Journal of Basic Medical Veterinary Juni 2022, Vol.11 No.

1, 37-48
Permatasari et al. Online pada https://e-journal.unair.ac.id/JBMV

The Effect of Mimosa Pudica Root Extract on Cerebrum Histopathological of

Rattus Norvegicus Induced with Naja Sputatrix Venom

Roselia Yuliani Permatasari1, Eka Pramyrtha Hestianah2, Djoko Legowo3, Kadek

Rachmawati4, Zainal Arifin5
1Veterinary Profession Program, 2Department of Veterinary Anatomy, 3Department of

Veterinary Pathology, 4Department of Basic Veterinary Science, 5Department of

Clinical Veterinary
Faculty of Veterinary Medicine, Universitas Airlangga
Corresponding author: [email protected]

ABSTRACT

The aim of this study was to know the effect of Mimosa pudica root extract on
histopathological appearance of Rattus norvegicus brain induced by Naja sputatrix
venom. Thirty rats were divided into 5 groups. There were 2 control groups and 3
treatment groups, which was given 250, 500, and 1000 mg/kg BW of Mimosa pudica
root extract orally. The first 7 days each group was adapted to the environment. On the
8th day, the treatment was started by injecting Naja sputatrix LD50 (0,13 BW)
IM in gluteus muscle, continued with giving Mimosa pudica root extract orally for the
treatment groups 5 minutes after venom injection. 6 hours after the last treatment, rats
were killed by cervical dislocation, injected with formalin 10% in the heart, then
necropsied. Histopathological evaluation was done to score brain damage based on
meningitis, perivascular cuffing, and necrotic cells using HE stain with 1000x
magnification. The result showed 1000 mg/kg BW dosage of Mimosa pudica root extract
can reduce brain damage based on meningitis, perivascular cuffing, and necrotic cells in
Rat (Rattus norvegicus) caused by Naja sputatrix venom and gave significant difference
(p < 0.05) among the treatment groups.
Keywords: Mimosa pudica, Naja sputatrix, snake venom, brain damage

Received: 17-02-2022 Revised: 28-04-2022 Accepted: 26-06-2022

INTRODUCTION
Snakebite envenoming is a global Venomous and poisonous snakes
public health problem of such size and are a significant cause of global
complexity that it deserves far more morbidity and mortality. They are found
attention from national and regional almost throughout the world, including
health authorities than it has been given many oceans and have evolved a variety
up until now. This environmental and of highly effective toxins and methods of
occupational disease affects mainly delivery. Their impact on humans is
agricultural workers and their children considerable, most current data suggest
in some of the most impoverished rural that they cause in excess of 3 million
communities of developing countries in bites per year with more than 150,000
Africa, Asia, Latin America and Oceania deaths, particularly in rural tropical
(World Health Organization, 2007). Asia areas (White, 2000).
is the continent where the majority of Snake bites and insect stings are
these bites take place, and also where most commonly encountered biotoxins
most deaths occur (Chippaux, 1998; (Mount, 1989). Snakes do not
Kasturiratne et al., 2008). attack/prefer not to bite animals unless

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they are disturbed / cornered. Among (Kamiguti et al., 2000). The nervous
the domestic animals, dogs are most system is a primary target for animal
frequently attacked and killed by the venoms as the impairment of its
snakes (Osweiler, 1996). Cattle and function results in the fast and
horses are also attacked while grazing. efficient immobilization or death of a
Sheep, goat, and pigs are occasionally prey (Osipov and Yutkin, 2012).
struck, while cat is not often attacked Histological changes in the brain
because of its greater caution and tissues following snake bites are not
superior agility when hunting (Shukla, widely documented compared to other
2009). organs. However, since snake venom is
Currently there are 29 recognized rich in neurotoxins significant
extant species of terrestrial cobras microscopical changes are likely to be
assigned to the genus Naja (Uetz and present in the brain tissue. Multiple
Hosek, 2015). Of these, 11 species are small foci of haemorrhages and
found in Asia, and 19 inhabit Afrika congestion of blood vessels have been
(Uetz and Hosek, 2015; Wallach et al., reported following administration of
2009). Cobra (Naja sp.) is characterized collubrid snake venom to rats which
by local necrosis, neurological paralysis was more marked with intravenous
and cardiotoxicity (Yap et al., 2011). injection of venom (Peichoto et al.,
Snake venoms are complex 2006).
mixtures containing predominantly Antivenoms immunotherapy is the
proteins and polypeptides and small only specific treatment against snake
amount of organic compounds and venom envenomation. There are various
minerals. Many of proteins exhibit side effects of antivenom such as
enzymatic activities, whereas the anaphylactic shock, pyrogen reaction
polypeptides include neurotoxins, and serum sickns. Most of these
cardiotoxins, myotoxins, and cytotoxins symptoms may be due to the action of
(Yap et al., 2011). Despite snake venoms high concentrations of non-
being a depot of target-specific toxins, immunoglobulin proteins present in
some of them may serve as drugs or commercially available hyper immune
prototypes for drug design, but the antivenom (Maya Devi et al., 2002).
management and neutralization of fatal Over the years many attempts have
snakebites are of priority. Antivenom is been made for the development of snake
the preferred and worldwide choice of venom antagonists from plant sources.
treatment for snakebites. Therefore, Several medicinal plants, which
polyvalent (prepared against the appear in old drug recipes or which
venoms of few snakes), bivalent have been passed on by oral tradition,
(prepared against the venoms of two are believed for the treatment of
snakes) and monovalent (prepared snakebite (Alam & Gomes, 2003). Mors
against the venom of one snake) et al. (1989) have found that β-sitosterol
antivenoms are currently available for successfully antagonises the
theurapeutic use (Chippaux et al., myotoxicity of South American Rattle
1991). Snake venom. Phytochemical analysis
Tissue changes following snake of M. pudica roots shows that the plant
envenomation depend on the species of contains ascorbic acid, crocetin, D-
snake responsible for the bite, the glucoronic acid, linoleic acid, linolenic
composition of its venom and also the acid, palmitic and stearic acids,
susceptibility of the tissue for a mimosine, D-xylose and β-sitosterols.
particular component of the venom

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In preliminary screening of water extraction), as Mahanta and

Mimosa pudica extract was found to Mukherjee (2001) did. The fresh Mimosa
exhibit anti-venom activity against pudica root left in the open air, dried
common sea snake (Enhydrina under the sun, and pulverized to a
schistosa) poisoning (Dnyaneshwar et powder. Four grams of the powder was
al., 2009). added into beaker glass and was added
Therefore, further work is 200 ml of distilled water, then stirred for
necessary for better understanding of about 3 hours at room temperature. The
the mechanism of venom inhibition. extract was filtered using muslin cloth
then concentrated at 40° C. Then it was
METHODS placed in freeze dry equipment.
This research conducted at the
Laboratory Animals Model at Veterinary Microscopic examination
Medicine of Airlangga University for the Histopathological slide
treatment of experimental animals. examination carried out using a light
Making of Mimosa pudica root extract microscope 100 times magnification of
was be done at the Laboratory of the objective lens to 10 different field of
Pharmacology, Veterinary Medicine view in a slide sample. The scoring was
Airlangga University. Rattus norvegicus be observe in meningens dan cerebrum.
brain histopathological representation The histopathologic scoring of vasculitis
was be observed at the Laboratory of modified from (Kennedy et al.,1997) as
Pathology Veterinary, at Veterinary follows:
Medicine of Airlangga University. Score
Implementation of this research was be 0 1 2 3 4
carried out from May 2016 to June
Meningitis None Mild Moderate Severe Severe
2016.
Perivascular None None Mild cuffing Prominent Promine
The experimental unit used in this Cuffing of some cuffing of nt of
study are healthy rat (Rattus norvegicus) vessels some most
vessels vessels
strain wistar with an average weight of
Necrosis Cell None 0-25% >25%-50%≤ >50%-75%≤ >75% cell
200 grams, maintained at the same cell cellnecrosis cellnecrosis necrosis
place and were given the same feed. necrosis

Experimental design
In this research used Rattus Data analysis
norvegicus which randomly taken and Data were analyzed with statistical
then put into the cage maintenance that test using Kruskal-Wallis and followed
has been marked (C (-), C (+), T1 T2 and by Mann-Whitney test to compare the
T3) by the same amount, each group treatment effect of each treatment.
contain 6 rats. Rat adapted and (Attched in appendix).
maintained for seven days in a cage,
these rat was given food and drink ad RESULTS AND DISCUSSION
libitum in the form of pellets and This experiment was conducted on
vegetables, replacing the litter when it’s 30 white rats (Rattus norvegicus). It
dirty. divided into 5 groups of treatment with 6
replications. And then after passing
Mimosa pudica extraction through a period of adaptation for 7 days,
Mimosa pudica root extraction the negative control (C-) was given
using water extraction method (normal aquadest 0,1ml then 10ml saline
solution 5 minutes later per-orally, then

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the positive control (C+) injected Naja Whitney test. Analytic result of total
sputatrix LD50 (0,13 BW) IM damage scoring is shown in the Table 1.
(musculus gluteus) and given saline
solution 10ml per oral 5 minutes later, Table 1. Meningitis Damage Value
(T1 injected Naja sputatrix LD50 (0,13 Treatment Mean ± SD
musculus gluteus) and
will be given 250 mg/kg BW Mimosa C- 0.350a ±0.104
pudica root extract per-oral in 5 minutes C+ 0.833c ±0.816
later, (T2) injected Naja sputatrix LD50
IM (musculus T1 0.783c±0.408
gluteus) and will be given 500 mg/kg BW T2 0.750bc±0.164
Mimosa pudica root extract per-oral in 5
T3 0.550b±0.187
minutes later (T3) were injected Naja
sputatrix LD50 The different superscript show there is
significant difference between treatment
(musculus gluteus) and will be given 1000
groups (p<0.05).
mg/kg BW Mimosa pudica root extract
per-oral in 5 minutes later. Then while
Statistical analysis of total damage
doing the necroption, rat was injected in Negative control (C-) shows significant
with formalin in the heart to maintain the
difference to other treatment groups.
brain stay on the shape. Positive control (C+) shows no significant
In this research, histological slide
difference to Mimosa pudica root extract
preparation were made from rat’s brain 250 mg/kg BW (T1) and Mimosa pudica
on day after the last treatment, by root extract 500 mg/kg BW (T2),while
fixating them in formalin 10%. The Mimosa pudica root extract 500mg/kg
making of histopathology slide took
BW (T2) shows no significant difference
approximately 2 weeks. Scoring of brain
to Mimosa pudica root extract 1000
slides were done using Modified of
mg/kg BW (T3). Yet Positive control
Kennedy (Kennedy et al., 1997).
shows significant difference to Mimosa
Histopathology examination of
pudica root extract 1000 mg/kg BW (T3).
white rat (Rattus norvegicus) brain given
The histopathological result was
Mimosa pudica root extract post Naja shown in Figure 1. It shows that there is
sputatrix venom injection was done
not any inflammatory cell in negative
microscopically by Hematoxylin Eosin control, C+ shows there is infiltration of
(HE) staining, using 1000 (100x) PMN cell. Treatment with Mimosa pudica
magnification. The variables observed root extract 250mg/kg BW (T1) shows
in this observation were necrosis cells,
that there are some infiltration of PMN
meningitis and perivascular cuffing.
cell. While treatment Mimosa pudica root
Meningitis is defined as
extract 500mg/kg BW (T2) shows that
inflammation of the membranes that there are some PMN cell and
surround the brain and spinal cord haemorrhagic. Treatment with Mimosa
(Schuhat et al.,1997). In this pudica root extract 1000mg/kg BW
experiment, meningitis can be found in
shows that numbers of inflammatory
piamater layer. cell is much reduced than prior
The result of meningitis scoring treatments.
was analyzed using Kruskal Wallis and
Perivascular cuffing scoring was
showing significant difference (p<0.05) analyzed using Kruskal Wallis and
between treatment groups, the analysis
showing significant difference (p<0.05)
was continued afterwards using Mann

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between treatment groups, the analysis Table 3. Necrotic Cell Damage

was continued afterwards using Mann Value
Whitney test. Analytic result of total
Treatment Mean ± SD
damage scoring is shown in the Table 2.
C- 0.400a±0.632
Table 2. Perivascular Cuffing Damage
C+ 3.900d ±0.167
Value
T1 3.316c±0.271
Treatment Mean ± SD
T2 2.216b±0.444
C- 0.316a ±0.117
T3 1.900b±0.209
C+ 3.716d ±0.147
The different superscript show there is
T1 3.367c±0.250 significant difference between treatment
groups (p<0.05).
T2 1.583b±0.386
T3 1.466b±0.265 From Figure 3 can be seen that,
The different superscript show there is histopathology in the negative control
significant difference between treatment (C-) contained no inflammatory cell
groups (p<0.05). infiltration, and it can be seen that the
size and shape of the cells is still
Statistical analysis of total damage relatively normal. In positive control (C+)
in Negative control (C-) and Positive shows that ganglion change to necrosis
control (C+) shows significant difference cell is more than 75%. And there is
to other treatment groups. Mimosa massive inflammatory cells in the blood
pudica 250 mg/kg BW (T1) shows vessel. Astrocyte, oligodendrocytes,
significant different to both treatment pyramid cells does necrosis.
groups (T2 & T3). While Mimosa pudica The histopathology of treatment 1
500 mg/kg BW (T2) shows no significant (Mimosa pudica root extract 250 mg/kg
difference to Mimosa pudica 1000 mg/kg BW) shows that the amount of necrosis
BW. cell is about ≥ 75% and there is
From Figure 2 can be seen that infiltration from inflammatory cell in the
there are necrotic cell and perivascular blood vessel.
cuffing. Histopathology of treatment 2
Necrosis has been defined as a type (Mimosa pudica 500 mg/kg BW) shows
of cell death that lacks the features of necrosis cell is less than 75%. But, some
apoptosis, and autophagy, and is massive inflammatory cells appears in
usually considered to be uncontrolled. the blood vessels.
(Pierre and Guido, 2006). Histopathology representation of
Necrotic cell scoring was analyzed treatment 3 (Mimosa pudica 1000 mg
using Kruskal Wallis and showing kg/ BW) is less massive than any other
significant difference (p<0.05) between treatment. It can be seen from
treatment groups, the analysis was appearance of total normal ganglion,
continued afterwards using Mann pyramid cell, astrocytes. The
Whitney test. Analytic result of necrotic inflammatory cells is rare to be found.
cell scoring is shown in the Table 1. The result of histopathological was
shown in Figure 3.
Based on statistical analysis using
Kruskal-Wallis test shows there are

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Figure 1. Picture of meningitis in the cortex cerebri in the brain. Showing: (1) Normal
meningens. (2) PMN Cell. (3) Haemmorhagic. HE staining, 1000x magnification.

Figure 2. Picture of perivascular cuffing in the cortex cerebri in the brain. Showing: (1)
Perivascular cuffing. (2) PMN Cell. HE staining, using 1000x magnification.

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Figure 3. Picture of nectrotic cell in the cortex cerebri in the brain. Showing: (1)
Normal cell. (2) Necrosis Cell. (3) PMN Cell. HE staining, 1000x magnification.

significant difference between treatment According to Mann-Whitney test,

groups (p<0.05), then the analysis Negative control (C-) shows significant
continued using Mann Whitney test. The difference to all treatment groups. The
result shown by observation and scoring result are negative control (C-) is 0.350,
of meningitis (inflammation of the positive control (C+) is 0.833, Mimosa
membranes). The result is so moderate pudica root extract 250mg/kg BW (T1)
between positive control (C+) and is 0.783, Mimosa pudica root extract
Mimosa pudica root extract 1000mg/kg 500mg/kg BW is 0.750, and Mimosa
BW. pudica 1000mg/kg BW is 0.550.
Cheng et al., (2008) said an α - Positive control (0.833) is
neurotoxins, key components of significant difference with Mimosa
neurotoxic venoms, recognize and bind pudica 1000mg/kg BW. It means
nAChRs, which are widely expressed in Mimosa pudica root extract 1000mg/kg
CNS including brain capillary endothelial BW can reduce the damage caused by
cells, which are the main constituent of Naja sputatrix venom. According to
the BBB. This property of α-neurotoxins Girish et al., (2004) Hyaluronidase and
might facilitate their penetration protease activities of the venom of some
through the BBB. Zhang et al., (2006) Indian snake (Naja naja, Vipera russelii
only 0.2% of the amount injected can and Echis carinatus) were found to be
reach the brain by subcutaneous. This inhibited by the root extracted in water.
means, snake venom succeed in In Joseph (2013), aqueous extract of
damaging brain even for meningitis is dried roots of M. pudica was tested for
not so severe. inhibitory activity on lethality,

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phospholipase activity, edema forming The presence of lymphocytes in

activity, fibrinolytic activity and vascular due to the success of the venom
hemorrhagic activity of Naja naja and that can penetrate the BBB into the
Bangarus caerulus venoms. The brain is considered a foreign substance,
aqueous extract displayed a significant so that there lymphocytes to fagocyt this
inhibitory effect on the lethality, snake venom. This is what causes
phospholipase activity, edema forming perivascular cuffing may occur. And also
activity, fibrinolytic activity and cobra venom factor (CVF) depleting
hemorrhagic activity. About 0.14 mg and complement system attenuates brain
0.16 mg of M. pudica extracts were able to edema in intracerebral hemorrhage.
completely neutralize the lethal activity Nam et al. (2010) showed that
of 2LD50 of Naja naja and Bangarus crocin and crocetin contain in Mimosa
caerulus venoms respectively. pudica provide protection against
According to statistical analysis neuroinflammation. This may explain
using Kruskal-Wallis test shows there the perivascular cuffing is lessen in
are significant difference between treatment 2.
treatment groups (p<0.05), then the Kruskall-Wallis statistical analysis
analysis continued using Mann Whitney test shows there are significant
test. The result shown by observation difference between treatment groups
and scoring of perivascular cuffing. The (p<0.05), then the analysis continued
statistic result shows Mimosa pudica using Mann Whitney test. The result
root extract 250 mg/kg BW (T1) 3.376 is shown by observation and scoring of
significant to both Mimosa pudica root necrosis cell.
extract 500 mg/kg BW (T2) 1.583, and The statistic result shows Mimosa
Mimosa pudica root extract 1000 mg/kg pudica root extract 250 mg/kg BW (T1)
BW (T3) 1.466. 3.316 is significant to both Mimosa
From the statistic data can be seen pudica root extract 500 mg/kg BW (T2)
that the effective dose for Mimosa pudica 2.216 and Mimosa pudica root extract
is 500 mg/kg BW. Because it significant 1000 mg/kg BW (T3) 1.900.
to T1 (3.376) and no significant to T3 From the statistic data can be seen
(1.466). This could happen because in that the effective dose for Mimosa pudica
dose 500 mg/kg BW it can lessen the is 500 mg/kg BW. Because it significant
necrosis higher than T1 and T3. to T1 (3.316) and no significant to T3
Negative control (C-) 0.316 and (1.900). This could happen because in
positive control (C+) are significant to all dose 500 mg/kg BW it can lessen the
treatment groups. It shows that Naja necrosis higher than T1 and T3.
sputatrix venom succeed in damaging In snake venom, there is a protein
and causing haemmorrhage the brain. that can penetrate through the blood
According to Peichoto et al., (2009) snake brain barrier (BBB) which is
venom is rich in neurotoxins significant phospholipases A2.Phospholipase A2
microscopical changes are likely to be and α-neurotoxins bind to postsynaptic
present in the brain tissue. Multiple α-neurotoxins to neuronal nicotinic
small foci of haemorrhages and acetylcholine receptors (nAChR),
congestion of blood vessels have been dendrotoxins - to voltage gated
reported following administration of potassium channels, neurotoxic
collubrid snake venom to rats which was phospholipases A2 – to presynaptic
more marked with intravenous injection membranes, and many other examples.
of venom. It makes the neurotransmitter to other

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organ is obstructed. It can cause necrotic cells and perivascular cuffing

respiratory failure and cardiac arrest. yet there isn’t any significant difference
α-neurotoxins, key components of in meningitis.
neurotoxic venoms, recognize and bind
nAChRs, which are widely expressed in REFERENCES
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