studies-on-neutralizing-effect-of-ophiorrhiza-mungos-root-extract-against-daboia-russelii-venom

Journal of Ethnopharmacology 151 (2014) 543–547
Contents lists available at ScienceDirect
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep
Studies on neutralizing effect of Ophiorrhiza mungos root extract
against Daboia russelii venom
S. Anaswara Krishnan a , R. Dileepkumar b,n , Achuthsankar S. Nair c , Oommen V. Oommen d
a Department of Zoology, University of Kerala, Thiruvananthapuram 695581, Kerala, India
b Centre for Venom Informatics, University of Kerala, Thiruvananthapuram 695581, Kerala, India
c Department of Computational Biology and Bioinformatics, University of Kerala, Thiruvananthapuram 695581, Kerala, India
d Kerala State Biodiversity Board, Pallimukku, Pettah, Thiruvananthapuram 695024, Kerala, India
article info
Article history:
Received 2 August 2013
Received in revised form
25 October 2013
Accepted 9 November 2013
Available online 23 November 2013
Keywords:
Ophiorrhiza mungos
Daboia russelii
Antisera
Yolk sac membrane
abstract
Ethnopharmacological relevance: The folklore or traditional therapy in southern India widely utilizes a
plethora of local herbs to treat the patients challenged with snake venom. Despite the widespread
implementation of antisera therapy, the local population of the country still relies on this century's old
medicinal formulas mainly due to the cost effectiveness, lesser side effects and also its cultural
acceptability. The present study aims to validate the neutralizing ability of one such traditionally
acclaimed antidote Ophiorrhiza mungos root extract against Russell's viper (Daboia russelii) venom in the
early developing chick embryos.
Materials and methods: The disc impregnated with venom, root extract or the combination of both was
placed on the yolk sac membrane preferably over the anterior blood vessel of 6th day chick embryo. The
neutralization/inhibition of venom-induced lethality or hemorrhage was achieved by incubating venom
and extract before being applied to the embryo. The membrane stabilizing properties of root extract was
estimated by HRBC lysis method. The preliminary phytochemical analysis was done to assess the phyto
constituents in the root extract.
Results: The LD 50 of Russell's viper venom in 6th day chick embryo was found to be 3 μg/μl. The
neutralising effect of root extract was achieved by pre-incubating venom with various concentrations of
extract and at the concentration of 10 μg/μl, 100% recovery of embryos was observed after 6 h of
incubation. Higher concentration of root extract showed remarkable results by completely abolishing
traces of hemorrhagic lesions induced by viper venom.
Conclusions: The above observations confirmed that the root extract of Ophiorrhiza mungos possess
potent anti snake venom neutralizing compounds, which inhibit the activity of viper venom. The chick
embryo, a new insensate model used in the present study is significant in venom research as it reduces
the ruthless suffering of higher mammalian experimental models.
& 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Despite of recent advances, snake envenoming is still a major
socio-medical and an economic dilemma of all tropical countries
including India. The annual estimate of ophidian accident in India
alone is more than 200,000 cases with 35,000–50,000 deaths
approximately (Chippaux, 1998; Bawaskar, 2004). Out of 3000
snake species are known to science, 30% are venomous and fatal to
humans (Cher et al., 2005). Snake envenoming causes a variety of
pathophysiological manifestations including severe local tissue
damage with myonecrosis, edema and hemorrhage, which may
result in irreversible lesions and even amputation of the affected
n Corresponding author. Tel.: þ919447830909; fax: þ914712308759.
E-mail address: dileepkamukumpuzha@gmail.com (R. Dileepkumar).
limb (Otero et al., 2002). The cocktail action of toxic proteins in the
venom which includes phospholipase A2, myotoxins, hemorrhagic
and coagulant factors, cytotoxins, cardiotoxins etc. coupled with a
series of biochemical events in victim's body assists in pathophysiology
following the envenomation.
Daboia russselli (Russell's viper) appears to be the most common
cause of fatal snake bite in southern India (Woodhams et al.,
1990). The most conventional clinical approach is the administration
of polyvalent anti-snake venom (ASV) prepared from sera of
horses or sheep. Unfortunately, this polyspecific ASV does not
provide adequate protection against venom-induced hemorrhages,
necrosis and nephrotoxicity and often produces serum reaction in
some patients (Sutherland, 1977; Corrigan et al., 1978; Stahel et al.,
1985; Alam and Gomes, 1998). In addition, antiserum development
in animals is highly expensive, time consuming and requires
ideal storage conditions (Cheng et al., 2001). Considering the
0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jep.2013.11.010

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A.S. Krishnan et al. / Journal of Ethnopharmacology 151 (2014) 543–547
limitations of ASV, scientific attention has turned back to systematic
investigation of plant-based tribal remedies for snakebite is
justified.
In India, the folklore health care system has deep rooted history
among tribal populations inhabiting different parts of the country.
The folklore medicinal system is based on natural products and/or
its derivatives for its therapeutic rationale, but the treasure of
knowledge is passed through generations without documentations,
except for a few (Perumal Samy and Ignacimuthu, 1998, 2000). Many
antisnake venom plants are recommended by the folklore remedy to
treat patients challenged with snake venom and it is claimed that a
surprising number of those herbal antidotes have considerable
therapeutic effect even in the advanced stage of venom toxicity.
Ophiorrhiza (Rubiaceae), is one such acclaimed antidote plant and
the genus Ophiorrhiza is represented by 49 species in India and
different species of the genus have been use in traditional medicines
against snake bite, stomatitis, ulcers and wound healing (Kirthikar
and Basu, 1975). The tribal groups inhabited in the southern India
have been extensively utilizing the roots of Ophiorrhiza mungos to
treat snake bitten patients (Anaswara Krishnan et al., 2013). The
present study is the first of its kind to examine the potential of
Ophiorrhiza mungos root extract to act as an antidote to neutralize
Russells viper venom using in-vitro and in-vivo methods.
2. Materials and methods
2.1. Venom collection
The venom was milked from two week fasted Russell's viper
(GO (Rt) No. 94/2009/F &WLD dated 25/02/2009) housed in the
Poojappura government serpentarium, Thiruvananthapuram, India.
The extracted venom was lyophilized and was kept at 20 1C until
further use. The venom concentration was expressed in terms of dry
weight.
2.2. Preparation of plant material
The whole plant of Ophiorrhiza mungos was collected from Kallar
region of south-western India (08130′N latitudeand076156′E longitude).
The botanical identification was performed in Department of
Botany, University of Kerala, Thiruvananthapuram, India and a voucher
specimen was deposited in the herbarium of the same department
under the number KUBH-5841. The hairy root was air-dried under
shade for four weeks and was pulverized with the aid of mortar and
pestle.
2.3. Extraction of plant material
A 20 g of air dried, finely powdered roots were added to 100 ml
distilled water, mixed thoroughly and boiled for 8 h in water bath
with continuous stirring. Thereafter, the solution was filtered and
the extracts were evaporated to dryness under reduced pressure.
The yield of the extract was calculated and stored at 0–4 1C until
further use.
2.4. Preliminary phytochemical analysis
Phytochemical tests were carried out on the methanolic, ethyl
acetate, diethyl ether, chloroform, benzene and n-butanolic extracts
of Ophiorrhiza mungos root using standard procedures to identify the
phytoconstituents as described by Onwukaeme et al. (2007).
2.5. In-vitro anti-snake venom activity
Anti-snake venom activity of Ophiorrhiza mungos was assessed
through inhibition of in-vitro Human Red Blood Corpuscles (HRBC)
lysis. The hypo-saline induced hemolysis was evaluated in-vitro by
the method of Roelofsen et al. (1971) and Balu and Alagesaboopathy
(1995). This method was modified in the present study by venom
induced hemolysis. Blood was collected from healthy human volunteers
by vein puncture. Heparin was used as an anticoagulant. The
collected blood was washed three times with saline. The preparation
of cell suspension was carried out as described by Murugesh et al.
(1981). Venom of Russell's viper was dissolved in physiological saline
solution to make a stock solution of 100 mg/ml. The different tubes
are filled with 1 ml of venom (100 mg/ml), 1 ml phosphate buffer
pH 7.4 and 1 ml of 1% HRBC and varying concentrations of aqueous
extract of Ophiorrhiza mungos (20, 40, 60 and 80 mg/ml). Control had
the same composition but was free of extract. The mixtures were
incubated at 37 1C for30minandthencentrifugedat1000rpmfor
3 min. The absorbance of the supernatant was measured at 540 nm
using a spectrophotometer (Systronics). The inhibition percent of
hemolysis was calculated by the following equation,
Inhibition % hemolysis ¼ A c
100
A c
A c
A t
absorbance of control ðwithout extractÞ
A t
absorbance of test ðwith extractÞ in venom solution
2.6. In-vivo <strong>studies</strong> in chicken embryo model
2.6.1. Shell-less egg preparation
Eggs were prepared using a standard method with slight
modifications (Dunn and Boone, 1976; Sells et al., 1997). In brief,
hatching eggs on day 1 were purchased from Regional Poultry
Farm, Thiruvananthapuram, Kerala, India and incubated until day
4at371C in a humid incubator. On day 4, each egg was wiped
with 70% ethanol, cracked out of its shell into a cling film
hammock and covered with a sterilized petri dish lid. Incubation
was continued until day 6 when the experiments were carried out.
Discs of 2 mm were cut from Whatman no. 1 filter paper using a
hand punch. Venom, root extract or mixture in a total volume of
2.0 ml was impregnated to each disc and was placed over anterior
vitelline vein on the yolk sac membrane. Control tests were carried
out using normal saline (0.9%) instead of venom.
2.6.2. Acute toxicity of root extract
Four groups of six embryos each were used per root extract
dilution (5, 10, 20 and 30 mg/ml) and a total volume of 2 ml was
impregnated to each disc with different concentrations were
placed over anterior vitelline vein on the yolk sac membrane of
the experimental embryos. Control group received 2 ml of saline
instead of the extract. The embryos were observed in hourly
intervals for 24 h for any lethality. Each test was carried out using
triplicate egg preparations.
2.6.3. Measurement of venom lethality
The venom was tested at different concentrations (0.5 to 5 mg/ml)
for finding the lethal toxicity of viper venom in the 6th day chick
embryo, using groups of six embryos for each venom dose. A total of
2.0 ml of venom was applied on the disc with different concentration
was placed on the yolk sac membrane preferably over the anterior
vitelline vein. Control groups received normal saline instead of
venom. The LD 50 was calculated with the confidence limit at 50%
probability by the analysis of death occurring within 24 h of venom
injection. All tests were carried out using triplicate egg preparations.

A.S. Krishnan et al. / Journal of Ethnopharmacology 151 (2014) 543–547 545
2.6.4. Measurement of antivenom activity
Venom sample (3 LD 50 ) was incubated with equal volume of
different concentrations (2.5, 5 and 10 mg/ml) of root extract at
37 1C for 30 min before being applied to the yolk sac membrane.
Positive control embryo received same amount of venom without
root extracts. A total of 2.0 ml volumes of each venom and
antivenom dilution were expanded to accommodate testing on
six eggs. Control tests using 2 ml of saline were also carried out.
The embryos were observed at 1, 2, 4 and 6 hourly intervals and
the number of survivors at 6 h was recorded for the analysis. The
death of the embryo was a clear end point with cessation of the
heart beat followed by submergence of the yolk sac membrane
into the yolk.
2.6.5. Measurement of hemorrhage
A corona of hemorrhage surrounding discs impregnated with
hemorrhagic venoms could be visualized after 2–4 h of incubation
at 37 1C. The concentration required to cause a hemorrhagic corona
2 mm wide was accepted as a standard hemorrhagic dose (SHD).
Neutralizing or inhibitory activity was determined by incubating one
SHD of venom with various concentrations (2.5, 5, 10 and 20 mg/ml)
of the root extract at 37 1C for 30 min. The mixture incorporating
one SHD, was applied to the disc which was then placed on the
membrane as previously described and left for 3 h to form a
hemorrhagic corona. The minimum amount of extract required to
abolish hemorrhage was recorded as the minimum effective neutralizing
dose (MEND). All tests were carried out using triplicate egg
preparations.
3. Results
3.1. Plant extract
The extract was dried under rotary vacuum evaporator and the
yield of the extract was found to be 1.38% w/w.
3.2. Phytochemical analysis
The results of preliminary phytochemical screening of different
extracts of Ophiorrhiza mungos revealed the presence of terpenes,
phenols, flavanoids, alkaloids, quinones, tannins, glycosides and
cardiac glycosides. The result also noted the absence of saponins in
any extract of root.
3.3. In-vitro anti-snake venom activity
In-vitro anti-snake venom activity was carried out by HRBC
membrane stabilization method. In the present investigation aqueous
extracts of the roots of Ophiorrhiza mungos at the concentration
of 20, 40, 60, and 80 mg/ml were used to evaluate the activity. These
extracts inhibit the hemolysis induced by Russell's viper venom at
lower concentration but decrease the percentage of inhibition when
the concentration of extract increases (Fig. 1).
3.4. Acute toxicity
Acute toxicity analysis of root extract showed that aqueous
extract was safe and no record of death up to 20 mg/ml of extract
treatment in the 6th day chicken embryos. At higher concentration
of root extract treatment, embryo lethality was observed within
the 6 h of experiment.
Inhibition %
Inhibition Percentage of HRBC lysis
20
b
18
16
Extract (20 µg/mL)
14
a
12
Extract (40 µg/mL)
10
c
Extract (60 µg/mL)
18.26
8
d Extract (80 µg/mL)
6 11.55
9.6
4
6.1
2
0
Experimental Groups
Fig. 1. In-vitro HRBC stabilization properties of Ophiorrhiza mungos root extract.
Percentage of Live embryos
120
100
80
60
40
20
0
100
3.5. Venom lethality
The 6th day embryo with a vascularised yolk sac displays a
primitive embryonic heart with normal blood circulation, the
arrest of which provides a clear end point for lethality test. The
LD 50 of Russell's viper venom in 6th day chick embryo was found
to be 3 μg/μl.
3.6. Anti venom efficacy
The neutralizing effect of root extract was achieved by preincubating
venom with various concentrations of extract. The
result exhibits the neutralization capacity of root extract on viper
venom lethality in a dose dependent fashion (Fig. 2). The heart
beat in embryos treated with venom was arrested within the first
hour of the experiment (Fig. 3A). It was noted that the root extract
at the concentration 10 μg/μl showed 100% recovery (Fig. 3B) of
embryos after 6 h of incubation when compared to the control.
The negative control group received saline alone was survived
during the experimental period (Fig. 3C).
3.7. Hemorrhagic activity
0
Survival rate Percentage
50
75
Viper venom at concentrations of 2 to 5 mg/μl produced hemorrhagic
coronas increasing from 1.0 mm to approximately 3.0 mm. The
standard hemorrhagic dose of 4 mg/μl produced approximately 2 mm
of hemorrhagic corona around the disc within 3 h (Fig. 3D). The viper
venom produced a corona of hemorrhage surrounding the disc with
100
Experimental groups
Negative control (Saline)
Positive control (9 µg/µl
Venom)
Treatment (2.5 µg/µl
Extract)
Treatment (5 µg/µl
Extract)
Treatment (10 µg/µl
Extract)
Fig. 2. Results of survival rate of embryos treated with the mixture of viper venom
and Ophiorrhiza mungos extract after pre-incubation.

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A.S. Krishnan et al. / Journal of Ethnopharmacology 151 (2014) 543–547
Fig. 3. (A) Embryo treated viper venom (3 LD 50 ) after 1st hour. (B) Embryo treated with pre-incubated viper venom with 10 mg/μl root extract after 6th hour. (C) Embryo
treated with saline (0.9%) after 6th hour. (D) Embryo treated with viper venom (4 mg/μl) alone shows haemorrhagic corona around the disc.
Table 1
Effect of Ophiorrhiza mungos root extract on viper venom-induced hemorrhage.
Extract/venom
concentrations
Salineþ4 mg/μl
venom
2.5 mg/μl Extractþ
4 mg/μl venom
5 mg/μl Extractþ
4 mg/μl venom
10 mg/μl Extractþ
4 mg/μl venom
20 mg/μl Extractþ
4 mg/μl venom
marked vaso-constriction in the positive control group, while the root
extract treatment neutralized the hemorrhagic lesion induced by
viper venom. The results showed that the Ophiorrhiza mungos root
extract was effective in neutralizing the hemorrhage induced by viper
venom and the minimum concentration of extract needed to abolish
the hemorrhage was recorded as 10 μg/μl (Table 1).
4. Discussion
Hemorrhagic
zone (mm)
Reduction
from
control (%)
MEND
(μg/μl)
2 – Alive
1 50 Alive
0.5 75 Alive
No
hemorrhagic
zone
No
hemorrhagic
zone
100 10 Alive
100 Alive
State of
embryo
in 3rd hour
The herbal components are a congregation of thousands of
novel molecules with high therapeutic potential. In recent years
there is a spurt in the popularity of herbal medicines mainly due to
its lesser side effects and good compatibility with the human body
and also due to the toxicity of allopathic medicines. India has one
of the richest plant's medical heritage in the world with approximately
20,000 medicinal plant species been recorded recently. The
traditional folklore medicinal system greatly explores numerous
plants or plant derived products for the snake bite treatment;
which remains an attractive research focus for alternative therapies.
Some of these species have been scientifically investigated
and proved for its anti-snake venom potency either in the crude
form or its isolated active principles. The bioactive components in
the herbal extract may bind with the various toxic components in
the venom and result in the detoxification of venom. The present
study is the first of its kind and the results provide an experimental
and scientific support for the use of Ophiorrhiza mungos
root in case of snake bite accident with viper venom. This
significant finding suggests that root extract may contain different
endogenous inhibitors of viper venom-induced lethality.
The preliminary phytochemical investigation of Ophiorrhiza mungos
root revealed the presence of flavanoids, cardiac glycosides and
phenolics. Phenolic compounds are the important constituents of
plants with anti snake venom potential. Flavonoids have been shown
to inhibit phospholipase A2, an important component in snake
venoms (Alcaraz and Hoult, 1985). The in-vivo study was conducted
in shell less egg culture that has been used in the recent decades for a
variety of toxicity <strong>studies</strong> (Rosenbruch, 1990; Herzinger et al., 1995).
This insensate model is useful in venom research especially for
viperidae venom as hemorrhage is considered to be one of its
principal lethal effects. The lethal concentration of 3 mg/μl produces
approximately 3 mm of hemorrhagic corona around the disc within
3 h of application. The disc applied with the pre-incubated venom
and increasing concentrations of extract mixture in the treatment
groups showed the elimination of venom induced hemorrhagic
lesion in a dose depended manner. This remarkable result shows
that the root extract of Ophiorrhiza mungos not only inhibits the

A.S. Krishnan et al. / Journal of Ethnopharmacology 151 (2014) 543–547 547
venom induced lethality but also completely reduced any trace of
hemorrhagic activity of viper venom. The activity of the crude extract
may be due to the presence of enzymatic inhibitors, chemical
inactivators, or immunomodulators present in its isolates. The results
of in-vitro study have shown that the root extract significantly inhibit
the hemolysis induced by viper venom. Hemolysis is one of the
principal lethal effects of snake envenomation, as the snake venom
contains phospholipase and haemolysin (Rosenberg, 1979), which act
on membrane associated phospholipids to liberate lysolecithin.
Lysolecitihin acts on the membrane of HRBC causing hemolysis
(Maeno et al., 1962). The percentage inhibition of hemolysis activity
against venom induced hemolysis is may be due to the stabilization
oftheproteininthemembraneofHRBC(Abe et al., 1991). Hence, it
may be suggested that the extract may interact with Russell's viper
venom and stabilize the protein in the membrane.
5. Conclusions
The demand and use of alternative medicine continues to grow
every year, still the treasure of traditional and cultural knowledge
are in peril. The outcome of the present study is encouraging and
intended to bridge the gap between traditional knowledge and the
science behind the anti-snake venom activity of Ophiorrhiza mungos.
The preliminary data indicates that the aqueous root extract have
the potential to directly neutralize the viper venom induced lethality
and hemorrhage in chick embryo model. The alternative model used
in the venom study eliminates the excessive suffering in venom
research by reducing the practice of conventional mammalian
experimental animals. Further researches on isolation of active
principles and the elucidation of exact mechanism of action responsible
for the observed biological activity could lead to the development
of a new natural antidote for snake envenoming.
Acknowledgments
This work was funded by Kerala State Council for Science,
Technology and Environment (KSCSTE), Govt. of Kerala, India. The
first author also would like to acknowledge the wet lab facilities of
SIUCEB, Department of Computational Biology and Bioinformatics,
University of Kerala and SAP facility at Department of Zoology,
University of Kerala.
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