolate showing the molecular mass (m/z) as 471.24 with Na+ adduct.

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(C) UV visible spectroscopic analysis showing the three peaks that represents the A, C and B
rings of flavonoid respectively. (D) FT-IR analysis that confirms the presence of C=O, C-O and
OH groups in flavonoid. (E and F) 1H-1H-homo nuclear NMR-COSY and TOCSY analysis

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respectively, showing the proton-proton couplings in flavonoid and sugar respectively. (G) 1H-
13
C-hetero nuclear HSQC-NMR spectroscopy showing carbon-proton couplings. (H) 1H-13C-

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hetero nuclear HMBC-NMR spectroscopy showing the glycoside linkage between C3 portion of
flavonoid and R1 portion of sugar moiety represented by a peak at 5.3 ppm (green circle).

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Figure 2: Molecular structure of the isolated flavonoid glycoside (with molecular formula
C21H20O11) identified as quercetin-3-O-rhamnoside (QR) showing the presence of quercetin
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flavonoid attached with rhamnose sugar through glycoside linkage.

Figure 3: Inhibition of Naja naja venom enzymes by quercetin-3-O-rhamnoside (QR). In all

assays, venom was pre-incubated with various concentrations of QR and then the specific
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enzyme activity was analyzed. (A) Protease inhibition analysis by densitogram; c-casein, v-
venom; (B) Hemorrhage inhibition, a-venom induced hemorrhage, b-venom induced hemorrhage
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at a ratio of 1:5 venom: quercetrin, c-hemorrhage inhibition at a ratio of 1:20, d- control injected
with saline showing no hemorrhage; (C) Phospholipase inhibition; (D) Edema inhibition; (E)
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Hyaluronidase inhibition; (F) Hemolytic inhibition.

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Figure 4: In vivo evaluation of QR against venom induced lethality and histopathological

observations of vital organs. (A) Evaluation of QR potential to protect the mice injected with 2
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LD50 of Naja naja venom. All the mice in a group injected with only venom were died at near
120 minutes while the mice co-injected with venom and QR showed significant increase in
survival time. (B) Histopathological observations of mice vital organs. Control; mice injected
with only saline showing normal tissue patterns in all vital organs. Venom; mice injected with 2
LD50 of Naja naja venom, showing severe damage, necrosis, infiltration and tissue degeneration
in the heart, liver, lungs and kidney. Venom+QR; mice co-injected with venom and QR showing
the reduced toxicity than that of venom injected. However, unlike control, the complete reversal
of all the venom induced toxicity was not seen even in presence QR at higher concentrations.

Figure 5: Comparative analysis of in vitro and in silico analysis of molecular interactions

between QR/Qn and PLA2 enzyme. (A) Molecular structures of Naja naja PLA2 (ribbon model)
and QR and Qn. (B) In vitro inhibition of phospholipase by QR and Qn. QR (red) showed dose
dependent inhibition of enzyme activity and maximum of 96% inhibition was achieved at a
concentration of 200 M. At the same concentration, Qn (black) showed only 42% inhibition of

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the enzyme. (C&D) Molecular docking analysis of PLA2-Qn and PLA2-QR complexes
respectively. Grey represents the enzyme structure while the QR/Qn was represented in red
color. PLA2-QR complex shows that QR is able to penetrate deep into the active site of an
enzyme comparing to Qn. (E&F) PyMole analysis of PLA2-Qn and PLA2-QR complexes. Apart
from hydrophobic interactions Qn was able to form two hydrogen bonds with His47 and Tyr63

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residues of an enzyme. Whereas QR in addition to hydrophobic interactions, is able to form four
hydrogen bonds with Tyr27, Cys44, Asp48 and Tyr63. (G) Root Mean Square Deviation

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analysis (RMSD) with a run of 25 ns. Black represents the native protein while red and green
represents the PLA2-Qn and PLA2-QR complexes respectively. Increase in deviation was

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observed between 13 and 23 ns of PLA2-QR complex that confirms the structural deviation upon
ligand binding unlike PLA2-Qn complex.

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Figure 6: Hydrogen bonds profile between PLA2-Qn (red) and PLA2-QR complexes (black)
under dynamic simulations of 25 ns. Increased number of hydrogen bonds was observed in
PLA2-QR over PLA2-Qn complex particularly between 13 and 23 ns.
MA
D
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Figure 1

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Figure 2
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Figure 3

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Figure 4

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Figure 5

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Figure 6 MA
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Tables

Table.1. Chemical shifts of 1H and 13C resonances, multiplicity and coupling constant values of
Quercetin-3-O-rhamnoside

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13 1
Carbon C chemical shifts H chemical shifts Multiplicity Coupling

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position (ppm) (ppm) constant (Hz)
C2 158.73 - - -
C3 136.08 - - -

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C4 (C=O) 179.46 - - -
C5 163.08 - - -
C6 100.63 6.19 d 1.38

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C7 168.26 - - -
C8 95.32 6.35 d 1.63
C9 159.09 - - -
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C10 105.27 - - -
C1 123.00 - - -
C2 116.19 7.35 d 1.76
C3 146.52 - - -
D

C4 149.97 - - -
C5 116.42 6.93 d 8.28
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C6 122.85 7.32 dd 8.28, 8.28

R1 (anomeric) 103.55 5.36 d 1.2
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R2 72.14 4.25 d 1.13

R3 72.05 3.78 dd 3.26, 3.26
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R4 73.30 3.35 d 2.26

R5 71.94 3.43 d 6.02
R6 (CH3) 17.67 0.96 d 6.15
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Table 2: Neutralization of venom lethality by QR administered intra peritoneal.

Group Venom: Dosage No of Survival Symptoms

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QR venom + QR mice time observed
(w/w) (mg/kg b.w) dead (min)

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I 1:00 0.9 + 0.0 4/4 140 7 Eye closure,
Limb paralysis

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Heavy breathing
II 1:10 0.9 + 9.0 4/4 157 4 Eye closure,
Limb paralysis
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Heavy breathing
III 1:20 0.9 + 18 4/4 235 20 Eye closure,
Limb paralysis
D

Heavy breathing
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IV 1:40 0.9 + 36 4/4 310 42 Eye closure,

Limb paralysis
Heavy breathing
P

V 1:80 0.9 + 72 4/4 442 38 Eye closure,

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Limb paralysis
Heavy breathing
VI 0:80 0.0 + 72 0/4 >24 No toxic symptoms
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observed
VII Saline 200 l 0/4 >24 No toxic symptoms
observed

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Table 3: Histopathological changes induced by snake venom in presence and absence of QR.

Group Venom: Dosage Tissue Histopathological observations

No. QR ratio mg/kg
body

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(w/w) weight

I 1:00 0.9 Heart Severe haemorrhage, infiltration

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Kidney Severe tubular degeneration and vacuolation

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in glomeruli

Liver Moderate vacuolated hepatocytes and

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Necrosis, infiltration of inflammatory cells,
hypertrophy, normal architecture disrupted

Lung Severe hemmorrhages and disrupted alveoli

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II 1:10 0.9 + 9 Heart Severe haemorrhage, sarcolysis

Kidney Moderate tubular degeneration and severe

D

hemmorrhage
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Liver Moderate necrosis, infiltration of

inflammatory cells, mild hypertrophy
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Lung Moderate hemmorrhages, infiltration of

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inflammatory cells

III 1:20 0.9 + 18 Heart Severe infiltration and minimal sarcolysis

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Kidney Moderate degeneration of tubules and

glomeruli and infiltration of inflammatory
cells

Liver Moderate focal necrosis of hepatocytes and

infiltration of inflammatory cells and mild
necrosis

Lung Moderate congestion and infiltration of

inflammatory cells

IV 1:40 0.9 + 36 Heart Hemorrhage, tissue degeneration

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Kidney Mild tubular degeneration and congestion of

blood vessels

Liver Mild hepatocellular necrosis and

degeneration, infiltration of inflammatory

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cells

Lung Mild congestion of blood vessels, edema

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and disruption of alveolar septa

V 1:80 0.9 + 72 Heart Mild sarcolyis

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Kidney Minimal degeneration of tubules

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Liver Mild hypertrophy, reduced necrosis and no
infiltration of inflammatory cells

Lung
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Reduced alveolar damage minimal
congestion of blood vessels and edema

VI 0:80 0 + 72 No abnormality was detected in any organs

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VII saline No abnormality was detected in any organs

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Table 4: Molecular docking analysis between Naja naja Phospholipase-A2 and QR/Qn using active site

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S. Ligand Binding No. of Residues Hydrogen Hydrogen Bond
No affinity H-bonds involved bond bond length
(kcal/mol) acceptor

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donor ()

1 Quercetin -7.9 2 His47, O5 (lig) N1(His47) 3.08

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(Qn)
Tyr63 O1 (lig) OH(Tyr63) 2.96

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2 Quercetin-3- -8.2 4 Tyr27, O7 (lig) O(Tyr27) 2.80

O-
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Cys44, O(Cys44) O6 (lig) 3.20
rhamnoside
docking. (QR) Asp48, O7 (lig) O2(Asp48) 2.89
D
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Tyr63 O3 (lig) OH(Tyr63) 2.81

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Highlights

The fractions of Euphorbia hirta methanolic extract was evaluated against Naja naja
venom enzymes

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One of the active fractions inhibited multiple enzymes of Naja naja venom in vitro

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The active principle was identified as Quercetin-3-O-rhamnoside (QR)

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Haemorrhage and edema causing activity of venom was also inhibited by QR ex vivo

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Further, its mode of enzyme inhibition was compared with its aglycon Quercetin against
PLA2 activity in silico. MA
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