ArticlePDF Available

Abstract and Figures

Background: Indigenous medicinal practice in Sri Lanka talks about powerful compounds extracted from native plants for treating venomous snake bites which are hardly documented in literature but are used by the indigenous doctors for thousand years. Objective: We screened the neutralizing ability of a herbal preparation practiced in indigenous medicine of Sri Lanka, consisting of Sansevieria cylindrica, Jatropha podagrica and Citrus aurantiifolia, for its ability to neutralize venom toxins of Naja naja (Common Cobra) and Daboia russelii (Russell's viper). Materials and methods: The venom toxicity was evaluated using a 5-day old chicken embryo model observing the pathophysiology and the mortality for six hours, in the presence or absence of the herbal preparation. The known toxin families to exist in snake venom, such as Phospholipase A2, Snake venom Metalloprotease, were evaluated to understand the mechanism of venom neutralizing ability of the herbal preparation. Results: The LD50 of D.?russelii venom, as measured using the 5-day old chicken embryo model, was 4.8???0.865 ug (R2?=?84.8%, P?=?0.079). The pre-incubation of venom with the herbal preparation increased the LD50 of D.?russelii venom to 17.64???1.35??g (R2?=?81.0%, P?=?0.100), showing a clear neutralizing action of D.?russelii venom toxicity by the herbal medicine. Whereas the pre-incubation of venom with the 1? venom neutralizing dose of commercially available polyvalent anti-venom serum shifted the LD50 venom only up to 5.5???1.35??g (R2?=?98.8%, P?=?0.069). In the presence of the herbal preparation, Phospholipase A2 activity of D.?russelii venom was significantly reduced from 9.2???10?3?mM?min?1 to 8.0???10?3?mM?min?1 and that of N.?naja from 2.92???10?2?mM?min?1 to 0.188???10?2?mM?min?1. Further, the pre-incubation of N.?naja venom with the herbal preparation significantly reduced its Metalloprotease activity from 0.069 units min?1 to 0.019 units min?1. Conclusion: The herbal preparation shows a clear neutralizing action against the toxicities of D.?russelii and N. naja venoms demonstrating the potential to be used as a plant based antidote for snake envenomation.
Content may be subject to copyright.
Characterization of Daboia russelii and Naja naja venom neutralizing
ability of an undocumented indigenous medication in Sri Lanka
Madhushika M. Silva
a
,
b
, Sampath S. Seneviratne
b
, Devaka K. Weerakoon
b
,
Charitha L. Goonasekara
a
,
*
a
Faulty of Medicine, General Sir John Kotelawala Defence University, Ratmalana, 10390, Sri Lanka
b
Department of Zoology, Faculty of Science, University of Colombo, Colombo, 03, Sri Lanka
article info
Article history:
Received 28 September 2016
Accepted 5 October 2016
Available online xxx
Keywords:
Daboia russelii
Naja naja
Venom
Neutralization
Herbal preparation
abstract
Background: Indigenous medicinal practice in Sri Lanka talks about powerful compounds extracted from
native plants for treating venomous snake bites which are hardly documented in literature but are used
by the indigenous doctors for thousand years.
Objective: We screened the neutralizing ability of a herbal preparation practiced in indigenous medicine
of Sri Lanka, consisting of Sansevieria cylindrica,Jatropha podagrica and Citrus aurantiifolia, for its ability
to neutralize venom toxins of Naja naja (Common Cobra) and Daboia russelii (Russell's viper).
Materials and methods: The venom toxicity was evaluated using a 5-day old chicken embryo model
observing the pathophysiology and the mortality for six hours, in the presence or absence of the herbal
preparation. The known toxin families to exist in snake venom, such as Phospholipase A
2
, Snake venom
Metalloprotease, were evaluated to understand the mechanism of venom neutralizing ability of the
herbal preparation.
Results: The LD
50
of D. russelii venom, as measured using the 5-day old chicken embryo model, was
4.8 ±0.865 ug (R
2
¼84.8%, P ¼0.079). The pre-incubation of venom with the herbal preparation
increased the LD
50
of D. russelii venom to 17.64 ±1.35
m
g(R
2
¼81.0%, P ¼0.100), showing a clear
neutralizing action of D. russelii venom toxicity by the herbal medicine. Whereas the pre-incubation of
venom with the 1venom neutralizing dose of commercially available polyvalent anti-venom serum
shifted the LD
50
venom only up to 5.5 ±1.35
m
g(R
2
¼98.8%, P ¼0.069). In the presence of the herbal
preparation, Phospholipase A
2
activity of D. russelii venom was signicantly reduced from
9.2 10
3
mM min
1
to 8.0 10
3
mM min
1
and that of N. naja from 2.92 10
2
mM min
1
to
0.188 10
2
mM min
1
. Further, the pre-incubation of N. naja venom with the herbal preparation
signicantly reduced its Metalloprotease activity from 0.069 units min
1
to 0.019 units min
1
.
Conclusion: The herbal preparation shows a clear neutralizing action against the toxicities of D. russelii
and N. naja venoms demonstrating the potential to be used as a plant based antidote for snake
envenomation.
©2016 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Publishing Services by
Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).
1. Introduction
Snake bites are proven to be a major health hazard in the tropical
belt especially affecting the rural communities and agricultural sector
in Asia, Africa, Oceania and Latin America. Recent studies show that
the annual envenoming cases around the world is as high as
421,000e1,841,00 0 [1] and the deaths may be as high as
24,000e94,000 [1].Thetruegurers of mortality could be even
higher as a proportion of the people affected do not seek formal
medical attention. The burden is mostly conned to the poorer
communities and mainly is an occupational hazard in farming and
agricultural communities [1,2]. Sri Lanka, a developing South Asian
country, falls among the countries of highest snakebite records [1e3].
Sri Lanka is inhabited by 102 species of snakes [4]; among the
mentioned snake species only 21 are considered highly venomous
and ve species as moderately venomous. From the highly venomous
species 14 are sea snakes and 2 are terrestrial species with very low
*Corresponding author.
E-mail addresses: madhushikas@gmail.com (M.M. Silva), charithalg@kdu.ac.lk
(C.L. Goonasekara).
Peer review under responsibility of Transdisciplinary University, Bangalore.
Contents lists available at ScienceDirect
Journal of Ayurveda and Integrative Medicine
journal homepage: http://elsevier.com/locate/jaim
http://dx.doi.org/10.1016/j.jaim.2016.10.001
0975-9476/©2016 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Publishing Services by Elsevier B.V. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Journal of Ayurveda and Integrative Medicine xxx (2016) 1e7
Please cite this article in press as: Silva MM, et al., Characterization of Daboia russelii and Naja naja venom neutralizing ability of an
undocumented indigenous medication in Sri Lanka, J Ayurveda Integr Med (2016), http://dx.doi.org/10.1016/j.jaim.2016.10.001
contact with humans, and the highest weight of the morbidity and
mortality are associated with snakebites of the highly venomous Naja
naja (Common cobra) and Daboia russelli (Russell's viper).
The only specic treatment currently available to snake venom
toxins is the hyper-immune globulins from snake venom immu-
nized horse [5]. But the cost of anti-venom does not make it a
readily accessible medication to tropical poorer regions. Therefore
around the world it is an emerging trend on experimenting other
possible antidotes for the snake envenomation. As a spectrum of
possibilities lie within many of eastern traditional medicines
extracted from plants, many of eastern herbal extracts are now
under the scientic limelight.
Several studies have explored efcacy of such remedies. Extracts
of Hydrocotyle javanica and Gloriosa superba gives 80e90% protec-
tion to mice treated with minimum lethal dose of venom (LD
99
)of
Naja nigricollis (Spitting Cobra) and has produced signicant
changes of membrane stabilization of human red blood cells
(HRBC) exposed to hyposaline-induced haemolysis [6]. In another
study, Andrographis paniculata and Aristolochia indica plant extracts
were tested for neutralizing activity against Echis carinatus (Saw-
scaled Viper) venom where both plant extracts have shown effec-
tive neutralization of venom induced lethal activity [7].Hibiscus
aethiopicus leaf extract completely stopped haemorrhagic activity
against the venom of Echis ocellatus (African Carpet Viper) and N.
nigricollis (Spitting Cobra) [8]. The methanolic root extracts of Vitex
negundo and Emblica ofcinalis extracts has signicantly antago-
nized the D. russellii and Naja kaouthia (Monocled Cobra) venom
induced lethal activity in both in vitro and in vivo studies with
neutralization of venom-induced haemorrhagic, coagulant, deb-
rinogenating and inammatory activities [9]. In another study,
snake venom neutralizing potential of Rauvola serpentina plant
extract was tested by in vitro and in vivo methods against D. russelli
venom. The in vivo assessment of LD
50
in D. russelli venom was
found to be 0.628
m
g/g. R. serpentina plant extract effectively
neutralized this venom lethality with an effective dose (ED) of
10.99 mg/3LD
50
of venom [10].
Sri Lanka being an oriental country inheriting a great indigenous
system of medicine, treating snakebites with herbal extracts is one
such practice that has been widely used by indigenous doctors.
Therefore, this study contributes towards the scientic evaluation
of the effectiveness of a traditional herbal preparation used in Sri
Lanka against N. naja and D. russelli venom toxicity and the char-
acterization of its ethnopharmacological properties. The practice of
this herbal preparation is conned to a late traditional medical
practitioner from the southern coastal region of the country, who
had inherited the medication through the family. The herbal
preparation had been applied as a topical treatment over the bite
site of the victims, who are at the early stages of envenomation by
N. naja and D. russelli. The consent of the medical practitioner's
family was obtained for the scientic evaluation of the herbal
preparation for this study. The ethical clearance was obtained from
the Institute of Biology, University of Colombo. To our knowledge,
this study provides the rst laboratory evidence for the venom
neutralizing ability of a herbal preparation from Sri Lankan indig-
enous medicine. Preliminary forms of this work were presented at
the 8th International Conference of General Sir John Kotelawala
Defence University and at the 2nd International Conference of
Traditional and Complementary Medicine [11,12].
2. Material and methods
2.1. Preparation of herbal extract
The herbal preparation was prepared by mixing together the
aqueous extracts, obtained by crushing 2.5 g each of leaves of
Sansevieria cylindrica, Jatropha podagrica, with a drop of the
extract of Citrus aurantiifolia fruit. S. cylindrica is also known as the
cylindrical snake plant, African spear or spear sansevieria. J.
podagrica is known by several English common names, including
Buddha belly plant, bottle plant shrub, gout plant, purging-nut,
Guatemalan rhubarb, and goutystalk nettlespurge. C. aurantiifolia
is known as the lime fruit. Fresh plant material of S. cylindrica, J.
podagrica and C. aurantiifolia were collected from home gardens
located off suburbs of Colombo in Kalutara district. The collected
species were identied and authenticated by taxonomists from
the Department of Plant Science, Faculty of Science, University of
Colombo Sri Lanka. The preparation was made as a fresh aqueous
extract on each day prior to testing. The volumes of the extracts
individually as well as in the mixture, and the pH of the nal
preparation, were measured at each preparation, in order to
maintain the consistency between preparations. The pH of the
nal preparation was 4.2.
2.2. Collection of venom samples
The venom samples of D. russelii and N. naja were collected from
captive animals housed in the herpeterium of Faculty of Medicine,
University of Colombo in September 2014. Samples from the two
species were pooled separately and were freeze dried and were
kept at 20
C until use in the experiments.
2.3. SDS polyacrylamide gel electrophoresis (SDS PAGE) of venom
The amount of protein in the freeze dried venom samples was
quantied by measuring absorbance at 280 nm wave length using
Bovine serum albumin (BSA) as a standard. A 35
m
g of freeze dried
venom of either D. russelii or N. naja, dissolved in sample buffer was
loaded on to a 12% polyacrylamide in the presence of Sodium
Dodecyl Sulphate and electrophoresed at 180V for 30 min [13].
Then the gels were stained with Coomassie R-250 for 45 min fol-
lowed by destaining with acetic acid and methanol, to visualize
venom protein groups after 12% SDS PAGE.
2.4. Statistical analysis
Statistical analysis was carried out using Minitab 17 software.
Sigmoidal dose-response curves for LD
50
was generated using
GraphPad Prism 4.03 (GraphPad Software,Inc.).
2.5. Chick embryo model for venom neutralizing activity of the
herbal preparation
Freeze dried venom of D. russelii and N. naja, at varying doses,
were reconstituted in PBS (7.4) to be impregnated on to 3 mm
diameter of Whatman no 1 lter papers and placed over the vi-
telline vein on the exposed yolk sac membrane of in vitro cultivated
5-day old chicken embryos [14]. Pathological symptoms induced by
each mentioned venom were closely monitored and recorded till
death over a period of six hours. The experiment was replicated
with each venom type incubated with the herbal preparation or the
anti-venom (as a positive control).
2.5.1. Analysis of snake venom and treatment with herbal
preparation and anti-venom
For the determination of LD
50
, venom on 5-day old chick em-
bryo, a gradient of 2
m
g, 4
m
g, 8
m
g, 16
m
g, 32
m
g, and 48
m
g of freeze
dried venom dissolved in distilled water was used in the above
procedure. The cut-off time for calculating number of embryo state
dead/alive was six hours after treatment.
M.M. Silva et al. / Journal of Ayurveda and Integrative Medicine xxx (2016) 1e72
Please cite this article in press as: Silva MM, et al., Characterization of Daboia russelii and Naja naja venom neutralizing ability of an
undocumented indigenous medication in Sri Lanka, J Ayurveda Integr Med (2016), http://dx.doi.org/10.1016/j.jaim.2016.10.001
The neutralizing ability of the D. russelii venom by the herbal
preparation was tested. It was determined by repeating the same
concentration gradient of venom which have been incubated with a
2
m
l of herbal preparation at 37
C for 15 min prior to application on
embryo, and comparing any shift in the LD
50
. Commercially avail-
able Anti-venom (Snake venom anti-serum IP by VINS bioproducts
limited) was used as the positive control in the 1neutralizing
dose (1167
m
g of anti-venom for 1
m
gofN. naja and D. russelii freeze
dried venom) as per the manufacturer's recommendations. Final
volume of each treatment added on the lter paper was always
maintained at 4
m
l. Results were statistically analyzed and the LD
50
was calculated using the Probit values [15] in Minitab 17.
2.6. Phospholipase A
2
(PLA
2
) neutralizing assay
The PLA
2
activity [16] was determined by egg yolk solution
diluted 50% by mixing egg yolk and 5 mM TBS (Tris Buffered Saline)
at pH 8 in 1:1 ratio. A 100
m
l portion of this diluent solution was
then mixed with 100
m
g of freeze dried venom reconstituted with
distilled water, alone or pre-incubated with 2
m
l herbal preparation,
at 37
C for 15 min. The nal volume of the venom was maintained
4
m
l. After 30 min incubation of the reaction mixtures the samples
were boiled in 100
C water for 2 min to stop the PLA
2
activity and
titrated with 12 mM NaOH in the presence of 4
m
l of phenol-
phthalein till the color of the mixture changes to slight pink. The
volume of titrated NaOH was recorded and the moles of liberated
fatty acids due to venom PLA
2
action on the phosphatidylcholine in
the egg yolk were calculated.
2.7. Proteolytic assay of Snake Venom Metalloprotease
A weight of 100
m
g of freeze dried N. naja venom or the venom
pre-treated with the herbal extract for 15 min at 37
C was incu-
bated in 1 ml of 50 mM TriseHCl (pH 8.5) containing 1e2mM
Calcium Chloride and 1% Casein for 30 min [17]. The reaction was
stopped by adding Trichloroacetic acid to a nal concentration of
1%. Then the mixture was centrifuged at 18,000 rpm for 5 min, and
the hydrolysis product of casein in the supernatant, produced due
to venom metalloprotease activity, was determined by measuring
absorbance at 280 nm.
3. Results and discussion
3.1. Visualization of the venom components using SDS PAGE
The venom proteins were separated on 12% SDS PAGE to identify
the protein families in venom. At least 12 prominent protein bands,
ranging from 3 to 188kD, were visible upon loading of 35
m
g of total
venom protein (Fig. 6). It was notable that, for same loading of
venom protein (35
m
g) from D. russelii or N. naja, the intensity of
protein bands of D. russelii being signicantly high. The composi-
tion of the venom varied signicantly among the two species.
Greater variation of the banding pattern of the two species was
signicant within 38 to 188kD region. Banding pattern from 3 to
28kD ranges was more likely to be shared among the two species.
Based on the molecular weights on SDS-PAGE, clusters of venom
protein bands could be categorized in to protein families as given in
the manual on snake vernom protein components [17]. When
compared with the characteristic venom protein proles given in
the above manual, these banding patterns revealed various protein
families, such as PLA
2
, phosphodiesterases, metalloproteases, etc.,
which could be recognized for the two venoms (Fig. 1).
3.2. Pathophysiological changes on the chick embryo by snake
venom toxins
The pathophysiological changes on the chick embryo upon
application of venom toxin, and with the treatment of the herbal
preparation, were observed. Application of 2
m
gD. russelii venom,
made the external capillary network of the embryo to show a clear
bleeding within 30 min. At this stage still the major blood vessels
remained intact (Fig. 2A and B). In the progressive stages, the size of
the blood vessels reduced and the heart beat started to drop with
bleeding in the capillary bed becoming very prominent and
intensied. Then death occurred within 1e2h(Fig. 2C). Incubating
the venom with herbal preparation reduced the above patho-
physiological effects. Rapid retraction of blood from the vascular
sac was not observed and the lack of bleeding from the capillary
was signicant. A slight drop in the heart rate could be observed
(Fig. 2D and E). However, the embryo could be kept alive till 24 h,
exceeding the 6 h experimental time (Fig. 2F). On the other hand,
the prescribed neutralizing dose of anti-venom (positive control)
did not show visible reduction of the effects generated by the
D. russelii venom.
The embryos applied with N. naja venom showed a different
pathophysiological pathway, as compared to what was observed
with D. russelii venom. With N. naja venom, the generation of a
localized hemorrhage near blood vessels under the disc impreg-
nated with venom was characteristic right after the application,
with no onset heart rate reduction (Fig. 3). In the presence of the
herbal preparation spreading of the localized haemorrhage around
the disc, impregnated with venom, was reduced with unaffected
heart beat (Fig. 2). Incubation of N. naja venom with prescribed
neutralizing dose of anti-venom did not prevent the appearance of
the hemorrhage in the positive control.
3.3. The analysis of the LD
50
of snake venom toxin on the chick
embryo and the effect of the treatment with herbal preparation
In order to quantify and compare the neutralizing ability of the
herbal preparation, the LD
50
values of D. russelii venom, alone and
after treating with the herbal extract or the anti-venom, were
calculated. This was done by calculating the percentage mortalityof
the 5-day old chick embryo at varying doses of D. russelii venom
(2
m
ge48
m
g) (Table 1).
For D. russelii venom, treated on the chick embryo, alone, a LD
50
value of 4.8 ±0.865
m
g of freeze dried powder of venom, which
contains a 667.2
m
g of total protein amount, was obtained (Fig. 4).
The LD
50
of D. russelii venom in the presence of 2
m
l of herbal
preparation was 17.64 ±1.35
m
g(Fig. 4) showing a marked increase
of nearly four times higher than the LD
50
of the venom alone. In the
presence of prescribed neutralizing dose of anti-venom, the LD
50
of
D. russelii venom could be increased up to 5.5 ±1. 3 5
m
g(Fig. 4).
The LD
50
values of the three tests showed that the neutralization
capacity of D. russelii venom by the herbal preparation is higher
than that of the commercially available anti-venom, at the recom-
mended neutralizing dose by the manufacturer (For 1
m
gofN. naja
and D. russelii freeze dried venom, neutralizing dose of Anti-venom
is 1167
m
g). The tested commercial anti-venom is a polyvalent
product raised against the Indian counterpart of D. russelii where
differences in venom could have arisen from the geographic vari-
ations, thereby making the anti-venom not as effective against the
local species. This has also been observed in clinical practice where
the commercial anti-venom being not very effective against snake
bites by D. russelii [18]; thus, stresses the need for alternative
treatment options for snake bites particularly against the D. russelii.
In this regard, the herbal preparation used in the current study is
M.M. Silva et al. / Journal of Ayurveda and Integrative Medicine xxx (2016) 1e73
Please cite this article in press as: Silva MM, et al., Characterization of Daboia russelii and Naja naja venom neutralizing ability of an
undocumented indigenous medication in Sri Lanka, J Ayurveda Integr Med (2016), http://dx.doi.org/10.1016/j.jaim.2016.10.001
promising, as it showed the clear presence of a potent neutralizing
compound that could counteract the toxicity of D. russelii venom.
3.4. The effect of delayed treatment with the herbal preparation on
its venom neutralizing ability
Having demonstrated the venom neutralizing activity by the
herbal preparation, it was interesting to nd out how delayed the
herbal treatment can be applied effectively, after the venom
toxicity is being ingested. In order to test this, the chick embryos
treated with a paper disk impregnated with 16
m
gofD. russelii
venom (zLD
50
of D. russelii in the presence of the herbal prepa-
ration) were subjected to treatment of either the 2
m
l of herbal
preparation or 10neutralizing dose of anti-venom, at varying
time intervals. Treatment was applied on to the same disk
impregnated with venom after 0 min, 5 min and 7 min.
Fig. 1. SDS PAGE of D. russelii and N. naja venom components. The separated protein components are categorized in to protein families according to their molecular weights
(denoted in boxes for D. russelii and in circles for N. naja). Note that the lower molecular weight components are predominant in N. naja venom, characteristic of elapid venom.
Fig. 2. Pathophysiological changes on the 5 day old chick embryo upon treating with 2
m
gofD. russelii venom. A) At 0 min B) After 30 min (Note: Visible signs of clear bleedings with
vessels of the capillary bed appearing blurred.At this stage still the major blood vessels remained intact.) C) Af ter 60 min (Note: Embryonic death with discoloration disappearance of blood
vessels). The effect of treating with herbal preparation on the venom toxicity eD) at 0 min E) In 1 h (Note: Rapid retraction of blood from the vascular sac was not observed and the lack
of the marginal hemorrhages and bleeding from the capillary was signicant.slight reduction of the thickness of the blood vessels). F) After 24 h (Note live embryo with normal vascular
network).
M.M. Silva et al. / Journal of Ayurveda and Integrative Medicine xxx (2016) 1e74
Please cite this article in press as: Silva MM, et al., Characterization of Daboia russelii and Naja naja venom neutralizing ability of an
undocumented indigenous medication in Sri Lanka, J Ayurveda Integr Med (2016), http://dx.doi.org/10.1016/j.jaim.2016.10.001
The embryos treated with venom alone caused death in
10 min. Treatment with herbal preparation or anti-venom given
after 5 min or 7 min increased the lifetime of the embryo to
25 min and 20 min respectively. Whereas, the embryos treated
with the herbal preparation and anti-venom at 0 min survived
for 6 h and 2 h respectively (data not shown). The observed
result is highly encouraging, where it showed that the herbal
extract could still be effective in treating snake bites, when the
treatment is applied with a time gap after the venom toxin
ingestion.
Fig. 3. Pathophysiological changes on the 5-day old chick embryo upon treating with 2
m
gofN. naja venom. A) After 10 min, (Note: the appearance of localized hemorrhage under the
disc impregnated with venom) B) After 25 min, (Note: blood vessels appeared to be reducing with withdrawal of blood from the vascular sac. This was followed by death, changing the color
of the embryo from healthy pink to pale white). The effect of treating with herbal preparation on the venom toxicity C) After 1 min, (Note: the reduction of the spread of the localized
hemorrhage beyond the disc) D) After 1 h, (Note: unaf fected heart beat and blood vessels and live embryo).
Table 1
Percentage mortality with varying doses of D. russelii venom.
Dose (
m
g) Log dose Number of affectd embrayos % mortality Probit value
Group 1 (D. russelii venom alone)
1 2 0.30103 0/3 0 3.47
2 4 0.60206 2/3 66.66 5.44
3 8 0.90309 2/3 66.66 5.44
4 16 1.20412 3/3 100 6.39
5 20 1.301 3/3 100 6.39
6 32 1.50515 3/3 100 6.39
7 48 1.681241 2/3 100 6.39
Group 2 (D. russelii venom in the presence of 2
m
l herbal preparation)
1 12 1.079181 0/4 0 3.46
2 16 1.20412 0/4 0 3.46
3 20 1.30103 1/5 80 5.84
4 22 1.342423 4/4 100 6.53
5 24 1.380211 4/4 100 6.53
Group 3 (D. russelii venom in the presence of 1neutralizing dose of anti-venom)
1 4 0.602059991 1/3 33.333 4.56
2 8 0.903089987 2/3 66.66 5.44
3 16 1.204119983 3/3 100 6.73
M.M. Silva et al. / Journal of Ayurveda and Integrative Medicine xxx (2016) 1e75
Please cite this article in press as: Silva MM, et al., Characterization of Daboia russelii and Naja naja venom neutralizing ability of an
undocumented indigenous medication in Sri Lanka, J Ayurveda Integr Med (2016), http://dx.doi.org/10.1016/j.jaim.2016.10.001
3.5. Direct analysis of PLA
2
activity inhibition
The effect on the Phospholipase activity, a known toxin in snake
venoms, of D. russelii and N. naja venoms was investigated using
fatty acid titration method. In this method, the fatty acids liberated
from the egg yolk lecithin by the proteolytic activity of venom PLA
2
was quantied by titrating with NaOH; thereby making the amount
of fatty acids liberated proportionate to the PLA
2
activity. The
amount of fatty acids released by PLA
2
from D. russelii was
0.276 mM, and the same from N. naja was 0.876 mM. In the pres-
ence of the herbal preparation, fatty acids released from D. russelii
venom dropped to 0.024 mM and from N. naja venom it dropped to
0.564 mM. The values show a clear decrease in the produced fatty
acid amounts from the egg yolk with pre-treatment of venom with
the herbal preparation, indicating an inhibition of PLA
2
enzyme
activity by the herbal preparation (Fig. 5).
Therefore, one possible mechanism for the observed neutral-
izing activity of the herbal preparation, as was evident with the
chicken embryos, could be through inhibiting PLA
2
enzyme in the
venom. PLA
2
is a venom toxin, which is known to induce a vast
range of pathological symptoms varying from neurotoxicity to
anticoagulation toxicity [17].
3.6. Proteolytic assay of snake venom metalloprotease (SVMPs)
Metalloproteases are a very important set of proteolytic en-
zymes found in venom that induces hemorrhages as it can digest
the proteins on the extracellular matrix. The observed hemorrhagic
activity around the disk impregnated with N. naja venom on the
chick embryo is consistent with its presence of metalloproteases in
the venom. The disappearance of the hemorrhage in the presence
of the herbal preparation is further consistent with the inhibition of
this metalloprotease activity by the herbal treatment.
Therefore, the activity of snake venom toxin, metalloprotease, in
the N. naja snake venom, was measured using an assay similar to
the PLA
2
assay. The hydrolysis products of casein, one of the sub-
strates of metalloproteases, were quantied as a measure of met-
alloprotease enzyme activity. In this assay N. naja snake venom
alone generated an activity of 0.069units/min. When incubated
with the herbal preparation it dropped to 0.019units/min, which
was almost the same as the activity shown by the negative control
test (0.013units/min) carried only with distilled water in the
absence of any venom (Fig. 6). Therefore the results showed a near
complete neutralization of N. naja venom metalloprotease activity
by the herbal preparation. Another mechanism of action for the
venom neutralizing activity by the herbal preparation could
therefore be through the inhibition of metalloprotease enzymes in
the N. naja venom.
4. Conclusion
The analyzed herbal preparation of S. cylindrica, J. podagrica and
C. aurantiifolia showed clear ability to neutralize the venom toxic-
ities of both D. russelii venom and N. naja. It further inhibited the
PLA
2
and metalloprotease enzyme activities of those venoms,
which give rise to a wide range of pathophysiological effects. Some
proteolytic activity by the herbal extract towards the venom pro-
teins was also evident, specically targeted on the protein families
with a molecular weight in the range of 28e188kD. Therefore the
neutralizing activity of the herbal preparation is suggestive to be
brought through the inhibition of PLA
2
or metalloprotease activities
and/or by the digestion of some of these protein toxins. This herbal
preparation highlights a potential treatment option for snake
venom bites as an alternative to currently practiced anti-venom
treatment. The current study therefore adds to the evidence for
alternative approaches for snakebite treatment using indigenous
knowledge on ethnopharmacology.
Author contributions
Madhushika M Silva eDeveloped the project proposal, con-
ducted all the experiments and drafted the initial manuscript.
Sampath S Seneviratne eGuided the project and corrected the
manuscript.
Devaka K Weerakoon eGuided the project.
Charitha L Goonasekara eGuided the project, supervised all
the experiments and corrected the manuscript.
Fig. 4. Dose response curve of D. russelii venom for mortality of 5-day old chick em-
bryos, following different treatments. Venom alone (blue line), Venom in the presence
of herbal preparation (red line) and Venom in the presence of 1neutralizing dose of
anti-venom (green line), LD
50
values for D. russelii, 4.8 ±0.865
m
g of freeze dried
powder, for D. russelii venom with 2
m
l of herbal preparation, 17.64 ±1.35
m
g, and for
D. russelii venom with anti-venom ¼5.5 ±1.35
m
g. (N ¼4)
Fig. 5. Phospholipase
2
(PLA
2
) activities of D. russelii and N. naja venom, in the presence
or absence of the herbal preparation. The enzyme activity is represented as the amount
of fatty acid released from egg yolk per minute. Note the decreased fatty acid release for
the herbal extract treated samples indicating inhibition of PLA
2
enzyme.(N¼3)
Fig. 6. SVMPs activity of N. naja venom in the presence and absence of herbal prep-
aration. The enzyme activity is represented in terms of the amount of hydrolyzed
product of 1% casein produced per minute. Note the reduction in the production of the
hydrolysis product of casein in the herbal extract treated samples to almost the same level
as in the negative control indicating complete inhibition of the enzyme (N ¼3).
M.M. Silva et al. / Journal of Ayurveda and Integrative Medicine xxx (2016) 1e76
Please cite this article in press as: Silva MM, et al., Characterization of Daboia russelii and Naja naja venom neutralizing ability of an
undocumented indigenous medication in Sri Lanka, J Ayurveda Integr Med (2016), http://dx.doi.org/10.1016/j.jaim.2016.10.001
Acknowledgement
The authors wish to thank the Department of Zoology, Univer-
sity of Colombo for providing necessary resources for the research
and Faculty of Medicine, Kotelawala Defense University, Ratmalana
for Laboratory facilities. The authors gratefully acknowledge Prof
Ariyaranee Gnanadasan for providing snake venom and Prof Rohini
Fernadupulle for providing Anti-venom samples.
References
[1] Kasthuriratne A, Wickremasinghe AR, de Silva N. The global burden of
snakebite: a literature analysis and modelling based on regional estimates of
envenoming and deaths. PLoS Med 2008;5:218.
[2] Sawroop S, Grab B. Snakebite mortality in the world. Bull World Health Organ
1954;10:35e76.
[3] Goonarathna C, De Silva P. Anti-venom for snakebite in Sri Lanka we need an
effective, low reactogenic, affordable and polyvalent AVS. The Ceylon Med J
2002;47:43e5.
[4] Wickramasinghe LJM. Recognising deadly venomus snakes from harmless
snakes of Sri Lanka. 1st ed. Colombo, Sri lanka: Ceylon Tea services PLC; 2014.
[5] WHO. Snakebites. 2009 [ONLINE] Available at: http://www.who.int/
neglected_diseases/diseases/snakebites/en/ [Last Accessed 3 April 2014].
[6] Kumarappan CI, Jaswanth A, Kumarasunderi K. Anti-haemolytic and snake
venom neutralizing effect of some Indian medicinal plants. Asian Pac J Trop
Med 2011 Sep;4(9):743e7.
[7] Meenatchisundaram S, Prajish, Parameswari G, Subbraj T, Michael A. Studies
on antivenom activity of Andrographis paniculata and Aristolochia indica plant
extracts against Echis carinatus venom. Internet J Toxicol 2008;6(1).
[8] Hasson SS, Al-Jabri AA, Sallam A, Al-Balushi MS, Mothana RAA. Antisnake
venom activity of Hibiscus aethiopicus L. against Echis ocellatus and Naja n.
nigricollis. J Toxicol 2010. Article ID 837864(8 pages).
[9] Kanoijia A, Chaudhari KS, Gotheca VK. Medicinal plants active against snake
envenomation. Int J Res Ayurveda Pharm 2012;3(3).
[10] James T, Dinesh MD, Uma MS, Vadivelan AS, Meenatchisundaram S,
Shanmugam V. In vivo and in vitro neutralizing potential of Rauvola
serpentine plant extract against Daboia russelli venom. Adv Biol Res 2013;7(6):
276e81.
[11] Silva MM, Seneviratne SS, Weerakoon DK, Goonasekera CL. Potential of an
herbal preparation used in the indigenous medicine to neutralize Naja naja
venom: example from wet zone, Sri Lanka. In: 8th International research
conference of Kotelawala Defence University, Colombo; 2015.
[12] Silva MM, Goonasekera CL, Seneviratne SS. Weerakoon DK.Daboia russelii
venom neutralizing ability of a herbal preparation used in indigenous medi-
cine in the wet zone of Sri Lanka. In: 2nd International conference of tradi-
tional and complementary medicine on health, Taiwan; 2015.
[13] Laemmli UK. Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature 1970 Aug 15;227(5259):680e5.
[14] Yalcin C, Shekhar A, Jinkya A, Rane A, Butcher JT. An ex-ovo chicken embryo
culture system suitable for imaging and microsurgery applications. J Vis Exp
2010 Oct;23(44):2154. http://dx.doi.org/10.3791/2154.
[15] Finney DJ. Probit analysis. Cambridge: Cambridge University Press; 1952.
[16] Guti
errez JM, Avila C, Rojas E, Cerdas L. An alternative in vitro method for
testing the potency of the polyvalent antivenin produced in Costa Rica. Tox-
icon 1988;26:411e3.
[17] Mackessy SP. Hand book of venoms and toxins of Reptiles. 1st ed. London:
CRC; 2009.
[18] Ariyarathnam CA, Sj
ostr
om L, Raziek Z, Kularatne SA, Arachchi RW,
Sheriff MH, et al. An open, randomized comparative trial of two antivenoms
for the treatment of envenoming by Sri Lankan Russell's viper (Daboia russelii
russelii). Trans R Soc Trop Med Hyg 2001;95(1):74e80.
M.M. Silva et al. / Journal of Ayurveda and Integrative Medicine xxx (2016) 1e77
Please cite this article in press as: Silva MM, et al., Characterization of Daboia russelii and Naja naja venom neutralizing ability of an
undocumented indigenous medication in Sri Lanka, J Ayurveda Integr Med (2016), http://dx.doi.org/10.1016/j.jaim.2016.10.001
... Existem vários relatos sobre o uso popular de plantas medicinais contra picada de serpente em todo o mundo, especialmente em regiões tropicais e subtropicais, como na Ásia, África e América do Sul (Silva et al., 2017;Upasani et al., 2018). Os povos rurais e tribais que vivem em áreas remotas dependem grandemente de medicamentos populares para o tratamento de picadas de quaisquer espécies de venenos (Sulochana et al., 2015). ...
... Atualmente, esses antídotos naturais utilizados na medicina popular tradicional ganharam ampla atenção de toxinologistas em todo o mundo, pois podem ser uma ferramenta para a projeção de inibidores contra as toxinas do veneno de serpentes. As principais vantagens das plantas antiofídicas são o baixo custo, fácil acesso, a estabilidade à temperatura ambiente e capacidade de neutralizar um amplo espectro de toxinas, incluindo o dano tecidual local (Butt et al., 2015;Silva et al., 2017). ...
Chapter
Full-text available
Accidents involving venomous snakes affect millions of people a year worldwide, resulting in a large number of deaths. In general, the inefficiency of antiophidic therapy means it can have disadvantages. In this respect, research involving the use of alternative methods, such as testing the potential of plants to neutralize snake venom, has becoming increasingly common. There are a number of reports on the use of medicinal plants for snakebites worldwide, especially in tropical and subtropical regions such as Asia, Africa and South America. Medicinal plants have long been used to counteract snake venom; a practice passed down through the generations in rural communities.The large variety of plant secondary metabolites is appealing to researchers and has led to the discovery of molecules that may be useful to human and animal health. As such, further research is needed to produce new therapies, including assessing target substances, formulating projects and improving current compounds to identify those that would best adapt to the specific needs of each location. KEYWORDS: Plant extract; Ophidism; Snake venom.
... Existem vários relatos sobre o uso popular de plantas medicinais contra picada de serpente em todo o mundo, especialmente em regiões tropicais e subtropicais, como na Ásia, África e América do Sul (Silva et al., 2017;Upasani et al., 2018). Os povos rurais e tribais que vivem em áreas remotas dependem grandemente de medicamentos populares para o tratamento de picadas de quaisquer espécies de venenos (Sulochana et al., 2015). ...
... Atualmente, esses antídotos naturais utilizados na medicina popular tradicional ganharam ampla atenção de toxinologistas em todo o mundo, pois podem ser uma ferramenta para a projeção de inibidores contra as toxinas do veneno de serpentes. As principais vantagens das plantas antiofídicas são o baixo custo, fácil acesso, a estabilidade à temperatura ambiente e capacidade de neutralizar um amplo espectro de toxinas, incluindo o dano tecidual local (Butt et al., 2015;Silva et al., 2017). (Molander et al., 2015). ...
... e shared protein families among different species indicate evolution from a common ancestry [28]. Observations of the current study are comparable with the earlier studies made on elapids and vipers [34][35][36][37][38][39]. So, SDS-PAGE can be used to screen an unknown questioned material to know if it is snake venom or not. ...
Article
Full-text available
Confirm and authentic identification of species is required for the implementation of wildlife laws in cases of illegal trafficking of snake venoms. Illegally trafficked snake venom might be misidentified with other drugs of abuse, and sometimes, the species of venom-yielding snake cannot be verified. Snake venoms from medically important snake species, Naja naja and Daboia russelii, were procured from Irula Snake Catcher’s Society, Tamil Nadu, India. Comparative analyses of both venoms were carried out using SDS-PAGE, LC-MS/MS, ICP-MS, and mtDNA analysis. The protein concentration of Naja naja and Daboia russelii venoms was 76.1% and 83.9%, respectively. SDS analysis showed a distinct banding pattern of both venoms. LC-MS/MS results showed proteins and toxins from 12 to 14 protein families in Naja naja and Daboia russelii venoms. Elemental analysis using ICP-MS showed a different profile of some elements in both venoms. mtDNA analysis of venoms using universal primers against Cyt b gene showed homology with sequence of Naja naja and Daboia russelii genes. The study proposed a template of various conventional and advanced molecular and instrumental techniques with their pros and cons. The template can be used by forensic science laboratories for detection, screening, and confirmatory analysis of suspected venoms of snakes. Clubbing of various techniques can be used to confirm the identification of species of snake from which the alleged venom was milked. The results can be helpful in framing charge-sheets against accused of illegal venom trafficking and can also be used to verify the purity and quality of commercially available snake venoms.
... Women of most of the tribal community had little knowledge of medicinal plants which act as antidotes against snakebite ( This study provided 41 species of antidote plants and their usage against snake bite. Among these plants, some plants were previously reported against snake bite (Prasad et al., 1987;Karuppusamy, 2009;Shanmugam et al., 2009;Revathi & Parimelazhagan, 2010;Jain et al., 2011;Alagesaboopathi, 2011;Nayak et al., 2020;Silva et al., 2017). Eight species of medicinal plant such as Ageratum racemosus, Brassica juncea, Centella asiatica, Cynodon dactylon, Lebelia nicotianifolia, Momordica dioica, Terminalia chebula, and Urginea indica were used for various ailments according to Gomes et al., 2010;Ayyanar et al., 2011, but the present study shows that herbs were the primary source of medicine followed by climber, tree, ...
... Crude venom was purchased from Irula Snake Catchers Society, Tamil Nadu, India. The crude venom was of Indian big four snakes i.e., Daboia russelii (LD 50 of 0.628 μg/g), Echis carinatus (LD 50 of 5 mg/kg), Naja naja (LD 50 of 0.7943 mg/kg) and Bungarus caeruleus (LD 50 of 0.2828 μg/g) [28][29][30][31]. HiLoad™ 16/600 Superdex™ 75 pg column was purchased from GE Healthcare life Sciences (Bucks, UK). ...
Article
Medicinal plants have always been used for snakebite treatment by traditional healers but they lack scientific evidence of action. However secondary metabolites of such plants have been explored and found to inhibit the toxic effect of venom proteins. Literature survey from 2003 to 2019 resulted in identification of 251 secondary metabolites with such properties. In silico docking studies of these metabolites with modelled structure of Daboxin P, a PLA2 from Indian Daboia russelii revealed that butein, mimosine and bakuchiol bind to Daboxin P with high affinity. Butein interacted with the catalytic triad but mimosine and bakuchiol interacted with the Ca2+ binding residues of Daboxin P. In vitro validation showed that the molecules inhibited the sPLA2 activity of Daboxin P. Interestingly, mimosine and bakuchiol could also neutralize the anti-coagulatory activity of Daboxin P. Further, it was observed that butein and mimosine could neutralize the PLA2 activity of Indian big four venoms dose dependently. On the other hand, mimosine and bakuchiol could also neutralize the pro/anti-coagulatory effect of big four crude venom. Thus, in this study, three molecules have been identified which can neutralize the PLA2 activity and pro/anti-coagulatory effect of Daboxin P as well as crude venom of big four.
... Snake bite is a major occupational health hazard and its burden is mostly confined to the poor communities in agricultural sector in Sri Lanka [10]. Antivenom is the only therapeutic agent in modern medicine available throughout the world for venom poison, but high cost does not make it readily accessible to the poorer community. ...
Article
Full-text available
Sri Lanka has unique heritage of own medical system handed down from generation to another over a period of 3,000 years. Native medical practitioners in different disciplines are inherited within a family tradition and their treatments are still effective and accepted by the Sri Lankan community. Nagaraja Guliya is one of traditional preparations prescribed as internal or external medicaments by the traditional physicians in the southern province in Sri Lanka since ten decades for poisons of animal origin. The study has been focused to standardize the herbo mineral formula in respect of quality, safety and analyze the potential of Nagaraja Guliya. The formulation consists of the eight herbo mineral ingredients; Aconitum ferox, Zingiber officinale, Myristica fragrans, Syzygium aromaticum, Mercury, Arsenic trisulfide, Copper sulphate and Luffa cylindrica. Authentication of the ingredients was carried out at Bandaranayaka Memorial Ayurveda Research Institute, Nawinna, Sri Lanka. Data has been gathered from Sri Lankan traditional manuscripts, Ayurveda authentic texts and different scientific journals. The results revealed that the Nagaraja Guliya contains secondary plant metabolites like Alkaloids, Tannins, Saponin and Phenols. The pH of the formulation was found to be 5.28 and is in acceptable range for oral administration and external application. Further, heavy metal contents of Mercury and Arsenic in the preparation was not reached to the harmful level to the human body. In Ayurveda view point; pharmacodynamic properties of the formula showed that maximum number of ingredients consist of Katu Rasa (pungent taste) Laghu Guna (Light property), Ushna Veerya (Hot in potency) and Katu vipaka with properties of pacification of Kapha and Vata dosha. Kushtagna, Vedanastapana, Raktashodaka, Shotahara and Vishahara. Hence, future studies should be planned to evaluate the existing data on traditional use of Nagaraja Guliya, along with experimental and clinical trials.
Article
Full-text available
Snakebite is an important cause of morbidity and mortality and is one of the major health problems in India. About 30000 to 40,000 persons die each year from venomous snake bite. Russell's viper or daboia (Viper russelli) appears to be the commonest cause of fatal snakebite in Southern India, Pakistan, Bangladesh, Sri Lanka, Burma and Thailand. Intravenous administration of anti-snake venom neutralizes the systemic actions, however, antiserum does not provide enough protection against venom induced hemorrhage, necrosis, nephrotoxicity and often develops hypersensitivity reactions. India has a rich tradition of the usage of medicinal plants. Many Indian medicinal plants are mentioned in Ayurvedic literature to treat snakebite victims and are used by many ayurvedic practioners as well as in rural areas by traditioners. So much research work has been conducted for anti-snake venom activity of herbal medicine as alternative for Anti Snake Venom. This article presents a review of such herbal drugs which are effectively neutralize the snake venom like vitex nigundo, Emblica officinalis, Hemidesmus indicus etc which were assayed in research laboratories. It is considered as a valuable source of natural products for development of medicines against venomous snake bite.
Article
Full-text available
To validate traditional claims of usefulness of the Indian plants in management of poisonous snakebite and evaluate the antivenom properties displayed by the alcoholic extracts of Andrographis paniculata (A. paniculata), Crateva magna (C. magna), Gloriosa superba (G. superba) and Hydrocotyle javanica (H. javanica). These plants were collected, identified and the extracts were prepared by using conventional Soxhlet ethanol extraction technique. The venom neutralization activity was accessed in mice (20-25g) and number of mortalities was observed against clinically important snake (Naja nigricollis) venom. Present study also deals with in vitro membrane stabilizing activity of these plants against hyposaline induced human red blood corpuscles (HRBC). Extracts of H. javanica and G. superba gave 80 % and 90 % protection to mice treated with minimum lethal dose of venom (LD(99)). These two plants showed significant neutralization effect against the venoms of Naja nigricollis venom. H. javanica and G. superba (25-100 mg/mL) produced significant changes of membrane stabilization of human red blood cells (HRBC) exposed to hyposaline-induced haemolysis. We conclude that probably due to presence of various phytochemicals plays an important role in the anti-venom potential of these Indian medicinal plants against Naja nigricollis venom. The above observations confirmed that A. paniculata, C. magna, G. superba and H. javanica plant extracts possess potent snake venom neutralizing capacity and could potentially be used as an adjuvants for antivenin therapy in case of snakebite envenomation, especially against the local effects of cobra venoms.
Article
Full-text available
Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.
Article
Full-text available
The objective of the study is to investigate whether the Hibiscus aethiopicus L. plant has neutralization activity against venoms of two clinically important snakes. The H. aethiopicus was dried and extracted with water. Different assays were performed to evaluate the plant's acute toxicity and its anti-snake venom activities. The results showed that H. aethiopicus extract alone had no effect on the viability of C(2)C(12) muscle cells, but significantly (P < .05) protected muscle cells against the toxic effects of E. ocellatus venom at 55, 150, and 300 mug/mL. The maximum protective effect of the extract was exhibited at 75 mug/mL. The extract significantly (P < .001) inhibited the cytotoxic effects of E. ocellatus venom at 300 mug/mL. All rabbits (n = 10) and guinea pigs (n = 10) were alive after the two weeks of given the lethal dosage 16 g/Kg of the H. aethiopicus extract herbal solution. No abnormal behaviour was observed of both groups of animals. All guinea pigs (n = 3) treated with venoms alone (5 mg/kg) died. However, all guinea pigs (n = 21) treated with venom (5 mg/kg) and the extract (400 to 1000 mg/kg) survived. Guinea pigs (n = 3) treated with Naja n. nigricollis venom alone (2.5 mg/kg) and guinea pigs (n = 21) venom with the extract (400 to 1000 mg/kg) died. The H. aethiopicus completely (100%) blocked the haemorrhagic activity of E. ocellatus in the egg embryo at 3.3 mg/mL of extract. These findings suggest that H. aethiopicus may contain an endogenous inhibitor of venom-induced haemorrhage.
Article
Full-text available
Envenoming resulting from snakebites is an important public health problem in many tropical and subtropical countries. Few attempts have been made to quantify the burden, and recent estimates all suffer from the lack of an objective and reproducible methodology. In an attempt to provide an accurate, up-to-date estimate of the scale of the global problem, we developed a new method to estimate the disease burden due to snakebites. The global estimates were based on regional estimates that were, in turn, derived from data available for countries within a defined region. Three main strategies were used to obtain primary data: electronic searching for publications on snakebite, extraction of relevant country-specific mortality data from databases maintained by United Nations organizations, and identification of grey literature by discussion with key informants. Countries were grouped into 21 distinct geographic regions that are as epidemiologically homogenous as possible, in line with the Global Burden of Disease 2005 study (Global Burden Project of the World Bank). Incidence rates for envenoming were extracted from publications and used to estimate the number of envenomings for individual countries; if no data were available for a particular country, the lowest incidence rate within a neighbouring country was used. Where death registration data were reliable, reported deaths from snakebite were used; in other countries, deaths were estimated on the basis of observed mortality rates and the at-risk population. We estimate that, globally, at least 421,000 envenomings and 20,000 deaths occur each year due to snakebite. These figures may be as high as 1,841,000 envenomings and 94,000 deaths. Based on the fact that envenoming occurs in about one in every four snakebites, between 1.2 million and 5.5 million snakebites could occur annually. Snakebites cause considerable morbidity and mortality worldwide. The highest burden exists in South Asia, Southeast Asia, and sub-Saharan Africa.
Article
Methanolic extracts of Andrographis paniculata and Aristolochia indica plants were tested for antivenom activity against Daboia russelli venom. Both plant extracts effectively neutralized the D. russelli venom induced lethal activity. About 0.15 mg of A. paniculata and 0.14 mg of A. indica plant extracts were able to completely neutralize the lethal activity of 2LD50 of D. russelli venom. Various pharmacological activities including edema, haemorrhagic, coagulant, fibrinolytic and phospholipase activities were studied and these pharmacological activities were significantly neutralized by both the plant extracts. The above observations confirmed that A. paniculata and A. indica plant extracts possess potent snake venom neutralizing capacity and could potentially be used for therapeutic purposes in case of snakebite envenomation.
Article
biochemists, toxicologists, physicians, clinicians and epidemiologists, and informed laypersons interested in the biology of venomous reptiles, the biochemistry and molecular biology of venoms, and the effects and treatment of human envenomation. This book examines the topic generally, provides an overview of the current taxonomy of these reptiles, explains the similarities and differences in the venom delivery apparatus in different groups of reptiles, reviews state-of-the-art knowledge about specific venom components and their action, and summarizes effects of envenomation and treatment in humans on different continents. Written by experts from 12 countries, the book has both a broad perspective and international relevance. Unlike previous books addressing venoms, this volume bridges several very different areas in modern biology and provides a synthesis of current knowledge about venoms and venomous reptiles. The wealth of illustrations, including an 8 page full color insert, present a view of reptile toxinology from the whole animal to the glands producing venoms to the molecular models and the mechanisms of actions of the toxins themselves. www.crcpress.com
Article
J. M. Gutiérrez, C. Avila, E. Rojas and L. Cerdas. An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica. Toxicon 26, 411 - 413, 1988. - The ability of several batches of polyvalent antivenom to neutralize indirect hemolytic activity of Bothrops asper venom was studied using a sensitive plate test. All samples of antivenom tested effectively neutralized this activity. A highly significant correlation was observed between neutralization of indirect hemolysis and neutralization of lethal activity. This simple and sensitive in vitro test could be used to monitor antibody levels in horses immunized to produce polyvalent antivenom.
Article
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.