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2012Autumn , 18No , 6 Volume Toxicology Iranian Journal of
1. Department of Biochemistry, University of Ilorin, Ilorin, Nigeria.
2. Department of Science Laboratory Technology, University of Jos, Jos, Nigeria.
*Corresponding Author Email: tomuyak@yahoo.com
Effects of Aqueous Stem Extract of Massularia Acuminata on
Some Liver Function Indices of Male Rats
Musa ToyinYakubu *1, Babasoji Percy Omoniwa 2
Received: 23.06.2012 Accepted: 17.07.2012
ABSTRACT
Background: Massularia acuminata has been claimed to be used in managing
several ailments in folk medicine and in some instances substantiated with
scientific data. This however has been without recourse to its safety. Therefore,
aqueous stem extract of M. acuminata was evaluated for its effects on some
function indices of the liver of male rats.
Methods: Sixty, male rats were grouped into 4 (A, B, C and D) such that Group A
(control) was orally administered 1cm3 of distilled water while those in groups B, C
and D received orally 1 cm3 of extract corresponding to 250, 500 and 1000 mg/kg
body weight respectively. Some biochemical parameters of liver function were
evaluated in the animals after 1, 7 and 21 daily doses.
Results: The extract significantly decreased (P<0.05) the activity of alkaline
phosphatase in the liver of rats throughout the experimental period. This decrease
was accompanied by corresponding increase in the serum enzyme. In contrast, all
the doses of the extract increased the activities of both the AST and ALT in the liver
and serum aspartate aminotransferase and alanine aminotransferase as well as
the concentrations of serum total bilirubin, protein and albumin.
Conclusion: This study has revealed that the aqueous stem extract of Massularia
acuminata at the doses of 250-1000 mg/kg body weight hampered the normal
functioning of the liver of male rats and is therefore not safe for oral consumption at
the doses investigated.
Keywords: Functional Indices, Hepatotoxicity, Massularia acuminata, Rubiacea,
Safety.
IJT 2012; 716-722
INTRODUCTION
Many of these indigenous plants
have been used by man since time
immemorial for curing various ailments
and thus lessening human sufferings
healthwise. They contain substances in one
or more of their organs with not only
chemopreventive and/or therapeutic effects
on ailments but can also be used for the
synthesis of useful drugs. These substances
referred to as bioactive agents include
alkaloids, flavonoids, phenolics,
anthraquinones, terpenes and saponins.
The continuous indiscriminate use of these
plants has therefore necessitated
investigations into their safety. One of such
plant is Massularia acuminata.
Massularia acuminata (family-
Rubiacea), is otherwise known as chewing
stick (English), Pako Ijebu (Yoruba-
Western Nigeria), and Igbo (Igbo-Eastern
Nigeria) is a medium sized shrub that
grows up to 5 m high. It is primarily found
in the understorey of the closed-forest of
western Africa. The leaves are large,
practically stalkless, elliptic and
acuminate. The flowers, usually red, borne
in short axillary cymes, appear around
January. The fruits which are narrowly
ovoid, beaked and yellowish-white in
Iranian Journal of Toxicology Musa ToyinYakubu et al
http://www.ijt.ir ; Volume 6, No 18, Autumn 2012 717
colour are 5 cm long. The stems are used
as chewing sticks in southern Nigeria (1).
The pulped roots are claimed to be used as
enema for dysentery, aphrodisiac and
anticancer. The fruit juice is also used as
antibiotic for eye drop in Sierra Leone (2).
The aqueous stem extract of M.
acuminata have been reported to contain
alkaloids (0.22%), saponins (1.18%),
anthraquinones (0.048%), flavonoids
(0.032%), tannins (0.75%) and phenolics
(0.066%) (3). Several studies have shown
that M. acuminata at concentrations less
than 10% is capable of inhibiting the
growth of Bacteriodes gingivalis, B.
assacharolyticus and B. melaninogenicus
(4-6). They have also reported that the
alkaloidal content of the plant extract have
anti-inflammatory activity and is effective
in preventing against gingivitis and
periodontitis. Furthermore, the androgenic
and gonadotropic effects as well as
aphrodisiac activity of the aqueous stem
extract of M. acuminata at the doses of
250, 500 and 1000 mg/kg body weight
have also been reported in separate studies
using male rats as model (3,7). Despite all
these studies, there is dearth of information
on the effect of the extract on some liver
function parameters of male rats.
Therefore, this study was designed to
provide information on the effect of
aqueous stem extract of Massularia
acuminata at the doses of 250-1000 mg/kg
body weight on some biochemical indices
of liver damage in male rats.
MATERIALS AND METHODS
Plant materials and authentication
The plants which were obtained from
herbsellers at Ijebu Ode, Ogun State,
Nigeria were authenticated at the Federal
Research Institute of Nigeria (FRIN)
Ibadan, Oyo State, Nigeria. A voucher
specimen (FHI107644) was deposited at
the Herbarium of the Institute.
Animals
Sixty, male albino rats(164.50 ± 9.20
g) of norvegicus strain were obtained from
the Small Animal Holding Unit of the
Department of Biochemistry, University of
Ilorin, Ilorin, Nigeria.
Assay kits
Assay kits for alkaline phosphatase
(ALP), aspartate aminotransferase (AST),
alanine aminotransferase (ALT), albumin,
total and conjugated bilirubin were
products of Randox Laboratories, Co-
Antrim, United Kingdom. All other
reagents used were of analytical grade and
were prepared in all glass distilled water.
Animal grouping
The rats were housed in standard
cages and allowed to acclimatize for 7
days. They were fed with normal rat pellet
and tap water throughout the experimental
period. Rats were grouped into 4: A, B, C
and D. Animals in Group A which served
as the control were orally administered
with 1cm3 of distilled water while those in
groups B, C and D received orally 1cm3 of
the extract corresponding to the doses of
250, 500 and 1000 mg/kg body weight
respectively. The administration was done
once daily. The animals were handled
according to the guidelines of European
Convention for the Protection of
Vertebrate Animals used for Experimental
and other Scientific Purposes (8). The 250,
500 and 1000 mg/kg body weight doses
were prepared by dissolving 2.05 g, 4.10 g
and 8.20 g of extract concentrate
respectively in 50 cm3 of distilled water.
The animals were sacrificed 24 hours after
1 (single dose), 7 (seven daily doses) and
21 (twenty one daily doses) days of
administration.
Preparation of aqueous stem extract of
Massularia acuminata
The stem of the plant was first
weighed after which it was sliced into
pieces and oven-dried at 40ºC to a constant
weight. This was then pulverized using an
electric grinding machine. A known
amount (500 g) was percolated in 1 litre of
Effects of Aqueous Stem Extract of … Iranian Journal of Toxicology
Volume 6, No 18, Autumn 2012; http://www.ijt.ir
718
distilled water with intermittent shaking
and kept in the refrigerator for 48 hours.
The solution was thereafter filtered using
Whatman No 1 filter paper and the filtrate
concentrated on a water bath to give a
yield of 25.50 g representing a percentage
yield of 5.10%.
Preparation of serum and liver
homogenates
The rats were anaesthetized in a jar
containing cotton wool soaked in diethyl
ether. When the animals became
unconscious, they were quickly brought
out of the jar and their jugular veins were
cut. Blood samples were collected into
sample bottles and allowed to clot at room
temperature for 20 minutes and thereafter
centrifuged at 1398 x g 15 minutes. Clear
colourless serum was collected with the aid
of Pasteur pipette. The animals were
quickly dissected and the liver removed,
blotted, weighed and homogenized in ice-
cold 0.25M sucrose solution (1:5w/v). The
homogenates were kept frozen and used
for the biochemical analysis within 24
hours of preparation.
Determination of liver function indices
The liver function indices were
determined as described for alkaline
phosphatase (E. C. 3.1.3.1) (9), aspartate
aminotransferase (EC 2.6.1.1) and alanine
aminotransferase (EC 2.6.1.2) (10), total
protein (11), total and conjugated bilirubin
(12) and albumin (13). All measurements
were done using spectronic 21 digital
Spectrophotometer (Bausch and Lomb,
Rochester NY).
Statistical analysis
Data were expressed as the mean ±
SEM of five replicates. The data were
subjected to statistical analysis using one-
way Analysis of Variance (ANOVA) and
complemented with Duncan’s Multiple
Range Test. Statistical difference was set at
p<0.05.
RESULTS
The extract significantly (P<0.05)
decreased the activity of ALP alkaline
phosphatase in the liver of rats throughout
the experimental period. This decrease was
accompanied by corresponding increase in
the serum enzyme (Table 1).
In contrast, the extract at all the
doses investigated significantly increased
the activities of both the AST and ALT in
the liver and serum of the animals. The
increase in serum enzyme activity
manifested at all the days of intervention
(Tables 2 and 3).
Table 1. Alkaline phosphatase activity of male rat liver and serum orally administered with
aqueous stem extract of Massularia acuminata
Liver Serum
Days after administration
Test Samples Doses
(mg/kg)body
weight 1 7 21 1 7 21
Control 1ml 33.25 ±
0.17a 33.16 ±
0.11a 32.20 ±
0.12a 5.93 ±
0.23a 5.96 ±
0.21a 5.95 ±
0.29a
M. acuminata
stem extract 250 19.40 ±
0.28b 20.10 ±
0.29b 27.10 ±
0.14b 8.04 ±
0.48b 8.65 ±
0.28b 8.55 ±
0.19b
“ 500 19.20 ±
0.16b 13.90 ±
0.16c 12.20 ±
0.32c 9.18 ±
0.16c 9.28 ±
0.07c 10.60 ±
0.06c
“ 1000 14.00 ±
0.14c 23.40 ±
0.16d 10.90 ±
0.50d 9.20 ±
0.12c 11.70 ±
0.19d 12.50 ±
0.74d
n=5± SEM; values carrying superscripts different from the control for each day are significantly different
(P<0.05).
Enzyme activities are expressed in UI
Iranian Journal of Toxicology Musa ToyinYakubu et al
http://www.ijt.ir ; Volume 6, No 18, Autumn 2012 719
Table 2. Aspartate aminotransferase activity of male rat liver and serum orally administered
with aqueous stem extract of Massularia acuminata
Liver Serum
Days after administration
Test Samples Doses(mg/kg)
body weight 1 7 21 1 7 21
Control 1ml 5.50 ±
0.20a 5.47 ±
0.29a 5.51 ±
0.17a 1.42 ±
0.11a 1.38 ±
0.18a 1.40 ±
0.11a
M. acuminata
stem extract 250 14.20 ±
0.41b 11.35 ±
0.23b 13.08 ±
0.13b 1.57 ±
0.05b 1.72 ±
0.03b 2.40 ±
0.01b
“ 500 14.00 ±
0.16b 11.50 ±
0.22b 18.05 ±
0.11c 1.57 ±
0.04b 1.75 ±
0.01b 4.50 ±
0.04c
“ 1000 17.50 ±
0.19c 18.25 ±
0.07c 24.00 ±
1.08d 1.75 ±
0.03c 2.91 ±
0.02c 5.80 ±
0.03d
n=5± SEM; values carrying superscripts different from the control for each day are significantly different
(P<0.05).
Enzyme activities are expressed in UI
Table 3. Alanine aminotransferase activity of male rat liver and serum orally administered
with aqueous stem extract of Massularia acuminata
Liver Serum
Days after administration
Test Samples Doses(mg/kg)
body weight 1 7 21 1 7 21
Control 1ml 4.48 ±
0.07a 4.46±
0.09a 4.45±
0.10a 0.50 ±
0.09a 0.51 ±
0.09a 0.48 ±
0.10a
M. acuminata
stem extract 250 5.71 ±
0.05b 6.82±
0.04b 8.39 ±
0.01b 0.48 ±
0.01a 1.40 ±
0.02b 1.04 ±
0.22b
“ 500 6.25 ±
0.03c 8.30±
0.02c 14.80 ±
0.71c 1.03 ±
0.03b 1.51 ±
0.05c 1.62 ±
0.36c
“ 1000 8.51 ±
0.01d 8.35±
0.04c 12.80 ±
0.36d 0.95±
0.04c 1.28 ±
0.71d 1.60 ±
0.08c
n=5± SEM; values carrying superscripts different from the control for each day are significantly different
(P<0.05).
Enzyme activities are expressed in UI
All the doses of the extract
significantly increased the concentration of
the total bilirubin in the serum of the
animals throughout the days of
intervention. These increases were
however not dose related (Table 4). This
pattern of increase was not the same for the
conjugated bilirubin in the serum of the
animals as the effect manifested only in the
1000 mg/kg body weight of the extract
treated animals (Table 4).The extract at the
doses of 250, 500 and 1000 mg/kg body
weight significantly increased the
concentration of total protein in the serum
of the animals (Table 5).
Similarly, the extract also increased
the serum albumin content in the animals
with the increase manifesting only in the
animals administered with a single dose
(day 1) of 500 and 1000 mg/kg body
weight treated animals whereas the seven
(day 7) and 21 daily administration (day
21) of all the doses of the extract
significantly increased the serum albumin
content of the animals (Table 5).
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Table 4. Serum total and conjugated bilirubin contents of the serum of male rats orally
administered with aqueous stem extract of Massularia acuminata
Serum total bilirubin Serum conjugated bilirubin
Days after administration
Test Samples Doses (mg/kg)
body weight 1 7 21 1 7 21
Control 1ml 2.00 ±
0.01a 1.96 ±
0.06a 1.98 ±
0.05a 1.00 ±
0.03a 1.04±
0.00a 1.00 ±
0.03a
M. acuminata
stem extract 250 3.00 ±
0.16b 3.60 ±
0.33b 6.60 ±
0.78b 1.00 ±
0.04a 0.98±
0.06a 1.00 ±
0.01a
“ 500 3.00 ±
0.74b 6.25 ±
0.64c 5.50 ±
0.41c 1.00 ±
0.07a 1.05 ±
0.01a 1.00±
0.06a
“ 1000 3.00±
0.02b 4.80 ±
0.01d 8.40 ±
0.01d 1.25 ±
0.36b 1.00 ±
0.07a 2.10 ±
0.03b
n=5± SEM; values carrying superscripts different from the control for each day are significantly different
(P<0.05).
Concentrations were expressed in µmol/L
Table 5. Serum total protein and albumin contents of the serum of male rats orally
administered with aqueous stem extract of Massularia acuminata
Serum total protein Serum albumin
Days after administration
Test Samples Doses
(mg/kg)
body
weight
1 7 21 1 7 21
Control 1ml 61.00 ±
0.71a 59.27±
1.51a 60.16 ±
0.84a 33.00 ±
0.00a 29.60 ±
3.51a 31.28 ±
2.02a
M. acuminata
stem extract 250 70.50 ±
0.57b 70.10 ±
0.12b 69.90 ±
0.28b 34.04 ±
0.04a 35.58±
0.10b 40.00 ±
0.64b
“ 500 70.20 ±
0.22b 70.50 ±
0.25b 65.00 ±
0.14c 36.30 ±
0.06b 43.75 ±
0.21c 44.60 ±
0.87c
“ 1000 65.00 ±
0.75c 70.70 ±
0.29b 69.00 ±
0.35b 39.00 ±
0.06c 35.50 ±
0.46b 40.30 ±
0.71b
n=5± SEM; values carrying superscripts different from the control for each day are significantly different
(P<0.05).
Concentrations were expressed in g/L
DISCUSSION
The liver is prone to xenobiotic-
induced injury because of its central role in
the metabolism of foreign compounds and
its portal location within the circulatory
system (14). The biochemical parameters
investigated in the present study are
indices routinely used to assess the normal
functioning of the liver. These functions
may be synthetic, secretory and excretory.
Enzyme pattern in the tissues may be used
to assess liver dysfunction and serum
enzyme may be used to corroborate the
physiological state of the organs.
ALP is located in the plasma
membrane and the membrane of the
endoplasmic reticulum (15). It is required
in certain amounts for proper functioning
Iranian Journal of Toxicology Musa ToyinYakubu et al
http://www.ijt.ir ; Volume 6, No 18, Autumn 2012 721
of organs (16). Therefore, the reduction in
the ALP activity of the liver accompanied
by corresponding increase in serum
enzyme suggest permeability changes
leading to leakage of the ALP from the
liver to the serum (17). Such pattern
indicates hepatotoxic effect of the aqueous
stem extract of M. acuminata at these
doses. Interestingly, the cytosolic enzymes
(AST and ALT) would have been expected
to follow the same trend as the ALP, but
the contrary is the case in the present
study. Although, the serum AST and ALT
were elevated corroborated the leakage,
but the reason behind the increase in these
liver enzymes is unclear. It is however
possible that the rate of induction of these
enzymes was far more than the amount that
leaked into the serum (18). All the same,
the reduction in the liver ALP as well as
the raised levels of AST and ALT are
suggestive of M. acuminata induced
hepatoxicity. It is also possible that these
enzymes in some organs not investigated
in the present study might have contributed
to the elevated levels of the transaminases
in the serum of the animals.
Assessment of albumin and protein
in the liver could be used as important
indicator of synthetic function of the
organs whereas bilirubin (total and
conjugated) could be used to assess the
excretory function of the liver (19,20).
Severe hemolysis causes the release of
more bilirubin into the blood which
manifests as elevated levels of
unconjugated and total bilirubin (21).
Unconjugated and total bilirubin can also
increase in the event of low bilirubin
conjugation (21). Therefore, the elevated
levels in the albumin, total protein and
bilirubin may be due to increase in the
functional activity of the organ. The extract
might have stimulated the liver to increase
the synthesis of the albumin and protein to
a level that far exceeded that required by
the animals. Similarly, the increase in the
total and conjugated bilirubin may not only
indicate obstruction of the biliary duct but
also suggest an effect on the normal
excretory function of the liver. This may
have consequential effect on the
conjugation process in the animals. Such
elevated levels of these biomolecules are
indicators of toxic effect of the extract on
the organ at the doses investigated.
CONCLUSION
The aqueous stem extract of
Massularia acuminata at the doses of 250,
500 and 1000 mg/kg body weight have
caused functional toxicity of the liver in
the animals. Therefore, the extract at these
doses is hepatotoxic and should be used
with caution.
ACKNOWLEDGEMENTS
The first author, YMT is grateful to
the financial support in form of research
grant (reference F/3977-1 and F/3977-2)
provided to him by International
Foundation for Science (IFS), Stockholm,
Sweden and Organisation for the
Prohibition of Chemical Weapons
(OPCW), The Hague, for co-sponsoring
parts of the grant.
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