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Toxin Reviews
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Neutralization of Bitis arietans venom-induced
pathophysiological disorder, biological activities
and genetic alterations by Moringa oleifera leaves
Babafemi Siji Ajisebiola, Solomon Rotimi, Ullah Anwar & Akindele
Oluwatosin Adeyi
To cite this article: Babafemi Siji Ajisebiola, Solomon Rotimi, Ullah Anwar & Akindele Oluwatosin
Adeyi (2021) Neutralization of Bitis�arietans venom-induced pathophysiological disorder, biological
activities and genetic alterations by Moringa�oleifera leaves, Toxin Reviews, 40:4, 847-858, DOI:
10.1080/15569543.2020.1793780
To link to this article: https://doi.org/10.1080/15569543.2020.1793780
View supplementary material Published online: 23 Jul 2020.
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RESEARCH ARTICLE
Neutralization of Bitis arietans venom-induced pathophysiological disorder,
biological activities and genetic alterations by Moringa oleifera leaves
Babafemi Siji Ajisebiola
a
, Solomon Rotimi
b
, Ullah Anwar
c
and Akindele Oluwatosin Adeyi
d
a
Department of Zoology, Osun State University, Oshogbo, Osun State, Nigeria;
b
Department of Biochemistry, Covenant University,
Ota, Osun State, Nigeria;
c
Department of Biosciences, COMSATS University Islamabad, Islamabad, Pakistan;
d
Animal Physiology Unit,
Department of Zoology, University of Ibadan, Ibadan, Nigeria
ABSTRACT
Bitis arietans venom (BAV) is known to cause various pathophysiological disorders by altering
the cellular inclusions and enzymatic activities of different organs. Moringa oleifera leaf has been
reportedly used for treatment of snake envenoming but there is no information on its neutraliz-
ing potentials against Bitis arietans venom. This study investigated the antivenin activity of etha-
nol crude extract of M. oleifera leaf on B. arietans envenomed rats and its inhibitory effects on
some biological activities of the venom. The lethal dose (LD
50
) of BAV was estimated at 1.5 mg/
kg
1
. BAV induced various toxic effects in the in vivo study however, treatment with M. oleifera
leaf extract (MOLE) ameliorated BAV-induced hypernatraemia and hypercalcemia. Acute anemia
observed in untreated envenomed rats was reversed after treatment with various concentrations
of MOLE with a significant (p<0.05) dose dependent increase in hematological indices. Liver
damage in untreated envenomed rats as indicated by higher concentration of serum liver
enzymes and higher concentration of antioxidant enzymes were significantly (p<0.05)
decreased in MOLE treated rats. Also, BAV exhibited hemorrhagic, hemolytic and coagulating
activities which were inhibited in a dose dependent manner by MOLE. A mild DNA fragmenta-
tion noticed in tissues of the heart of untreated envenomed rats was ameliorated in the MOLE
treated rats. Results obtained in this study indicated that M. oleifera leaf have antivenin activity
against B. arietans venom induced toxicities and underscores its use in folk medicine for the
treatment of snake bites.
ARTICLE HISTORY
Received 13 February 2020
Accepted 5 July 2020
KEYWORDS
Snakebite; antivenin;
Moringa oleifera; Albino
Wista rats; DNA
fragmentation; Bitis arietans
1. Introduction
Snakebite is a neglected public health problem that
causes tens of thousands of morbidity and mortality
each year mostly in rural communities of tropical
regions and sub-Sahara Africa (World Health
Organization 2010). Globally, up to 5.4 million snake-
bite incidence occur yearly with an estimate of 1.8
and 2.7 million cases of envenoming, resulting in
81 000–138 000 mortality and 400 000 cases of per-
manent disabilities (Guti
errez et al. 2017). In West
Africa (Nigeria), 320 000–330 000 DALYs (Disability-
Adjusted Life Years) are lost to snakebite envenoming
yearly (Habib et al. 2015).
Snakebite envenoming is a medical emergency that
results from contact with snake venoms which contain
complex mixtures of many enzymes, non-enzymatic
polypeptide toxins and nontoxic proteins with a wide
spectrum of biological activities (Badr et al. 2012).
Snake venom is regarded as the most complex of all
animal toxins containing different enzymatic and non-
enzymatic components (Gomes et al. 2010). The path-
ophysiologic basis for morbidity and mortality is the
disruption of normal cellular functions which depends
on the qualitative and quantitative distribution of dif-
ferent enzymes and toxins in snake venoms (Awadh
et al. 2014).
In Nigeria, incidence of snakebite is mostly common
in northern region and venomous species responsible
for snake bite fatalities in these areas are Echis ocella-
tus, Bitis arietans, Naja haje, Causus maculatus and
Naja nigricollis (Yusuf et al. 2015). Bitis arietans also
called puff adder is a viper, a specie considered to be
of high medical importance in Nigeria (Yusuf et al.
2015) and their venom is one of the most toxic of the
Viperidae family (Barlow et al. 2013). Bitis arietans
occupies densely populated habitats throughout the
savannah areas of sub-Saharan Africa and in Nigeria
(World Health Organization 2010, Barlow et al. 2013).
CONTACT Babafemi SijiAjisebiola dbabslinks@yahoo.com Department of Zoology, Osun State University, Oshogbo, Osun State, Nigeria
Supplemental data for this article can be accessed here.
ß2020 Informa UK Limited, trading as Taylor & Francis Group
TOXIN REVIEWS
2021, VOL. 40, NO. 4, 847–858
https://doi.org/10.1080/15569543.2020.1793780
Bitis arietans are widely observed to contribute to the
estimated 43,000 deaths from snakebite reported in
Africa yearly (Kasturiratne et al. 2008) this makes B.
arietans a significant public health concern.
Bitis arietans venom (BAV) is a rich source of protein
toxins exhibiting different biological effects such as
hemorrhagic, cytotoxic, hemolytic, myotoxic and pro-
teolytic activities on envenomed victims or prey
(Biswajit and Sivaraman 2013). The venom of this spe-
cie is highly potent and typically interferes with the
hemostatic system resulting into a severe local and
systemic effects such as hemorrhages, swelling, hypo-
tension, bradycardia, etc (Fernandez et al. 2014). Such
pathophysiological and biological effects have been
documented in animal models using BAV (Brink and
Steyler 1974, Nagel 2012, Fernandez et al. 2014).
Antivenin is the only available antidote to snakebite
poisoning (Williams et al. 2010), but antivenin often
induces numerous clinical side effects after use com-
bined with scarcity and high cost (World Health
Organization 2010). Also, report abounds that antive-
nins only neutralizes venom toxins to stop further
damage, but do not reverse the damage already done
(Gomes et al. 2007). Interestingly, researchers have
shifted their attention to natural products from plants
capable of neutralizing toxic effects of snake venoms
primarily due to their safety, effectiveness, cultural
preferences, accessibility, low cost and dependence on
neighboring forests (Dal Belo et al. 2008, Kumar and
Khamar 2010).
Moringa oleifera Lam. is a medicinal plant com-
monly grown throughout Southern Asia (Tejas et al.
2012) and in some parts of Nigeria. Numerous
pharmacological actions of M. oleifera leaf have been
reported (Tejas et al. 2012). Traditionally, M. oleifera
plant have been an alternative therapy for the treat-
ment of snakebites in most part of the world (Kumar
and Khamar 2010) including Nigeria (Adeyi et al.
2020). Also, earlier study has reported the antivenin
potentials of M. oleifera leaf against Naja nigricollis
venom (Adeyi et al. 2020). However, this study further
investigated the antivenin efficacies of ethanol leaf
extract of M. oleifera against B. arietans venom using
in vivo and in vitro models.
2. Materials and methods
2.1. Venom collection
Lyophilized Bitis arietans venom was obtained from
the Department of Veterinary Physiology and
Pharmacology, Amadu Bello University, Zaria, Nigeria.
It was stored at 2–4C prior to this study.
2.1.1. Animals
A total number of 103 male albino Wistar rats weigh-
ing between 100–140 g were obtained from animal
house of Department of Zoology, University of Ibadan,
Nigeria. Animals were acclimatized for two weeks in
pathogen free cages at room temperature, feed with
grower pelletized feed and given water ad libitum.
Fourty rats were used for the Lethal dose test (LD
50
)
of B. arietans venom and M. oleifera leaf extract. Thirty
rats were used for the antivenin experiment while the
remaining thirty three rats were used for the antihea-
morrhagic assay.
2.1.2. Determination of lethal dose (LD
50
)ofB. arie-
tans venom (BAV)
Lethal Dose test was carried out as described by
(Spearman-Karber 1978) with modifications. Twenty
male albino Wista rats were randomly divided into
four groups (n¼5). Group A was injected intraperito-
neally (i.p) with 0.2 ml of saline (control) while group
B, C and D were injected (i.p) with 0.2 ml of 0.8, 1.6,
3.2 mg/kg
1
of the venom in normal saline respect-
ively. Rats were observed for 48 h and mortalities were
recorded. The LD
50
of the venom was calculated with
confidence limit at 50 % probability using probit ana-
lysis of death occurring within 48 h after the venom
administration. All animal experiments in this study
complied with the National Institutes of Health publi-
cation on guide for the care and use of laboratory ani-
mals, 8th edition, 2011.
2.1.3. Snake venom antiserum
Polyvalent antivenin used for this study was EchiTAb-
Plus ICP made of horse immunoglobulin. The anti-
venin was produced in Instiudo Clodomiro Picado,
University of Coasta Rica, Coasta Rica. The antivenin
was produced for the treatment of envenoming by
Sub-Sahara African snakes of Echis ocellatus,Bitis arie-
tans and Naja nigricolis. This three species have the
most reported cases of snakebite in Nigeria (Yusuf
et al. 2015).
2.2. Collection of plant material
Fresh leaves of M. oleifera were collected from the
Botanical Garden of the University of Ibadan, Nigeria.
The plant leaves were identified and authenticated by
a taxonomist at the herbarium of Department of
Botany, University of Ibadan, Nigeria, where a voucher
specimen was deposited (Voucher No: UIH-22442).
848 B. S. AJISEBIOLA ET AL.
2.2.1. Preparation of the plant extract
The fresh leaves were air dried for four weeks, pow-
dered using Super-Master electric blender (Model no:
SMB-2977) and sieved with 2 mm filter cloth to
remove the leaf stalks and 0.002 kg of the powdered
leaf of M. oleifera was soaked in 3 liters of ethanol for
72 h. Cold maceration method (World Health
Organization 1998) was employed for the extraction of
plant material. The content was filtered through a
Whatman filter paper (grade 1) (Model no: F1000-01A,
made in China) lined funnel into 3000 ml laboratory
borosilicate glass Erlenmeyer conical flask. The filtrate
was concentrated to a paste using Borosilicate Glass
Rotary evaporator (Model no: 129152962) at 40 C
and stored in a properly labeled bottle as M. oleifera
Leave Extract (MOLE) and stored in the refrigerator
at 4 C.
2.2.2. Lethal dose (LD
50
) determination of M. olei-
fera leave extract (MOLE)
Lethal dose test was determined with modification to
previous method (Lorkes 1983). Twenty male albino
wistar rats were randomly divided into four groups
(n¼5). Group 1 was given only saline water (control)
while group 2, 3, 4 were administered orally with 300,
600 and 900 mg.kg
1
of the crude extract dissolved in
saline respectively. The animals were observed
for 48 h.
2.3. Qualitative and quantitative phytochemical
screening of MOLE
MOLE was screened for qualitative and quantitative
phytochemicals constituents for the detection of sapo-
nins, alkaloids, tanins, flavonoids, terpenoids, anthra-
quinones and cardiac glycosides using standard
methods (Trease and Evans 1989, AOAC 2005).
2.4. In vivo neutralization of BAV
2.4.1. Experimental groups
Thirty male albino Wista rats were divided randomly
into six groups (n¼5) for this study. Group 1 was
injected with normal saline (Normal control) while
group 2 was untreated envenomed rats (venom con-
trol). Groups 3, 4, 5 and 6 were envenomed rats
treated with 0.2 ml of 200, 400, 600 mg/kg
1
of MOLE
and 0.2 ml of standard polyvalent antivenin
respectively.
2.4.2. Venom injection and treatments
Rats in each group were envenomed by a single intra-
peritoneal injection of 0.2 ml of 1.5 mg/kg
1
(LD
50
)of
BAV dissolved in 1 ml of saline. Each dose of MOLE
was dissolved in 1 ml of saline and administered orally
to envenomed rats treated with extract while anti-
venin was injected intravenously to the antivenin
treated rats. Animals were treated 1 h post envenom-
ing for seven consecutive days and mortalities
were recorded.
2.4.3. Blood sample and organs collection
After treatment for a week, blood samples were col-
lected from the experimental rats through the veins
into Ethylene diamine tetraacetic acid (EDTA) bottles.
The animals were then sacrificed based on guides
(Rowett 1977). The liver and the heart of the rats were
harvested and each organ cuts into two equal halves.
Each half were stored in 10% formalin for histopatho-
logical studies and in the freezer (20 C) for bio-
chemical assay and DNA analysis.
2.5. Measurement of hematological indices
The blood samples were centrifuged for five minutes;
results were read on the hematocrit reader for Packed
Cell Volume (PCV), White Blood Cell (WBC) Red Blood
Cell (RBC), white blood counts differentials, hemoglo-
bin level and platelet (Baker and Silverton 1985).
2.6. Blood chemistry analysis
Blood plasma obtained after centrifugation was used
for the estimation of total protein using buret method
(Gornal et al. 1949). Also, serum albumin (ALB) was
tested by the bromocresol green method (McPherson
and Everad 1972). Serum creatinine levels were deter-
mined according to standard kit method (Bartels
et al. 1972) while sodium and calcium levels were esti-
mated spectrophotometrically using standard kits
(Maruna 1958).
2.7. Biochemical assay
Alkaline Phosphatase (ALP) and Gamma-glutamyltrans-
ferase (GGT) activity was carried out according to the
method of Schmidt and Schmidt (Schmidt and
Schmidt 1963), while method described by Reitman
and Franked (Reitman and Franked 1957), was
adopted for Aspartate Amino Transferase (AST) and
Alanine aminotransferase (ALT) assay.
TOXIN REVIEWS 849
2.8. Antioxidants assay
Superoxide dismutase (SOD) and peroxidase enzyme
activity was carried out as described by Marklund and
Marklund (Marklund and Marklund 1974), and
Fergusson and Chance (Fergusson and Chance 1955),
respectively. Also, method of Ellman (Ellman 1959),
was used to carry out Glutathione assay while gluta-
thione-S-transferase (GST) activity was carried out
using method described by Habig et al. (Habig
et al. 1974).
2.9. Biological studies
2.9.1. Neutralization of venom-induced hemorrhage
by MOLE
The Minimum Hemorrhagic Dose (MHD) of BAV was
determined (Theakston and Reid 1983) with modifica-
tion using male Albino wistar rats. Thirty three rats
were randomly divided into 11 groups (n¼3), 5 of
the groups (a normal control and four venom treated
groups) were used for the MHD determination while
6 groups were used for the antiheamorrhagic study
(a normal control, venom control, antivenin group
and 3 extract treated groups). For the MHD, 0.1 ml of
varying concentrations of the venom from a serial
dilution (5.0, 2.5, 0.25 and 0.625 mg) of 10 mg in nor-
mal saline was injected intradermally into the rats.
The Minimum Hemorrhagic Dose was defined as the
least amount of venom which when injected intrader-
maly (i.d.) into rats results in a hemorrhagic lesion of
10 mm diameter in 3 h. Anti-hemorrhagic activity was
determined with modification to previous methods
(Theakston and Reid 1983). A fixed amount of venom
(3 MHD) was mixed with different doses of MOLE
(100, 200, 300 mg/kg
1
) while polyvalent antivenin
wasusedasstandard.Themixturesofvenom/
extracts and venom/antivenin were incubated at
37 C for 1 h and 0.1 ml of the mixture was injected
intradermaly into rats. The hemorrhagic lesion was
estimated after 3 h and was expressed in percent-
age inhibition.
2.9.2. Neutralization of venom-induced red blood
cell lyses
This was carried out using 20 ml of citrated bovine
blood (Gomes and Pallabi 1999). Erythrocytes were
washed with 5 ml of saline (0.9%) by centrifugation
(2400 rpm) for 10 min and this was done 10 times to
obtain a free packed cells. After repeated washings
with saline, a 10% cell suspension was prepared. The
0.2 ml of 10 mg of BAV in 1 ml of saline was mixed
with 1 ml of 10% cell suspension in saline and 0.2 ml
of various dilutions of MOLE (100, 200, 300 mg/ml)
and polyvalent antivenin (0.2 ml) in each separate
tubes. All mixtures are in triplicates. The mixtures were
incubated at 37 C for 60 min and reaction was
stopped by adding 3 ml of chilled phosphate buffer
saline (PBS pH 7.2). The tubes were centrifuged at
2400 rpm for 10 min and absorbance of the super-
natant was measured at 540 nm. Supernatant of tube
treated with 3 ml distilled water was taken as 100%
lyses and serves as control. Percentage inhibition was
calculated using the formula
%Inhibition ¼Control–Test Sample 100 Control
2.9.3. Neutralization of venom-induced coagulation
Coagulant activity was assayed using citrated bovine
plasma (Gomes and Pallabi 1999). 0.2 ml of various
amounts of BAV (5.0, 2.5, 0.25 and 0.625 mg) from a
serial dilution of 10 mg of venom in 1 ml of phosphate
buffer solution (PBS, pH 7.2) was added to 0.2 ml of
bovine citrated plasma and 0.2 ml of CaCl
2
. All mix-
tures are in triplicates. The mixtures were incubated at
37 C and coagulation time was recorded and the min-
imum coagulant dose (MCD) was determined as the
venom dose, which induced clotting of plasma within
60 s (Theakston and Reid 1983). Plasma incubated with
PBS alone served as control. In neutralization assays
constant amount of venom (3 MCD) was mixed
with 0.2 ml of various dilutions of MOLE (100, 200,
300 mg/ml) and polyvalent antivenin (0.2 ml) separ-
ately. Mixtures are in triplicates. The mixtures were
incubated for 30 min at 37 C. Then 0.2 ml of mixture
was added to 0.2 ml of citrated plasma and calcium
chloride (CaCl
2
) and clotting times recorded. In control
tubes plasma was incubated with either venom alone
or plant extracts alone. Neutralization was expressed
as effective dose (ED), defined as the ratio ll antivenin
(plant extracts)/mg venom at which the clotting time
decreased three times when compared with clotting
time of plasma incubated with three MCD of
venom alone.
2.10. Histopathological studies
Conventional method using paraffin-wax sectioning
and hematoxylin-eosin staining were adopted for the
histological observations (Carleton et al. 1980).
2.11. DNA fragmentation assay using agarose gel
electrophoresis
Extraction of DNA from organs was carried out to
measure apoptotic DNA fragmentation. The presence
850 B. S. AJISEBIOLA ET AL.
of DNA ladder was determined (Wlodek et al. 1991).
Extraction of DNA was done according to the method
of (Aljanabi and Martines 1997). 20 mg of liver and
heart tissue in eppendorf tubes were lysed with 600 ml
buffer (50 mM NaCl, 1 mM Na2EDTA, 0.5% SDS, PH 8.3)
and gently shaked. The mixture was incubated over-
night at 37 C then, 20 ml of saturated NaCl was added
to the sample, shaked and centrifuged at 12 000 rpm
for 10 min. the supernatant was transferred to new
eppendorf tubes and then DNA precipitated by 600 ml
cold isopropanol. The mixture was inverted several
times till fine fibers appear, and then centrifuged for
5 min. at 12 000 rpm. The supernatant is removed and
the pellets were washed with 500 ml 70% ethyl alcohol
then centrifuged at 12 000 rpm for 5 min. After centri-
fugation the alcohol was decanted or tipped out and
the tubes plotted on Whatman filter paper to be dry.
The pellets were resuspended in 50 ml or appropriate
volume of TE buffer (10mM Tris, 1 mM EDTA, PH 8).
The resuspended DNA was incubated for 3060 min
with loading mix (RNase þloading buffer) and
then loaded into the gel wells. A gel was prepared
with 2% agarose containing 0.1% ethidium bromide
(200 mg/ml). The DNA samples were mixed with loading
buffer (0.25% bromophenol blue, 0.25% xylene cyanole
FF and 30% glycerol) and loaded into the wells (20 ml
of DNA/lane) with a standard molecular- sized ladder
marker (Pharmacia Biotech, Centennial Avenue
Piscataway, NJ, USA.). The gel was electrophoresed at a
current of 50 mA for 1.5h using the submarine gel elec-
trophoresis machine. The DNA was visualized and pho-
tographed with illumination under UV light.
3. Statistical analysis
Data were expressed as the Mean ± Standard devi-
ation. Significant differences between all experimental
groups were tested using ANOVA and Duncan mul-
tiple tests using the SPSS computer software (IBM
SPSS Statistics for Windows, IBM Corp. Armonk, NY,
USA), version 20. A value of (p<0.05) was considered
statistically significant.
4. Results
4.1. Lethal dose (LD
50
) test of the venom
The LD
50
of the venom was estimated at 1.5 mg/kg
1
.
Also, 24 h post, 25, 50 and 100 % mortalities were
recorded in Group B, C and D respectively, this groups
were envenomed with 0.8, 1.6, 3.2 mg/kg
1
of BAV.
4.2. Lethal dose (LD
50
) test of M. oleifera extract
Death was not recorded across the experimental
groups after observation for 48 h an indication that
various doses of the extract used do not exhibit a
lethal response. This result means that the Lethal dose
of the extract is >900 mg/kg which is the highest con-
centration tested. Various doses of the extract used
for this study was within the non-toxic range based
on the findings from the acute toxicity study.
4.2.1. Qualitative and quantitative phytochemical
analysis of M. oleifera extract
The results of the qualitative and quantitative phyto-
chemical screening showed that ethanol MOLE contains
saponins, alkaloids, flavonoids, tanins, terpenoids and
cardiac glycosides, while the quantitative phytochemical
compositions of the extract were saponins (22 %); alka-
loids (6 %), Tanins (4.6 %) and flavonoids (8.4 %).
4.3. Mortalities of the envenomed animals during
treatment with M. oleifera extract
Mortalities were recorded post envenoming in the
experimental groups. Group 2 (venom control) and
group 3 (envenomed and treated with 200 mg/kg
1
of
the extract) recorded 40 % mortality while 20 % mor-
tality was observed in group 4 (envenomed and
treated with 600 mg/kg
1
of the extract) and group 6
(envenomed and treated with 0.2 ml of antivenom).
Observations and behavioral responses noticed post
envenoming includes; bleeding on the site of venom
injection, restriction of movement within their cages,
dizziness and increased water consumption.
4.4. Haematological parameters
The results of the PCV, Hb, RBC and the platelet counts
of group 2 (venom control) were significantly (p<0.05)
lower compared normal control and treated groups
(Table 1). Treatment with the plant extract improved
the blood parameters of the extract treated rats (group
3, 4, 5). Comparing the effectiveness of the different
doses of the extract and antivenin, hematology indices
were best improved in group 5 envenomed and
treated with 600 mg/kg
1
of the extract. However,
MCV, MCH, MCHC values of the entire experimental
groups were significantly (p<0.05) not different.
4.4.1. White blood cell and differentials
Counts for the WBC, lymphocytes neutrophils and mono-
cytes were significantly (p<0.05) not different in all the
experimental groups. The results showed that WBC and
TOXIN REVIEWS 851
lymphocytes counts were reduced in group 2 (venom
control), however, a marked increase in the WBC and the
lymphocytes were observed in all treatment groups.
4.5. Blood chemistry analysis
The calcium concentration in the serum was signifi-
cantly (p<0.05) higher in the venom control com-
pared to all the treatment groups (Table 2). However,
group 5 treated with 600 mg/kg
1
of the extract
showed a significantly (p<0.05) lower value compared
to other extract treated groups. Albumin concentra-
tion showed a significantly (p<0.05) lower value in
the venom control compared to all treated groups.
Total protein, globulin, blood urea nitrogen, creatinine
and sodium were significantly (p<0.05) not different
in all the experimental groups.
4.6. Biological enzyme indices
Changes in the levels of serum parameters show that
AST and GGT were significantly (p<0.05) not different.
A higher level of ALP was recorded in the venom con-
trol compared to the extract and antivenin treated
groups. Also, significantly (p<0.05) higher level of ALT
was recorded in the venom control compared to all
the treatment groups.
4.7. Antioxidant enzymes
Results of the oxidative stress status of the serum in
response to B. arietans venom showed that the con-
centration of the SOD (Table 3) was significantly
(p<0.05) higher in the venom control compared to
group 3,4 and 6 which were treated with 200 and
400 mg/kg
1
of the extract and antivenin respectively.
Group 5 envenomed and treated with 600 mg/kg
1
of
the extract recorded a significantly (p<0.05) higher
concentration of SOD compared to other treated
groups. GST, GSH and peroxidase values were signifi-
cantly (p<0.05) not different in all the experimen-
tal groups.
Table 1. Heamatological parameters of B. arietans-envenomed rats after treatment with ethanol leaf extract of M. oleifera.
Treatment group PCV (%) Hb (g/dl) RBC (10
6
ml) Platelet (10
3
ml) MCV (femtoliter) MCH (pg) MCHC (g/dl)
1 (normal control) 41.67 ± 3.51
b
13.73 ± 0.99
b
6.98 ± 0.39
b
75.67 ± 19.04
a
5.97 ± 0.18
a
19.85 ± 0.51
a
33.29 ± 0.47
a
2 (venom control) 34.00 ± 5.66
a
11.25 ± 2.05
a
5.82 ± 0.69
a
58.50 ± 7.78
a
5.83 ± 0.28
a
19.27 ± 1.25
a
33.05 ± 0.53
a
3 (venom þ200mg/kg) 37.00 ± 141
ab
12.25 ± 0.35
ab
6.38 ± 0.25
ab
61.50 ± 40.31
a
5.81 ± 0.01
a
19.22 ± 0.19
a
33.11 ± 0.31
a
4 (venom þ400mg/kg) 39.33 ± 0.58
b
13.23 ± 0.25
b
6.54 ± 0.08
b
79.00 ± 5.00
a
6.01 ± 0.01
a
20.22 ± 0.19
a
33.64 ± 0.28
a
5 (venom þ600mg/kg) 41.33 ± 1.53
b
13.83 ± 0.49
b
6.94 ± 0.39
b
83.00 ± 23.43
a
5.96 ± 0.11
a
19.66 ± 0.65
a
33.47 ± 0.29
a
6 (venom þantivenom) 34.30 ± 2.87
a
11.42 ± 1.23
a
5.35 ± 0.17
a
82.33 ± 23.35
a
5.87 ± 0.15
a
19.30 ± 0.55
a
33.00 ± 0.46
a
Data are expressed as means ± S.D, (n¼3). Values in the same column with different superscript are considered significant (p<0.05); PCV: Packed cell
volume, HGB: Hemoglobin; RBC: Red blood cell, MCV: Mean Corpuscular volume, MCH: Mean corpuscular hemoglobin, MCHC: Mean Corpuscular
Hemoglobin Concentration.
Table 2. Blood chemistry of the envenomated rats after treatment with ethanol leaf extract of M. oleifera.
Parameters Group 1 Group 2 Group 3 Group 4 Group 5 Group 6
Total Protein (mg/L) 7.44 ± 0.21
a
7.35 ± 0.71
a
7.30 ± 0.42
a
7.60 ± 0.51
a
7.33 ± 0.35
a
7.35 ± 0.31
a
Albumin (mg/L) 3.68 ± 0.20
ab
3.50 ± 0.14
a
3.35 ± 0.21
a
4.10 ± 0.38
b
3.73 ± 0.20
ab
3.68 ± 0.17
b
Globulin (mg/L) 3.90 ± 0.31
a
3.85 ± 0.71
a
3.4500 ± 0.92
a
3.50 ± 0.30
a
3.58 ± 0.52
a
3.68 ± 0.30
a
Albumin-Globulin ratio(md/dl) 0.90 ± 0.71
ab
0.85 ± 0.71
a
1.35 ± 0.78
b
1.13 ± 0.12
ab
1.03 ± 0.21
ab
0.93 ± 0.10
ab
Blood urea nitrogen(mg/dl) 15.00 ± 1.60
a
14.50 ± 0.71
a
14.00 ± 0.00
a
15.70 ± 2.31
a
14.30 ± 1.90
a
13.50 ± 0.60
a
Creatinine (mg/dl) 0.72 ± 0.30
a
0.50 ± 0.14
a
0.50 ± 0.00
a
0.77 ± 0.40
a
0.70 ± 0.30
a
0.60 ± 0.82
a
Calcium (mEq/L) 12.50 ± 0.58
ab
12.90 ± 0.21
b
11.60 ± 1.60
ab
10.90 ± 2.19
ab
10.13 ± 0.80
a
12.10 ± 1.60
ab
Sodium (mEq/L) 143.4 ± 10.30
a
147.0 ± 5.70
a
143.0 ± 18.40
a
139.3 ± 2.52
a
140.5 ± 2.40
a
144.3 ± 5.30
a
Data are expressed as means ± S.D, (n¼3). Values in the same column with different superscript are considered significant (p<0.05).
Group 1: Normal control (water only), Group 2: Venom control (venom only), Group 3: Treatment (venom and 200 mg/kg), Group 4: Treatment (venom
and 400 mg/kg), Group 5: Treatment (venom and 600 mg/kg), Group 6: Treatment (venom and 0.2 ml antivenom).
Table 3. Effects of MOLE on antioxidant enzymes activity of BAV envenomed rats.
Treatment group GST (U/L) Px (U/L) SOD (U/mL) GSH (mM)
1 (normal control) 5.99 ± 1.69
a
24.23 ± 13.75
a
5.09 ± 1.52
a
0.28 ± 0.03
a
2 (venom control) 3.59 ± 1.69
a
18.67 ± 0.65
a
7.85 ± 5.21
ab
0.29 ± 0.03
a
3 (venom þ200 mg/kg extract) 9.58 ± 6.78
a
16.20 ± 10.69
a
6.69 ± 1.85
a
0.36 ± 0.13
a
4 (venom þ400 mg/kg extract) 9.58 ± 6.78
a
22.69 ± 11.13
a
4.14 ± 0.74
a
0.41 ± 0.19
a
5 (venom þ600 mg/kg extract) 2.39 ± 0.00
a
23.15 ± 6.11
a
15.83 ± 5.41
ab
0.34 ± 0.06
a
6 (venom þ0.2 ml antivenom) 5.99 ± 5.08
a
21.60 ± 2.62
a
6.86 ± 3.48
a
0.29 ± 0.02
a
Data are expressed as means ± S.D, (n ¼3). Means with similar superscript on the same column are not significantly different
(p<0.05). GST: Glutathione Transferase; Px: Peroxidase; SOD: Superoxide Dimustate; GSH: Glutathione.
852 B. S. AJISEBIOLA ET AL.
4.8. Biological studies
4.8.1. Antihemorrhagic activity of M. olei-
fera extract
Various concentrations of the venom showed visible
hemorrhagic spot. The Minimum Hemorrhagic Dose
(MHD) was 0.625 mg/ml after intradermal injection of
different amount of BAV. There was 100 % hemor-
rhage in the venom control while treatment with
MOLE and polyvalent antivenin effectively reduced
hemorrhage in the treatment groups (Table 4) but
there was no complete inhibition.
4.8.2. Antiheamolytic activity of the extract of
M. oleifera
BAV showed a direct hemolysis on the bovine red
blood cells. There was complete hemolysis in the nor-
mal control. Venom control showed above 80 % hem-
olysis, the medium and high dose of MOLE showed
above 50 % inhibition of hemolysis while the polyva-
lent antivenin showed 90 % inhibition (Table 5).
4.8.3. Coagulation studies
The minimum coagulating dose (MCD) of BAV was
0.65 mg/ml. Result of anticoagulant assay showed that
venom control clotted at 125 s. All venom/extract mix-
tures clotted below 100 s. High dose (300 mg) of
MOLE clotted plasma at 43 s. Also, antivenin group
clotted at 40 s. MOLE showed significant reduction in
coagulating time in a dose dependent manner
(Figure 1).
4.9. Results of the histopathological examination
of the liver and the heart of the envenomed
treated rats
The histopathological examination of the liver of nor-
mal control rats appeared with normal hepatocytes.
Slides prepared from the liver of venom control
revealed observable structural deformities as organs
showed a multifocal single-cell hepatocellular necrosis,
Kupffer cell hyperplasia with intra-cytoplasmic brown
pigments and a mild congestion of central veins.
However, these effects were ameliorated in the extract
and antivenin treated rats. Slides prepared from the
heart of normal control rats showed a normal appear-
ance of cardiomyocytes with no observable lesions
but slides from the heart of venom control showed a
multiple foci of hemorrhages (extravasations of red
blood cells). The structural defects observed were
ameliorated in extract and antivenin treated groups.
4.10. DNA fragmentation assay results of the liver
and the heart of the envenomed treated rats
The DNA of the liver of rats in the control group (lane
1) was observed normal and showed no fragmentation
(Figure 2(a)). Also, DNA fragmentation was not noticed
in the venom control (lane 2) including the extract
treated groups (lanes 3, 4, 5, 6). Furthermore, the heart
of rats in control group (lane 1) showed a normal
DNA (Figure 2b). A mild DNA fragmentation was
noticed in the venom control (lane 2) but this effect
was ameliorated in all the extract treated groups
(lanes 3, 4, 5, 6).
5. Discussion
In this study, LD
50
of BAV through intraperitoneal
route was 1.5 mg/kg
1
higher compared to LD
50
of
0.87 mg/kg
1
(IP route) reported in other studies
(Oukkache et al. 2014). Variations in enzymatic compo-
sitions of venoms are responsible for differences in
their toxicity level due to factors such as age, diet,
geographical location and sex (Currier et al. 2010).
Snake bites have been reported to exert their effects
on cells through the action of various biologically
active components of snake venoms (Tamieti et al.
Table 4. Inhibition of hemorrhagic activity of the venom by
M. oleifera extract.
Treatment Groups Percentage inhibition (%)
Group 1 No Lesion
Group 2 No inhibition
Group 3 25 ± 1.73
Group 4 35 ± 1.62
Group 5 51 ± 1.73
Group 6 64 ± 1.73
Data are expressed as means ± SD of three individual experi-
ments. p<0.05.
Group 1: Rats injected with 0.2 ml of Normal saline. Group 2: Rats
injected with 0.2 ml of the venom. Group 3: Rats injected with 0.2 ml of
venom/100 mg extract, Group 4: Rats injected with 0.2 ml venom/ 200 mg
extract. Group 5: Rats injected with 0.2 ml of venom/300 mg extract.
Group 6: Rats injected with 0.2 ml of venom/polyvalent antivenom.
Table 5. Inhibition of hemolytic activity of the venom by M.
oleifera extract.
Treatment Groups Percentage inhibition (%)
Group 1 0
Group 2 16 ± 1.00
Group 3 32 ± 1.53
Group 4 52 ± 1.15
Group 5 68 ± 1.53
Group 6 90 ± 1.58
Data are expressed as means ± SD of three individual experi-
ments. p<0.05.
Group 1: Mixture of distilled water/citrated RBCs (Normal control).
Group 2: Mixture of venom/citrated RBCs (venom control). Group 3:
Mixture of venom/citrated RBCs/100 mg of crude extract. Group
4: Mixture of venom/citrated RBCs/200 mg of crude extract. Group 5:
Mixtures of venom/citrated RBCs/300 mg of crude extract. Group 6:
Mixtures of venom/citrated RBCs/standard polyvalent antivenom.
TOXIN REVIEWS 853
2007). Bitis arietans is a viper with a cytotoxic and
hemotoxic venom (World Health Organization 2010)
containing many enzymes majorly snake venom met-
alloproteinases (SVMPs) which is responsible for rapid
local and systemic hemorrhage after envenoming
(Currier et al. 2010). Report abound that the main tar-
get of metalloproteinase is the platelet (Currier et al.
2010). Metalloproteinase inhibit platelet interaction
with collagen particularly type IV collagen, a major
component of basement membrane and Willebrand
Factor (vWF) by various mechanisms through targeting
platelet receptor or their ligands. This process leads to
the degradation of the extracellular matrix proteins
which compromise the blood vessel wall integrity,
undoubtedly contributing to systemic bleeding
(Kamiguti 2005). In this study, BAV induced acute
anemia and thrombocytopenia in envenomed rats,
this effects may be due to spontaneous and prolong
bleeding as a result of the hemorrhagic effects of the
venom as observed after B. arietans in human and ani-
mals. Previous studies have reported such hemato-
logical disturbances using viper venom (Echis
Ocellatus) (Onyeama et al. 2013). Treatment with etha-
nol MOLE resulted into a dose-dependent reverser of
Normal
control
Venom
control
100 mg
extract/ve
nom
200 mg
extract/ve
nom
300 mg
extract/ve
nom43
0.2 ml
polyvalen
t
Anveno
m/venom
Clong Time in
Seconds 31 125 98 67 43 40
0
20
40
60
80
100
120
140
Figure 1. Coagulating Activity of ethanol leaf extract of M. oleifera.
Data are expressed as means ± S.D. of three individual experiments. p<0.05.
Group 1: Distilled water/citrated plasma (Normal control). Group 2: venom/citrated plasma (Positive control). Group 3: venom/citrated plasma/100mg
extract. Group 4: venom/citrated plasma/200mg extract Group 5: venom/citrated plasma/300 mg extract. Group 6: venom/citrated plasma/0.2 ml polyva-
lent antivenin.
Figure 2. Effects of M. oleifera extract on the DNA of the liver and heart of after rats envenomed with B. arietans venom
Envenomation. (a) No DNA fragmentation of the liver (lane 2). (b) Mild DNA fragmentation of the heart (lane 2).
M: DNA markers
Lane 1: Normal control group, Lane 2: venom control group; Lane 3: Envenomated rats treated with 200mg/kg of the extract; Lane 4: Envenomated rats
treated with 400mg/kg of the extract; Lane 5: Envenomated rats treated with 600 mg/kg of the extract; Lane 6: Envenomated and treated with 0.2 ml
of antivenin.
854 B. S. AJISEBIOLA ET AL.
anemia and thrombocytopenia induced by the venom
with a significant increase in RBC, Hb and a marked
increase in the platelet counts. Studies have reported
improvements in heamatological indices in enve-
nomed rats treated with M. oleifera extract (Adeyi
et al. 2020). Further, MOLE has been reported as hea-
matinics in the treatment of anemia (Otitoju et al.
2014, Mun’im et al. 2016). The improved hematology
may be due to blood boosting effects of the plant
extract which may be a positive factor in boosting
blood counts in anemia induced in snakebite patients.
Although, the mechanisms behind the plant extract
stimulating erythropoesis is yet unknown. Lower val-
ues of hematological indices obtained in the antivenin
group may indicate that antivenin only detoxifies the
venom toxins with no reversal on damages caused by
BAV as previously reported (Gomes et al. 2010). Also,
there was a decrease in MCH values in untreated rats
but high dose of the extract increased the MCH values
after treatment, such observation has been docu-
mented in rats envenomed with a viper venom (Echis
ocellatus) and treated with plant extract (Onyeama
et al. 2012). White blood cells are effectors of immune
systems and the principal function of WBC as phago-
cytes is to defend against invading microorganisms or
xenobiotics (Otitoju et al. 2014). The decrease in WBC
obtained in the venom control which recorded a
dose-dependent increase upon treatment with MOLE
may suggest that the extract combated the venom
directly without cells of the immune system producing
effectors cells.
Reduction in total serum proteins, albumin and
globulin could be due to disturbances in renal func-
tion as well as hemorrhages in some internal organs.
High sodium and calcium in serum of untreated rats
may be due to acute nephropathy following viper
bites (Meier and Stocker 1991). Liver is a major produ-
cer of most plasma proteins and their total levels in
the blood are regulated by liver function (Rosalki
1974). Elevated activities of AST and ALT in serum are
indicative of cellular leakage and loss of functional
integrity of cell membrane in the liver an indication of
hepatoxicity. Studies have reported such physiological
disorder caused by BAV (Ibrahim and Al-Jammaz 2001)
however, high enzyme activity was ameliorated after
treatment with MOLE and polyvalent antivenin.
In this study, BAV induced oxidative stress by
enhancing SOD enzymes activities in venom control
rats. SOD is considered as the primary antioxidant
enzymes since they are involved in direct elimination
of reactive oxygen species and play an important role
in body defense mechanism against deleterious effects
of oxygen free radicals in biological systems (Al-
Quraishy et al. 2014). Oxidative stress observed in
venom control rats may be as a result of excessive
reactive oxygen species generation or failure of the
cellular antioxidant system (Awadh et al. 2014). MOLE
decreased SOD activities in extract and antivenin
treated rat but high dose of MOLE (600 mg/kg)
increased SOD activities which could be a response by
antioxidant enzymes to alleviate oxidative stress in
this group. Although, antioxidant properties of M. olei-
fera leaves has been documented (Suphachai 2014).
Glutathione is the cell’s natural antioxidant which
destroys free radicals formed in cells and this plays a
crucial role in the detoxification process. Decreased
GSH in untreated rats supported the previous inter-
pretation of the consequences of GSH deficiency
which causes oxidant damage (Awadh et al. 2014).
Lower MHD (0.625 mg/ml) of BAV indicated high
hemorrhagic activity caused mainly by metalloprotei-
nase enzymes which are abundantly found in viper
venom (Mukherjee 2008). Heamorrhage observed in
venom control rats was inhibited in a dose dependent
manner by MOLE and antivenin but there was no
complete inhibition an indication the extract reduced
leakage of blood from the blood vessels which is in
tandem with previous reports on antiheamorrhagic
effects of M. oleifera extract (Adeyi et al. 2020). BAV
induced a direct hemolytic activity on bovine erythro-
cytes which can be attributed phospholipase A
2
(PLA
2
)
enzymes present in the venom. PLA
2
is an ubiquitous
enzyme that specifically catalyze hydrolysis of mem-
brane phospholipids to release lysophospholipids and
free fatty acids, namely arachidonic acid which pro-
vides substrate for eicosanoids biosynthesis that
modulate acute and chronic inflammation resulting in
blood lyses (Rajesh et al. 2017). MOLE showed a dose
dependent inhibition of the hemolysis induced by BAV
in all the treated samples compared to venom control.
This observation may indicate possible inhibition of
PLA
2
in BAV.
Hemostatic disturbances including spontaneous
bleeding has been reported following B. arietans enve-
noming (Warrell et al. 1975). Snake venom proteins
affect hemostasis process either by prolonging or
shortening blood clotting time. BAV prolonged coagu-
lation in venom control an indicative of poor pro-
coagulant effects which corroborated previous studies
(Fernandez et al. 2014). However, clotting time was
shortened in all MOLE treated samples including the
antivenin samples which may suggest that MOLE has
procoagulant effects.
TOXIN REVIEWS 855
Also, various pathological defects observed in the
histology examination of the liver and heart of the
untreated rats were ameliorated when treated with
MOLE with various signs of tissue regenerations
noticed. DNA fragmentation was not observed in the
liver of venom control rats but mild effects was
noticed in the heart which may suggest that BAV is
capable of inducing genetic alteration. DNA fragmen-
tation is regarded as a marker for apoptosis and three
mechanisms are involved in the apoptotic process: a
receptor-ligand mediated mechanism, a mitochondrial
pathway, and a mechanism in which the endoplasmic
reticulum plays a central role (Francischetti et al.
1997). Researchers have documented morphological
alterations on DNA in envenomed rats using Echis pyr-
amidum venom which is a viper (Awadh et al. 2014).
Phytochemical screening of MOLE revealed the
presence of flavanoids, terpenoids, cardiac glycosides,
alkaloids, tanins and saponins which agrees with previ-
ous studies (Sankar 2012). MOLE phytochemical con-
stituents may be responsible for inhibiting BAV toxic
effects. Several researchers have documented inhib-
ition of snake venom’s toxins using plant extract
(Mukherjee et al. 2008, Amagon, 2012, Gopi et al.
2016, Akah et al. 2019, Adeyi et al. 2020) and some
attributed the antivenom effects of these plant
extracts to their bioactive constituents such as quer-
cetin (Al Asmari et al. 2016, Gopi et al. 2016), flavo-
noids (Amagon, 2012) and alkaloids (Mukherjee et al.
2008). However, much investigations have not been
carried out on the neutralizing mechanisms of their
actions but in some cases, a direct interaction with
catalytic sites of enzymes or with metal ions which are
essential for enzymes activities may be involved
(Borges et al. 2005, Nunez et al. 2005).
6. Conclusion
In this study, B. arietans venom induced various toxic
effects in vitro and in vivo but ethanol leave extract of
M. oleifera showed ameliorated effects on the damage
caused by the venom toxins to some extent which
support the traditional use of M. oleifera in the treat-
ment of snakebite. Currently, studies to isolate and
characterize the active components of the plant
extract that may be responsible for inhibiting the
venom toxins are ongoing in our research laboratory.
Disclosure statement
All authors declared no conflict of interest.
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