Volume 7, Issue 12, December – 2022 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Snake-Antivenom Activities of Aqueous

Extracts of Amaranthus spinosus L. Against
Naja subfulva Venom
Kennedy K. Yego1, Eliud N.M. Njagi2, George O. Orinda3, Joseph K. Gikunju4
1.
Department of Biochemistry, Microbiology, and Biotechnology, Kenyatta University
2.
Department of Biochemistry, Microbiology, and Biotechnology, Kenyatta University
3.
Department of Biochemistry, Microbiology, and Biotechnology, Kenyatta University
4.
Department of Medical Laboratory Sciences

Jomo Kenyatta University of Agriculture and Technology

Abstract:- The present management regime of snake bites The current crisis in the supply of antivenom to Sub-
requires the use of anti-venom immunoglobulins (Igs). Saharan Africa has prompted a need to look for alternative
However, these anti-venoms have the limitations of being antivenoms that are either synthetic or natural, which will
expensive, requiring cold storage facilities, and having substitute or complement the animal-derived anti-venoms
problems of hypersensitivity reactions in some [4]. Herbal remedies for snakebite treatment are readily
individuals. Amaranthus spinosus plant medicine has available in rural areas, but most of these phytochemicals
traditionally been used in managing snake bites in Uasin- used in various parts of the world against snake envenomation
Gishu County, Kenya. However, its efficacy has not been are seldom studied [5]. This study, therefore, aims at
scientifically validated. Therefore, this study aimed to determining in vivo and in vitro, using the mouse model, the
determine in vivo and in vitro the efficacy of the medicinal efficacy and safety of Amaranthus spinosus medicinal plant
plant against Naja subfulva venom using the mouse model, used to treat snake bites in parts of Uasin-Gishu County,
agarose-erythrocyte-egg yolk gel plate, and human- Kenya.
citrated plasma methods. The antivenom studies suggest
that the aqueous plant extracts possess antivenom activity II. MATERIALS AND METHODS
against N. subfulva venom both in vivo and in vitro. In
conclusion, this study confirmed that aqueous extracts of A. Collection and Identification of Medicinal Plants
Amaranthus spinosus were effective in neutralizing in vivo Whole plant parts were collected from their natural
and in vitro snake venom activity of Naja subfulva. habitats using a local name (Chepkerta-Nandi) and plant
identity authenticated as per the International Code of
Keywords:- Median effective dose (ED50), Median lethal dose Botanical Nomenclature by an acknowledged authority in
(LD50), Phospholipase A2, Toxicity, Phytochemicals. taxonomy from the East African herbarium at the National
Museums of Kenya. Coordinates for the location of collection
I. INTRODUCTION points for the plant were taken and recorded as
E35.1381903N.5550996.
Snake envenomation is a significant medical concern in
tropical countries and many other parts of the world. B. Preparation of Plant Extracts
Globally, the incidence of clinically significant snakebite One hundred grams (100 g) of shade-dried powdered
envenomation has been calculated at above 421000 and plant materials were mixed with 2000 ml double distilled
20000 deaths annually [1]. The only validated and water in 2500 ml screw-caped conical flasks and kept in a
recommended therapy for treating snake bite envenomation water bath at 60⁰C for 2h. The extracts were then filtered by
effectively is through antivenoms; a serum separated from the using a muslin cloth and then re-filtered using Whatman filter
blood of hyperimmunized horses [2]. Antivenom neutralizes paper No. 1. The filtrate was then stored in a refrigerator at
the effects of snake venom envenomation stopping further 40C until they were freeze-dried with a lyophilizer. Freeze-
damage to the body, but it does not reverse the damage dried materials were weighed and kept in a freezer at -200C
already done. Other limitations of snake antivenom therapy until use.
include its unavailability, high cost, inadequate storage
facilities in developing countries, and difficulty in identifying C. Venom and Experimental Animals
the snakes. Some individuals may also suffer from an Lyophilized snake venom powder of Naja subfulva was
immediate hypersensitivity reaction after antivenoms obtained from Bio-Ken snake farm, Watamu, Malindi, and
treatment [3]. stored at 4⁰C. Swiss albino mice of either sex (18-20 g) were
used for efficacy and toxicity studies. The mice were bred

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Volume 7, Issue 12, December – 2022 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
under standard laboratory conditions of 12 hours exposure to Aliquots of 10 µl of the mixtures were added to wells in
light, 25 ± 2ºC temperature, and 35-60 % humidity in the agarose-egg yolk-sheep erythrocyte gels. Control samples
animal houses of the Departments of Biochemistry, contained venom without plant extracts. Plates were then
Microbiology and Biotechnology and Medical Laboratory incubated at 37⁰C for 20 h. Neutralization was expressed as
Sciences (MLS) of Kenyatta University and Jomo Kenyatta the plant extracts/venom’s MIHD mixture that reduced the
University of Agriculture and Technology respectively. They diameter of the hemolytic halo by 50 % when compared to
were fed with standard mice pellets from Unga Feeds Limited the effect induced by venom’s MIHD alone.
and provided with water ad libitum.
 Procoagulant Activity
D. In Vivo Venom-Neutralization Potency Tests of the The procoagulant activity was assayed according to the
Aqueous Plant Extracts method described by Theakston & Reid [7] and as modified
The only validated means of assessing venom toxicity by Laing et al. [8]. Various amounts of venom dissolved in
and antivenom neutralizing efficacy by manufacturers and 100 µL PBS (pH 7.2) were added to human citrated plasma
regulatory authorities is by determining venom lethality at 37⁰C. Coagulation time was recorded and the minimum
(LD50) and antivenom neutralizing capacity (ED50) [6]. coagulation dose (MCD) was determined as the venom dose
which induced clotting of plasma within 60 seconds. Plasma
 Venom Median Lethal Dose (LD50) Assay incubated with PBS alone served as a control. In
The median lethal dose (LD50) of Naja subfulva venom neutralization assays, a constant dose of venom was mixed
was assayed as per the method developed by. Various doses with various dilutions of plant extracts. The mixtures were
of venom dissolved in 0.2 ml of physiological saline were then incubated for 30 minutes at 37⁰C. 0.1 mL of the mixture
injected into the tail vein of mice, using groups of 5 mice for was then added to 0.3 mL of citrated plasma and the clotting
each venom dose. The LD50 was estimated by probit analysis times were recorded. In control tubes, plasma was incubated
at a 50% probability of deaths occurring within 24h of venom with venom or plant extracts. Neutralization was expressed as
injection. an effective dose (ED), defined as the plant extracts/venom
dose (MCD) at which the clotting time increased three times
 Aqueous Extracts Median Effective Dose (ED50) Assay when compared with the clotting time of plasma incubated
The venom-neutralizing potential of aqueous extracts of with two times MCD of venom alone.
the five selected plants was determined against 2LD50
(“challenge dose”) of Naja subfulva venom. Various doses of F. Qualitative Phytochemical Screening of the Aqueous
plant extracts (100, 200, 300, 400, and 500 mg/kg body Plant Extracts
weight) were mixed with 2LD50 of Naja subfulva venom The presence or absence of phytochemicals in the
sample in a total of 0.2 ml and incubated at 37⁰C for 30 aqueous plant extracts was carried out using standard
minutes and injected intravenously into mice. Control mice methods [10, 11].
received a mixture of venom “challenge dose” with
physiological saline solution alone to confirm that the venom G. Data Management and Statistical Analysis
“challenge dose” induces 100 % lethality. Five mice were Data were recorded in the Laboratory notebook and then
used for each antivenom dose. The median effective dose entered into Excel Spreadsheet for cleaning after which it was
(ED50) was calculated from the number of deaths occurring exported to MINITAB 18 for statistical analysis LD50 and
within 24h of venom/antivenom mixture injection by probit ED50 were determined by probit analysis.
analysis. The lower the ED50 value, the higher the
neutralizing ability of the antivenom [8]. H. Ethical Approval
This study was approved by Kenyatta University Ethics
E. In Vitro Venom-Neutralization Potency Tests of the Review Committee and licensed by the National Commission
Aqueous Plant Extracts for Science, Technology, and Innovation (NACOSTI) of
Kenya (license number NACOSTI/P/22/15100).
 Phospholipase A2 Activity
Phospholipase A2 activity was determined using an III. RESULTS
indirect hemolytic assay on an agarose-erythrocyte-egg yolk
gel plate as per the methods described by Gutiérrez et al.[9]. A. Lethality of Naja Subfulva Venom and in Vivo
Increasing doses of N. subfulva venom were added into 3mm Neutralization Potency of the Five Aqueous Plants
wells in agarose gels (0.8 % in PBS, pH 8.1) containing 1.2 Extracts in Mice
% sheep erythrocytes, 1.2 % egg yolk as a source of lecithin The lethality of Naja subfulva venom after its
and 10 mM CaCl2. Slides were then incubated at 37⁰C intravenous injection in mice through the tail vein is reported
overnight and the diameters of the hemolytic halos were in Table 1. Results indicate that after intravenous injection of
measured. Control wells contained 15 µl of physiological various doses of Naja subfulva venom in mice via the tail, the
saline. The minimum indirect hemolytic dose (MIHD) LD50 of the venom was found to be 0.99 µg/g body weight
corresponds to a venom dosage, producing a hemolytic halo (Figure 1). The neutralization potency of the aqueous extract
of 11 mm diameter. The efficacy of plant extracts in of A. spinosus was determined by mixing various doses of the
neutralizing the Phospholipase A2 activity was determined by plant extract with 2LD50 (2 µg/g body weight) of venom
mixing a constant dose of venom (MIHD) with various doses sample and incubating the mixture at 37⁰C for 30 minutes
of plant extracts and incubating for 30 minutes at 37⁰C. before intravenous injection into mice. Results indicate that

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Volume 7, Issue 12, December – 2022 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
the ED50 obtained for the aqueous extracts was 87.53mg/kg Survival Plot for Mice killed
body weight (Table 2; Figure 2). Normal - 95% CI
Probit Data - ML Estimates

1 00 Table of Statistics
Table 1: Median lethal dose (LD50) of Naja subfulva venom Mean 1 .9421 7
StDev 0.400064
in mice 80
Median 1 .9421 7
IQR 0.539678

Group Dose (µg/g Mortality % Log

60
body weight) (24hr) Death dose

Percent
1(Control) 0.2 ml saline 0/5 0 - 40

2 2.5 5/5 100 0.40 20

3 2.0 5/5 100 0.30

0
4 1.5 3/5 60 0.18 1 .0 1 .5 2.0 2.5 3.0

5 1.0 2/5 40 0.00 Log dose

6 0.5 1/5 20 - Fig 2: The median effective dose (ED ) for Amaranthus
50
0.30 spinosus

Mean of log dose (Log ) = 1.94217

10

1.94217
ED = 10 =87.53mg/kg body weight
50

B. In vitro venom neutralization potency of the five aqueous

plants extracts

 Phospholipase A2 Activity
1.5µg of N. subfulva venom produced 11mm diameter
hemolytic halos, which is considered 1U (U/10μg) as well as
the Minimum Haemolytic Dose (MHD). This indicates that
N. subfulva venoms possess the enzymes (phospholipase A2)
that can lyse sheep RBCs. Incubating different concentrations
Fig 1: SNAKE VENOM LD of aqueous extracts of Amaranthus spinosus from 100-500mg
50 with 1.5µg of N. subfulva venom, it was found that the extract
prevented hemolysis by a minimum of 57.3% at 100mg/kg to
Mean of log dose (Log10) = -0.0051612 a maximum of 66.4% at 500mg/kg ranging from 4.7±0.08 to
3.7±0.10mm diameter (Tables 3 and 4)
-0.0051612
LD = 10 = 0.99µg/9g body weight Table 3: The effect of N. subfulva venom (PLA2) induced
50
hemolysis on sheep RBCs plate (n₌3)
Venom(µg) Haloes(mm)
Table 2: Effect of intravenous administration of a 0 (control) 3.00±0.00
preincubated mixture of 2LD50 (2 µg/g body weight) Naja 2.5 13.00±0.20
subfulva venom and the aqueous plants extract to mice 2.0 12.10±0.20
Group Dose (mg/kg Mortality % Log 1.5 11.03±0.25
body (24 hr) Survival dose 1.0 7.03±0.12
weight) 0.5 6.03±0.06
extract
Amaranthus Table 4: The effect of aqueous extract of Amaranthus
spinosus spinosus on N. subfulva venom (PLA 2) induced hemolysis
1 0 (venom 5/5 0 - on sheep RBCs plate
Control only) Treatment
2 100 2/5 60 2.00 Venom (1.5µg) + Hallos in mm % inhibition
3 200 1/5 80 2.30 extract
4 300 1/5 80 2.48 100mg/kg 4.7±0.08 57.30
5 400 0/5 100 2.60
200mg/kg 4.6±0.06 58.20
6 500 0/5 100 2.70
300mg/kg 4.4±0.10 60.00
400mg/kg 3.9±0.13 64.50
500mg/kg 3.7±0.10 66.40

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Volume 7, Issue 12, December – 2022 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
 Procoagulant Activity requires premixing of the venom with the test antivenom
No activity was observed when Naja subfulva venom before intravenous injection into the animal [16]. This,
was tested for procoagulant activity using the protocols however, does not replicate the clinical situation where the
described by Theakston and Reid [7]. events occur at different times, that is, envenomation
followed by antivenom therapy. Following this protocol,
C. Phytochemical Screening neutralization studies on the plant extracts showed that the
Phytochemical analysis of aqueous plant extracts aqueous extracts of Amaranthus spinosus exhibited varying
revealed the presence of alkaloids, tannins, terpenoids, and abilities to neutralize the lethality induced by N. subfulva
saponins in aqueous Amaranthus spinosus. The results are venom in a dose-dependent manner. Since snake venom is
replicated in table 4 mainly made up of proteinaceous compounds (approximately
90-95%) mainly enzymes, the most likely mechanism for the
Table 4: Phytochemical analysis of aqueous extracts of plant extracts’ ability to neutralize N. subfulva venom could
Amaranthus spinosus be due to the ability of the extract’s triterpenoids, terpenoids,
Phytochemicals Amaranthus spinosus polyphenolic, tannins and tannin like substances to bind
venom proteins resulting in inhibition and precipitation of the
Alkaloids +++ venom proteins [17].
Tannins ++
Cardiac ++ Naja subfulva venom showed the presence of
glycosides phospholipase A2 enzymes by producing hemolytic haloes in
Steroids - indirect hemolytic assays due to the formation of
phospholipid hydrolysis products such as lysophospholipids
Triterpenoids -
and free fatty acids which are lytic. Different doses of the
Flavonoids +++ aqueous extracts inhibited PLA2 in a dose-dependent manner.
Terpenoids + This inhibition may be due to the extracts’ phytochemicals
Phenols ++ such as flavonoids, phenols, terpenoids, and quinonoid
Flavones - activity which have been documented to deactivate snake
Saponins ++ venom constituents [18]. Plant secondary metabolites react
KEY: +++ High presence; ++ Moderate presence; + Trace; - and sequester phospholipases and pose a hindrance in the
Negative results binding of the latter to their target site (s) hence reversing the
anticoagulation action of phospholipase A2 [19].
IV. DISCUSSION
The procoagulant activity was studied using human-
Snakebite is a major public health hazard that leads to a citrated plasma but no coagulation of the plasma was
high mortality rate worldwide. World Health Organization observed. This was in agreement with studies done by
(WHO) estimates that 2.5 million snakebites occur Suntravat [20], which showed that N. subfulva venom had no
worldwide each year and out of these, 125,000 are fatal [12]. significant activity on both procoagulant and anticoagulant
Snake antivenoms are currently the only validated antidotes activities. Naja subfulva toxic phospholipase A2 probably
for snake envenomation, but they have several limitations affects coagulation due to its interaction with plasma
which include hypersensitivity reactions among some phospholipids.
patients and inefficient supply systems leading to their
unavailability in rural areas. Although the use of plants V. CONCLUSIONS
against the effects of snake bites has long been recognized,
more scientific attention has only been given to this area of i) The aqueous plant extracts of Amaranthus spinosus
research in the last 20 years [13]. The present study examined demonstrated significant antivenom activity both in in vivo
antivenom potential, phytochemical composition, mineral and in vitro assays.
elements composition, and acute and sub-acute toxicity ii) Qualitative phytochemical analysis of the aqueous plant
effects of aqueous extracts of Amaranthus spinosus. The most extracts showed the presence of tannins, cardiac glycosides,
important step in determining the antivenom potential of phenols, triterpenoids, terpenoids, saponins, alkaloids, and
plant extracts is the pre-clinical testing using in vivo and in flavonoids that could have contributed to the plant’s ability to
vitro methods to assess their neutralizing potential against a neutralize snake venom.
wide range of venom effects [14].
DATA AVAILABILITY
The only validated protocol for assessing venom
toxicity and antivenom-neutralizing potency by both The quantitative and qualitative data used to support the
manufacturers and regulatory authorities is the test for findings of this study are included in the article.
determining venom lethality (LD50) and antivenom-
neutralizing capacity (ED50) [15]. In this study, therefore, in CONFLICT OF INTEREST
vivo and in vitro pharmacological tests for venom lethality,
procoagulant activity, and phospholipase activity (PLA2) The authors declare that there is no conflict of interest
caused by Naja subfulva venom were investigated. The regarding the publication of this paper.
design for the conventional antivenoms rodent ED50 tests

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Volume 7, Issue 12, December – 2022 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
FUNDING STATEMENT [12]. Ramos H.R., Ho P.L. (2015). Developing Snake
Antivenom Sera by Genetic Immunization: A Review,
The authors wish to state that no funding/grant was in Gopalakrishnakone P., Faiz A., Fernando R.,
received from any organization or any other supporting body Gnanathasan C.A., Habib A.G., Yang C.C. Clinical
for this research at any stage. The research was financed by Toxinology in Asia Pacific and Africa, Toxinology. 2;
the researcher’s own savings and resources. 401–414. Springer Dordrecht.
[13]. Rita P., Datta A.K., Mandal N., Benoy G.K, Sandip
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