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Toxicology Reports 1 (2014) 667–673

Contents lists available at ScienceDirect

Toxicology Reports
journal homepage: www.elsevier.com/locate/toxrep

Inhibition of Naja naja venom enzymes by the methanolic


extract of Leucas aspera and its chemical profile by GC–MS
Kadiyala Gopi, Kadali Renu, Gurunathan Jayaraman ∗
School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore 632014, Tamil Nadu, India

a r t i c l e i n f o a b s t r a c t

Article history: Purpose: The present investigation was aimed at evaluating the anti-ophidian properties of
Received 16 May 2014 ethnomedicinal herb Leucas aspera against Indian cobra, Naja naja venom enzymes.
Received in revised form 24 July 2014 Methods: Methanolic extract of Leucas aspera was evaluated, in vitro, for its ability to inhibit
Accepted 24 August 2014
the major enzyme activities of Naja naja venom including protease, phospholipase A2 ,
Available online 29 August 2014
hyaluronidase and hemolytic factors. The type of phytochemicals present in the extract
was analyzed. Also, the major phytoconstituents in the extract was determined by gas
Keywords:
chromatography–mass spectrometry (GC–MS).
Leucas aspera
Results: Venom protease and hyaluronidase activities (two isoforms) were completely
Naja naja
Protease (100%) neutralized by the L. aspera methanolic extract at ratio of 1:50 w/w (venom:
Hyaluronidase plant extract) and venom hemolytic activity was also completely neutralized at a ratio
Hemolysis of 1:80 w/w by the plant extract. However, the extract failed to neutralize phospholipase
PLA2 A2 activity even at the highest concentration used. Phytochemical analysis revealed the
presence of alkaloids, acidic compounds, flavonoids, steroids and cardiac glycosides in
the extract. GC–MS analysis indicated that a total of 14 compounds were present in the
extract. The major bioactive constituents were found to be 6-octadecenoic acid (32.47%), n-
hexadecanoic acid (25.97%), and 17-octadecen-14-yn-1-ol (14.22%) along with the minor
constituents, sitosterol (2.45%) and stigmasterol (2%), which was previously reported to
exhibit antivenom activity.
Conclusion: The results obtained demonstrate for the first time that the methanolic extract
of Leucas aspera possesses anti-venom activity and could be considered as a potential source
for the anti-ophidian metabolites.
© 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction their low cost and availability [1]. In India there are about 54
million indigenous people of different ethnic groups using
Since time immemorial man has been dependent on medicinal plants for the treatment of various types of ail-
plant sources for food and medicine. Recently, there has ments, including snakebites [2]. Snakebite is often readily
been renewed attention and interest in the use of tradi- treated by traditional herbal medicines by rural population,
tional medicine. The World Health Organization (WHO) especially in tropical and subtropical countries including
estimated that nearly 80% of the population depends on India.
herbal medicines to meet primary health care needs due to It is estimated that 1.2 to 5.5 million snakebites occur
globally, with 94,000 deaths occurring annually [3]. In India
alone, 35,000–50,000 deaths were reported every year
∗ Corresponding author. Tel.: +91 4162202573. [4,5]. The major victims are agriculturists and their families
E-mail address: gjayaraman@vit.ac.in (G. Jayaraman). living in rural areas of the country. Snake venom is complex

http://dx.doi.org/10.1016/j.toxrep.2014.08.012
2214-7500/© 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
668 K. Gopi et al. / Toxicology Reports 1 (2014) 667–673

mixture of enzymes, carbohydrates, proteins and several 2.3. Venom and chemicals
other small molecules. Among all enzymes present in the
venom, proteases, phospholipases, and hyaluronidases are Lyophilized snake venom (N. naja) was purchased from
reported to be medically important in developing effec- Irula snake catcher’s Industrial co-operative society limited
tive antidotes [6]. Proteases are known to degrade extra (Chennai, India). The venom was dissolved in 0.9% saline
cellular matrixes and allow other venom components and centrifuged at 675 × g for 10 min and the supernatant
to access the local tissue. Phospholipases are multifunc- was used for the study. Hyaluronic acid was purchased
tional enzymes that degrade membrane phospholipids and from Sigma Aldrich (US). All other reagents and solvents
release arachidonic acid which is a precursor molecule in were of high quality analytical grade and were purchased
causing inflammation by cyclooxygenase (COX) or lipooxy- from SD Fine chemicals/Sisco Research Laboratory Pvt
genase (LOX) pathways. Hyaluronidases are spreading Ltd/HiMedia, Mumbai.
factors which facilitate diffusion of venom toxins from the
site of bite into general circulation and thereby increase 2.4. Caseinolytic inhibition
the severity of envenomation. The only treatment avail-
able since 1895 is animal derived polyclonal antisnake Protease activity was determined using 1% casein as
venom (ASV) therapy. Despite of its wide usage, due to high substrate in 1% agarose plates according to the method
seasonal and biochemical variations in the venom compo- described previously [9]. 50 ␮g of N. naja venom was pre-
sition, records indicate that ASV developed against big four incubated with various concentrations of plant extract for
venoms is imprecise and less effective [7] in the treatment 1 h at 37 ◦ C. Briefly, the pre-incubated samples were loaded
of snakebite victims. In addition to its high cost, hypersen- into 3 mm diameter wells of casein-agarose plates (0.1 M
sitive reactions were also reported from the ASV treated Tris–HCl buffer, pH 8.0) and incubated overnight at 37 ◦ C.
victims [8]. Also, the quantity of the ASV produced does Plates were then stained with Coomassie Brilliant Blue-R
not meet the demand. Therefore interest in exploring the 250 for 1 h and destained with methanol:water:acetic acid
snake venom antidotes from plant sources has increased. (50:40:10). The percentage of protease inhibition was cal-
Hence the present study was aimed to validate the anti- culated by measuring the zone of clearance in the presence
ophidian activity of the commonly used traditional herbal of plant extract. Zone of clearance values obtained for the
plant Leucas aspera against Naja naja venom enzymes. venom in the absence of the plant extract served as control.
In addition to the plate assay, SDS-PAGE was also per-
formed to evaluate the protease inhibition by the plant
2. Materials and methods
extract. Briefly, 50 ␮g of N. naja venom was pre-incubated
with various concentrations of plant extract for 1 h at 37 ◦ C.
2.1. Plant collection
These samples were further incubated with 15 ␮l of 1%
casein (dissolved (w/v) in 0.1 M Tris–HCl buffer, pH 8.0,
The present investigation was carried out by conducting
37 ◦ C). After 30 min, the reaction was stopped by adding
surveys and questionnaires with tribal people and Iru-
SDS loading buffer (containing 50 mM Tris HCl pH 6.8, 10%
lars living in Irularpatti, Vallimalai, Alamalrengapuram in
(v/v) glycerol, 2% (w/v) SDS, 100 mM ␤-mercaptoethanol
Vellore district of Tamil Nadu, India and Bangarupalyam,
and 0.1% (w/v) bromophenol blue). SDS-PAGE of the casein
Punganur and Chittoor in Andhra Pradesh, India. Tradi-
treated with venom in presence and absence of plant
tional healers, called ‘Vaidyars’ from different indigenous
extract was then performed according to the method
groups were also approached for the documentation of
described previously [10] using 15% acrylamide gel.
the medicinal plants used in the treatment of snakebite.
The plant species collected was identified by a Botanist Dr.
2.5. Phospholipase (PLA2 ) inhibition
Madhavachetty, Department of Botany, Sri Venkateswara
University, Tirupati, Andhra Pradesh and a voucher spec-
PLA2 inhibition was evaluated using egg yolk as sub-
imen (voucher no: 1200) was deposited in the same
strate in 1% agarose plates according to the method
department.
described by Gutierrez et al. [11]. 50 ␮g of N. naja venom
was pre-incubated with various concentrations of plant
2.2. Plant extraction extract for 1 h at 37 ◦ C. The pre-incubated samples were
then loaded into 3 mm diameter wells of 1% agarose plates
Five hundred grams of shade dried powdered plant containing 0.6% egg yolk and 5 mM CaCl2 followed by
material was extracted with 1000 ml of methanol for 24 h overnight incubation at 37 ◦ C. The PLA2 inhibition was cal-
with continuous stirring. The extraction was repeated for culated by measuring the zone of clearance in the presence
3 days by changing the solvent every 24 h. The extract and absence of plant extract. PLA2 activity of venom in
was concentrated using rotary vacuum evaporator under absence of plant extract served as control.
reduced pressure at 40 ◦ C. Known quantity of the dried
extract was subsequently dissolved in 10 mM phosphate 2.6. Hemolytic inhibition
buffered saline (PBS pH 7.4). Centrifugation at 10,800 × g
(for 10 min) was performed for any insoluble. The clear The hemolytic inhibition assay was carried out as per
solution was used for the enzyme inhibition studies. The the method described previously [12], with slight modifi-
venom activity in absence of plant extract served as the cations. 100 ␮g of N. naja venom was pre-incubated with
negative control. various concentrations of plant extract in a volume of
K. Gopi et al. / Toxicology Reports 1 (2014) 667–673 669

200 ␮l PBS for 1 h at 37 ◦ C. Briefly, human erythrocytes and enzymes in N. naja snake venom. Snake venom metallo-
10 mM PBS (pH 7.4) were mixed (1:8 v/v) and 100 ␮l of proteases, phospholipase A2 (PLA2 ) and hyaluronidases are
this suspension was incubated with pre-incubated venom the key enzymes involved in tissue necrosis and extracel-
samples for 2 h at 37 ◦ C. The reaction was stopped by adding lular matrix degradation [6,15,16]. Thus, inhibition of these
1 ml of ice cold PBS and centrifuged at 2500 rpm for 10 min enzymes is generally considered to be the rate limiting step
at 4 ◦ C. The amount of hemoglobin released in the super- in the management of snakebite. Therefore, in the present
natant was measured at 540 nm. study, methanolic extract of L. aspera was evaluated for its
potential to inhibit these major enzymes.
2.7. Hyaluronidase inhibition assay
3.1. Protease inhibition
Hyaluronidase inhibition assay was performed accord-
ing to the method of Girish et al. [13] with minor Agar well diffusion assay indicated that the methanolic
modifications. The substrate gel for zymogram was extract of L. aspera completely inhibited the proteolytic
prepared by incorporating hyaluronic acid at a final con- activity of the venom at a ratio of 1:50 (w/w) venom: plant
centration of 0.17 mg/ml into 10% SDS-polyacrylamide gel extract (Fig. 1). Also, SDS-PAGE analysis indicated that
matrix. 100 ␮g of N. naja venom was pre-incubated with 50 ␮g of N. naja venom was sufficient to hydrolyse casein
different concentrations of plant extract for 1 h at 37 ◦ C. All within the incubation period of 30 minutes at 37 ◦ C. Similar
samples were prepared under non-reducing conditions for to the plate assay results, venom caseinolytic activity was
electrophoresis. After electrophoresis, the gel was soaked completely inhibited by the plant extract at a ratio of
subsequently in 50 ml of 5% Triton X-100 for 1 h and then in 1:30 w/w venom: plant extract. Fig. 2 demonstrates dose
0.05% Triton X-100 for 30 min followed by distilled water
for 1 h. The gel was equilibrated in 0.1 M sodium acetate
buffer (pH 5.0) containing 0.15 M NaCl for 48 h at 37 ◦ C. The
gel was then washed with distilled water and stained with
0.1% alcian blue solution for 2 h. Destaining was carried out
with 5% acetic acid until clear translucent bands appeared
against dark blue background.

2.8. Phytochemical analysis

In order to obtain insight into the type of phytochemi-


cals present in the extract, analysis was performed as per
the methods described by Kokate et al. [14].
Fig. 1. Dose dependent inhibition of protease activity of N. naja venom by
2.9. Gas chromatography–mass spectrometry analysis L. aspera methanolic extract. 50 ␮g of N. naja venom was pre-incubated
with various concentrations of the plant extract (w/w) for 1 h at 37 ◦ C. The
protease activity was assyed by plate method. Venom protease activity in
Chemical composition of the methanolic extract of L. absence of plant extract was considered as 0% inhibition. Values represent
aspera was analyzed using a GC–MS GCD-HP1800A system the mean ± S.D of replicates.
(Hewlett-Packard, USA) equipped with a split/splitless cap-
illary injection port. The column size was 30 m × 250 ␮m.
An electron ionization system (Quadruples analyzer; mass
range, 10–425 amu) with ionization energy of 70 eV was
used for GC–MS detection. Each of these steps was carried
out under high vacuum (10−4 to 10−8 Torr). Helium was
used as a carrier gas at a constant flow rate of 1 ml/min
and an injection volume of 2 ␮l was employed (split ratio
of 10:1). Injector and mass transfer line temperatures were
set at 250 ◦ C and 280 ◦ C, respectively and fragments were
scanned from 50 to 600 Da. The components of extract were
identified by comparison with the NIST database.

3. Results and discussion

Fig. 2. Inhibition of N. naja venom caseinolytic activity by L. aspera


Crude extracts of plants are being traditionally used
methanolic extract. 50 ␮g of N. naja venom was pre-incubated with var-
in the treatment of wide variety of diseases including ious concentrations of plant extract (w/w) for 1 h at 37 ◦ C. The protease
snakebite envenomations. However, in most cases the effi- activity was assayed in SDS-PAGE method. Lane 1: 50 ␮g of venom; Lane
cacy of this traditional treatment regimen is unproven. In 2: casein; Lane 3: casein incubated with venom; Lane 4 to 12: samples
containing casein incubated with venom in presence of different concen-
the present study, the methanolic extract of L. aspera, one of
trations of plant extract i.e. 1:05, 1:10, 1:20 to 1:80 (w/w venom: plant
the herbal plants widely used as an antidote by many rural extract) respectively. Arrows indicate the major bands of casein. Lane 1–3
populations was evaluated for its efficacy to inhibit selected served as controls.
670 K. Gopi et al. / Toxicology Reports 1 (2014) 667–673

Fig. 3. Effect of L. aspera methanolic extract on Naja naja venom Fig. 4. Dose dependent inhibition of N. naja venom hemolytic activity by
phosholipaseA2 (PLA2 ) activity. 50 ␮g of N. naja venom was pre-incubated L. aspera methanolic extract. 50 ␮g of N. naja venom was pre-incubated
with various concentrations of plant extract (w/w) for 1 h at 37 ◦ C. PLA2 with various concentrations of plant extract (w/w) for 1 h at 37 ◦ C. The
activity in absence of plant extract was considered as 100% activity. Values hemolytic activity in absence of plant extract was considered as 0% inhi-
represent the mean ± S.D of replicates. bition. Values represent the mean ± S.D of replicates.

dependent inhibition of venom caseinolytic activity by L.


3.3. Hemolytic inhibition
aspera methanolic extract. Snake venom proteases majorly
affect hemostasis and cause systemic hemorrhage. The
L. aspera methanolic extract inhibited the hemolytic
most probable mechanism involved in the inhibition
activity of the venom, at a ratio of 1:80 w/w (Fig. 4).
of these proteases by plant extracts could be due to
Hemolytic activity is another distinct feature of cobra
the chelating property of phenolic components present
venoms greatly induced by multicomponents including
in the extract. It is reported that, phenolic compounds
metalloproteases, PLA2 , and more specifically, cardiotoxins
form hydrogen bonds and strongly bind to the histidine
and cytotoxins of venom [26,27]. Though, L. aspera did not
residues present in Zn2+ binding motifs of metallo pro-
show inhibition on venom PLA2 , it completely protected
teases, resulting decrease in the hydrolytic activity of
the hRBC from direct hemolytic activity of N. naja venom.
the enzyme [17]. Melo et al. [18] and Soares et al. [19]
As cardiotoxins and cytotoxins also induce lysis of human
suggested that the plant extracts would have compounds
erythrocytes [28], it is possible that the phytochemicals in
that bind to divalent metal ions, which are required for
the plant components could inhibit these toxic proteins in
enzymatic activities. As the presence of proper metal ion
the venom.
coordination is a pre-requisite for the hydrolytic activity
of metalloproteases, any metabolite that can weaken the
protease-metal ion interaction will result in inhibition of 3.4. Hyaluronidase inhibition
the proteolytic activity.
100 ␮g of N. naja venom exhibited two clear bands
3.2. Phospholipase inhibition of hyaluronidase isomers (in 10% SDS-PAGE zymogram
under non reducing conditions). L. aspera exhibited com-
In contrast to protease inhibition, the extract com- plete inhibition of both the isoforms at a ratio of 1:50 w/w
pletely failed to neutralize venom PLA2 activity even venom-plant extract (Fig. 5). Local tissue damage associ-
at highest concentration of extract used (Fig. 3). PLA2 ated with snakebite envenomation is due to the combined
causes cardiotoxicity, myotoxicity, pre or postsynaptic action of metalloproteases along with hyaluronidases and
neurotoxicity, edema, hemolysis, hypotension, convul- is reported to continue even after antisnake venom (ASV)
sion, inhibition of platelet aggregation and anticoagulation treatment [29]. Hyaluronidases are a class of endo-␤-
[20–22]. Snake venoms are especially rich in group I glycosidases that degrade the hyaluronic acid in the cell
(Elapidae) and II (Viperidae) PLA2 s. These enzymes are wall and facilitate easy diffusion of venom toxins into cir-
Ca2+ dependent and hydrolyse the 2-acyl ester bonds culation, which would otherwise take much longer time to
of membrane glycerophospholipids producing free fatty diffuse [30,31].
acids and lysophospholipids which are key components of
inflammatory process. Timely administration of ASV could 3.5. Phytochemical analysis
neutralize the systemic effects caused by these myotoxic
enzymes but provides no protection against local toxicities Qualitative phytochemical analysis revealed the pres-
induced by the venom components. Hence, screening of ence of acidic compounds, alkaloids, flavonoids, tannins,
herbal antidotes to averse snake venom induced toxic- saponins and cardiac glycosides as major components
ity is of great importance in the snakebite management in the methanolic extract of L. aspera (Table 1). The
[23]. Hemidesmus indicus root extract had been formerly anti-venom activity of plants that are widely used against
reported against Daboia russellii and Naja kaouthia snake snake venom induced toxicity is majorly due to the
venom induced toxicities in vivo. Lupeol acetate and 2- presence of various secondary metabolites that bind
hydroxy-4-methoxy benzoic acid isolated from methanolic directly to venom components and neutralizes their
root extract of H. indicus was demonstrated to have anti- effect. Flavonoids and alkaloids isolated from differ-
PLA2 activity against viper and cobra venoms [24,25]. ent plant species demonstrated anti-inflammatory and
K. Gopi et al. / Toxicology Reports 1 (2014) 667–673 671

Table 1
Qualitative analysis of the phytochemicals present in methanolic extract of L. aspera.

Phytochemical Test name +/− Phytochemical Test name +/−

Alkali compounds Litmus paper (Red → Blue) − Tannins Ferric chloride test +
Acidic compounds Litmus paper (Blue → Red) + Proteins Biuret test −
Alkaloids Mayers test + Steroids Salkowski test +
Tannic acid test +
Hagers test + Triterpenoids Salkowski test −
Carbohydrates Molisch’s test + Saponins Froth test +
Fats and fixed oils Glycerin test + Cardiac glycosides Keller kiliani test +

+ Present, − absent.

hyaluronidase activities of N. naja venom. Presence of


these active secondary metabolites in the extract could
have contributed for plants anti-venom activity.

3.6. GC/MS and qualitative phytochemical analysis

In order to obtain information on the major metabo-


lites present in the extract, GC–MS was performed.
Analysis revealed the presence of 6-octadecenoic acid
(32.47%), n-hexadecanoic acid (25.97%), 17-octadecen-
14-yn-1-ol (14.22%), methyl 13-octadecanoate (5.33%),
tetradecanoic acid, 10,13-dimethyl-methyl ester (4.51%),
methyl 5-6-octadecadienoate (3.98%), gamma-sitosterol
(2.45%), stigmasterol (2%) as the major phytoconstituents.
Table 2 describes the phytochemicals identified in the
Fig. 5. Inhibition of N. naja venom hyaluronidase activity by L. aspera extract along with their molecular weight and formula.
methanolic extract. 100 ␮g of N. naja venom was pre-incubated with Previous reports indicated that tetradecanoic acid binds to
various concentrations of plant extract (w/w) for 1 h at 37 ◦ C. The
catalytic site of (Bothrops neuwiedi) venom PLA2 through
hyaluronidase activity was assayed in substrate gel SDS-PAGE zymogram
under non-reducing conditions. Lane 1 and 8: 100 ␮g of N. naja venom hydrogen bonding between ASN27, CYS and HIS47 amino
in absence of plant extract, indicating the presence of two isoforms of acid residues and also by hydrophobic interactions with
hyaluronidase; Lane 2 to 7: 100 ␮g of venom in presence of different con- neighboring amino acid residues such as LEU2, LEU5,
centrations of plant extract i.e. 1:05, 1:10, 1:25, 1:50, 1:75 and 1:100 (w/w CYS2, TYR21, PRO17, GLY6, LYS7, ALA18, ILE9, LEU111
venom: plant extract), respectively, showing dose dependent inhibition of
hyaluronidase activity of both the isoforms. Arrows indicate two isoforms
and PRO113 present in the catalytic site. Such compet-
of venom hyaluronidase. itive binding of this molecule is expected to inhibit the
PLA2 activity of the toxin [32]. Also, hexadecanoic acid was
demonstrated to have anti-inflammatory activity (Table 3)
anti-hemorrhagic effects against various snake venoms. through competitive inhibition of PLA2 [33]. However, in
Some of them are flavonoid-like (Quercetin, kaempferol, the present study the PLA2 activity of N. naja venom was
luteolin, myricetin)/alkaloid-like (aristolochic acid and not inhibited by the methanolic extract of L. aspera, even
ajmalin) and have revealed strong inhibitory potential though GC–MS analysis indicate the presence of these fatty
against snake venom hyaluronidases [For recent review, acids. Sequence analysis indicated (Gopi and Jayaraman,
6]. In the present study, L. aspera methanolic extract unpublished results) that the amino acid residues present
showed potent inhibition on protease, hemolytic and in binding site of the enzyme are significantly different at

Table 2
Phytochemicals identified in the methanolic extract of L. aspera.

S. no. RT Phytochemical name Mol. formula Mol. (wt) Peak area (%)

1 14.56 Heptane-1-methanesulfonic acid,7,7-dimethyl-2-oxo C10 H16 O4 S 232 1.52


2 14.83 3-Methyl-2-(2-oxopropyl) furan C8 H10 O2 138 1.54
3 17.71 Tetradecanoic acid,10,13-dimethyl-methyl ester C17 H34 O2 270 4.51
4 18.18 n-Hexadecanoic acid C16 H32 O2 256 25.97
5 19.33 Methyl 9-cis,11-trans-octadecadienoate C19 H34 O2 294 1.70
6 19.39 Methyl 13-octadecenoate C19 H36 O2 296 5.33
7 19.61 Methyl 5-6-octadecqdienoate C19 H34 O2 294 3.98
8 19.84 6-Octadecenoic acid C18 H34 O2 282 32.47
9 20.08 17-Octadecen-14-yn-1-ol C18 H32 O 264 14.22
10 20.23 2-(Fecnch-2-yl) fenchane C20 H34 274 1.36
11 25.48 [1,1-Bicyclohexyl]-4-carboxylic acid, 4 -pentyl-4-pentylphenyl ester C29 H46 O2 426 1.38
12 28.69 Stigmasterol C29 H48 O 412 2.00
13 29.36 Gamma-sitosterol C29 H50 O 414 2.45
14 29.82 Benzene,1-methyl-2-(1-methyl-2-methylenecyclopentyl) C14 H18 O 202 1.49
672 K. Gopi et al. / Toxicology Reports 1 (2014) 667–673

Table 3
Phytochemicals identified from methanolic extract of L. aspera by GC–MS and their reported bioactivity.

S. no. Phytochemical Compound nature Bioactivity Reference

1 Heptane-1-methanesulfonic acid,7,7-dimethyl-2-oxo Sulfonic acid ester Not reported –


2 3-Methyl-2-(2-oxopropyl) furan Isoprene Strong anti-oxidant [34]
3 Tetradecanoic acid,10,13-dimethyl-methyl ester Myristic acid Anti-inflammatory PLA2 inhibition [32]
4 n-Hexadecanoic acid Saturated fatty acids Anti-inflammatory PLA2 inhibition [33]
5 Methyl 9-cis,11-trans-octadecadienoate Unsaturated fatty acids Maintains the cell integrity [36]
6 Methyl 13-octadecenoate Unsaturated fatty acids Maintains the cell integrity [36]
7 Methyl 5-6-octadecqdienoate Unsaturated fatty acids Maintains the cell integrity [36]
8 6-Octadecenoic acid Saturated fatty acids Synthetic inhibitor of neurotoxins [35]
9 17-Octadecen-14-yn-1-ol Alcohol Not reported
10 2-(Fecnch-2-yl) fenchane Terpenes Anti-snake venom [6]
11 [1,1-Bicyclohexyl]-4-carboxylic Esters Not reported –
acid,4 -pentyl-4-pentylphenyl ester
12 Stigmasterol Phytosterol Anti-snakevenom [37]
13 Gamma-sitosterol Phytosterol Anti-snake venom [37]
14 Benzene,1-methyl-2-(1-methyl-2-methylenecyclopentyl) Not reported –

these positions and therefore resulted in no inhibition of methanolic extract (Table 3) could help in preventing the
the enzymatic activity of phospholipase. oxidative damage caused upon snake envenomations. The
Also, plants having higher the amounts of isoprene derivatives of octadecenoic acid were reported to be very
derivatives were demonstrated to have strong antioxi- strong synthetic inhibitors of neurotoxins [35]. The unsat-
dant activity by preventing the free radical formation urated fatty acids found in the L. aspera methanolic extract
[34]. Antioxidants are known to play an important role would help in maintaining the cell integrity which in-
in controlling the neurotoxicity. Hence, the presence of turn prevents the distribution of venom components from
isoprene 3-methyl-2-(2-oxopropyl) furan in the L. aspera the bite site [36]. Apart from isoprene derivatives, octade-
canoic acid derivatives in the extract would also help
in inhibiting snake venom toxins and their neurotoxic-
ity. Hemorrhage is majorly caused by the combined effect
of proteases and hyaluronidases. Sitosterol and stigmas-
terol were demonstrated to inhibit viper venom induced
lethality, neurotoxicity and hemorrhage [37]. In the present
study, protease and hyaluroniade activities were com-
pletely inhibited in a dose dependent manner. This could
be due to the strong anti-venom activity of the phytoste-
rols (sitosterol and stigmasterol) present in the extract.
Fig. 6 represents some of the major bioactive components
identified from L. aspera methanolic extract. In addition,
these phytochemicals were previously demonstrated for
their hyaluronidase inhibition activity by either forming
an inhibitor-hyaluronic acid complex that restricts the
substrate availability to the enzyme or direct binding to
the enzyme active site that affects the enzyme activity
[38–40]. Hence, the presence of these multiple bioac-
tive (anti-snake venom) compounds in the extract could
have contributed for its efficient antivenom activity in the
present study. Based on the finding of the present study, it is
demonstrated that methanolic extracts of L. aspera, possess
antivenom activity against N. naja venom enzymes and
supports their traditional use in herbal medicine against
snakebites.

Conflict of interest

None.

Fig. 6. Molecular structures of major bioactive phytochemicals from Transparency document


L. aspera methanolic extract identified by GC–MS analysis. (1) 6-
octadecenoic acid, (2) n-hexadecenoic acid, (3) 17-octadecen-14-yn-1-ol,
(4) methyl-13-octadecenoic acid, (5) tetradecenoic acid, (6) sitosterol, (7) The Transparency document associated with this article
stigmasterol. can be found in the online version.
K. Gopi et al. / Toxicology Reports 1 (2014) 667–673 673

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(2005) 2625–2641.
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be found, in the online version, at doi:10.1016/j.toxrep. [21] C.L. Ownby, Structure, function and biophysical aspects of the
2014.08.012. myotoxins from snake venoms, J. Toxicol. Toxins Rev. 17 (1998)
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