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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.
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.
Table 1
Qualitative analysis of the phytochemicals present in methanolic extract of L. aspera.
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.
Table 2
Phytochemicals identified in the methanolic extract of L. aspera.
S. no. RT Phytochemical name Mol. formula Mol. (wt) Peak area (%)
Table 3
Phytochemicals identified from methanolic extract of L. aspera by GC–MS and their reported bioactivity.
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.
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