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Research Article
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Sub-chronic Study of Aqueous Stem Bark Extract of Senna siamea in Rats |
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A. Mohammed,
S.B. Mada
and
H.M. Yakasai
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ABSTRACT
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Senna siamea is a plant commonly used in traditional medicine to treat
hypertension, malaria and diabetes mellitus. The aim of this study was to investigate
the sub-chronic toxicity of aqueous extract from the stem bark of S. siamea
male Wistar rats. The extract was orally administered for seven weeks at 200,
400, 800 and 1600 mg kg-1 body weight. The results obtained showed
that the extract significantly (p<0.05) increases the body weight and feed
intake of the rats. Hematological parameters (PCV, Hb, platelets, WBC and RBC)
were not significantly (p<0.05) affected by the extract. But the levels of
serum liver enzymes (Alkaline Phosphatase (ALP), Alanine Aminotransaminase (ALT),
Aspartate Aminotransaminase (AST) were significantly different (p<0.05) from
the normal control group. In contrast, no significant change was observed in
the total protein and albumin levels in the treated group compared to the normal
group. Similarly, serum glucose, triglycerides, cholesterol and the markers
of kidney function (creatinine, urea, potassium, sodium and chloride) did not
differ significantly (p<0.05) from the normal control group. The quantitative
determination of saponins, alkaloids, total polyphenolics and flavonoids in
(g/g) were found to be 0.07±0.01, 0.05±0.02, 0.92±0.05
and 0.06±0.01. These results may explain the use of S. siamea stem
bark in folk medicine due its less toxic effect.
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Received: July 09, 2012;
Accepted: September 08, 2012;
Published: October 13, 2012
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INTRODUCTION
Medicinal plants are extensively used in treatment and management of diseased
conditions, especially in developing countries. This is due to result of their
wide availability, less toxicity and more affordable alternative to synthetic
drugs. Toxicity studies on medicinal plants or extracts from them usually determine
the level of safety particularly during the development of drugs (Jaijoy
et al., 2010). Senna siamea Lam. (Irwin and Barneby-Cassia
siamea Lam.) (Fabaceae, Caesalpiniaceae) (Doughari and
Okafor, 2008), or Thailand shower (Aliyu,
2006), is a native of tropical Asia, introduced and now naturalized in Africa.
This plant reaches up to 15-20 cm tall, with about 30 cm diameters. Bark is
grey or light brown, smooth but becoming slightly fissured with age (Von
Maydell, 1986). S. siamea is claimed to be used to treatment of disease
conditions such as diabetes, insomnia, hypertension, asthma, constipation and
dieresis (Hill, 1992). S. siamea, fresh young
flowers and/or young leaves have been used as vegetables in Thailand (Otimenyin
et al., 2010). The stem bark extract was locally reported to have
analgesic and anti-inflammatory effects (Ntandou et al.,
2010) and antimalarial effect (Odugbemi et al.,
2007).
Most of the toxicity studies carried out on S. siamea was on the leaf
and root extracts, little information on S. siamea stem bark toxicity
is currently available. The purpose of the present study is to investigate sub-chronic
toxicity of the aqueous stem bark extract S. siamea in rats.
MATERIALS AND METHODS
Collection, identification and extraction of plant materials: The fresh
stem bark of the S. siamea was collected from the Zaria Local Government
garden, Zaria, Kaduna State, Nigeria in March, 2008. The plant was identified
and authenticated by botanist in the Biological Sciences Department, Ahmadu
Bello University, Zaria, Kaduna State, Nigeria. The stem bark was sorted to
eliminate any dead matter and other unwanted particles. The stem bark was air-dried
for 2 weeks and then ground into fine powder using pestle and mortar. A total
of 400 g of the ground powder was soaked in 2 L of distilled water for 48 h
at room temperature. The mixture was filtered into conical flasks with Watman
filter paper (No. 1). The filtrate was dried at a temperature of 30°C for
10 h.
Experimental animals: Sixty five healthy male Wistar rats (weighing
about 100-160 g) bred in the animal house of Department of Pharmacology, Faculty
of Pharmaceutical Sciences, Ahmadu Bello University, Zaria-Nigeria, were used
for this study. They were fed with feeds (Vital feeds, Nigeria) and tap water
ad libitum. The experiment was approved by the ethical committee on the
use of experimental animals of the Ahmadu Bello University, Zaria, Nigeria.
Methodology
Acute toxicity study: The acute toxicity (LD50) was carried out
by Lorke (1983) Method. The method involved two phases
of which eighteen (18) rats were grouped in to three groups of six rats each.
They received 10, 100 and 1000 mg kg-1 body weights of the extracts.
In the second phase twelve rats were grouped in to three groups of for rats
each, and they received 1500, 2900 and 5000 mg kg-1 body weights.
The rats were observed daily for any signs of toxicity, throughout the period
of study.
Sub-chronic study: Thirty five were used for sub-chronic study. The
rats were randomly grouped into five groups of seven animals in each group.
Group 1 served as control and received distilled water while Groups 2, 3, 4
and 5 received the aqueous extract of the stem bark of S. siamea 200,
400, 800 and 1600 mg kg-1, respectively. The dosages used were established
from the previous study. Rats received their doses once daily orally, for 7
weeks. The rats were observed daily for any signs of toxicity, throughout the
period of study.
Throughout the treatment period, body weights and feed intakes were recorded
weekly. At the end of the treatment, all the animals were fasted for 8 h. Blood
samples were collected from rats
tail into heparinized blood sample bottles and then the rats were anesthetized
with ether and sacrificed by drawing blood from the inferior vena cava. Samples
collected were used for the determination of hematological and biochemical parameters.
Determination of hematological parameters: Packed Cell Volume (PCV)
of each sample was determined using a Hawksley micro-haematocrit centrifuge
(McGovern et al., 1955). Erythrocytes (RBC) and
total leucocytes (WBC) were counted using the improved Neubauer haemocytometer
as describe by Otimenyin et al. (2009).
Determination of biochemical parameters: Biochemical analysis of serum
samples were conducted using reagents kits (Randox kits) by the following methods:
AST and ALT (Reitman and Frankel, 1957), ALP (King,
1965), bilirubin (Malloy and Evelyn, 1937), creatinine
(Bartels et al., 1972), cholesterol (Stein,
1987), triglyceride (McGowan et al., 1983),
total protein and albumin (Lowry et al., 1951),
urea (Fawcett and Scout, 1960), glucose by glucose oxidase
method (Beach and Turner, 1958). Sodium, potassium and
chloride were analyzed using flame photometer (model Gallen-Kamp FGA-300-C).
Quantitative determination of phytochemicals: Quantitative determination
of phytochemicals was carried out for total phenolics (Edeoga
et al., 2005), flavonoids (Bohm and Koupai-Abyazani,
1994), alkaloids and saponins (Obadoni and Ochuko, 2001).
Statistical analysis: Data are presented as Mean±Standard deviation
(SD) and analyzed using Analysis of Variance (ANOVA) and Duncan post hoc
test and significance was determined at p<0.05.
RESULTS
The results obtained from the present study indicated that the oral LD50
of the aqueous extract of stem bark of S. siamea was found to be greater
than 5000 mg kg-1. The extract showed no sign of toxicity for the
time periods and no death was recorded.
For sub-chronic study, the result indicated that the aqueous stem bark extract
of S. siamea did produce significant (p<0.05) increase in body weight
from second week to seventh week, especially with group 5 (Fig.
1). It was also observed that all the groups significantly (p<0.05) increased
in their feed-intake habit when compared with that of normal control group (Fig.
2).
The results for phytochemical screening (Table 1), indicated
that the amount of total polyphenolics (g/g) was the highest (0.92±0.05),
while the amounts of saponins, flavonoids and alkaloids were found not to differ
significantly (p<0.05) from each other.
Hematological parameters analyzed (Table 2) showed that,
all the treated groups did not differ significantly (p<0.05) from the normal
control group (group 1) up to the maximum dose of 1600 mg kg-1 body
weight. The PCV slightly increases by 1.7% in group 2, but decreased in other
treated groups.
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Fig. 1: |
Growth curves of rats receiving the aqueous stem bark extract
of S. siamea for 7 weeks |
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Fig. 2: |
Feed intake of rats receiving S. siamea for 7 weeks |
Table 1: |
Quantitative amount of phytochemical constituents of Senna
siamea stem bark extract |
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Values are of Mean±SD of triplicates determinations |
Table 2: |
Effect of aqueous stem bark extract of S. siamea on
haematological parameters in rat |
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All values are Mean±SD of seven replicates, Values
with different superscripts along a row are statistically different at p<0.05,
n = 7, D: Percentage decrease compared to control, I: Percentage increase
compared to control |
Group 3 recorded the highest decrease of 2.5% when compared with that of group
1. The Hb content of group 2 and 4 recorded similar percentage (%) decrease
serum levels (6.0 and 5.9%, respectively) which are not statistically significant
(p<0.05) from group 1. Group 3 Hb levels had an increase of 2.9%, higher
than that of group 5 (0.3%). All the treated groups recorded a decrease in RBC
levels, with the exception of group 4, which has an increase of 4.2% versus
group 1. WBC levels of all the treated groups indicated an increase, with group
4 having the highest of about 13.8%. The platelets levels of group 2 decrease
by 9.0% when compared to group 1. Unlike the other treated groups that showed
an increase in platelets contents which did not differ significantly (p<0.05).
Moreover, result on serum liver enzymes showed that AST, ALT and ALP were statistically
significant (p<0.05) in comparison to the normal control group (Table
3). AST and ALP levels of all the treated groups recorded a decrease, with
group 2 and 3 having the highest % decrease in AST and ALP of 4.7 and 8.0%,
respectively. The ALT levels of group 3 significantly increase by 1.4%, while
the other treated groups recorded a decrease in ALT levels, with group 5 having
the highest decrease of 7.5%. Group 3 and 4 showed an increase in bilirubin
and total protein levels of 16.1 and 9.6%, respectively.
Table 3: |
Effects of the aqueous stem bark extract of S. siamea
on serum markers of liver damage |
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All values are Mean±SD of seven replicates, Values
with different superscripts along a row are statistically different at p<0.05,
n = 7, D: Percentage decrease compared to control, I: Percentage increase
compared to control |
Table 4: |
Effects of the aqueous stem bark extract of S. siamea
on serum markers kidney function |
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All values are Mean±SD of seven replicates, Values
with different superscripts along a row are statistically different at p<0.05,
n = 7, D: Percentage decrease compared to control, I: Percentage increase
compared to control |
Table 5: |
Effects of the aqueous stem bark extract of S. siamea
on serum glucose, cholesterol and triglyceride |
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All values are Mean±SD of seven replicates. Values
with different superscripts along a row are statistically different (p<0.05).
n = 7, D: Percentage decrease compared to control |
The remaining other groups recorded a % decrease when compared with group
1. In all the treated groups there is an increase in the levels of the albumin
contents when compared to group.
Also, no significant (p<0.05) difference in the serum levels of markers
of kidney functions was recorded (Table 4). In all the parameters
analyzed, group 2 recorded a slight % increase while group 5 showed a % decrease
in the levels of markers of kidney functions, with the exception of group serum
chloride levels (1.4% increase).
In Table 5, the serum glucose, cholesterol and triglycerides
levels of all the treated groups recorded a % decrease which did not differ
significantly (p<0.05) from that of group 1. The highest % decrease recorded
was serum cholesterol of group 5 (8.7%), while the lowest was the triglycerides
levels of group 4 (0.04%).
DISCUSSION
The therapeutic effect of most medicinal plants is indisputable but their toxicities
sometimes limit their clinical uses. Hence, the toxicity profile of most plants
must always be considered especially as the doses and dosing regimens of their
preparations are not usually determined. The result of this finding is quite
promising as the stem bark extract of S. siamea was relatively non toxic
to rats at oral doses up to 5000 mg kg-1. The increase in body weight
observed, which cuts across all the groups might have resulted from the increased
in the feed intake habit, probably stimulated by the extract. This contradict
with that of C. sieberiana stem bark extract, which resulted in a slight
reduction on body weight of the treated groups (Obidah et
al., 2009). But the result of this study agreed with the findings of
Otimenyin et al. (2010). This might be likely
due to similar content of phytochemicals, as the plants activity depends
on its chemical contents (Kwada and Tella, 2009).
The result also indicated no gross physiological alterations in the body system
as no significant (p<0.05) effect were noticed on the hematological parameters
analyzed (Table 2). The slight physiological fluctuations
on hematological parameters observed did not correspond with that of the S.
siamea leaves extract (Chavalittumrong et al.,
2003). This possibly might be due short study period or the high levels
of some hepatotoxicants, such as barakol, reported on the leaves extract (Padumanonda
and Gritsanapan, 2006).
In detecting liver damage the determination of enzyme levels such as AST, ALT
and ALP is largely used. Necrosis or membrane damage releases the enzyme into
circulation and hence it can be measured in the serum. Elevated levels of serum
enzymes are indicative of cellular leakage and loss of functional integrity
of cell membrane in liver (Palanivel et al., 2008).
The extract has shown no much effect on the serum enzymes of liver function
(Table 3), this might be attributed to the fact that, the
liver was not greatly damaged by the extract due to short time period, to cause
a significant increase on the serum levels of liver enzymes and bilirubin into
the blood.
Serum total protein is a complex mixture containing a number of components
which differ in properties and function. Liver is the organ mainly responsible
for formation of plasma albumin (Obidah et al., 2009).
Decrease in serum total protein and increase serum urea and creatinine levels
are associated with liver and renal failure (Chawla, 1999).
Urea represents the major way of total urinary nitrogen excretion. The treated
groups in this study exhibited no significant increase on serum urea and creatinine
that might suggest the level of safety of the extract on kidney. This corresponds
with the previous findings on roots extract of S. siamea (Otimenyin
et al., 2010).
The extract also showed no toxic effect on the serum levels of glucose and
lipids profile throughout the experimental period. This might be associated
to less toxic effect of the extract on liver, as liver is plays a significant
role in metabolism of cholesterol and other lipids compounds Many toxic effects
of medicinal plants extract depend basically on many factors such as route of
administration and the dose administered (Singh and Devkota,
2003). Intraperitoneal administration of extracts provides more clear toxicity
levels than the oral route, usually due to some inherent factors that limit
absorption in the gastrointestinal tract. Thus, further work is recommended
for intraperitoneal administration of the extract and chronic toxicity study,
to fully establish its safety.
CONCLUSION
In conclusion, the crude aqueous stem bark extract of S. siamea showed
no significant effect on most of the parameters evaluated. The finding suggests
that the stem bark extract of S. siamea, is relatively not toxic and
further work should be carried out on its chronic toxic effect.
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