Biokemistri Volume 33, Number 1

ISSN 0795-8080
VOLUME 33, NUMBER 1

MARCH, 2021
BIOKEMISTRI
An International Journal Published by the
Nigerian Society for Experimental Biology
ii
BIOKEMISTRI
An International Journal Published by the Nigerian Society for Experimental Biology
Editor-in-Chief
Prof. Clement O. Bewaji, Department of Biochemistry, Faculty of Life Sciences, University of
Ilorin, Ilorin, Nigeria.
Editorial Advisory Board

Dr-Mrs. R. A. Adisa, Department of Dr. Yun-gen Miao, Key Laboratory of
Biochemistry, College of Medicine, Animal Virology, College of Animal
University of Lagos, Lagos, Nigeria. Sciences, Zhejiang University, Hangzhou
310029, China.
Prof. I. S. Afolabi, Department of
Biochemistry, Covenant University, Ota, Dr. Saleh A. Mohamed, Department of
Nigeria. Biochemistry, Faculty of Science, King
Abdulaziz University, Jeddah 21589, Saudi
Dr. N. Afzali, Department of Animal
Arabia.
Science, Faculty of Agriculture, Birjand
University, Birjand, Iran. Dr. Shazia Nouren, Department of
Chemistry and Biochemistry, University of
Dr. R. O. Arise, Department of
Agriculture, Faisalabad-38040, Pakistan.
Biochemistry, Faculty of Life Sciences,
University of Ilorin, Ilorin, Nigeria. Dr. Farwa Nurjis, National Institute for
Biotechnology and Genetic Engineering,
Demiray Dr. Hatice Demiray, Department of
Faisalabad-38000, Pakistan.
Biology, Ege University, Izmir (Bornova),
Turkey. Prof. Ganiyu Oboh, Department of
Biochemistry, Federal University of
Prof. Evans C. Egwin, Department of
Technology, Akure, Nigeria.
Biochemistry, Federal University of
Technology, Minna, Nigeria. Prof.-Mrs. Henrietta Oboh, Department of
Medical Biochemistry, Faculty of Basic
Dr. Hossam S. El-Beltagi, Department of
Medical Sciences, University of Benin,
Biochemistry, Faculty of Agriculture, Cairo
Benin City, Nigeria.
University, Giza, Egypt.
Prof.-Mrs. O. A. Odunola, Department of
Prof. O. Farombi, Department of
Biochemistry, Faculty of Basic Medical
Biochemistry, Faculty of Basic Medical
Sciences, University of Ibadan, Ibadan,
Sciences, University of Ibadan, Ibadan,
Nigeria.
Nigeria.
Dr. Raphael John Ogbe, Department of
Dr. A. Igunnu, Department of Biochemistry,
Veterinary Physiology, Pharmacology and
Faculty of Life Sciences, University of
Biochemistry, University of Agriculture,
Ilorin, Ilorin, Nigeria.
Makurdi, Nigeria.
Prof.-Mrs. A. T. Oladiji, Department of
Biochemistry, Faculty of Life Sciences,
University of Ilorin, Ilorin, Nigeria.
Dr. Mehdi Rajabi, Department of Process Dr. N. J. Siddiqi, Department of
Chemistry, Kinentia Bioscience LLC, 7 Biochemistry, College of Science, King
University Place, Rensselaer, New York Saud University, Riyadh-11495, Saudi
12144, USA. Arabia.
Dr. Sara Alaei Shehni, Department of Dr. Farzana Siddique, Department of Food
Biology, Alzahra University, Tehran, Iran. Technology, PMAS-Arid Agriculture,
Rawalpindi, Pakistan.
Dr-Mrs. K. O. Shittu, Department of
Biochemistry, Federal University of Prof. M. T. Yakubu, Department of
Technology, Minna, Nigeria. Biochemistry, Faculty of Life Sciences,
University of Ilorin, Ilorin, Nigeria.
Dr. Olutayo Sunday Shokunbi, Department
of Biochemistry, Babcock University, Ilisan
Remo, Nigeria.
BIOKEMISTRI
Volume 33 Number 1 Contents March, 2021
BKR 2020080/33101
Selected liver and kidney function indices of Wistar rats fed with Ficus exasperata vahl
leaf-based diet
O. Soji-Omoniwa and H. O. B. Oloyede.............................................................................................. 1
BKR 2020084/33102
Hypolipidemic potentials of methanol extracts of Vernonia colorata
I. N. Eke-Ogaranya, I. I. Ijeh and A. C. Nnamudi ............................................................................... 15
BKR 2020085/33103
Antigenotoxic and hepatoprotective activities of ethanol extract of the leaf of Eclipta alba
in sodium arsenite-induced toxicity
O. A. Odunola, N. O. Fashina, I. M. Iloba, M. A. Gbadegesin, A. M. Adegoke and O. J.
Olugbami ............................................................................................................................................. 27
BKR 2020095/33104
Gamma-sitosterol-rich fraction from the methanolic extract of Ficus exasperata restores
pathophysiological alterations in alloxan-induced diabetic rats
A. O. Adeyi, G. O. Izu, O. L. Erukainure and Md. S. Islam ................................................................. 39
BKR 2020096/33105
Methanolic extract of Garcinia kola elicits diuretic activity with alteration in circulating
electrolyte concentration in male Wistar rats
K. S. Olaniyi, N. T. Akinnagbe, C. L. Atuma, H. Mahmud, C. O. Badejogbin, T. B.
Agunbiade, O. C. Akintayo and O. J. Sanya ........................................................................................ 53
BKR 2020066/33106
Biochemical changes in rats exposed to crude oil and the antioxidant role of Allanblackia
floribunda stem bark
S. O. Olubodun, D. K. Fayemi and O. A. Osagie ................................................................................. 67
BKR 2020093/33107
Safety evaluation of Dialium guineense fresh fruit pulp meal-based diet
R. A. Oyegoke and A. T. Oladiji .......................................................................................................... 77
vi
O. Soji-Omoniwa & H.O.B. Oloyede
0795-8080/2021 $10.00 + 0.00

Biokemistri
Vol. 33, No. 1, March 31, 2021 © 2021 Nigerian Society for Experimental Biology
Printed in Nigeria http://journals.niseb.org.ng
Also available online at http://www.bioline.org.br/bk
BKR 2020080/33101
Selected liver and kidney function indices of Wistar rats fed

with Ficus exasperata Vahl leaf-based diet
Omolola Soji-Omoniwa* and Hussein Oyelola Bukoye Oloyede
Department of Biochemistry, University of Ilorin, P.M.B. 1515, Ilorin, Kwara State, Nigeria
*Corresponding author E-mail: lolasojiomoniwa@gmail.com; Telephone: 07034205898
(Received November 24, 2020; Accepted February 4, 2021)
ABSTRACT: In a previous study, treatment of diabetes mellitus with Ficus exasperata leaf-based diet (FELD)
significantly reduced the level of blood glucose and improved glucose utilization in diabetic rats. It is therefore
important to ascertain safety of consumption of FELD in healthy rats. Twenty-four rats were randomly selected into
4 groups of 6 animals each namely: C [control rats fed diet without Ficus exasperata leaf (FEL)], F1, F2, and F3
[experimental groups fed diets containing 10 %, 30 % and 50 % FEL respectively]. Rats were allowed access to the
compounded feed ad libitum for 7, 14 and 21 days. They were sacrificed at the end of the experiment and serum
collected for biochemical assays. Total protein, albumin, globulin, bilirubin (total and conjugated), urea, creatinine,
electrolytes, alkaline phosphatase (ALP), alanine amino transferase (ALT), aspartate amino transferase (AST), liver
and kidney histopathology were evaluated. Results showed no significant difference (p<0.05) in albumin, globulin,
bilirubin, electrolytes, ALP, AST and ALT of rats fed with test diets compared to control. Also, there were no changes
in the liver and kidney histoarchitecture of rats fed with different proportions of FELD compared to control. The
increased urea and creatinine concentrations of rats fed with 30 and 50 % FELD at the end of days 7 and 14 reversed
to normal at the end of day 21 of the study. Therefore, results of this study suggest that consumption of 10, 30 and 50
% Ficus exasperata leaf-based diet for 21 days did not impair the selected liver and kidney functions indices of the
treated rats.
Keywords: Ficus exasperata, Liver function, kidney function, leaf-based diet, albumin
Introduction
Ficus exasperata Vahl belongs to the mulberry family and is popularly known as fig plant (Oladosul
et al., 2009). It is widespread in tropical Africa. In Nigeria, there are more than 45 different species of Ficus
(Adewole et al., 2011; Mbakwem-Aniebo et al., 2012). Ficus exasperata leaf is commonly called ewe ipin
in Yoruba language, kawusa in Nupe, ameme in Edo and anwerenwa in Igbo languages in Nigeria (Ijeh and
Ukweni, 2007). Traditionally, the decoction and infusion of the leaves are used to treat diabetes mellitus,
ulcers, dysentery and hypertension amongst others (Joseph and Raj, 2010). Fresh leaves of the plant are
regularly included during the milling or pounding stage of palm oil production by natives of Nigeria to
improve the quality and stability of palm oil (Umerie et al., 2004).
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Biokemistri Volume 33, Number 1 (2021)
In previous studies, proximate compositions, mineral contents and fatty acids profile of Ficus
exasperata leaf were studied with a view to investigating its nutraceutical potentials. Report showed that it
is rich in fiber, protein and carbohydrate, low moisture and fat contents (Falade et al., 2004; Bello et al.
2014). It is also a rich source of potassium, calcium, linoleic acid and oleic acid. Phytochemical analysis of
the leaves and stem extracts also showed that it contains flavonoids, tannis, saponins, alkaloids and
cyanogenic glycosides (Ijeh and Ukweni, 2007; Soji-Omoniwa, and Oloyede, 2018).
In recent years, the use of non-culinary herbs in food products had risen (Jenkins et al., 2008; Peng et
al., 2010; Ansari and Kumar, 2012; Soji-Omoniwa, 2018). Herbs consist of leaves, flowers, stems and roots
from a variety of herbaceous plants used either in fresh or dried form (Oxford dictionary of English, 2010:
Allaby, 2012). Some herbs are used for culinary purposes, e.g. basil, bay leaves, marjoram and thyme.
Others are generally used for non-culinary purposes only, e.g. Echinacea, evening primrose, St John’s wort,
Ginkgo biloba and Ficus exasperata. Some herbs may be used for both culinary and non-culinary purposes,
for example ginger and garlic (Allaby, 2012). This technology of incorporating medicinal or non-culinary
herbs into diet offers great promise for disease management.
For instance, Peng et al. (2010) fortified bread with grape seed extract (GSE), which contains catechins
and proanthocyanidins. These metabolites possess strong antioxidant and free radical scavenging activity.
The GSE- fortified bread elicited a significant antioxidant activity than blank bread. Also, Jenkins et al.
(2008) extracted glucomannan, a glucose-mannose polysaccharide from the tuber roots of Amorphophallus
konjac and incorporated it into dough of biscuits. Consumption of these biscuits reduced glycemic index
significantly by 74% in healthy human volunteers and by 63% in participants with diabetes mellitus.
Similarly, Ficus exasperata leaves were incorporated into locally-sourced food ingredients to produce Ficus
exasperata leaf-based diet (FELD). Treatment of diabetes mellitus with FELD significantly reduced the
level of blood glucose and improved glucose utilization in diabetic rats compared to control (Soji-Omoniwa
and Oloyede, 2018).
Despite the extensive use of Ficus exasperata leaf, it is important to assess the safety of Ficus
exasperata leaf-based diet for consumption by evaluating it effect on some selected indices of liver and
kidney function of wistar rats.
Materials and Methods
Plants collection and authentication

Fresh leaves of Ficus exasperata were collected in February, 2018 from Odo Eri in Kogi State, Nigeria.
The specimen sample was prepared and deposited for identification and authentication at the Herberium
unit of the Department of Plant Biology, University of Ilorin, Ilorin, Nigeria. A voucher number
UILH/001/883 was thereafter issued.
Experimental animals
Wistar rats of norvegicus strain, weighing between 100 g to 150 g were obtained from the animal
holding unit of the Department of Biochemistry, University of Ilorin, Ilorin, Nigeria. The rats were housed
in well ventilated cages and allowed to acclimatize to animal house conditions for 7 days. They were fed
with normal rat pellet and tap water prior to the commencement of the experiment.
Ethical clearance
Ethical clearance for the study was obtained from the University of Ilorin Ethical Committee with
protocol identification code UERC/LSC 067.
Plant preparation
Fresh leaves of F. exasperata were washed and air dried to a constant weight. They were thereafter
pulverized into fine powder using an electronic blender, oven treated using laboratory oven at 110oC for 6
mins and then stored in an air tight container in a refrigerator prior to analyses.
2
Feed ingredients
Yellow corn, maize husk, soy bean oil, soy bean grain and sucrose (sugar) were purchased from local
markets in Ilorin, Kwara State, Nigeria. The corn and soy bean were stored in air tight containers prior to
the commencement of the experiment. The soy bean oil was a product of Kewalram Nigeria Limited,
Nigeria. Vitamin and mineral mix, D-methionine and L- lysine were purchased from local vendors in Ilorin,
Nigeria.
Feed preparation and administration to experimental rats

Corn starch was prepared by rinsing and soaking 10 kg of yellow corn in 20 L of distilled water for 72
hours. It was then ground and sieved using muslin cloth. The filtrate was stored in a sac and drained under
heavy weight for 6 hours, after which it was sun dried to constant weight.
Maize husk was used as the cellulose source and it was prepared by sun drying 10 kg husks for 3 days
and then ground using commercial grinder.
The soy bean grain (7 kg) flour was prepared by first removing the bean coat using a commercial
grinder. Thereafter, it was ground to smooth texture. The Corn starch, maize husk, sucrose, soybean flour,
vitamin/mineral mix, DL-methionine and L- lysine were thoroughly mixed together in the various
proportions indicated in Table 1. Soybean oil (40 ml) and distilled water (1000 ml) were added slowly to
the mixed ingredients until the mixture became a paste. The paste was then pelletized using a wire mesh
and then oven dried at 40oC to a constant weight to form the control feed. The test feeds were prepared in
a similar manner to the control feed. However, 100, 300 and 500 grams of the pulverized F. exasperata
leaves were added to the mixed ingredients as shown in Table 1. The prepared feeds were then administered
to rats ad libitum for a period of 7, 14 and 21days.
Animal grouping
Twenty-four (24) albino rats were randomly selected into 4 groups of 6 animals each as indicated
below for each study period (days 7, 14 and 21):
C - Control rats fed diet without Ficus exasperata leaves (FEL)

F1 - Rats fed diet containing 10 % w/w Ficus exasperata leaves
Table 1: Feed Formulation of Ficus exasperata leaf-based diet
Ingredients (g/kg) Control 10% 30% 50%
Corn starch 512 412 212 12

Cellulose 40 40 40 40
Sucrose 100 100 100 100
Soybean 250 250 250 250
Soybean Oil 40 40 40 40
Vitamin/mineral mix 50 50 50 50
D- Methionine 4 4 4 4
L-lysine 4 4 4 4
F. exasperata leaves - 100 300 500
3
Animal sacrifice and tissue collection

Rats were sacrificed 24 hr after days 7, 14 and 21 of consuming FELD. They were anaesthetized with
diethyl ether and sacrificed by incising the jugular vein using a scalpel. Blood samples were collected into
plain sample bottles for biochemical analysis. After sacrifice, the rats were dissected the livers and kidneys
were then isolated.
The isolated tissues were cleansed with cotton wool to remove blood stains, weighed and immediately
stored in ice cold phosphate buffer. They were then homogenized in an ice-cold phosphate buffer (0.1 M,
pH 7.4) (1:5w/v). The homogenates were stored in the freezer (-4oC) until required for further analysis.
Serum was collected by allowing blood sample to stand at room temperature for 30 mins to form clot. The
supernatant which is the serum was collected using Pasteur pipette.
Biochemical parameters
Liver function indices

Total and conjugated bilirubin, albumin and globulin concentrations were determined by using the
method described by Teitz (1995). Total protein concentration was determined using biuret method.
Alkaline phosphatase (ALP) activities in the liver and serum were assayed using the method described by
Wright et al. (1979). The method described by Reitman and Frankel (1957) was used for assaying the
activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the liver and serum.
Kidney function indices

Serum urea concentration was determined by using the method of Fawcett and Scott (1960).
Serum creatinine and electrolytes (phosphate and calcium ion) concentrations were determined using
the method described by Tietz (1995). Sodium ion, potassium ion, chloride and bicarbonate concentrations
were determined using Trinder (1951), Henry (2001), Sink and Neal (2009) and Bergmeyer (1987).
Histopathology study
Histopathological examination of the liver and kidney was carried out using the method described by
Krause (2001).
Statistical analysis
Data were expressed as mean of 6 determinations ± SEM. The data were subjected to statistical
analysis using the IBM® statistical package for social sciences (SPSS) software version 20. All significant
differences were determined by one way analysis of variance (ANOVA). Post hoc multiple comparisons
were done using Duncan's multiple range test. The level of significance was set at p < 0.05 (confidence
level = 95 %).
Results
Liver function indices

The serum total protein of rats fed with 50 % FELD was significantly reduced (p<0.05) compared to
the control group at the end of days 7, 14 and 21 of the study. The rats fed with 10 and 30 % FELD were
however not significantly different (p>0.05) compared to the control. There was no significant difference
(p>0.05) in results of serum albumin and globulin for all the experimental rats compared to control (Table
2). Similarly, total and conjugated bilirubin concentrations, alkaline phosphatase, alanine amino transferase
and aspartate amino transferase activities for all the experimental rats were not significantly different
(p>0.05) to the control (Table 3, Figures 1, 2, 3).
4
Table 2: Serum total protein, albumin and globulin concentrations of rats fed with Ficus exasperata Leaf-based Diet
Serum total protein (g/dl) Serum albumin (g/dl) Serum globulin (g/dl)
Groups
Day 7 Day 14 Day 21 Day 7 Day 14 Day 21 Day 7 Day 14 Day 21
C 8.31 ±0.26a 6.65 ± 0.06a 6.61 ± 0.02a 5.50±0.20a 5.52 ± 0.10a 5.36 ± 0.05a 2.81 ± 0.10a 1.13 ± 0.10a 1.25 ± 0.05a
F1 8.30 ±0.17a 6.58 ± 0.07ab 6.61 ± 0.02a 5.55±0.15a 5.48 ± 0.15a 5.28 ± 0.01a 2.75 ± 0.15a 1.10 ± 0.15a 1.32 ± 0.02a
F2 7.98±0.25ab 6.68 ± 0.05a 6.54 ± 0.03ab 5.56±0.22a 5.56 ± 0.12a 5.35 ± 0.07a 2.42 ± 0.30a 1.12 ± 0.08a 1.19 ± 0.09a
F3 7.49 ±0.26b 6.49 ± 0.06b 6.47 ± 0.05b 5.32±0.24a 5.30 ± 0.14a 5.32 ± 0.06a 2.17 ± 0.15ab 1.19 ± 0.10a 1.08 ± 0.15a
Values are expressed as mean of 6 determinations ± S.E.M. and those with different superscripts down the column are statistically different (p < 0.05)
C- Control Rats fed with feed without Ficus exasperata leaf; F1- Rats fed with 10 % Ficus exasperata leaf-based diet; F2- Rats fed with 30 % Ficus exasperata
leaf-based diet; F3- Rats fed with 50 % Ficus exasperata leaf-based diet
5

A significant increase (p<0.05) in serum urea concentration of rats fed with 30 and 50 % FELD was
recorded at the end of days 7 and 14 of this study compared to control (Table 4). However by day 21, there
was no significant difference (p>0.05) in the urea concentration of the test diets compared to control.
Similarly, serum creatinine concentration of groups fed with 30 and 50 % FELD increased
significantly (p<0.05) compared to control at end of days 7 and 14, while there was no significant difference
(p>0.05) in the results at the end of day 21 of the study. Serum electrolytes concentrations, kidney and
serum ALP activities were not significantly different (p>0.05) to control at the end of the experimental
period.
Histopathological study
Photomicrograph of both the liver and kidney of rats fed with FELD showed intact liver and kidney
architecture for all the experimental groups. No degenerative changes and inflammation was observed in
the livers. For the kidneys, the glomerula and tubules appeared normal, and there was no evidence of tubular
necrosis.
Table 3: Serum total and conjugated bilirubin concentration of rats fed with Ficus exasperata leaf-
based diet
Day 7 Day14 Day21

total conjugated total conjugated total conjugated
bilirubin bilirubin bilirubin bilirubin bilirubin bilirubin
Group (µmol/l) (µmol/l) (µmol/l) (µmol/l) (µmol/l) (µmol/l)
C 8.50 ± 0.05a 0.02 ± 0.00a 8.80 ± 0.26a 0.02 ± 0.00a 8.98 ± 0.31a 0.02 ± 0.00a
F1 9.00 ± 0.02a 0.02 ± 0.00a 8.98 ± 0.11a 0.02 ± 0.01a 9.47 ± 0.26a 0.02 ± 0.01a
F2 9.22 ± 0.21a 0.02 ± 0.00a 9.55 ± 0.05a 0.02 ± 0.00a 9.61 ± 0.04a 0.02 ± 0.00a
F3 9.32 ± 0.04a 0.02 ± 0.00a 9.56 ± 0.06a 0.02± 0.00a 9.66 ± 0.56a 0.02 ± 0.00a
Values are expressed as mean of 6 determinations ± S.E.M. and those with different superscripts down the column are
statistically different (p < 0.05)
C- Control Rats fed with feed without Ficus exasperata leaf; F1- Rats fed with 10 % Ficus exasperata leaf-based diet;
F2- Rats fed with 30 % Ficus exasperata leaf-based diet; F3- Rats fed with 50 % Ficus exasperata leaf-based diet
6
80
iAlkaline phosphatase activity
(Nm/min/mg protein) 70
60
50
C
40
F1
30 F2
20 F3
10
Liver Serum Liver Serum Liver Serum
Day7 Day14 Day21
Days of administration
Figure 1: Liver and serum alkaline phosphatase activities of rats fed with Ficus exasperata leaf-based
diet
F2- Rats fed with 30 % Ficus exasperata leaf-based diet; F3- Rats fed with 50 % Ficus exasperata leaf-based diet.
2500
Alanine amino transferase activity (U/I)
2000
1500
C
1000 F1
F2
500
F3
0
Day7 Day14 Day21
Figure 2: Liver and serum alanine amino transferase activity of rats fed with Ficus exasperata leaf-
based diet
7
1200
Aspartate amino transferase
activity (U/I) 1000
800
600 C
F1
400
F2
200 F3
0
Day7 Day14 Day21
Figure 3: Liver and serum aspartate amino transferase activity of rats fed with Ficus exasperata leaf-
based diet
Table 4: Serum urea and creatinine concentrations of rats fed with Ficus exasperata leaf-
based diet
Urea (mg/dl) Creatinine (mg/dl )

Groups
Day 7 Day 14 Day 21 Day 7 Day 14 Day 21
C 59.89±0.35a 54.61 ± 0.50a 52.21± 0.76a 0.18±0.04a 0.24 ± 0.02 a
0.78 ± 0.02a
F1 58.98 ± 0.36a 56.44 ± 0.33a 55.87± 5.04a 0.74±0.03ab 0.28 ± 0.02a 0.82 ± 0.03a
F2 76.77 ±0.30b 58.89 ± 0.12b 50.83 ± 0.30a 1.49±0.04bc 1.22 ± 0.04b 1.00 ± 0.03a
F3 123.97±0.18c 60.32 ± 2.02b 51.66 ± 0.22a 2.08 ±0.03c 1.28 ± 0.03b 1.19 ± 0.04a
8
Figure 4: Kidney and serum alkaline phosphatase activity of rats fed with Ficus exasperata leaf-based
diet
Values are expressed as mean of 6 determinations ± S.E.M. and those with different superscripts down the
column are statistically different (p < 0.05)
C- Control Rats fed with feed without Ficus exasperata leaf; F1- Rats fed with 10 % Ficus exasperata leaf-
based diet; F2- Rats fed with 30 % Ficus exasperata leaf-based diet; F3- Rats fed with 50 % Ficus
exasperata leaf-based diet
Discussion
The present study evaluated the effect of Ficus exasperata leaf-based diet on some selected liver and
kidney function indices of wistar rats. Liver and kidney are important organs that perform a central role in
metabolism. The liver is responsible for the uptake, metabolism, conjugation, and excretion of various
endogenous and foreign substances. It also perform immunological function, as the reticuloendothelial
capacity of the liver plays a role in phagocytosis, and clearance of microorganisms and endotoxins from
the portal blood. The kidney on the other hand maintains total body homeostasis through excretion of
metabolic wastes and regulation of intracellular fluid volume, electrolyte composition, and acid-base
balance (Ukoha et al., 2015). Hence, ensuring that any therapeutic agent developed does not impair the
functions of these organs is very important.
The non-significant alteration in the albumin, globulin and bilirubin concentrations as well as the ALP,
ALT and AST activities observed in this study, suggests there is no impairment in both the enzymic and
non-enzymic parameters of the liver function at the end of days 7, 14 and 21. However, the cause of the
significant reduction in the total protein concentration of group fed with 50 % FELD for days 7, 14 and 21
observed was not clear, because there was no concomitant alteration in albumin or globulin concentrations
of the rats. The level of total protein in the blood is normally a relatively stable value, reflecting a balance
in loss of old protein molecules and production of new protein molecules. However, total protein may
decrease in conditions where production of albumin or globulin proteins is impaired, such as severe liver
disease (Busher, 1990).
9
Table 5: Serum cation concentrations in rats fed with Ficus exasperata leaf-based diet
Na+ (mmol/l) K+ (mmol/l) Ca2+ (mmol/l)
Groups
C 109.28± 2.74a 108.00±1.20a 109.22±1.40a 3.61±0.27a 3.68±0.20a 3.60±0.30a 2.51±0.02a 2.50±0.10a 2.52±0.01a
F1 109.63±2.40a 109.24±2.00a 109.20±1.00a 3.81±0.27a 3.80±0.24a 3.81±0.26a 2.51±0.10a 2.52±0.02a 2.51±0.02a
F2 108.34±2.32a 109.62±1.00a 108.22±2.02a 3.53±0.02a 3.58±0.18a 3.56±0.17a 2.49±0.02a 2.48±0.12a 2.50±0.02a
F3 109.28±1.74a 108.00±1.84a 108.24±2.24a 3.61±0.27a 3.61±0.28a 3.60±0.26a 2.51±0.02a 2.51±0.10a 2.51±0.01a
Table 6: Serum anion concentrations in rats fed with Ficus exasperata leaf-based diet
Groups PO43- (mg/dl) Cl- (mmol/l) HCO3- (mmol/l)

C 7.69±0.39a 7.68±0.38a 7.68±0.39a 79.26±1.18a 78.26±1.15a 78.30±1.20a 32.16±3.43a 33.23±2.24a 32.20±2.20a
F1 7.66±0.40b 7.64±0.80a 7.66.0.41a` 80.36±1.59a 78.40±1.08a 79.00±1.89a 33.32±1.57a 33.20±2.00a 33.00±1.18a
F2 7.72±0.47ab 7.70±0.45a 7.70±0.44a 81.11±0.81a 79.05±0.80a 78.42±1.15a 35.37±4.11a 34.02±1.18a 32.00±2.00a
F3 7.69±0.39a 7.70±0.36a 7.70±0.42a 79.26±1.18a 79.08±1.18a 77.02±2.28a 32.16±3.43a 33.20±2.02a 33.34±1.00a
10
11
12
The kidney function parameters evaluated in this study shows a significant increase in the serum urea
and creatinine concentrations of rats fed with 30 and 50 % FELD for 7 and 14 days respectively. This
suggests there might be an initial assault in the excretory function of the kidneys, which was later reversed
at the end of day 21 of this study. Urea is synthesized in the body of many organisms as part of the urea cycle,
either from the oxidation of amino acids or from ammonia (Walter, 2004). It is found soluble in the blood and
is excreted by the kidney as a component of urine. Creatinine on the other hand, is a break-down product
of creatine phosphate in muscle and is usually produced at fairly constant rates by the body. It is freely
filtered at the glomerulus and is not reabsorbed by the tubules. The level of serum creatinine is the most
commonly used indicator of renal function. A rise in blood creatinine levels is observed only with marked
damaged to functioning nephrons (Kaplan and Pesce, 1996; Gross et al., 2005).
The results of the serum electrolytes and alkaline phosphatase activities were not altered compared to
the control in this study. This further corroborated reversal of the initial assault on the kidney function of
groups fed with 30 and 50 % FELD respectively. Also, this findings was further supported by the results of
the histopathology assessment of the liver and kidney which showed intact tissues architecture. The non-
toxic effect of FELD observed in this study is in agreement with reports of some previous researchers that
have rendered the leaf of F. exasperata relatively safe for possible human consumption (Sowemimo et al.,
2007; Ogbonnia et al., 2011).
Conclusion
The results of this study suggest that consumption of 10, 30 and 50 % Ficus exasperata leaf-based diet
for 7, 14 and 21 days did not impair the selected liver and kidney functions indices of experimental rats.
Therefore, it might be safe for consumption.
References
Adewole, S.O., Adenowo, T.K., Thajasvarie, N. and Ojewole, J.A.O. (2011). Hypoglycaemic and hypotensive effects
of Ficus exasperata vahl. (Moraceae) leaves aqueous extract in rats. African Journal of Traditional,
Complementary and Alternative medicines, 8: 275 - 283.
Allaby, M. (2012). A Dictionary of Plant Sciences. Oxford University Press
Ansari, M.M. and Kumar, D.S. (2012). Fortification of Food and Beverages with Phytonutrients. Food and Public
Health, 2 (6): 241 – 253
Bello, M. O., Abdul-hammad, M., Adepoju, A. J., Esan, O. A. and Tiamiyu, A. A (2014). Nutritional Composition
and Fatty Acids Profile of Ficus exasperata Fruit and Fruit Oil. Journal of Natural Sciences Research, 4 (2): 25 -
29.
Bergmeyer, H. U. (1987). Methods of enzymatic analysis, third edition, vol. VII. Pp 572.
Busher, J. T. (1990). Serum Albumin and Globulin. In: Walker, H. K, Hall, W. D., Hurst, J. W., editors. Clinical
Methods: The History, Physical and Laboratory Examinations. 3rd edition. Boston:Butterworths. Chapter
101. Available from: https://www.ncbi.nlm.nih.gov/books/NBK204/
Falade, O. S., Dare, A. F., Bello, M. O., Osuntogun, B. O. and Adewusi, S. R. A. (2004). Varietal changes in proximate
composition and the effect of processing on the ascorbic acid content of some Nigerian vegetables. Journal of
Food Technology, 2: 103 - 108.
Fawcett, J. K. and Scott, J. E. (1960). A rapid and precise method for the determination of urea. Journal of Clinical
Chemistry, 13 (2): 156 - 159.
Gross, M. L., Amann, K. and Ritz, E. (2005). Nephron number and renal risk in hypertension and diabetes. Journal
of the American society of Nephrology, 16 (1): S27 – S29.
Henry, J. B. (2001). Clinical Diagnosis & Management by Laboratory Methods, 20th Edn.
Ijeh, I. I. and Ukweni, A. I. (2007). Acute effect of administration of ethanol extracts of Ficus exasperata vahl on
kidney function in albino wistar rats. Journal of Medicinal Plant Research, 1 (2): 027 – 029.
Jenkins, A. L., Jenkins, D. J. A., Wolever, T. M. S., Rogovik, A. L., Jovanovski, E., Bozikov, V., Rahelic, D. and
Vuksan, V. (2008). Comparable postprandial glucose reductions with viscous fiber blend enriched biscuits in
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healthy subjects and patients with diabetes mellitus: Acute randomized controlled clinical trial. Croatian Medical
Journal, 49: 772 - 782.
Joseph, B. and Raj, S.J. (2010). Phytopharmacological and phytochemical properties of three Ficus species - An
overview. Int J Pharma Bio Sci, 1:246-53.
Kaplan, L. A. and Pesce, A. J. (1996).Clinical Chemistry: Theory, Analysis, Correlation. 3rd ed. St. Louis,
MO: Mosby, Pp 1064.
Krause, W. J. (2001). The art of examining and interpreting histologic preparations: A student handbook. Partheton
publishing group, UK, Pp 566.
Mbakwem-Aniebo, C., Onianwa, O. and Okonko, I. O. (2012). Effects of Ficus Exasperata Vahl on common
dermatophytes and causative agent of Pityriasis versicolor in Rivers State, Nigeria. American Journal of
Dermatology and Venereology, 1 (1): 1 – 5.
Ogbonnia, S.O., Mbaka, G.O., Anyika, E.N., Emordi, J.E. and Nwakakwa, N. (2011). An evaluation of acute and
subchronic toxicities of a Nigerian polyherbal tea remedy. Pak J Nutr, 10:1022-8.
OladosuI, A., Zubair, M.F., Ali, M.S. and Olawore, N.O. (2009). Anticandidal activity of volatile compounds from
the root bark of Ficus exasperata Vahl-Holl (Moraceae). JEOBP, 12:562-8.
Oxford dictionary of English (2010). Herb. Oxford University Press (3rd ed.) p. 819.
Peng, X., Ma, J., Cheng, K. W., Jiang, Y., Chen, F. and Wang, M. (2010). The effects of grape seed extract fortification
on the antioxidant activity and quality attributes of bread. Food Chemistry, 119: 49 - 53.
Reitman, S. and Frankel, S. (1957). A colorimetric method for determination of serum glutamate oxaloacetate and
glutamic pyruvate transaminase. American Journal of Clinical Pathology, 28: 56 - 58.
Sink, T. D. and Neal, J. W. (2009). Stress response and posttransport survival of hybrid striped bass transported with
or without clove oil. North American Journal of Aquaculture, 71: 267 - 275.
Soji-Omoniwa, O. and Oloyede, H. O. B (2018). Normoglycaemic Action of Ficus exasperata vahl. Leaf-Based Diet
on Fructose and Streptozotocin-induced Diabetic Rats. Nigerian Journal of Biochemistry and Molecular Biology,
33(1&2):18-31.
Sowemimo, A. A., Fakoya, F. A., Awopetu, I., Omobuwajo, O. R. and Adesanya, S. A. (2007). Toxicity and mutagenic
activity of some selected Nigerian plants. Journal of Ethnopharmacology, 113: 427 - 432.
Tietz, N. W. (1995). Clinical Guide to Laboratory tests. 3rd ed. Philadelphia. WB. Saunders, Pp. 268 - 273.
Trinder, P. (1951). A rapid method for the determination of sodium in serum. Analytical Chemistry, 76: 596 - 599.
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the kidney following chronic consumption of Hibiscus sabdariffa. Advances in biology. 1-5.
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in local processing methods. Bioresource Technology, 94: 307 - 10.
Walter, F. B. (2004). Medical Physiology: A Cellular and Molecular Approach. Elsevier Saunders, Pp. 837.
Wright, R., Albert, K. G. M., Marran, S. and Millwardsandler, G. H. (1979). Liver and biliary disease. W.B. Sanders
Company Limited, London.
14
I. N. Eke-Ogaranya et al.
0795-8080/2020 $10.00 + 0.00

Biokemistri
BKR 2020084/33102
Hypolipidemic potentials of methanol extracts of Vernonia

colorata
Ijeoma Nina EKE-OGARANYA1,2, Ifeoma Irene IJEH2 and Anthony Chibuzor
NNAMUDI*1
1
Department of Biochemistry, Faculty of Basic Medical Sciences, PAMO University of Medical Sciences, Port
Harcourt, Rivers State, Nigeria
2
Department of Biochemistry, College of Natural Sciences, Michael Okpara University of Agriculture, Umudike, Abia
State, Nigeria
*Author for correspondence: Tel: 07032869195; E-mails: anthonynnamudi@gmail.com; annamudi@pums.edu.ng
ABSTRACT: This study was designed to evaluate the effect of concomitant feeding of high fat diet (HFD) and
administration of methanol extracts of Vernonia colorata (MEVC) on lipid profile and body weight changes in Wistar
albino rats. Thirty male rats aged between 10-12 weeks were used for this study. Animals received food and water ad
libitum. Graded doses of the extract were dissolved in Dimethyl sulphoxide (DMSO) and administered orally on a
daily basis. Body weight was measured weekly while plasma lipid components were measured at the end of the study
which lasted for 10 weeks. The findings of this study revealed that concomitant feeding of high fat diet and
administration of methanol extracts of Vernonia colorata resulted in significantly (p<0.05) lower plasma
triacylglycerol, cholesterol and LDL-cholesterol but higher HDL-cholesterol concentrations when compared to the
high fat diet only and high fat diet + DMSO groups. The lipid lowering effects of methanol extracts of Vernonia
colorata was similar to Orlistat. Administration of Vernonia colorata also resulted in dose-dependent decrease of
22.2% and 15.8% in body weight gain relative to 12.5% decrease in the Orlistat group. The findings of this study
provide convincing evidence for the hypolipidemic and anti-obesity potentials of methanol extracts of Vernonia
colorata. This suggests that the plant may be useful in weight loss regimen, attenuating dietary obesity and also serve
as a potential drug lead in the search for natural products for the treatment of diseases associated with dyslipidemia.
Keywords: Vernonia colorata, high fat diet, obesity, dyslipidemia
Introduction
Obesity is gradually becoming a disease of global concern as higher values of body mass index (BMI)
correlates with increasing prevalence of cardiovascular diseases, hypertension and diabetes.1 Since genetic
predisposition and the consumption of energy rich foods are the commonest pathogenetic factors, many
therapies will therefore target to achieve weight reduction through dietary modulation.1,2 Dyslipidemia is a
major risk factor for several non-communicable diseases as it significantly increases the risk of obesity,
15
atherosclerosis, cardiovascular diseases, cerebrovascular diseases, hypertension and type 2 diabetes

mellitus.3 Dyslipidemia describes a group of metabolic anomalies characterized by any or all of the
following: elevated total cholesterol, elevated low-density lipoprotein cholesterol (LDL-c), elevated
triacylglycerol and decreased high-density lipoprotein cholesterol (HDL-c).4,5,6 Expectedly, it contributes
significantly to increased risk of atherosclerotic cardiovascular disease in diabetes 7 thereby constituting a
significant public health challenge.
The high cost which results in its unaffordability by majority of people in sub Saharan Africa in
addition to its associated undesirable side effects constitute a double jeopardy for currently available lipid-
lowering synthetic drugs. This has led to the continued dependence on plants with folkloric use in
ethnomedicine as alternatives to these synthetic drugs.8 Moreover, there is a global dependence on
medicinal plants in healthcare.9 This implies that there is an urgent need to evaluate the scientific basis of
the efficacy of these plants in certain health conditions. Correspondingly, there has been an upsurge in
scientific reports demonstrating positive biological activity, thus providing scientific evidence for their
efficacy. This has resulted in a steady increase in the use of plant extracts for the treatment of a wide variety
of diseases.10 Medicinal plants synthesize and store up secondary metabolites such as alkaloids, sterols,
terpenes, flavonoids, saponins, glycosides, cyanogenics, tannins, resins, lactones, quinines and volatile oils.
These secondary metabolites and other chemical constituents present in medicinal plants account for their
medicinal potency and efficacy.11,12
Vernonia colorata is a medicinal plant, belonging to the Asteraceae family 13 and Vernonia genus.
Other common members of that genus include V. amygdalina and V. calvoana. They are all eaten locally
as leafy vegetables. Vernonia colorata is a perennial shrub of 3.5-8m high that is found throughout Central
and West Africa. Vernonia colorata is similar to Vernonia amygdalina in appearance and nutrient content,
but has broader, wildly hairy leaves and it is less bitter-tasting than Vernonia amygdalina.14 Hence, it can
be described as sweet bitter leaf due to its characteristic non-bitter taste.15 The leaves can be eaten fresh or
in semi-processed form and they are used as accompaniment to various indigenous staples or as a spice in
food.16 There are available reports on the effects of various extracts of Vernonia species on blood lipids 17
although reports on concomitant feeding of high fat diet and administration of V. colorata are not readily
available. This present study is therefore aimed at evaluating the possible effects of concomitant feeding of
high fat diet and administration of V. colorata on lipid profile and body weight changes.
Collection and Identification of Plant

Fresh mature leaves of Vernonia colorata (Figure 1) were harvested from the farms in Forestry
Research Institute, Ahiaeke, Abia State. The leaves were identified and authenticated by Mr. Ibe Ndukwe,
a Taxonomist at the Herbarium unit of the Department of Forestry and Environmental Management,
Michael Okpara University of Agriculture, Umudike, Abia State. A voucher specimen (FHI 4873-Vernonia
colorata) was deposited at the herbarium.
Preparation of Methanol Extracts of Vernonia colorata

The fresh leaves were plucked, sorted, rinsed in clean tap water and air dried in the absence of sun
light at room temperature. The plant was pulverised and stored in air-tight containers. The pulverised
Vernonia colorata (500g) was packed into a glass column and was saturated with methanol. This was
allowed to stand for 48 hours to allow for complete extraction of methanol-soluble phytochemicals. The
tap was opened at the end of the extraction process and the extract collected into pre-weighed beakers. The
methanol was removed by evaporation to recover the crude extract.
16
Figure 1: Vernonia colorata showing its leaves and fruits.
Standard Drug
The standard drug Orlistat (Xenical Pharmaceuticals, Japan) was obtained from Blessed Pharmacy, 30
Lagos Street, Umuahia, Nigeria. It was reconstituted in distilled water and administered orally at a dose of
5.14 mg/kg body weight which was simulated from the human regimen.2
Feed Formulation
The basal diet and high fat diet was prepared with basic feed materials, following standard protocol.
The components of the diet is presented in Table 1. The high fat diet was formulated such that 35 % of the
total energy in the diet came from fats according to the method of Egedigwe et al.18 All materials used for
diet formulation were purchased from reputable vendors in Umuahia, Abia State, Nigeria. The components
of the diet were weighed out into a bowl and thoroughly mixed to obtain a dough-like consistency. They
were thereafter made into pellets by extrusion through an improvised device. The pellets were oven-dried
at 40oC and stored in air-tight containers to avoid rancidity.
Experimental Animals
Thirty male albino rats of the Wistar strain aged between 10-12 weeks obtained from the Animal
Breeding Unit of the Faculty of Veterinary Medicine, University of Nigeria, Nsukka were used for this
study. The rats were transported to the Animal House of the College of Natural Sciences, Michael Okpara
University of Agriculture Umudike, Abia State where the study was carried out under controlled
temperature (25-28 ℃) and 12 hour light/dark cycle. The animals were allowed to acclimatize, receiving
food and water ad libitum. The study protocol followed the guidelines of the National Research Council for
the care and use of laboratory animals.19
17
Table 1: Composition of Diets
Concentration (g/1000 g of feed)

Item High Fat Diet Basal Diet
Maize 388.80 388.80
Egg yolk powder 58.39 0.00
Groundnut cake 133.93 133.93
Cray fish 21.58 21.58
Vitamin mix 3.97 3.97
Mineral mix 15.89 15.89
Bone meal 19.87 19.87
Cellulose 3.97 3.97
Palm kernel oil 69.53 0.00
Palm oil 69.53 69.53
Corn starch 214.54 214.54
Experimental Design
The rats were randomized into six groups of 5 rats each. The rats in the six groups had similar average
body weights at the onset of the study. The six groups received feed and extract as follows:
Group I: Basal Diet

Group II: High Fat Diet + 1000 mg/kg V. colorata
Group III: High Fat Diet + 200 mg/kg V. colorata
Group IV: High Fat Diet + DMSO
Group V: High Fat Diet + Orlistat (5.14 mg/kg body weight)
Group VI: High Fat Diet only
A portion of the methanol extract of Vernonia colorata (10 g) was dissolved in 10 ml of 5 % Dimethyl
sulphoxide to give 100 mg/ml and was administered at 1000 and 200 mg/kg body weight. Oral
administration of the extract was done daily and the rats were fed the formulated diet for 10 weeks. In order
to avoid spoilage, fresh diet was compounded for each group on a weekly basis. Following an overnight
fast, the rats were euthanized at the end of 10 weeks of treatment and whole blood was collected by cardiac
puncture with sterile needle into heparinized tubes and centrifuged at 5,000 rpm for 10 minutes. Plasma
was separated and stored at a temperature of -4 ℃ until required.
Biochemical Analysis
The enzymatic colorimetric endpoint methods were used to determine the plasma concentrations of
total cholesterol, triacylglycerols and high density lipoprotein (HDL)-cholesterol.20,21,22 Low density
lipoprotein (LDL)-cholesterol concentration was estimated by difference according to the Friedewald
equation.23
Body Weight Measurement

Body weight was determined on a weekly basis based on the daily measurements and increases or
decreases in body weight was obtained for each group. This was calculated as the difference in animal body
weight on the day of measurement and weight at onset of the study (Daymeasurement−Day0) according to the
method of Zhou et al.24
18
Statistical Analysis
Descriptive statistics was carried out on the data generated and results were expressed as the Mean ±
Standard Deviation. Significant differences between groups were separated by one way ANOVA and
Duncan’s multiple comparison test. Data analysis was done using Statistical Package for the Social
Sciences (SPSS) version 20.0 (SPSS Inc Chicago IL) while body weight curve was plotted using Microsoft
Excel 2007 (Microsoft Corporation US). A p-value < 0.05 was considered statistically significant.
Results
Effect of concomitant feeding of high fat diet and administration of methanol extract of Vernonia
colorata on lipid profile:
Effect on Total Cholesterol concentration:

The administration of methanol extract of Vernonia colorata to high fat diet fed albino rats resulted in
a dose dependent significant (p<0.05) decrease in the mean total cholesterol of the animals in the treated
group relative to the group that received only high fat diet (Figure 2).
Figure 2: Total cholesterol concentration of rats concomitantly fed high fat diet and
administered methanol extract of Vernonia colorata.
19
Effect on Triacylglycerol concentration:

Figure 3 shows that the administration of methanol extract of Vernonia colorata resulted in a
significant (p<0.05) decrease in triacylglycerol concentration of animals that received extracts relative to
the high fat diet control group.
Figure 3: Triacylglycerol concentration of rats concomitantly fed high fat diet and
Effect on HDL cholesterol concentration:

The administration of methanol extract of Vernonia colorata caused significant (p<0.05) increase in
HDL cholesterol concentration in the treated groups relative to the control group (Figure 4).
Effect on LDL cholesterol concentration:

There was a significant (p<0.05) decrease in LDL cholesterol concentration upon concomitant feeding
of high fat diet and administration of methanol extract of Vernonia colorata relative to the high fat diet
control group (Figure 5).
Effect of concomitant feeding of high fat diet and administration of methanol extract of Vernonia
colorata on body weight changes:
Concomitant feeding of high fat diet and administration of methanol extracts of Vernonia colorata
resulted in respective decrease of 22.2 % and 15.8 % in body weight of animals that were administered
1000 mg/kg and 200 mg/kg of methanol extract of Vernonia colorata relative to 12.5 % decrease in the
Orlistat group (Figure 6).
20
Figure 4: HDL cholesterol concentration in rats concomitantly fed high fat diet and
Figure 5: LDL cholesterol concentration in rats concomitantly fed high fat diet and
21
MEVC 1000mg/kg
250
MEVC 200mg/kg
BASAL DIET
HFD
200
ORLISTAT MEVC =
Methanol
extracts of
V. colorata
Body Weight (g)
HFD =
150 High Fat
Diet
100
50
0
1 2 3 4 5 6 7 8 9 10
Weeks
Figure 6: Effect of concomitant feeding of high fat diet and administration of methanol
extracts of Vernonia colorata on body weight of rats.
Discussion
Increased consumption of high fat foods rich in cholesterol and triacylglycerols has been implicated
in the elevation of plasma cholesterol level and has emerged as a strong risk factor for metabolic diseases
such as, obesity, diabetes and cardiovascular diseases.25,3 The use of diet to regulate plasma lipid level is
now gaining an enormous acceptance worldwide. This underscores the recent focus on medicinal plants in
many dietary studies as scientists seek to explore the wide range of therapeutic benefits of such medicinal
plants.1,2
In this present study, it was observed that there was a decrease in plasma cholesterol level in rats
administered methanol extract of Vernonia colorata when compared with rats that were fed high fat diet
only. The dose dependent decrease in cholesterol levels were observed in both groups concomitantly fed
and administered methanol extracts of Vernonia colorata at both 1000 mg/kg and 200 mg/kg doses. This
finding which is in agreement with previous findings 26,17 may be attributed to the repertoire of
phytochemicals present in the methanol extract of Vernonia colorata leaves.18 Such phytochemicals include
but are not limited to saponins, sesquiterpenes, lactones and flavonoids.27
Rats that were fed only high fat diet showed higher plasma levels of triacyglycerols than rats that were
treated with extracts of Vernonia colorata. The findings of this study indicated that the administration of
Vernonia colorata to experimental animals effectively reduced triacylglycerol levels. These findings agree
22
with previous findings that have reported hypolipidemic effects of extracts of Vernonia species such as V.
amygdalina in normolipidemic rats.26,17
The prevention and control of dyslipidemia is important in achieving a reduction in the burden of
cardiovascular diseases.5 Research evidence has shown that polyphenols and saponins contribute to the
prevention of diseases related to lipid derangements.28 The phytochemical analysis of Vernonia colorata
has revealed the presence of dietary fiber, saponins and polyphenols.29 Antioxidants prevent oxidative stress
caused by free radicals that are produced via lipid peroxidation. Therefore, by terminating chain reactions
triggered by these free radicals, polyphenols and saponins contained in Vernonia colorata may prevent the
onset of cardiovascular diseases. Fundamentally, polyphenols and saponins contained in Vernonia colorata
are known to stimulate cholesterol lowering activity by eliciting resin-like action, thereby reducing the
enterohepatic circulation of bile acids. This process ensures the conversion of more cholesterol into bile
acids, thus resulting in the reduction of total plasma cholesterol level due to increased excretion.18
In this present study, the administration of graded doses of methanol extract of Vernonia colorata
caused significant elevation of HDL cholesterol in the treated groups relative to the control group. This
finding is consistent with a previous report.30 The findings of a dose-dependent elevation of HDL-
cholesterol in this study is indicative of a positive modulation of lipid profile in high fat diet fed rats. This
is because of the anti-atherogenic role which HDL cholesterol plays in promoting reverse cholesterol
transport and ensuring that cholesterol from peripheral tissues return to the liver to be either excreted or
reused.31 Therapeutic interventions that raise HDL-cholesterol are highly encouraged, as higher levels have
been correlated with reduced risk of conditions such as metabolic syndrome and future risk of
cardiovascular diseases.32
Elevated LDL cholesterol in the blood plasma is considered detrimental, as their increased
concentration can potentiate glucose intolerance 33 and cause lipid accumulation in the arteries. This leads
to an increase in the adherence of circulating monocytes to the arterial endothelial cells where they
differentiate to form macrophages and become converted to foam cells which can exacerbate
atherosclerosis.34 The findings of this present study revealed that administration of methanol extracts of
Vernonia colorata administered at both 1000 mg/kg and 200 mg/kg significantly lowered LDL cholesterol
concentrations of rats that were concomitantly fed a high fat diet. This finding is consistent with an earlier
study which reported that incorporation of Vernonia colorata in the diets of normolipidemic rats had a
lowering effect on serum low density lipoprotein (LDL) cholesterol concentration at both 5 % and 10 %
dietary incorporation levels.15
Administration of methanol extract of Vernonia colorata at 1000 mg/kg and 200 mg/kg significantly
(p<0.05) down regulated weight gain in animals fed high fat diet relative to the untreated group that was
fed high fat diet only. This is consistent with previous reports.35,2 The dose dependent steady loss in weight
could have been as a result of the presence of anti-nutritional factors such as saponins in Vernonia colorata
which may reduce lipid distribution and nutrient availability by delaying the absorption of fats in the
intestines.36,30
Conclusion
This study showed that the administration of methanol extracts of Vernonia colorata to animals fed a
high fat diet had beneficial effects on the plasma lipid profile and body weight changes of the animals. The
findings of this study therefore provide convincing evidence for the hypolipidemic and anti-obesity
properties of methanol extracts of Vernonia colorata. This suggests that the plant may be useful in weight
loss regimen, attenuating dietary obesity and also serve as a potential drug lead in the search for natural
products for the treatment of diseases associated with dyslipidemia.
23
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lipoproteins separated by three different methods. Clin. Chem. 23(5): 882-884.
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23. Friedewald, W.T., Levy, R.I. and Frederickson, D.S. (1972). Estimation of the concentration of LDL cholesterol
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and Martin, R.J. (2011). Dietary whey protein decreases food intake and body fat in rats. Obesity (Silver Spring).
19(8): 1568-1573. doi: 10.1038/oby.2011.14.
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Hypolipidemic effect of ethanolic extract from the leaves of Hibiscus sabdariffa L. in hyperlipidemic rats. Acta.
Pol. Pharm. 67(2): 179-184.
26. Adaramoye, O.A., Akintayo, O., Achem, J. and Fafunso, M.A. (2008). Lipid-lowering effect of methanolic
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27. Ijeh, I.I. and Ejike, C.E.C.C. (2011). Current perspectives on the Medicinal Potentials of Vernonia amygdalina
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A., Salehi, P., Hamburger, M. and Yassa, N. (2012). In vitro α-glucosidase inhibitory activity of phenolic
constituents from aerial parts of Polygonum hyrcanicum. Daru. 20(1): 37.
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Conventional Leafy Vegetables consumed in Cameroun. Parkistan J. Nutr. 6(6): 712-717.
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colorata leaves. Niger. J. Biochem. Molecular Biol. 29(1): 34-43.
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microvascular dysfunction: interventional strategies. J. Inflamm. 7:54.
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Disease. Cardiovasc. Drugs Ther. 33: 207-219. https://doi.org/10.1007/s10557-018-06846-w.
33. Singh, A.K., Singh, N.K., Agrawal, N. and Gopal, K. (2011). Obesity and dyslipidemia. Int. J. Biol. Med. Res.
3(2): 824-828.
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Their Potential to Promote or Prevent Atherosclerosis. The UCLA USJ. 22: 1-17
35. Atangwho, I.J., Ebong, P.E., Egbung, G.E., Eteng, M.U. and Eyong, E.U. (2007). Effect of Vernonia amygdalina
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36. Ijeh, I.I. and Obidoa, O. (2001). Effect of dietary incorporation of two varieties of Vernonia amygdalina on mean
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26
O. A. Odunola et al.
0795-8080/2021 $10.00 + 0.00

Biokemistri
BKR 2020085/33103
Antigenotoxic and hepatoprotective activities of ethanol

extract of the leaf of Eclipta alba in sodium arsenite-induced
toxicity
Oyeronke A. ODUNOLA1*, Nelson O. FASHINA2, Ifeanyi M. ILOBA1, Michael A.
GBADEGESIN1, Ayodeji M. ADEGOKE1, Olorunjuwon J. OLUGBAMI1
1
Cancer Research and Molecular Biology Laboratories, Department of Biochemistry, College of Medicine,
University of Ibadan, Ibadan. Nigeria.
2
African Indigenous Knowledge Production Unit, Faculty of Arts, University of Ibadan, Ibadan, Nigeria.
*Correspondences to: ronodunola@yahoo.com and aygoke@yahoo.com, Tel.: +234 8023387152
ABSTRACT: Arsenic pollution in developing countries poses a major health hazard to humans and animals, thus the
search for potent remedies. The medicinal use of Eclipta alba in the management of some ailments such as ulcer,
diarrhoea, constipation, and pile have been documented. We therefore scientifically explored the effects of the
ethanol leaf extract of E. alba (ELEA) in sodium arsenite (SA) induced geno-hepatotoxicity using male Wistar rats.
Thirty-five (35) rats were randomised into seven groups of five animals each. Group I was treated with distilled
water only while groups II to VII had various levels of treatments with ELEA (200mg/kg body weight) and/or SA
(5.0mg/kg body weight) for 14 days. We evaluated both the preventive and therapeutic effects of ELEA. The
activities of serum transaminases, γ-glutamyl transferase and alkaline phosphatise were evaluated, liver histological
analysis and histomorphometry were also monitored as additional markers for hepatotoxicity. Micronucleus
induction assay was used to assess genotoxicity and kidney histology to monitor the effect of ELEA on the kidney.
Serum transaminases/aminotransferases, γ-glutamyl transferase and alkaline phosphatise, frequency of
micronucleated polychromatic erythrocytes (mPCEs) as well as hepatic cell/mm2 were significantly (p˂0.05)
increased by SA, while PCV, HB and RBC counts decreased significantly (p<0.05) but administration of ELEA
significantly reversed these parameters close to normal. Ethanol extract of E. alba leaves exhibits some protective
effect and may serve as a potent remedy in sodium arsenite induced hepatotoxicity and genotoxicity.
Key words: Arsenite, Genotoxicity, Hepatotoxicity, Herbal, Micronucleus, Transaminases.
Introduction
Inorganic arsenic compounds are linked with higher risks of cancer and other health challenges1.
Some of the common trivalent inorganic arsenic compounds include: arsenic trioxide, sodium arsenite and
27
arsenic trichloride2. Exposure to arsenic is regarded as a major public health concern due to its
carcinogenic potential3.
According to Sharma et al. (2014), the exposure to inorganic arsenic is most likely via drinking
water and various foods4. Arsenic polluted drinking water from both human-induced and naturally
occurring sources have affected millions of people globally5. Due to absence of taste, odour and colour,
exposure to arsenic cannot be detected and avoided by a layman4. Several regions of the world have been
affected by arsenic polluted drinking-water in levels ranging from tens to even thousands of micrograms
per litre, primarily in Bangladesh, China, West Bengal (India)6.
It has been demonstrated that arsenic induced genotoxicity is associated with point mutation on the
HPRT locus in lymphocytes from adults chronically exposed to arsenic via drinking water for over 10
years, regardless of the presence of skin lesions and chromosomal aberrations 7. Arsenic exposure also
induces micronucleated cells both in urothelial cells and cells from oral mucosa, and most of the
infertility problems and birth defects observed in human populations exposed to arsenic are associated
with genotoxicity8,9.
Plants have played very important therapeutic roles in maintaining and enhancing the quality of
human health for thousands of years. In recent times, there is an increasing focus on plant research and
this has gained momentum all over the world and evidence has shown the immense potential of medicinal
plants in the treatment and management of various health concerns10. Eclipta alba (syn. Eclipta prostata)
commonly known as false daisy is a specie of plant in the family asteraceae with characteristic medicinal
properties. In Nigeria, E. alba known as ewe arojoku (Yoruba) and agbirigba ozara (Igbo) is used locally
in many herbal formulations for the treatment of ulcer, insomnia, urinary tract infection, pile, labour
pains, headaches, sight and hearing abnormalities, and skin disorders11. Extracts and other concoctions
made from the plant have been reported to be used in folk and traditional medicine for alopecia,
cancer/tumour, gastrointestinal disorders, skin diseases and blood/pus in urine and wound healing 12. The
plant was known to possess significant antidiabetic, hepatoprotective, anaphylaxis, analgesic, anti-
inflammatory and immunomodulatory activity13. Several properties of E. alba have been studied
including its effect on CCl4-, galactosamine- and phalloidin-induced liver damage in rats. The present
study was designed to investigate the antigenotoxic and hepatoprotective potency of ethanol extract of E.
alba in sodium arsenite exposure in male Wistar rats.
Chemicals and Reagents for Analysis

Kits for aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl
transferase (γGT) and alkaline phosphatase (ALP) were obtained from Randox Laboratories, UK. Sodium
arsenite (NaAsO2; BDH Chemicals Ltd Poole England) was used. Other chemicals and reagents used in
this study were of analytical grade and are products of Sigma Chemical Co. St. Louis, MO., USA.
Preparation of Ethanol leaf extract of Eclipta alba (ELEA)

Fresh leaves of E. alba were bought from Bode market in Ibadan, Nigeria and were identified at the
Department of Botany, Faculty of Science, University of Ibadan, Nigeria by Mr. Esimekhnai, Donatus.
The leaves were air dried, weighed and ground to powdered form. The powdered sample was extracted
using 70% ethanol. The extracts were further concentrated with a rotary evaporator at 40 °C to yield a
solid residue at the Department of Pharmaceutical Chemistry, University of Ibadan.
Experimental Protocols and Treatments

Thirty-five (35) male Wister albino rats weighing 100-120 g were bought from the Animal House,
Department of Physiology, Faculty of Basic Medical Sciences, University of Ibadan, Nigeria. The rats
were kept in the experimental animal house, Department of Biochemistry, University of Ibadan at 29 ± 2
°C and were fed with rat pellets (Vita feeds, Ibadan, Nigeria) and with water ad libitum, 12 h light/dark
28
cycle. All the animals were handled in adherence to the guide for the care and use of experimental
animals, as specified by the National Institute of Health (NIH publications number 85–93 revised in
1985). The rats were acclimatized for one week and then randomly divided into seven experimental
groups of five rats per group.
Group I: Serve as the negative control and received distilled water only.
Group II: Rats treated with sodium arsenite (SA) 5.0 mg/kg body weight every other day for 14 days.
Group III: Rats treated with 200 mg/kg body weight of ethanol leaf extract of Eclipta alba (ELEA) daily.
Group IV: Rats treated with SA 5.0 mg/kg body weight every other day and 200 mg/kg body weight
ELEA daily simultaneously
Group V (natural recovery): Rats treated with SA 5.0 mg/kg body weight followed by water for another
14 days.
Group VI (post-treatment): Rats treated with SA 5.0mg/kg body weight followed by 200 mg/kg body
weight of ELEA for another 14 days.
Group VII (pre-treatment): Rats treated with 200 mg/kg body weight of ELEA followed by 5.0 mg/kg
body weight of SA for another 14 days.
Except otherwise stated, all treatments were done by gavages for 14 days. Sodium arsenite was
administered at 5 mg/kg body weight (1/25th of the oral LD50)14.
Liver function enzymes assays
Aminotransferases activities
Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were
investigated according to the method previously described15 with the use of diagnostic kits. Briefly, this
method involves the reaction of pyruvate, the product of transamination reaction catalysed by ALT or
AST, with 2, 4 -dinitrophenyl hydrazine which give rise to deep coloured hydrazone which is read at 546
nm with the aid of a spectrophotometer (Spectronic-20).
γ-glutamyl transferase activity (γGT)
The γGT was investigated in the serum by employing the reconstituted γGT diagnostic reagent
according to the previously described method16. Briefly, this involves the transfer of glutamyl group from
a glutamyl peptide (L-γ-glutamyl-p-nitroanilide) to another peptide (glycylglycine), in a reaction
catalyzed by γGT, thereby yielding a cleavage product (pnitroaniline) which is read at 405 nm thus
making a direct kinetic determination of γGT activity a possibility.
Alkaline Phosphatase (ALP) Activity
The ALP activity was determined according to the optimized method previously described17.
Alkaline phosphatase catalyzes the hydrolysis of p-nitrophenylphosphate into phosphate and p-
nitrophenol which absorbs UV light at 405nm, thus making a direct kinetic determination a possibility.
p-nitrophenylphosphate + H20 ALP -→ phosphate + p-nitrophenol
Micronucleus (MN) assay

The femurs from the experimental animals were removed and bone marrow was aspirated with a
needle and syringe. The microscopic slides of the bone marrows were prepared following the procedure
previously described18. The slides were thereafter fixed in methanol, air-dried, pre-treated with May-
Grunwald solution, and air-dried again. The dried slides were thereafter stained in 5% Giemsa solution
and induced in phosphate buffer for 30 seconds. The slides were then rinsed in distilled water and air-
dried. The air-dried slides were mounted and scored with a microscope for micronucleated polychromatic
erythrocytes (MPCEs) according to the established standard procedure at x40 magnification.
29
Liver and Kidney histological analysis

Liver sections from the rats were fixed in 4 % p-formaldehyde and thereafter washed in phosphate
buffer pH 7.4 at 4 oC for 12 h. After dehydration, the tissue was embedded in paraffin, and then cut into
sections, 5 µm thickness were stained with haematoxylin and eosin staining method19, and then evaluated
under a microscope at the Pathology Department, University of Ibadan, Ibadan, Nigeria.
Hepatic Cell Analysis

Hepatic cell per mm2 analysis was investigated by counting the numbers of cells on stained slides
prepared from the liver; this was done under a Nikon light microscope at x40 with the aid of a grid and
tally counter.
Haematological analysis
Prior to sacrifice, blood was collected from experimental rats via ocular puncture in heparinized
bottles for haematological analysis at the Department of Veterinary Medicine, University of Ibadan
according to the method previously described20.
Data analysis
Results are expressed as mean ± Standard deviation. Differences between the groups were analysed
by one-way analysis of variance (ANOVA) with the aid of Statistical Package for Social Sciences (SPSS)
software, SPSS Inc., IBM, Standard version 20.0.0. P values <0.05 were considered statistically
significant for differences in mean.
Results
Effect of ELEA on SA-induced hepatotoxicity in male albino Wister rats

The activities of serum enzymes increased significantly (p<0.05; Table 1) in groups II and V rats
treated with SA only compared with the control (groups I). The ELEA only treated rats showed
approximately the same levels of the serum enzymes compared to the control treated with water only.
Almost similar levels were observed in the SA-only treated rats (groups II and V). Pre- and post-treatment
with ELEA (groups VI and VII) reduced the activity of the liver enzymes as compared to the SA only
treated rats, with the rats pre-treated with ELEA (group VI) alone being statistically significant (p<0.05;
Table 1).
Table 1 Serum activities of aspartate amino transferase, alanine amino transferase, gamma glutamyl
transferase and alkaline phosphatase in sera of rats treated with ELEA and SA.
Group ALT AST ALP GGT
I-water 13.45 ± 1.56a 23.19 ± 2.83a 155.48 ± 29.77a 10.42 ± 1.16a

II-SA 27.68 ± 3.22b 50.58 ± 3.75b 451.72 ± 28.73b 25.09 ± 1.77b
III-ELEA 13.57 ± 2.53a 24.06 ± 3.09a 173.88 ± 27.18a 9.26 ± 4.01a
IV-SA+ELEA 13.21 ± 2.06a 36.38 ± 7.18a 270.48 ± 152.99a 15.82 ± 6.38
V-SA+water 26.01 ± 2.36b 49.27 ± 5.90b 571.32 ± 172.12b 30.49± 13.72b
VI-SA+ELEA 17.74 ± 1.49a 38.55 ± 7.49a 398.36 ± 14.16a 13.51 ± 2.41a
VII-ELEA+SA 23.57 ± 1.43 43.04 ± 3.98 440.68 ± 56.38 23.55 ± 6.69
Values are expressed as mean ± SD. a= the mean difference is significant (p< 0.05) when compared with
group II. b= the mean difference is significant (p< 0.05) when compared with group I.
30
Effect of ELEA and SA on the frequency of induction of micronucleated polychromatic

erythrocytes (mPCEs) in bone marrow cells.
As expected, there is a significant increase (p<0.05; Fig. 1) in the number of mPCEs in the bone
marrow cells of rats treated with SA alone (groups II and V) as compared with the control which received
water only (group I). There was no observed difference in the number of mPCEs between the ELEA only
treated rats (group III) as compared to the control (group I). A significant reduction (p˂0.05; Fig. 1) in
mPCEs were observed in rats treated with SA+ELEA simultaneously (group IV), as compared with SA
only group. Pre- and post-treatment with ELEA (groups VI and VII) showed significant reduction
(p<0.05; Fig. 1) in the frequency of mPCEs by at least 2.14-fold decrease when compared with the SA-
only treated groups (groups II and V).
Frequency of induction of mPCEs
7
Number of mPCEs/1000Pces
b
6
b
5
4
3 a
2 a a
a
a
1
0
water SA ELEA SA+ELEA SA+water SA+ELEA ELEA+SA
Treatment Groups
Fig 1 Frequency of micronucleated polychromatic erythrocytes induction in the bone marrow cells of rats
exposed to ELEA and SA.
a= the mean difference is significant (p< 0.05) when compared with group II
b= the mean difference is significant (p< 0.05) when compared with group I
Histomorphometry (Hepatic cell/mm2)

The hepatic cells per mm2 increased significantly (p<0.05; Fig. 2) in SA-only rats (groups II and V)
when compared with the control and ELEA-only treated rats (groups I and III). No statistical significant
difference was observed between the positive control groups II treated with SA only and the recovery
group V. There was an observed significant decrease (p<0.05; Fig. 2) in the rate of cell proliferation of
rats treated simultaneously with ELEA+SA (group IV) when compared with the SA-only group. Pre- and
post-treatment with ELEA (group VI and VII) show a slight decrease in the hepatic cells per mm2
compared to the SA-only groups, though not significant.
31
Figure 2: Hepatic Cell per millimeter sq. analysis of rats treated with ELEA and sodium arsenite
Effect of ELEA on haematological parameters

SA was observed to significantly decrease (p<0.05; Table 2) the RBC, Hb and PCV levels in the
SA-only treated rats compared to the control (group I) fed with water only, while conversely, the
lymphocyte count was significantly increased (p<0.05; Table 2) in SA-only rats compared to the control.
ELEA administration significantly increased (p<0.05; Table 2) the RBC, Hb and PCV levels and at the
same time, significantly reducing (p<0.05; Table 2) the lymphocyte count.
Table 2 Evaluation of PCV, HB, LYMPH, WBC, and RBC levels of rats treated with ELEA and
sodium arsenite (SA)
Group PCV (%) HB (g/dl) RBC WBC (cells/µl) LYMPH (%)

(million
cells/µl)
I- H2O 37.50±1.73a 14.43±0.56a 7.14±0.49a 5437.50±912.13 50.25±9.64a
b
II-SA 31.00±3.37 11.58±0.86b 5.25±0.54b 4925.00±830.25 70.25±6.85b
a a
III-ELEA 39.25±1.50 14.48±0.46 7.52±0.33a 6162.50±1058.60 54.75±6.34a
IV-SA+ELEA 40.75±1.50a 15.83±0.57a 7.85±0.40a 3250.00±727.37 59.25±5.62a
V-SA+water 33.25±4.43b 11.78±1.62b 6.09±0.87b 3750.00±907.62 73.25±3.50b
VI-SA+ELEA 41.25±2.22a 14.08±0.99a 7.95±0.44a 4687.50±949.91 60.25±12.58a
VII- 42.5±1.29a 13.68±0.93a 7.61±0.26a 3025.00±843.9 64.75±6.40a
ELEA+SA
Values are expressed as mean ± SD.
Packed cell volume (PCV), Heamoglobulin (HB) Lymphocytes (LYMPH), white blood cells (WBC), and
Red blood cells (RBC) levels of rats treated with ELEA and sodium arsenite (SA).
32
Effect of ELEA on the histology of the liver cells during SA toxicity
33
Effect of ELEA on the histology of the kidney during SA toxicity
Discussion
The understanding of the physiological processes of arsenic metabolism and the biochemical
pathways, can be used to design treatments of arsenic toxicity21. The effects of ethanol leaf extract of
Eclipta alba (ELEA) were studied as a preventive and treatment alternative for sodium arsenite (SA)
toxicity. Because cells demonstrate the ability for repair upon insult, we attempted to make a distinction
between the natural cellular recovery mechanisms on exposure to SA and pre- and post-treatment effects
of ELEA. Exposure to inorganic arsenic compounds results in adverse health effects including renal and
hepatic diseases22. The increased activity of AST, ALT, ALP and γGT are indicators of liver injury23,24.
The activities of serum enzymes were increased significantly (p<0.05; Table 1) in the positive control
(group II) and natural recovery (group V) that were treated with SA only as compared with the negative
34
control which received water only and ELEA treated rats. This observation is consistent with observations
from our laboratory on the induction of hepatotoxicity and oxidative stress upon exposure to SA25,26.
Pre-treatment with ELEA before exposure to SA (i.e. groups VI) significantly reduced liver enzymes
activities when compared to the SA only/positive control group. This shows the potential protective effect
the ELEA on SA induced hepatotoxicity in albino rats. This decrease in the activities of serum enzymes
may be due to the presence of wedelolactone and associated intrinsic phytochemicals in ELEA with
potential antihepatotoxic properties27. Wagner et al. (1986) also confirmed that the coumestan component
of E. alba, wedelolactone and demethylwedelolactone, are most likely responsible for the potent anti-
hepatotoxic activities28. The significant reduction in the levels of ALT, AST, γGT and ALP are good
indicators of hepatoprotective functions29 suggesting that ELEA contains active phytochemicals with the
potential to reduce the hepatotoxic effects of SA and possibly restore the hepatocytes physiology in
arsenic toxicity. No significant difference was observed in groups II and V (natural recovery) rats treated
with SA only, showing the likelihood of the absence of natural recovery upon exposure to SA.
Chronic arsenic toxicity which occurs as a result of drinking arsenic polluted water is one of the
worst health hazards in history30. Exposure to SA polluted drinking water has been shown to cause
alteration in chromosomal segregation which may lead to cancer30. The micronuclei assay was developed
to easily detect in vivo chromosomal aberration in bone marrow cells than the traditional cytogenetic
methods.
The approximately 2-fold increase observed in the frequency of mPCEs in the SA-treated rats is an
indication of chromosomal damage. This observation is consistent with earlier reported observation in our
laboratory on the genotoxic properties of sodium arsenite26,31. The increased frequency of induction of
mPCEs observed in the SA-only treated rats may be due to arsenic generated free radicals that can attack
DNA leading to chromosomal breakage. However, most studies have reported SA as a potent mutagen or
carcinogen32,33. In all, ELEA caused a decrease in the frequency mPCEs showing the potential of the E.
alba extract to repair chromosomal damage and protect against carcinogenesis and suggesting a potential
protective and therapeutic effect of the ELEA treatment. This may be because E. alba possesses
antioxidant activities that helped in scavenging the free radicals and reactive oxygen species generated by
SA34.
Herbs that are rich in important phytochemicals have become a reference point for treatment of
various toxicities. This has brought about novel concepts have appeared with the trend, such as
nutraceuticals, phytonutrients, and phytotherapy35. The phytochemical present in E. alba will likely
contribute positively to maintaining wellbeing, promoting health, and modulating immune function for
disease prevention. The E. alba has a great potential in clinical therapy due to its potential to reduce side
effects that accompany chemotherapy or radiotherapy36.
The haematological parameters were used to assess the efficacy of ELEA in the prevention and
treatment of SA-induced toxicity. The RBC, Hb and PCV levels were significantly reduced (p<0.05;
Table 2) in the SA-only treated rats (both positive control and natural recovery groups) compared with the
negative control group fed with water only. This observed erythrocytopenia is possibly due to depression
of bone marrow activity which had been reported previously37,38,39. No significant recovery was observed
for the blood parameters in the recovery group. Our findings showed that the administration of ELEA
enhanced the levels of RBC, PCV and Hb concentration, thereby reversing the alterations in
erythropoiesis observed during SA intoxication.
The increase in PCV, Hb and RBC levels because of the administration of ELEA conforms to
previous reports40,41 of restoration of blood parameters in animals exposed to toxicants. In addition, pre-
and post-treatment with ELEA increased the PCV, Hb and RBC levels while reducing the lymphocyte
count, showing the potential protective and therapeutic properties when compared to the SA-only group.
The observed increase is possibly an indication of the release of the lymphocytes from lymphoid tissues
as a protective response when challenged with SA. The ELEA showed the potential to suppress the toxic
effects of SA and thus, probably protecting against impairment or cellular transformations because of the
toxicant.
35
The biochemical observation above is supplemented with histopathological examination of the liver
and kidney sections. The results show a moderate periportal and extensive cellular infiltration and
fibrosis, and diffuse tubular degeneration with protein casts in the experimental animals treated with SA
only (groups II and V) in both liver and kidney, respectively. This illustrates the possible contribution of
SA toxicity to the induction of hepatic and adrenal tumours as previous reported30. No visible lesion was
seen in the kidney of the rats treated with ELEA and SA simultaneously (group IV), while a moderate
periportal and extensive cellular infiltration was observed in the liver of animals that had SA+ELEA
simultaneously.
Pre- and post-treatment with ELEA (groups VI and VII) showed no visible lesion in the liver
showing its potential protective and curative effect in SA toxicity most especially in the liver cells. The
cells/mm2 assay was carried out to assess the rate of cell proliferation. This was used as an index for
measuring the tumorigenic potential of a compound since unregulated cell proliferation can be a marker
for carcinogenesis. The significant increase by at least 1.53 folds observed in the cells as a result of the
SA toxicity was probably due to the promotion of cell proliferation in the experimental rats treated with
SA only. Post-treatment with ELEA (group VII) reduced the rate of cell proliferation when compared to
the SA-only treated rats. This further supports the earlier findings that ELEA may serve as a potent
remedy in SA induced cell proliferation.
Conclusion
Arsenic pollution of drinking water in developing countries is a major health concern for man and
animals. Ethanol leaf extract of Eclipta alba exhibited some potential protective effect and can probably
suppress sodium arsenite-induced hepatotoxicity and genotoxicity in rats. Further studies are
recommended on the molecular mechanisms of E. alba on sodium arsenite toxicity.
Conflicts of Interest
There is no conflict of interests associated with this study.
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0795-8080/2021 $10.00 + 0.00

Biokemistri
BKR 2020095/33104
Gamma-sitosterol–rich fraction from the methanolic extract of

Ficus exasperata restores diabetes associated pathophysiological
alterations in an alloxan-induced diabetic rats
Gloria Otito Izu1, Akindele Oluwatosin Adeyi1*, Ochuko Lucky Erukainure2, Md.
Shahidul Islam3
1
Animal Physiology Unit, Department of Zoology, University of Ibadan, Ibadan, Nigeria
2
Department of Pharmacology, University of the Free State, Bloemfontein 9300, South Africa
3
Biomedical Research Lab, Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal
(Westville campus), Durban 4000, South Africa
*Author for Correspondence: delegenius@yahoo.com
(Received December 21, 2020; Accepted February 4, 2021)
ABSTRACT: Ficus exasperata has been reported to have hypoglycemic and antidiabetic effects which are even
better than some standard antidiabetic drugs. However, key compounds behind these effects are still unknown. This
study was therefore designed to isolate and conduct preliminary characterization of the components of F. exasperata
that are responsible for its antidiabetic effects. Methanol extract of F. exasperata was partitioned using ethyl acetate
and n-hexane when ethyl acetate extract was found to have more hypoglycemic potential in animal model. Hence,
the ethyl acetate fraction was used for the in vivo study. Adult male rats were divided into 4 treatment and one
control groups (n=5). Diabetes was induced by a single intraperitoneal injection of alloxan (150mg/kg body weight).
The effects of the fractions and a standard antidiabetic drug (glibenclamide) on blood glucose, haematological
parameters, liver enzymes, lipid profile and histopathology of some organs were studied thereafter. All treated rats
responded positively to treatment with the fraction and hyperglycemia was reversed within 7 days of treatment.
Treatment with the fraction induced significantly better (p<0.05) haematopoetic values and lower hyperlipidemia
than the standard antidiabetic drug. The degrees of diabetes related degeneration in the pancreas, kidney, liver and
heart of the treated groups were significantly lower compared to the rats treated with glibenclamide. The fraction
contained 7 compounds and the most prominent compound was gamma-Sitosterol with a percentage of 25.49. The
results of this study suggest that Ficus exasperata (Ethyl Acetate fraction) is a good candidate for the treatment of
diabetes mellitus.
Keywords: Diabetes mellitus, Ficus exasperata, hypoglycemic, histopathology, rats
Abbreviations
DM: Diabetes mellitus; T1D: Type 1 diabetes; T2D: Type 2 diabetes; HDL: High Density Lipoprotein; LDL: Low
Density Lipoprotein; TLC: Thin layer chromatography ;GC-MS :Gas Chromatography–Mass Spectrometry; PCV:
Packed Cell volume; Hb: Hemoglobin; WBC: White blood cell; RBC: Red blood cell; MCH: Mean corpuscular
Hemoglobin; MCV: Mean corpuscular volume; MCHC: Mean corpuscular hemoglobin concentration; AST:
39
Aspartate Transaminase; ALT: Alanine Transaminase; ALP: Alkaline Phosphatase; GGT: Gamma Glutamyl
Transferase; BILI: Bilirubin; HP:Hepatocytes; CCR: Creatinine Clearance Rate; GFR: Glomerular Filtration Rate.
Introduction
Diabetes mellitus (DM) is a metabolic disorder of multiple aetiologies characterized by chronic

hyperglycaemia, glycosuria and negative nitrogen balance. Pathophysiology of diabetes involves a
complex cascade of several interrelated mechanisms resulting from lack of insulin secretion from the beta
cells of pancreas and desensitization of insulin receptors at cell surface with disturbances of carbohydrate,
fat and protein metabolism [1]. The global prevalence of diabetes in adults has been increasing over the
recent decades. The International Diabetes Federation (IDF) estimated the global prevalence was 151
million in 2000 [2] which has been increased to 425 million by 2017 [3]. This is expected to increase by
48% in 2048 with a total of 629 million diabetics [3].
The etiology of DM has been associated with the inability of the pancreatic cells to secret insulin,
regarded as type 1 diabetes (T1D) or inability of the body to utilize the insulin synthesized by the
pancreatic β-cells, a condition termed type 2 diabetes (T2D) [4]. Occurrence of the former has been
linked to genetic, environmental and immunological factors (T1D), with daily injection of insulin as the
major treatment [5].
In recent years, there has been a growing interest in the plant-based natural anti-diabetic medicines
not only due to their lower cost but also for their less or no side effects. This became more apparent
following the recommendation of World Health Organization [6] regarding the development and
evaluation of better pharmacological agents for improving insulin secretion, enhancing insulin sensitivity,
preventing pancreatic beta cells destruction, promoting beta cells regeneration and ameliorating pathways
that lead to the various complications of diabetes [6]. In many Nigerian communities, various herbs are
being used in the treatment and management of DM [7]. Amongst such herbs is Ficus exasperata.
Ficus exasperata is a commonly used medicinal plant for various ailments. Different parts of the
plant are used for the folkloric treatment of ulcers, anaemia, piles, jaundice, haemorrhage of the nose and
mouth, DM and various diseases of the blood [8]. Studies by [9] reported that the aqueous extract of F.
exasperata exhibited better hypoglycemic effects than glibenclamide in experimentally induced diabetic
rats. Additionally, treatment with the extract increased the values of PCV, Hb and RBC compared to the
untreated rats and rats treated with glibenclamide. Concentrations of total cholesterol, triglyceride, HDL-
/LDL-cholesterol ratio and CRI were also markedly reduced when diabetic groups were treated with the
extract compared to those treated with glibenclamide. From the results of this study, [9] suggested the
isolation and characterization of the active components of the plant that may be responsible for these
effects.
Hence, the current study was designed to examine the effects on restoring diabetes related alterations
of F. exasperata ethyl acetate fraction in an alloxan-induced diabetes model of rats and characterization
of the bioactive compounds that maybe responsible for its hypoglycemic as well as antidiabetic activities.
Collection of plants materials and extract preparation

Fresh leaves of F. exasperata were collected from the University of Ibadan, Ibadan, Nigeria during
the month of December 2014. The plant materials were identified and authenticated by Mr. Donatus,
Department of Botany, University of Ibadan, Nigeria. A sample specimen was deposited at the University
herbarium, after assigning a voucher number UIH - 22438.
The leaves were shade-dried and grounded to coarse powder using an industrial mill. The blended
sample (2849.45g) was extracted with methanol by soaking in 10L of the extracting solvent for 5 days.
40
A. O. Adeyi et al.
The obtained solution was decanted, filtered with Whatmann filter paper (no. 1) and concentrated in
vacou with a rotary evaporator (Heidolph, Germany).
The methanolic extract was further fractionated with n-hexane and ethyl acetate. Both fractions
obtained were concentrated using above-mentioned vaccum rotary evaporator.
Qualitative phytochemical screening of the extract

The procedure described by [10] was adopted for the determination of tannins, cardiac glycosides,
anthraquinones, terpenoids, alkaloids, saponins, flavonoids in the methanolic extract of the leaf of F.
exasperata.
In vivo hypoglycemic studies

A preliminary in vivo study was conducted with the methanolic extract and ethyl acetate and n-
hexane fractions to identify the extract or fraction with best hypoglycemic effect.
Characterization of compounds from F. exasperata fraction
The ethyl acetate fraction having the best hypoglycemic effect in diabetic rats was further separated
by column chromatography. The fraction was successfully eluted with stepwise gradient of n-hexane and
ethyl acetate solvent system (100: 0; 97: 3; 95:5; 90: 10; 85: 15; 80: 20; 70: 30; 50: 50) on a silica gel
column. Fractions obtained were checked on thin layer chromatography (TLC) plates. Dark green sticky
eluents were obtained in Hexane, Ethyl Acetate solvents in the ratio 90: 10. This fraction was used for
further studies. It was checked on thin layer chromatography (TLC) and showed a single spot and used for
an in vivo study. It was also subjected to gas chromatography–mass spectrometry (GC–MS) analysis. The
analysis was performed on a JEOL GCMATE II GC–MS system in EI/CI mode equipped with a
split/split less injector (220°C), at a split ratio of 1/10, using a VF-1MS fused-silica capillary column (30
m × 0.25 mm i.d.; film thickness: 0.25 mm). The oven temperature was programmed from 60°C to 280°C
in 5 min at an increment rate of 4°C/min and held at the temperature for 10 min. Helium was used as a
carrier gas at a flow rate of 0.8 ml/min.
Animals
Adult male Wistar rats weighing between 120 to 140g were obtained from the Central Animal House
of the Department of Anatomy, University of Ibadan, Nigeria. They were transported to the animal house
of the department of Zoology, University of Ibadan, Nigeria where the animal study was conducted. The
animals were kept in rat cages at room temperature (25-27oC) where food (Ladokun commercial pellet)
and water was given to them ad libitum. They were allowed to adapt for a week prior to the induction of
diabetes. The protocol for this study was approved by the Animal Care and Use Committee of the
University of Ibadan.
Animal experimental design

The animals were randomly divided into four groups of 5 animals as follows:
Group A: Non-diabetic animals treated with distilled water

Group B: Diabetic animals treated with glibenclamide 10 mg/kg
Group C: Diabetic animals treated with 200mg/kg of the 90:10, hexane:ethyl acetate fraction
Group D: Diabetic rats treated with distilled water only.
Induction of diabetes mellitus

After an overnight fast, diabetes mellitus was induced in the rats by a single intraperitoneal injection
of 0.2 ml ofalloxan in normal saline at a dose of 150mg /kg body weight. After a period of 48 hours, the
blood glucose level of the animals was checked using a portable glucometer (Accu-chek, Roche
Diagnostics, Mannheim, Germany).Blood glucose levels above 200mg/dl were considered as diabetic and
used for the further experiment.The intervention trial was lasted for 7 days when the blood glucose level
of rats were measured daily during the entire intervention period.
41
All experiment was conducted according to the National Institute of Health guidelines of care and
use of laboratory animals (NIH 1985).
Sacrifice and organ collection

At the end of the experiment, rats were fasted overnight. Blood samples were collected from the
retro orbital venous plexus of conscious animals by using heparinized capillary tubes. A portion of whole
blood was used for the haematological analysis. The remainingblood samples were allowed to clot and
then centrifuged at 2300 rpm for 10 min to obtain serum. The serum samples were preserved at -30oC
until further analysis of biochemical parameters.The animals were then sacrificed by cervical dislocation
and dissected as described by [11]. Urine was collected from their bladder after dissection into sample
bottles for biochemical analysis. The liver, heart, kidneys, and pancreas of each animal were collected and
a small piece of each was stored in 10% formalin solution for histopathological studies.
Haematological studies
The haematological parameters such as Packed Cell volume (PCV), Hemoglobin (Hb), White blood
cell (WBC), Red blood cell (RBC), Mean corpuscular Hemoglobin (MCH), Mean corpuscular volume
(MCV) and Mean corpuscular hemoglobin concentration (MCHC) were measured in the whole blood
according to the methods described by [12].
Determination of serum biochemical parameters

The levels of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase
(ALP), gamma glutamyl transferase (GGT) and total bilirubin in the serum was determined using Randox
test kits (Randox Laboratories Ltd., Crumlin, England) according to manufacturer’s protocols.
Serum lipid profile

Total cholesterol, HDL-cholesterol and triglycerides in the serum were determined using
commercially available kits (Randox Laboratories Ltd., Crumlin, England). Low density lipoprotein
(LDL) cholesterol was calculated according to the following formula:
LDL-Cholesterol = [Total cholesterol – (HDL-cholesterol + TG/5)]
Where TG/5 is equivalent to the concentration of VLDL-cholesterol.
Urine analysis
The albumin concentration in the urine sample was determined by using a commercially available
reagent (Bromocresol green solution), the urea concentration of the urine samples was determined using
the urease – Berthelot (enzymatic) colorimetric method, while the creatinine content was determined
using commercially available kits (Randox Laboratories Ltd., Crumlin, England).
Data obtained were expressed as mean ± SEM. Significant difference between test and control
groups was determined by using one way analysis of variance(ANOVA)and p <0.05 were considered as
significantly different(SPSS for Windows, version 16.0, USA)
Results
Phytochemicals in methanolic extract

Phytochemical screening of the methanolic extract of F. exasperata leaf revealed the presence of
alkaloids, saponins, flavonoids and phenolics while tannins, anthraquinones, cardiac glycosides and
steroids were not detected (Table 1).
42
A. O. Adeyi et al.
Table 1: Qualitative phytochemical constituents of methanolic extract of F. exasperata leaves
Phytochemicals Inference
Alkaloids +
Saponins +
Tannins +
Anthraquinones +
Cardiac glycosides _
Flavonoids +
Terpenoids +
Key: + = Detected; - = Not detected
GC-MS analysis
GC-CM analysis of the 90:10, hexane:ethyl acetate fraction revealed the presence of 6 compounds,
with gamma Sitosterol being the most abundant (35.87%) as shown in Figure 1 and Table 2.
Figure 1 GC-MS identified compounds in 90:10 hexane : ethyl acetate fraction
43
Table 2: GC-MS identified compounds in 90:10 hexane:ethyl acetate fraction
Pk# RT Library/ID CAS# Quality

Area%
1 15.953 2.15 2-Pentadecanone, 112036 000502-69-2 96
6,10,14-trimethyl
2 16.805 11.52 Hexadecanoic acid, 113690 000112-39-0 98

methyl ester
3 18.574 3.45 11-Octadecenoic 133708 052380-33-3 99

acid, methyl ester
4 18.814 1.59 Octadecanoic acid, 135381 000112-61-8 98

methyl ester
5 21.452 15.21 Tetracosane 164289 000646-31-1 96
6 35.877 25.49 gamma.-Sitosterol 199879 000083-47-6 99
Blood glucose concentration

The blood glucose concentrations prior to the induction of diabetes were between 61 and 78.50
mg/dl (Figure 2). Marked increase in blood glucose concentrations (ranging from 267 to 400mg/dl) were
recorded after alloxan injection, indicating an induction of DM. Treatment of diabetic rats with the
fraction and glibenclamide caused marked reduction in glucose concentration after each day of treatment.
Rats treated with the fraction had significantly (p< 0.05) lower blood glucose levels compared to rats
treated with glibenclamide. Hyperglycemia was reversed in all treated rats by the 6th day of treatment.
Figure 2: Blood glucose concentration in different animal groups during the intervention period
44
A. O. Adeyi et al.
Haematological parameters
There was no significant difference (p < 0.05) in the values of the PCV, Hb and platelet count of the
untreated group when compared with the treated groups (Table 3). The MCV and MCH values of the
untreated group were significantly lower (p < 0.05) than the treated group. There was however no
significant difference in MCHC levels in all the groups.
Table 3: The levels of various haematological parameters indifferent animal groups at the end of
the study
Treatment PCV (%) Hb RBC Platelet MCV MCH MCHC

Groups (g/dL) (cell/L) (105µL) (femtoliter) (pg) (g/dL)
A 44.50±0.5a 14.90±0.3a 7.31±0.05a 9.9±6.5a 60.92±0.3a 20.39±0.51 33.48±0.3a

c
B 42.00±4.0a 14.05±1.5a 7.16±0.6a 8.9±22.5b 60.69±2.5a 19.59±1.25 33.40±0.5a

b
C 44.50±1.5a 14.90±0.6a 6.65±0.6b 9.3±7.0a 67.31±3.8a 22.52±1.2c 33.48±0.2a

D 42.50.±1.5a 14.00±0.5a 7.74±0.4a 8.6±5.5a 55.15±4.7b 18.17±1.6a 32.95±0.0a
Data are presented as mean ± SEM (n ≤ 5); Values in the same column with different superscript letters are
significantly different from each other group of animals (p<0.05).
Group A, Non-diabetic animals treated with distilled water only; Group B,Diabetic animals treated with
glibenclamide 10 mg/kg bw;Group C, Diabetic animals treated with 200mg/kg of 90:10 hexane:ethyl acetate
fraction;Group D, Diabetic rats treated with distilled
Levels of various blood cells

The values of the WBC, lymphocytes and neutrophils were significantly higher (p<0.05) in the
untreated group than in the treated and control groups (Table 4). However, there was no significant
difference (p<0.05) in the counts of monocytes. The eosinophils count in the untreated rats was
significantly lower (p<0.05) than the treatment groups.
Table 4: Levels of various blood cellsin different animal groups at the end of the study
Treatment White Blood Lymphocytes Neutrophils Monocytes Eosinophils

groups Cell (103µL) (%) (%) (%) (%)
A 3.6±1.5a 62.00±2.0d 34.00±3.0a 3.0±0.0a 2.0±0.0a
B 5.5±1.3b 62.00±6.0d 33.00±5.0a 2.0±1.0b 3.0±0.0b
C 5.7±1.0b 61.00±2.0d 36.00±4.0a 2.0±1.0b 3.0±1.0b
D 6.9±2.2c 65.50±3.5d 29.50±4.5b 2.0±1.0b 2.0±1.0a
Values are shown as mean±SEM (n ≤ 5); Values in the same column with different superscript letters are
significantly different from each other group of animals (p<0.05).
Liver function tests

The results of the liver function tests are presented in Table 5.The result shows no significant
difference (p<0.05) in AST level of the untreated, glibenclamide and fraction treated diabetic rats (group
B-D). There was also no significant difference (p<0.05) in the activities of ALT in the plasma of the
untreated and fraction treated group but was significantly reduced (p<0.05) in the glibenclamide treated
group.
45
There was a significant reduction (p<0.05) in the concentration of alkaline phosphatase (ALP) in the
treated group (C) compared to the diabetic control group (B). The total bilirubin concentration of the rats
treated with the fraction was reduced significantly (p<0.05) compared to all other groups (Table 5).
Table 5: Levels of liver function related parameters in the serum of different rat groups at the end of the
study
Treatment AST ALT ALP GGT Total Bilirubin

groups (mg/dL)
(IU/L)
A 38.00±2.0c 30.00±1.0a 109.00±1.0b 0.15±0.05a 0.40±0.1c

B 40.50±0.5a 29.50±0.5b 111.50±11.5c 0.20±0.00b 0.40±0.1c
C 41.00±5.0a 30.50±2.5a 109.00±4.0b 0.25±0.05b 0.35±0.05b
D 44.50±1.5a 33.00±1.0a 122.50±1.5a 0.30±0.06c 0.45±0.05c
Values are shown as mean±SEM (n ≤ 5); Values in the same column with different superscript letters are
significantly different from each other (p<0.05).
Group A,Non-diabetic animals treated with distilled water only; Group B,Diabetic animals treated with
fraction; Group D, Diabetic rats treated with distilled water only; AST, aspartate transaminase; ALT, alanine
transaminase; ALP, alkaline phosphatase; GGT, gamma glutamyl transferase.
Serum lipid profile

Table 6 shows the results of serum total cholesterol, triglyceride, LDL-cholesterol and HDL-
cholesterol levels in the serum of different experimental groups. It was observed that the levels of total
cholesterol, triglyceride and LDL-cholesterol were reduced significantly (p<0.05) in the treated group (C)
compared to the untreated group (D). However, the HDL cholesterol level was significantly increased
(p<0.05) in treated group compared to the untreatment group (Table 6).
Table 6: Serum lipid profile in different animal groups at the end of the study
Treatmen Total Triglycerides HDL- LDL-

t groups cholesterol cholesterol cholesterol
(mg/dL)
A 58.50±7.5b 41.00±1.0c 38.50±0.5a 11.80±0.4c

B 63.00±0.0a 43.00±2.0c 33.00±6.0a 21.40±0.6b
C 60.50±7.5a 40.50±2.5c 30.00±6.0a 22.40±1.0b
D 72.00±1.0c 52.50±3.5b 27.00±7.0b 34.50±0.7a
Values are shown as mean ± SEM, n ≤ 5. Values within a column having different superscript letters are
significantly different from each other group of animals, p<0.05.
Group A,Non-diabetic animals treated with distilled water only;Group B,Diabetic animals treated with
fraction;Group D, Diabetic rats treated with distilled water only; HDL, high density lipoprotein; LDL, low density
cholesterol.
46
A. O. Adeyi et al.
Urine Analysis
The urea, creatinine and albumin levels in the urine of all animal groups are shown in Table 7. In this
study, the level of urea was significantly decreased in the urine of fraction treated group compared to all
other groups. There was no significant difference (p<0.05) in the levels of creatinine and albumin among
the diabetic groups.
Table 7: The levels of renal function related parameters in the urine of different animal groups at the end
of the study
Treatment Urea Creatinine Albumin

groups (mg/dL)
A 9.25±0.45a 38.50±1.50c 2.15±0.05a
B 11.20±1.00b 26.50±1.50b 2.15±0.05a
C 8.85±0.75c 29.00±1.00b 2.55±0.05a
D 12.15±0.65d 25.50±1.50b 2.40±0.10a
Values are mean ± SEM, n ≤ 5. Values within a column having different superscript letters are significantly different
from each other group of animals, p<0.05.
Group A, Non-diabetic animals treated with distilled water only;Group B,Diabetic animals treated with
fraction;Group D, Diabetic rats treated with distilled water only.
Histopathological Studies
Pancreas
The pancreatic tissue of the diabetic control group showed normal appearance of the islets of
Langerhans scattered throughout the tissue (Fig. 3A). That of the diabetic group treated with plant
fraction showed mild vacoulation of the islets of Langerhans (Fig. 3B). Untreated diabetic rat shows
abnormal and fewer islets of Langerhans that are barely seen (Fig. 3C)
A B C
Figure 3. (A) Pancreas of control rat showing normal appearance of the islet of langerhans ×400 H and E;
×400 H and E; (B) pancreas of alloxan-induced diabetic rat treated with plant fractionshowing mild
vacoulation of the islets of Langerhans ×400 H and E. (C) pancreas of untreated alloxan-induced diabetic
rat, showing marked degeneration of the Islets of Langerhans.
The Liver
Histopathological examination of the liver of the control group appeared normal with a proper
arrangement of the hepatocytes (Fig. 4A). That of the diabetic group alloxan-treated with the plant
47
fraction revealed a mild degeneration of the hepatocytes (Fig. 4B), the untreated diabetic group showed
severe degeneration of the hepatocytes (Fig.4C).
A B C
Figure 4. (A) Liver of control rat, showing normal arrangement of the hepatocytes with no visible lesion.
×400 H and E; (B) liver of alloxan-induced diabetic rat treated with the plant fraction revealed a mild
degeneration of the hepatocytes ×400 H and E. (C) Liver of untreated alloxan-induced rat showing severe
degeneration of the hepatocytes (HP) with numerous vacuolations. ×400 H and E
The kidney
Histopathology of the kidney of the control groups showed normal appearance of the organ with no
visible lesions (Fig. 5A). The glomeruli are also surrounded by narrow and normal bowman’s spaces. The
kidney diabetic rat treated with the plant fraction showed normal glomerulus with mild vacuolations of
the tissue (Fig. 5B). The glomerular spaces in the treated group were noticed to show some signs of
regenerations in them, with their glomerular spaces appearing narrower than that of the diabetic untreated
rat (Fig. 5C).
A B C
Figure 5. (A) Kidney of normal rat showing normal appearance of the glomeruli. ×400 H and E; (B)
kidney of alloxan-induced rats treated with plant fraction showing normal glomerulus with mild
vacoulations of the tissue. ×400 H and E (C) kidney of untreated alloxan-induced diabetic rat showing
vacoulation of the kidney ×400 H and E
The heart
A normal appearance of the endothelium which is supported by a layer of collagenous tissue was
observed in the histological examination of the heart of the control group (Fig. 6A). In the diabetic
untreated group (E), marked areas of degeneration of the myofibres with diffuse vacoular degenerations
of the myocytes were revealed (Fig. 6C). Also, a depletion of the cardiac blood tissues was visibly
observed. Whereas, that of the diabetic treated groups revealed a normal appearance of the myocytes and
likewise, a form of regeneration was identified in the cardiac muscle / tissues (Fig. 6B).
48
A. O. Adeyi et al.
A B C
Figure 6. (A) Heart of normal rat showing normal appearance of the endothelium. ×400 H and E; (B)
Heart of alloxan-induced rats treated with plant fraction showing regeneration of the cardiac tissue. ×400
H and E (C) Heart of untreated alloxan-induced diabetic rat showing severe degeneration of the myocytes
×400 H and E
Discussion
Diabetes is referred to as a chronic disease marked by high levels of sugar in the blood [13]. The
geometric increase in glucose concentration observed in all rats after a single intraperitoneal injection of
alloxan monohydrate confirmed the induction of diabetes. Alloxan induces “chemical diabetes” in a wide
variety of animal species by damaging the insulin secreting pancreatic β-cell of the islets of Langherhans,
resulting in reduced synthesis and release of endogenous insulin characteristically similar to type 1
diabetes in humans [14].
The phytochemicals present in the methanol extract of F. exasperata showed flavonoids, tannins,
saponin, alkaloids, terepenoids and anthraquinone. Flavonoids is one of the most diverse and widespread
group of natural compounds, the presence of hydroxyl groups confers scavenging ability and also plays an
important role in preventing lipid peroxidation [15]. Saponins are glycosides of triterpenes, steroids or
alkaloids. Saponins may have a glucagon decreasing effect and may enhance glucose utilization lowering
blood glucose as well as stimulate insulin release from the pancreas [16]. Tannin is composed of a central
glucose molecule derivatized at its hydroxyl groups with one or more galloyl residue and in the presence
of copper ions; act as an antioxidant suppressing hydroxyl radical formation [17]. Anthraquinones
derivatives have also been found to play an important role in the treatment of tumors, diabetes, ulcer and
cancer [18]. Thus, the phytochemical constituents indicate that the methanol extract of F. exasperata
could have potentials to be an antidiabetic agent which is in agreement with a previous study [9].
The preliminary study showed that the ethyl acetate fraction of the methanolic crude extract has the
best hypoglycemic effect when compared to the methanolic crude extract and the n-hexane fraction in
alloxan-induced diabetic rats. The components were therefore separated using column chromatography.
Fractions received from the column chromatography at ratio 90:10 ethyl acetate to n-hexane was used to
treat alloxan-induced diabetic rats in the main study. Results obtained from this study show that the
fraction significantly (P<0.05) lowered blood glucose levels of diabetic rats (Figure 2). This action of the
fraction on blood glucose in diabetic rats is similar to that of Gilbenclamide (10mg/kg bw), a potent
hypoglycemic agent, and suggested that the fraction contain active principles with potent hypoglycemic
property. The fractions may have achieved this hypoglycemic property via increased insulin secretion,
increased peripheral utilization of glucose, inhibition of endogenous glucose production or by inhibition
of intestinal glucose absorption as reported in some previous studies [19,20]. This result supports the
hypoglycemic and antidiabetic potential of the F. exasperata for the treatment of diabetes mellitus.
49
In diabetes, the value of PCV, Hb, RBC, MCV, MCH and MCHC are reduced due to lyses of blood
cells caused by reactive oxygen species (ROS) and the resulting oxidative stress [21,22,23] leading to
anaemia [24]. This was the trend observed in this study (Table 3). However, fraction treatment caused
significant (p<0.05) amelioration in the value of these parameters such that it brought about a significant
increase in the MCH value, a factor that measures the rate of erythrocyte synthesis. It therefore can be
deduced that extract was able to reverse the lytic effect of ROS and so reduced or rather completely
prevented oxidative stress thereby giving room for the regeneration of erythropoietic cells, a process
mediated by erythropoietin secretion from the bone marrow [21,25]. The overall effect is the restoration
of the oxygen carrying capacity of the RBC as a result of the inhibition of the process of lipid
peroxidation in the membrane of RBC [26].
Changes in TWBC have been associated with insulin resistance and complications of CVD [22,23].
The result of this study showed a significant (p<0.05) increase in the value of TWBC in the diabetic
control group which reduced significantly on fraction treatment. This may be interpreted to mean the
fraction’s ability to restore insulin sensitivity to the cells [23].
The liver is known to play an important role in the metabolism of carbohydrate and so its cellular
integrity can be compromised in diabetes and related disease [27]. Therefore, some liver function tests
were performed to assess its pathological condition. Prominent among these tests are the analysis of AST,
ALT, ALP activities and Bilirubin levels. Results show that the plant fraction reduced ALP activities and
the level of total Bilirubin. Reduction of ALP activity as shown by fractions treatment is suggestive of the
fractions’ ability to protect the cell from cytotoxic injury. Bilirubin is a breakdown product of blood with
biological and diagnostic values [28]. Mild decrease in bilirubin levels in the treated group compared to
untreated group has been proposed to have a null protective effect on cells [29].
The diabetic control groups had elevated total cholesterol, triglycerides, decreased high density
lipoprotein cholesterol (HDL-cholesterol). The results agreed with [30], who reported that high levels of
triglycerides, LDL-cholesterol and low levels of HDL-cholesterol have been associated with heart
disease, insulin resistance and diabetes mellitus. On the other hand, HDL is often referred to as ‘Good
Cholesterol’ with high levels associated with a decreased risk of myocardial infarction. HDL removes
cholesterol from non-hepatic tissues to liver through the process known as reverse cholesterol transport
[31]. The plant fraction therefore has shown hypolipidemic effect in diabetic rats. This is in agreement
with the results of [9].
The significant increase in urine creatinine for both normal and treated diabetic rats is a strong
indication of the positive impact treatment with of the fraction may have on the glomerular filtration rate.
Creatinine levels in blood and urine are usually used to calculate the creatinine clearance rate (CCR)
which reflects the glomerular filtration rate (GFR). The GFR is important clinically because it is a
measure of the renal function. High creatinine level in the urine indicates the ideal, while a low urine
creatinine level may indicate malfunctioning of the kidneys. GFR is so important in assessing the
excretory function of the kidneys [32]. The ability of the fraction of the methanolic leaf extract of F.
exasperata to increase the levels of creatinine, reduce the level of urea and albumin in urine suggest that
the fraction may ameliorate diabetic nephropathy.
Alloxan monohydrate has been described as a toxic glucose analogue which selectively destroys the
insulin-producing beta cells of the pancreas [33]. Mild vacuolations of the islets was however observed in
pancreas of rats treated with the plant extract fractions which is probably indicative of the ability of the
extract to restore the degenerations cause by alloxan-induced diabetes mellitus this is in agreement with
[34,35]
The potentials of the extract to ameliorate the pathological effects of diabetes mellitus was
demonstrated as rats treated with the plant fraction showed mild degeneration of liver compared to rats
treated with glibenclamide.
An ameliorative property of the fraction is seen as the kidney of rats treated with the fraction show
normal glomeruli with mild vacuolation while the histology of diabetic heart section of the treatment
groups (B &C) show no alteration with the cytoarchitecture similar to that of normal control. The fraction
50
A. O. Adeyi et al.
has ameliorative effect on the heart of the diabetic rat. The section of the heart of diabetic control showing
myocytes with their intercalated discs and interdigitations.
It is worthy to note that treatment with the plant extract fraction has proved to be effective in the
reduction of blood glucose levels, is capable of stimulating blood cell formation (erythropoiesis) and
confer protection to hepatocyte against cell injury due the effect of oxidative stress it has hypolipidemic
potentials and has also showed ameliorative and restorative effects on structures during diabetic
complications. The GC-MS result shows that the most prominent compound in this plant fraction is β
sitosterol which has been reported to exhibit anti-inflammatory activity in carragenaan paw oedema
model [36] and in mice induced by phorbol derivative [37].
In conclusion, this study demonstrates the hypoglycaemic and hypolipidemic potentials of F.
exasperate in T1D model of rats, which can be attributed to the synergistic effect of the identified
compounds particularly gamma-sitosterol in the fraction. This further gives credence to the use of the
plant in the management of diabetes and its complications.
Consent for publication – All authors consented for publication
Competing interests – No Competing interest
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Methanolic extract of Garcinia kola elicits diuretic activity

with alteration in circulating electrolyte concentration in
male Wistar rats
Kehinde S. Olaniyi1,4*, Nifesimi T. Akinnagbe1,4, Chukwubueze L. Atuma1,4, Hadiza
Mahmud1,4, Caroline O. Badejogbin2, Toluwani B. Agunbiade3, Oloruntoba C. Akintayo4,
and Olurotimi J. Sanya4
1
Cardio/Repro-metabolic & Microbiome Research Unit, Department of Physiology, College of Medicine and Health
Sciences, Afe Babalola University, Ado-Ekiti, 360101, Nigeria.
2
Department of Physiology, Benjamin Carson School of Medicine, Babcock University, Ilishan-Remo, 121003,
Nigeria.
3
Department of Medical Microbiology and Parasitology, College of Medicine and Health Sciences, Afe Babalola
University, Ado-Ekiti
4
Department of Physiology, College of Medicine and Health Sciences, Afe Babalola University, Ado-Ekiti, 360101,
Nigeria.
*Author to whom correspondence should be addressed. Email: kennethnitty2010@gmail.com;

olaniyisk@abuad.edu.ng
Tel: +2348034392745; ORCID iD: 0000-0002-8229-9688
(Received December 30, 2020; Accepted February 4, 2021)
ABSTRACT: Objective: Garcinia kola is a kolaviron-containing nut native to tropical African and cultivated
extensively in the new world tropics. Several studies have reported its effects on body weight and reduction in body
fat without undesirable side effects as well as on gastric secretion. This study was designed to investigate the diuretic
activity of methanolic extract of Garcinia kola (MEGCO) in male Wistar rats. Design and Method: Adult male
Wistar rats were randomly allotted into control (distilled water, po), Furosemide (po), MEGCOA (200 mg/ kg),
MEGCOB (400 mg/ kg), MEGCOC (600 mg/ kg), groups with 5 rats/group. The extract was prepared as previously
described and the treatment lasted for 4 weeks. Urine electrolytes, serum electrolytes, serum uric acid, urea and
creatinine as well as liver ALT and AST were assayed using standard colorimetric method. Urine volume and diuretic
indices were also monitored. Results: Treatment of all rats with different doses of Garcinia kola did not significantly
alter body weight but increased kidney weight (400 mg and 600 mg/kg doses) and increased urine volume, urine Na+,
K+ and Cl- concentration, did not alter serum Na+, but 600 mg/kg of MEGCO increased serum K+ and Cl-
concentration. In addition, 600 mg/k of MEGCO increased hepatic ALT but not AST activity, increased serum
creatinine, urea and uric acid concentration when compared with control and furosemide-treated groups. Conclusion:
The present study demonstrates that methanolic extract of Garcinia Kola, particularly 600 mg/kg dose causes diuresis,
natriuresis and kaliuresis, but put the animals at risk of renal toxicity and electrolyte imbalance.
Keywords: Diuresis; Garcinia kola, potassium; renal toxicity, sodium.
53
Introduction
It is noteworthy from several studies that modern medicine cannot be regarded as a realistic treatment
option for a significant proportion of the world population, particularly developing and underdeveloped
countries (Anna et al. 2019). In the 21st century, the influence of plant-based pharmaceuticals is evident and
the use of herbs as therapy for various diseases is widespread. The efficacy of some of these herbs in disease
treatment has been confirmed by several studies. About 80% of the world population relies on herbs and
plant-based medication for primary care (Buba et al. 2016). Garcinia Kola is a potent medicinal plant found
in West and Central Africa. This plant has been dubbed “Wonder Plant” due to the medicinal importance
of every part of the plant (Ekene et al. 2014). The Garcinia Kola can be described as a medium-large tree,
which grows up to 15-17 meters in height (Iwu, 1993). The fruit is smooth and it weighs about 30 – 50
grams; it is 5-10cm in diameter and a single fruit could have 1-4 seeds (Juliana et al. 2006).
Garcinia Kola is of cultural significance among all Nigerian tribes particularly the Yoruba and Igbo
tribes of Nigeria (Atilade, 2004). To natives of the Yoruba land it is known as “Orogbo” and the Igbos call
it “Aku ilu‟ (Dalziel, 1937). It is commonly served to visitors at naming ceremonies, weddings, and other
social events; it is a sign of peace and acceptance of guests (Ekene et al. 2014). The bitter chew-sticks
common to West Africans are products of the roots of the Garcinia Kola plant (Otor et al.2001).
Furthermore, the increase in the commercial value of Garcinia Kola has made natives of communities
where it is endemic keen-on its cultivation. Its commercialization has been known to have a significant
impact on the standard of living of people in rural communities. The production and sale of Garcinia Kola
is usually a family business in rural communities and the proceeds are earmarked towards domestic
expenditure like feeding, school fees, and family ceremonies (Atilade, 2004). Garcinia Kola is believed to
cleanse the digestive system and excessive consumption does not cause abdominal problems (Buba et al.
2016). A mixture of ground Garcinia Kola seeds and honey is used to make a traditional cough medication
(Buba et al. 2016). Garcinia Kola has been employed as an antidote for snake bites, therapy for cough, and
vomiting (Buba et al. 2016). It enjoys notoriety in Africa as a poison antidote and has a plethora of
traditional medical applications (Konziase, 2015; Buba et al. 2016).
The phytochemical constituents of Garcinia Kola seeds include; flavonoids, saponins, tannins,
phenols, glycosides, and alkaloids (Adesuyi, 2012). Although the seeds have been deemed safe for
consumption, anti-nutrients such as oxalate and phytate were observed (Konziase, 2015). Flavonoids are
the most abundant phytochemicals in Garcinia Kola seeds; they are antioxidants with low molecular weight
which scavenge free radicals and convert them to harmless molecules and also influence several aspects of
immune cell activation (Nworu et al. 2008). They also provide protection for the Central Nervous System
against oxidative and excitotoxic stress (Ijomone et al. 2012). Kolaviron is a major active component of
Garcinia Cola seeds; it is riched in bioflavonoids that consists of GB-1, GB-2, and Kola flavanone
(Tchimene et al., 2015). Kolaviron is known to possess sedative and anti-inflammatory properties; it acts
both centrally and at the periphery and this legitimizes its traditional use as a pain reliever and anti-
inflammatory (Anna et al. 2019). In a recent study, kolaviron was administered to a fruit fly (Drosophila
melanogaster) and it was observed that the fly had a protracted life-span which was due to its antioxidative
and anti-inflammatory properties (Farombi et al. 2018). Many of the properties of kolaviron have been
elucidated in animal models, including antioxidant, hepatoprotective, anti-inflammatory, anti-malarial,
anti-microbial, anti-diabetic, anti-ulcer, anti-cancer, anti-asthma, anti-arthritis and anti-hypertensive (Buba
et al. 2016; Quadri et al. 2019).
Diuretics have been known to be effective in treating hypertensive patients, it has been observed that
it reduces systolic and diastolic blood pressures in hypertensive patients. Administration of diuretics in
combination with other agents forms the basis of therapy for many hypertensive patients (Shah et al. 2004).
The natriuretic property of diuretics is what causes the decrease in total body sodium; potent diuretics act
at a site where a large quantity of sodium is normally reabsorbed. Consequently, the amount of sodium
excreted in the urine and accompanying fluid loss can be enhanced immensely with these agents by
54
increasing the dose (Shah et al. 2004). Sodium excretion and fluid loss play an important role in the
management of edema and hypertension (Koushik et al. 2014). Herbal medicine is an essential source of
diuretics, mono and poly-herbal preparations have been employed as diuretics. According to an
extrapolation, more than 650 mono and poly-herbal preparations in the form of decoction, tincture, tablets,
and capsules from more than 75 plants are in clinical use (Chopra et al. 1986). Herbs were popularly used
as traditional therapy for some renal diseases and a lot of plants have been reported to show potent diuretic
activity (Koushik et al. 2014).
There have been several studies that investigate the efficacy of herbal medicine as diuretics. Garcinia
Kola is also suggested to act as an effective loop diuretic (Quadri et al. 2019). However, studies
investigating the diuretic potentials of Garcinia Kola and in particular its effect on electrolyte balance are
limited. Therefore, the present study was designed to investigate the diuretic activity of methanolic extract
of Garcinia Kola (MEGCO) and its effects on the circulating electrolytes in male Wistar rats.
Plant collection and authentication

Garcinia Kola seed were sourced locally in Ado-Ekiti, Nigeria, and authenticated by a botanist in the
Department of Agricultural science, Afe Babalola University where a voucher specimen was documented.
Drug and chemicals

Furosemide 40 mg manufactured by (Mancare Pharmaceuticals Pvt. Ltd) marketed as a diuretic drug
was used as the standard drug. Assay kits for Biochemical parameters were purchased from Randox by the
use of appropriate kits agent; Teco Laboratories at Ado-Ekiti.
Plant extraction
The methanolic extraction of Garcinia cola seed was prepared as previously described by Tendel et
al. (2011). Fresh Garcinia kola seed were collected and washed with clean water, cut into small pieces and
air dried under shade. 2.55 kg of dried Garcinia cola seeds were pulverized with an Electric pulverizer into
fine powdered seed (1.95kg) with 3000 cm3 of methanol, macerated in a polytron Homogenizer for 48
hours and the solution was filtered under vacuum using Buchner funnel and Whatman No.1 filter paper
(Whatman International Ltd, Maidstone, UK). The filtrate was evaporated under reduced pressure using a
rotary evaporator and later freeze dried in a lyophilizer (Ilshin Lab. Co.Ltd, Seoul, Republic of Korea). The
extract obtained was kept in a desiccator prior to use.
Animal care and management

Twenty-five (25) adult male Wistar rats weighing 160-200g were used for this study. They were
purchased from the Animal House of the College of Health Science, Afe Babalola University, Ado-Ekiti
where the study was carried out. The rats were housed in plastic cages under natural light/dark cycle in the
laboratory and allowed to have access to standard rat chow (ABUAD Farm, Nigeria) and water ad libitum.
They were allowed to acclimatize in the laboratory for two weeks before the commencement of the study.
The investigation was conducted in accordance with the National Institutes of Health Guide for the Care
and Use of Laboratory Animals and the protocol was approved by the Institutional Ethical Review Board
of Afe Babalola University, and every effort was made to minimize both the number of animals used and
their suffering.
Grouping and administration

The animals were allotted into five (5) groups of n=5 namely: control, Furosemide, MEGCOA,
MEGCOB and MEGCOC groups. Control group received vehicle (distilled water), Furosemide group
received 20 mg/kg of Furosemide, MEGCOA group received 200 mg/kg of MEGCO, MEGCOB group
55
received 400 mg/kg of MEGCO and MEGCOC group received 600 mg/kg of MEGCO. The administration
was carried out by oral gavage and it lasted for 4 weeks.
Diuretic study
Diuretic potential of methanolic extract of Garcinia Kola was determined following the method of
Lipchitz et al., 1943. The rats were allowed to acclimatized in metabolic cages fabricated by (Central
Technological Laboratory and Workshops (CTLW), Afe Babalola University, Ado-Ekiti, according to
Ohasu R Model; Ohasu, Pine Brook, New Jersey, USA) for one week before the commencement of the
study. For urine collection, the rats were fasted overnight with free access to water after which their bladders
were emptied by gentle of the pelvic area and by pulling the base of the tails.
Prior to the day of sacrifice urine samples were collected 2 hours intervals for 6 hours. Urine volume
and electrolyte concentrations were determined using appropriate biochemical kits. The urine volume was
expressed in ml.
Evaluation of diuretic activity (Lipschiz Test)

Urine volume (m1) and Na+, K+ and Cl- concentration (mEq/L) in the urine were determine and
various indices for diuretic action were calculated (Lipschitzet al., 1943). The ratio of urine volume of the
experimental group to that of the control group was taken as a measure of the diuretic index for any given
dose of the extract.
Diuretic Index = Urine Volume of Test group (Ve)/Urine volume of Control Group (Vc)
Indices of 1.0 and more are regarded as a positive effect or potent diuretics. The diuretic activity is
considered to be positive if the diuretic index values are greater than 1.50, moderate if the values are
between 1.00 and 1.50, mild if the values lie in between 0.27 and 1.00 and there is no diuretic activity if the
values is <0.72 (Abdala et al. 2008). Since diuretic index is prone to variability, a parameter knows as
Lipschiz value was calculated. To obtain the Lipschiz value, urine volume of the extract treated rats was
compared to that of the group that receive the standard drug (Furosemide) (Mukherjee, 2002).
Lipchitz value (or diuretic activity) = Urine Volume of Test Group (Ve)
Urine Volume of standard Group (vr)
Sodium Assay
Sodium was assayed in accordance with the method described by (Trinder, 1951). To 1.0ml of uranyl
acetate 2.1mM and magnesium acetate 20 mM in ethyl alcohol, 50 u of the sample (urine) was added. The
test tubes were shaken vigorously for proper mixing for 3 minutes and then centrifuge at 1500
revolution/minutes for 10 minutes to obtain a clear supermarket and 50 potassium ferrocyanide, non-
reactive stabilizers, and fillers were added. Absorbance was recorded at 550nm against blank containing
distilled water. Concentration (cone) of sodium was calculated using this formula:
Calculation:
Abs. of unknown 𝑚𝐸𝑞 𝑚𝐸𝑞

𝑋 𝐶𝑜𝑛𝑐. 𝑜𝑓 𝑆𝑇𝐷 ( ) = 𝐶ℎ𝑙𝑜𝑟𝑖𝑛𝑒 𝐶𝑜𝑛𝑐. ( )
Abs. of STD 𝐿 𝐿
Abs. = Absorbance, S = Sample, STD = Standard (150 mE/L)
Potassium Assay
Potassium was assayed according to the method of (Terri and Sesin, 1958). To 1.0ml of sodium
tetraphenylboron 2.1mM, 0.01ml (10μ) of samples (urine) was added. The resulting solution was mixed
and incubated at room temperature for 3 minutes. Absorbance was then recorded at 500 mm against blank
containing distilled water. Concentration of potassium was calculated using this formula:
56
Calculation:
Abs. of unknown
𝑋 𝐶𝑜𝑛𝑐. 𝑜𝑓 𝑆𝑇𝐷 (𝑚𝐸𝑞/𝐿) = 𝐶ℎ𝑙𝑜𝑟𝑖𝑛𝑒 𝐶𝑜𝑛𝑐. (𝑚𝐸𝑞/𝐿)
Abs. of STD
Abs. = Absorbance, S = Sample,

STD = Standard (150mE/L)
Chloride assay
Chloride was assayed according to the previous method of (Skeggs and Hochstrasser, 1964). To 1.5ml
of mercuric nitrate o.o58mM, mercuric thiocyanate 1.75mM, mercuric chloride 0.74mM and ferric nitrate
22.3m M, 0.01(10μ) of sample (urine) was added. Absorbed was the recoded at 480 nm against blank
containing distilled water. Concentration of chloride was calculated using this formula:
Calculation;
Abs. of unknown 𝑚𝐸𝑞 𝑚𝐸𝑞
𝑋 𝐶𝑜𝑛𝑐. 𝑜𝑓 𝑆𝑇𝐷 ( ) = 𝐶ℎ𝑙𝑜𝑟𝑖𝑛𝑒 𝐶𝑜𝑛𝑐. ( )
Abs. of STD 𝐿 𝐿
Where, Abs. = Absorbance, S = Sample, STD = standard (100mEq/L)
At the end of two weeks of the study period, the rats were sacrificed using sodium pentobarbitone (50
mg/kg ip) and blood sample was collected through cardiac puncture into heparinized bottle, centrifuged and
plasma was collected for biochemical analysis.
Body weight
The initial body weights of rats were determined and recorded before the commencement of the study,
and the final weights of the rats were also monitored before sacrifice using a Camry weighing balance to
assess the weight gain or loss in each group.
Urine volume
The volume of urine was measured using clean and dried measuring cylinder.
Biochemical assay
Blood sample was collected by cardiac puncture and draw into separate heparinised bottle and
centrifuged at 3000 rpm for 15 minutes at 4oC, using centrifuge (Centurium Scientific, Model 8881). The
plasma obtained was draw into separate plain bottle for biochemical assay. Also, the liver was isolated,
weighed and sliced into sections of about 1g on dry ice and then placed in 5mL of ice-cold phosphate
buffered saline. The tissue was homogenized on ice. The homogenate was then centrifuge at 1500xg for 15
minutes. The clarified supernatant was frozen stored for biochemical analysis.
Serum creatinine assay

Creatinine concentration was assayed by the method of (Bartels and Bohmer, 1972). To 1.0 ml of piric
acid and sodium hydroxide, 0.1ml (10) of samples was added. Absorbance was then recorded at 492nm
against blank and standard containing 0.1 ml of distilled water and standard reagent respectively.
Concentration of creatinine was calculated using this for formula:
57
Calculation:
A2 –A1 =∆A sample or ∆A standard
Concentration of creatinine in the tissue
∆A sample 𝑚𝑔
𝑋 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝐶𝑜𝑛𝑒. ( ) =
∆A standard 𝑑𝑙
Where;
A2 =Absorbance reading after 30 minutes, A2 = Absorbance reading after 2 minutes.
∆A sample =Absorbance reading of standard
Standard conc. = standard concentration in mg/dl (2.07mg/dl)
Renal urea assay

Urea concentration was assayed by the method of (Chaney and Marbach, 1962). To 100μ1 of sodium
nitropusside 100μ of samples was added. The resulting solution was incubated at 37 0C for 10minutes.
Then, 2.50 ml of Phenol (reagents II) and Sodium hypochlorite (reagents III) was added to the incubated
mixture. This was further incubated 37oC for 15 minutes. Absorbance was then recorded at 546 mn against
blank and standard containing 10μ of distilled water and standard solution respectively. Concentration of
urea was calculated using this formula:
∆A sample 𝑚𝑔
Calculation: 𝑋 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝐶𝑜𝑛𝑒. ( ) =
∆A standard 𝑑𝑙
Where;
A sample = Absorbance reading of sample
A standard = Absorbance reading of standard.
Stand Conc. = standard concentration in mg/dl(80.77mg/dl)
Hepatic AST and ALT

Activities of liver ALT and AST were determined according to the method of Duncan (1994).
Serum uric acid

Serum uric acid was determined as previously described (Kageyama, 1971).
All data were expressed as means ± SEM. Statistical group analysis was performed with SPSS, version
22 of statistical software. One-way analysis of variance (ANOVA) was used to compare the mean values
of variables among the groups. Bonferroni’s test was used to identify the significance of pair wise
comparison of mean values among the groups. Statistically significant differences were accepted at p <
0.05.
Results
Effect of methanolic extract of Garcinia kola on body weight in male Wistar rats
The administration of methanolic extract of Garcina cola did not alter the body weight when compared
with control and furosemide-treated rats as shown in the table below.
58
Table 1: Effect of methanolic extract of Garcinia kola on body weight in male Wistar rats
GROUPS INITIAL WEIGHT (g) FINAL WEIGHT(g) WEIGHT GAIN(g)

CONTROL 174.0±18.5 249.1±21.0 75.2±25.2
FRD 173.8±17.1 226.6±5.0 52.8±4.5
MEGCOA 162.4±36.6 192.4±49.9 30.0±6.1
MEGCOB 162.8±11.4 159.4.±7.2 (3.5±2.5)
MEGCOC 169.4±25.3 168.2±26.3 (1.2±16.4)
Data are expressed as mean±S.E.M. n=5. Data were analysed by one-way ANOVA followed by Bonferroni post hoc
test. (Not significant at p<0.05 vs. control).
Effect of methanolic extract of Garcinia kola on kidney and liver weight in male Wistar rats
As shown in table 2, the administrations of 400 mg or 600 mg/kg body weight dose of methanolic
extract of Garcinia cola significantly increased the kidney weight but not alter the liver weight adjusted for
body weight when compared with control and furosemide-treated groups. However, the dose of 200 mg/kg
body weight did not affect kidney and liver weight compared with control and furosemide-treated groups.
Table 2: Effect of methanolic extract of Garcinia cola on kidney and liver weight in male Wistar rats
GROUPS Kidney weight (g/100g b.w) Liver weight (g/100g b.w)

CONTROL 0.30±0.02 1.47±0.74
FRD 0.40±0.01 1.95±0.61
MEGCOA 0.45±0.03 0.99±0.76
MEGCOB 0.63±0.03*# 1.45±0.71
MEGCOC 0.86±0.03*# 1.89±0.59
Data are expressed as mean±S.E.M. n=5. Data were analysed by one-way ANOVA followed by Bonferroni post hoc
test) (Significant at *p<0.05 vs. control, #p<0.05 vs. FRD).
Effect of methanolic extract of Garcinia kola on urine volume in male Wistar rats
Administration of methanolic extract of Garcina cola significantly increased the urine volume after 4
hrs in furosemide and MEGCOC-treated group compared with control. The urine volume for MEGCOC-
treated group was significantly higher than furosemide treated group. The urine volume of furosemide and
all the extract-treated groups were significantly higher compared with control group after 6 hrs of urine
collection.
59
Table 3: Effect of methanolic extract of Garcinia kola on urine volume in male Wistar rats
GROUPS URINE VOL (ml) URINE VOL (ml) URINE VOL (ml)
(2 HOURS) (4 HOURS) (6 HOURS)
CONTROL 0.2 ± 0.1 0.08 ± 0.1 0.05±0.1
FRD 0.25 ± 0.025 0.2 ± 0.025* 0.9±0.025*
MEGCOA 0.20 ± 0.04 0.1 ± 0.04 0.3 ±0.04*#
MEGCOB 0.24 ± 0.1 0.1 ± 0.1 0.4 ±0.1*#
MEGCOC 0.30 ± 0.01 0.3 ± 0.01*# 0.8 ± 0.01*#
Data were analysed by one-way ANOVA followed by Bonferroni post hoc test. (*p<0.05 vs. Control; #p<0.05 vs.
FRD).
Diuretic indices for administration of methanolic extract of Garcina kola in male Wistar rats
According to Lipchitz’s test, Indices of 1.0 and more are regarded as a positive effect or potent
diuretics. The diuretic activity is considered to be positive if the diuretic index values are greater than 1.50,
moderate if the values are between 1.00 and 1.50, mild if the values lie in between 0.27 and 1.00 and there
is no diuretic activity if the values is <0.72 (Abdala et al., 2008). Since diuretic index is prone to variability,
a parameter knows as Lipchitz‘s value was calculated by adjusting for the standard (furosemide). As shown
in Table 4 comparing the value obtained with Lipchitz’s value, all the extract-treated groups (200 mg, 400
mg and 600 mg) show diuretic activity. However, only 600 mg/kg body weight shows diuretic activity
when adjusted for furosemide (standard).
Table 4: Diuretic indices for administration of methanolic extract of Garcina kola in male Wistar rats
Adjusted for control Adjusted for furosemide

MEGCA MEGCB MEGCC MEGCA MEGCB MEGCC
1.82 2.24 4.24 0.44 0.55 1.04
Effect of administration of methanolic extract of Garcina kola on urine Na+ concentration in male
Wistar rats
There was a significant decrease in urine sodium (Na) concentration of MEGCOA (400 mg) –treated
group after 2 hours of urine collection when compared with control. However, administration of methanolic
extract of G. cola also led to significant increase in urine sodium concentration when compared with
furosemide-treated groups. There was significant increase in furosemide-treated groups and extract-treated
groups after 4 hours of urine collection compared with control. Sodium urine concentration significantly
increases in extract-treated groups compared with furosemide groups after 4 hours of urine collection. After
6 hours of urine collection, the urine Na concentration significantly increases in furosemide and extract-
treated groups. However, a decreased was observed in urine sodium concentration of 200mg and 400mg
compared with furosemide-treated groups.
60
Table 5: Effect of administration of methanolic extract of Garcina kola on urine Na+ concentration
in male Wistar rats
Urine Na+ Conc. (mEq/L) (2 Urine Na+ Conc. (mEq/L) Urine Na+ Conc. (mEq/L)
HOURS) (4HOURS) (6 HOURS)
70.05 ± 2.5 45 ± 2.5 25.57±2.5
56.19 ± 3.1* 55.01 ± 3.1* 64.25±3.1*
66.2 ± 1.5# 68.76 ± 1.5*# 57.91 ±1.5
79.94 ± 3.2*# 61.99 ± 3.2*# 36.85±3.2*#
69.6 ± 4.0* 62.96 ± 4.0*# 61.13 ± 4.0*
FRD).
Effect of administration of methanolic extract of Garcina kola on urine Cl concentration in male Wistar
rats
There was a significant decrease in urine chloride concentration in the furosemide/extract-treated
groups (200 mg and 400 mg) after 2 hours of urine collection but no significant change in MEGCOC (600
mg) compared with control. However, MEGCOB (400 mg) and MEGCC (600 mg) were significantly
higher than furosemide-treated groups. No significant change in urine chloride concentration. After 6 hours
of urine collection there was significant decrease in urine chloride concentration compared with control
group. However, no significant alteration in urine chloride concentration in all the extract-treated groups
when compared with furosemide-treated group as shown in Table 6.
Table 6: Effect of administration of methanolic extract of Garcina kola on urine Cl concentration in

male Wistar rats
GROUPS Urine Cl- Conc. (mEq/L) Urine Cl- Conc. (mEq/L) Urine Cl- Conc.
(2 HOURS) (4HOURS) (mEq/L) (6 HOURS)
CONTROL 122.31 ± 5.2 173.72 ± 5.2 159.6 ±5.2
FRD 72.03 ± 8.5* 150.16 ± 8.5 123.16 ±8.5*
MEGCOA 68± 6.0* 152.49 ± 6.0 155.93 ±6.0
MEGCOB 98.32±10.0*# 144.35 ± 10.0 138.19±10.0
MEGCOC 121.75± 2.0 161.86 ± 2.0 137 ± 2.0*
FRD).
Effect of administration of methanolic extract of Garcina kola on urine K+ concentration in male Wistar
rats
The potassium urine concentration significantly increased in all the extract-treated groups compared
with control and furosemide-treated groups after 2 hours of urine collection. The urine potassium
concentration also significantly increased in all the extract-treated groups compared with control and
furosemide groups after 4 hours of urine collection. Furosemide-treated group and extract treated groups
significantly increased when compared with control groups. However, the urine potassium was significantly
higher in MEGCOC (600 mg)-treated group compared with furosemide group as shown in Table 7.
61
Table 7: Effect of administration of methanolic extract of Garcina kola on urine K+ concentration in

male Wistar rats
GROUPS Urine k+ Conc. (mEq/L) Urine k+ Conc. (mEq/L) Urine k+ Conc. (mEq/L)
(2 HOURS) (4HOURS) (6 HOURS)
CONTROL 11.16 ± 1.2 10.79 ± 1.2 11.27 ±1.2
FRD 11.33 ± 1.9* 11.39 ± 1.9 16.98 ±1.9*
MEGCOA 16.2± 1.5*# 17.61 ± 1.5*# 17.61 ±1.5*
MEGCOB 17.61±0.9*# 15.61 ± 0.9*# 18.61±0.9*
*# *#
MEGCOC 17.61± 2.0 18.61 ± 2.0 26.13 ± 2.0*#
Data were analysed by one-way ANOVA followed by Bonferroni post hoc test. (*p<0.05 vs. Control; #p<0.05 vs
FRD).
Effect of administration of methanolic extract of Garcina kola on serum K+, Na+ and Cl- in male Wistar
rats
Administration of methanolic extract of Garcinia Kola did not significantly alter the serum Na+
concentration compared with furosemide and control groups. In addition, the administration of MEGCO
did not alter serum potassium concentration except for MEGCOC-treated group when compared with
control and furosemide treated groups. Similarly, administration of MEGC at 400 mg and 600 mg/kg body
weight led to significant increase in serum Cl- level compared with control and furosemide-treated groups,
whereas 200mg/kg body weight did not alter the serum Cl- level when compared with control but
significantly increased when compared with furosemide-treated groups.
Table 8: Effect of administration of methanolic extract of Garcina kola on serum K+, Na+ and Cl- in
male Wistar rats
GROUPS Serum K+ Conc. Serum Na+ Conc. Serum Cl- Conc.

(mmol/l) (mmol/l) (mmol/l)
CONTROL 4.24± 0.12 84.79± 4.2 69.23± 1.5
FRD 3.03 ± 0.5 76.89 ± 8.5 54.82 ± 3.5*
MEGCOA 3.63± 0.7 80.25± 6.0 71.68± 2.0*#
MEGCOB 4.49± 0.2 79.66± 10.0 93.50± 1.2*#
MEGCOC 4.70± 0.1*# 81.15± 2.0 83.89± 2.0*#
FRD).
Effect of administration of methanolic extract of Garcina kola on serum uric acid, urea and creatinine
concentration in male Wistar rats
Serum uric acid has been documented as a pro-inflammatory biomarker. The administration of
MEGCO (600 mg/kg) did not alter the serum uric acid concentration except in MEGCOC-treated group
where a significant increase was observed with control and furosemide groups. In addition, administration
of MEGCO significantly increased serum urea concentration in all the extract-treated and furosemide
groups compared with control. However, serum urea level of MEGCOC (600 mg)-treated group is
significantly higher compared with furosemide-treated groups. Likewise, administration of MEGCO did
62
not significantly altered the serum creatinine level except at 600 mg-treated groups that significantly
increased when compared with furosemide and control groups as shown in table 9.
Table 9: Effect of administration of methanolic extract of Garcina kola on serum uric acid, urea and
creatinine concentration in male Wistar rats
GROUPS Serum Uric acid Serum Urea Conc. Serum Creatinine Conc.
Conc. (mg/dl) (mmol/l) (µmol/l)
CONTROL 6.47± 0.3 1.33± 0.16 35.75± 5.0
FRD 4.56 ± 0.8 3.63 ± 0.3* 36.33 ± 3.0
MEGCOA 4.70± 0.7 4.09 ± 0.56* 46.63± 4.0
*
MEGCOB 5.40± 0.2 4.70± 0.2 46.82± 3.2
MEGCOC 7.28± 0.1*# 4.79± 0.1*# 50.69± 0.9*#
FRD).
Effect of administration of methanolic extract of Garcina kola on hepatic AST and ALT activities in
male Wistar rats
Administration of MEGCO did not significantly altered hepatic AST activity when compared with
control and furosemide treated groups. In addition, methanolic extract of Garcina cola significantly
increased hepatic ALT activity at 400 mg and 600 mg/kg when compared with furosemide-treated group.
However, the extract-treated groups were not significantly different from control except 600 mg-treated
group that was significantly higher than control group.
Table 10: Effect of administration of methanolic extract of Garcina kola on hepatic AST and ALT
activities in male Wistar rats
GROUPS AST (u/g prot.) ALT (u/g prot.)

CONTROL 9.58 ± 0.90 4.30 ± 0.50
FRD 7.52 ± 1.20 3.20 ± 0.40
MEGCOA 7.26 ± 0.70 3.04 ± 0.7
MEGCOB 9.11 ± 0.50 5.22 ± 0.20#
MEGCOC 9.5 ± 0.80 9.50 ± 0.95*#
FRD).
Discussion
This study examined the potential diuretic activity of Garcinia Kola using a Wistar rat model. This
assessment involved the comparison of the volume of urine excreted and circulating electrolytes of animal
that were administered with a methanolic extract of Garcinia Kola and animals that were administered with
Furosemide, a standard diuretic. This study demonstrated that an effective dose of Garcinia Kola induced
diuresis and altered electrolyte balance. Administration of 400 mg/kg and 600 mg/kg of the extract
significantly increased urine volume after at 6 hours. The diuretic indices were tested by the Lipchitz’s
method and the tests demonstrated that all the extract-treated groups (200 mg/kg, 400 mg/kg and 600
63
mg/kg) showed diuretic activity. However, the diuretic index of 600 mg/kg was higher (1.04) when adjusted
for furosemide (standard). The results also show that 400 mg/kg and 600mg/kg of the extract significantly
increased the concentration of Na+ and K+ in the urine. The 600 mg/kg concentration of the extract altered
the Cl- concentration in an inverse manner, thereby significantly reducing its concentration in the urine.
Furthermore, 400 mg/kg and 600 mg/kg of the extract did not alter the serum Na+ concentration but the 600
mg/kg significantly increased the serum K+ concentration. It was also observed that 400 mg/kg and 600
mg/kg of the extract also increased serum Cl- concentration. In addition, administration of 600 mg/kg of the
extract significantly increased serum uric acid, urea and creatinine. The 600 mg/kg concentration of the
extract also increased the alanine transaminase activity but did not alter aspartate transaminase activity in
the tissues.
Since the basic function of a diuretic is the removal of excess water from the body by increasing urine
formation and excretion, the marked increase in urine volume of animals treated with Garcinia Kola relative
to control underscores the efficacy of Garcinia Kola as a diuretic agent. The Lipchitz’s test has been used
over the years as a common test of the potency of diuretics (Mejo et al. 2020). Consequently, this study
employed Lipchitz’s test and the results demonstrated that all extract treated groups showed diuretic
activity. However, in order to emphasize the efficacy of the extract as a diuretic the Lipchitz’s value was
calculated while adjusting for furosemide (Standard diuretic). It was observed that the group treated with
600 mg/kg of the extract was higher (1.04) when adjusted for furosemide (Standard diuretic). Although the
presence of the bioflavonoid group in Garcinia Kola gives it several pharmacokinetic advantages like the
survival of first pass metabolism which inactivates most monomeric flavonoids, (Olaleye et al. 2000), the
consequent increase in bioavailability that comes from an increase in dose may be the reason for the dose
dependent effect.
In this study the change in urine volume was not associated with body weight change. However, there
was a significant increase in the kidney weight in groups treated with higher concentrations of the extract
(400 mg/kg, 600 mg/kg). Relative organ weight has been indicted as an index of the toxic effects of a
chemical compound (Quadri et al. 2019). Therefore, the increase in kidney weight could imply renal
hypertrophy, which is an indication of renal toxicity. This is consistent with recent study by Quadri et al.
that suggested that higher doses of kolaviron (active substance in Garcinia kola) caused derangement in
biomarkers of renal function urine volume, serum creatinine, urinary albumin/protein (Quadri et al. 2019).
One of the salient attributes of diuretics is that they reduce reabsorption of sodium and chloride at different
parts of the nephron, as a result increasing urinary sodium and water losses (Hunt et al. 2009, Dickstein et
al. 2010). The excretion of sodium in the urine (natriuresis) which was observed in the groups treated by
the extract underscores the natriuretic potential of of Garcinia Kola. Therefore, this study suggests that
effective dose of the extract can be an adjuvant treatment for hypernatremia and hypertension and this
supports previous studies by (Adaramoye, 2012, Akomolafe et al. 2017). Bolstering this assertion is the
observation of the increase in the urine potassium concentration of groups treated with the extract compared
to control. Studies have also shown that the risk of developing hypertension increases with reduce urine
potassium excretion (Quadri et al. 2019). Therefore, this present study suggests that the methanolic extract
of Garcinia Kola causes diuresis, natriuresis and kaliuresis.
Garcinia Kola did not affect the serum sodium concentration and this observation corroborates a past
study by (Agada and Braide, 2009). Likewise, the treatment only affected serum potassium concentration
in groups that were treated with 600mg/kg of the extract. There was a significant decrease in serum
potassium concentration compared to the control and furosemide groups, which implies that administration
of 600 mg/kg of the extract causes potassium-dependent electrolyte depletion. In addition, the serum
chloride concentration was significantly increased in all the extract treated groups compared with control
and furosemide groups, which is inconsonance with earlier studies (Agada and Braide, 2009). The
administration of Garcinia Kola also altered serum uric acid concentration at 600 mg/kg. Uric acid has been
earlier documented as a biomarker of proinflammatory response (Camilo et al. 2020). The present study
indicates that in addition to diuresis, natriuresis and kaliuresis, Garcinia Kola also promotes
proinflammatory responses at a dose 600 mg/kg. However, there was no significant change in serum uric
acid concentration at 200 mg/kg and 400 mg/kg. Although serum urea was significantly increased in all the
64
treated groups compared to control, creatinine was only higher in the groups treated with 600 mg/kg of the
extract compared to control. This suggests that the administration of Garcinia Kola could predispose the
experimental animals to renal toxicity. Previous studies have used increased urea levels to indicate renal
toxicity, this is because it is water soluble and an increase in the serum levels of urea indicates that the
capacity of the kidneys to expel this substance has been reduced (Meotti et al. 2003).
ALT (Alanine Transaminase) and AST (Aspartate Transaminase) are liver function markers that
indicate tissue or cellular toxicity (Meotti et al. 2003, Saad et al. 2018); Administration of Garcinia Kola
increased hepatic ALT at 400 mg/kg and 600 mg/kg and as a consequence, at these doses the animals are
predisposed to tissue toxicity. This could be due to the fact that ALT concentration is higher in the liver of
rats while AST can be found in a myriad of tissues, although AST concentrations are usually higher in
muscular tissues and the liver (Uko et al. 2001). Therefore, the high ALT levels could be an indication of
hepatotoxicity in groups treated with higher concentration of the extract. In addition, at 200 mg/kg there
was no alteration in the AST and ALT activities which imply the non-toxicity of lower doses to the liver
and this is consonant with a previous study by Kalu et al. (Kalu et al. 2016). Therefore, this study suggests
that the apparent toxicity of the methanolic extract of Garcinia Kola is dose dependent. The findings of this
study suggest that Garcinia Kola can be employed as a potential diuretic agent and can serve as adjuvant
therapy for conditions like hypernatremia, hyperkalemia, and hypertension.
Conclusion
In conclusion, the effective dose (600 mg/kg) of the methanolic extract of Garcinia Kola caused
diuresis, natriuresis and kaliuresis but put the animals at risk of renal toxicity and electrolyte imbalance.
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S. O. Olubodun et al.
0795-8080/2021 $10.00 + 0.00

Biokemistri
BKR 2020066/33106
Biochemical changes in rats exposed to crude oil and the

antioxidant role of Allanblackia floribunda stem-bark
Stella O. Olubodun*, Dele K. Fayemi and Ododo A. Osagie
Department of Medical Biochemistry, University of Benin, PMB 1154, Benin City, Nigeria.
Emails: stella.olubodun@uniben.edu, delefayemi1@gmail.com, augustine.osagie@uniben.edu

*
Correspondence: stella.olubodun@uniben.edu, +234 8023411948.
(Received November 14, 2020; Accepted March 8, 2021)
ABSTRACT: The role of Allanblackia floribunda stem-bark on oxidative stress and biochemical changes induced by
crude oil were investigated in rats. Thirty albino rats of both sexes, were randomly grouped into five with six rats in
each group. A group which served as normal control had no treatment, a second group was orally administered crude
oil at a dose of 5 ml/kg body weight, every other day for 28 days, a third group received 50 mg/kg A. floribunda stem-
bark extracts, another two groups were orally administered crude oil at a dose of 5 ml/kg body weight and received A.
floribunda stem-bark extracts (25 and 50 mg/kg) using oral dosing needle, every other day for 28 days. The results
showed that administration of 5.0 (ml/kg bw), crude oil resulted in a significant increase in serum malondialdehyde
(MDA) levels, and induced significant alterations in the activities of plasma superoxide dismutase (SOD), peroxidase
(POX) and catalase (CAT) in the rats. A significant increase was also observed in the activities of alkaline
phosphatases (ALP), alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH)
and increase the level of uric acid and total protein. The biochemical changes in antioxidant enzymes and other marker
enzymes may be due to oxidative stress and/or adaptive responses.
The rats that were simultaneously treated with crude oil and A. floribunda extracts however, maintained relatively no
significant (p>0.05) biochemical changes in the serum and hepatic cells when compared with the control. The non-
significant (p>0.05) changes recorded may be due to the antioxidant role of A. floribunda. The results shows that crude
oil induces oxidative stress and biochemical changes in serum and liver in the rats. The crude oil treated rats needle-
dosed with A. floribunda showed the probable therapeutic and antioxidative role of A. floribunda in crude oil oxidative
stress.
Keywords: Antioxidant, Biochemical changes, Crude oil, Allanblackia floribunda, Oxidative stress
Introduction
Normal metabolic cells, tissues, and organs produce activated chemical species known as reactive
oxygen species (ROS), an array of highly reactive molecules produced in enzymatic or non-enzymatic
reactions through sequential reductions of oxygen (1). Reactive oxygen species (ROS) are responsible for
toxic effects in the body through various tissue damages. The ability to maintain the production and
degradation of generated ROS is contained in the cell. However, when excess generation or excess
67
degradation of ROS occurs, it may lead to abnormal oxidative damage (1, 2). Unfavourable conditions such
as environmental/industrial toxicant-induced ROS could overwhelm the antioxidant defenses, leading to
increased lipid peroxidation and oxidative damage (3). High concentration of ROS can result in non-
controlled oxidation in cells, which is referred to as oxidative stress. Oxidative stress is defined as a serious
imbalance between the production of ROS and antioxidant defense and this situation (ROS-attack) can
cause damage to cellular macromolecules, including proteins (protein oxidation), membrane lipids (lipid
peroxidation), carbohydrates and DNA (4, 5).
Crude oils contain hydrocarbons, some heavy metals and other chemicals (6, 7). It is the major form
of income for the Nigerian economy and is produced in the Niger-Delta region where we have recurrent oil
spills. These spills pollute the environment and pose treats to both aquatic and terrestrial organisms (5, 8).
Heavy metals as well as crude oil poisoning has been reported to produce ROS and other free radicals which
induce oxidative stress and peroxidation of lipids when plants and animals are exposed to them (1, 6, 7).
Various researchers have reported increased incidence of organ damage in both chemical and environmental
stresses (2, 9).
It has been estimated that about three million lives may be saved each year when plants in form of
fruits, vegetables, spices and herbs are increased in diet. This they proposed will help overcome and/or
ameliorate toxic effects of contaminants/pollutants (10).
Nigeria, like other Africa countries, is blessed with various arrays of medicinal plants. Majority of the
people of Nigeria do not use these medicinal plants even though some people do rendering most of these
plants under-utilized. Diverse plant species possesses nutritional and phytochemical compounds which are
important in maintaining general well-being. They possess antioxidative potentials and are able to prevent
or reduce damages such as lipid peroxidation, oxidative damage to membrane, etc (11).
Allanblackia floribunda Oliv is one of a plant species of the nine species of the genus Allanblackia
and belongs to the family, Clusiaceae. It is commonly known as tallow tree in English Language. It is found
in a wide range of habitats and distributed in moist evergreen tropical rainforests across Africa. A.
floribunda is a tree commonly seen in the Niger-Delta Region of Nigeria, especially in abandoned forests
(acidic soils) with rainfall as high as 2400 mm. It is a seasonal fruit found during the rainy season in some
countries but are available all seasons in Nigeria. It is an indigenous fruit tree that has been recently
recognised as containing valuable edible oil, used for the manufacture of margarine, soap, chocolate,
ointment and food products. As a medicine, the pounded bark, roots, leaves and/or seed oils is applied as
herbal remedy in treatment of various illnesses (12, 13). The medicinal values of the plant lies in their
phytochemicals, which includes polyphenols, flavonoids, saponins, tannins and glycosides, important for
antioxidant, anti-inflammatory and analgesic activities (14, 15).
Some researchers reported the free-radical scavenging activity and anti-inflammatory property of A.
floribunda (16). Ayoola et al (16) also reported that the leaves in A. floribunda have hypoglycemic activity
in alloxan-induced diabetic rats. They inferred that the hypoglycemic activity may be due to the ability of
Allanblackia antioxidant molecules such as flavonoids, tannins to reduce cell defense against oxidative
stress through the free radical scavenging effect of the plant extracts.
Crude oil contamination has been on the increase in the past decade. The need to investigate the
impacts of crude oil on living systems and their probable toxicity can therefore not be over emphasized.
Also, since the growing focus to follow systematic research methodology to evaluate scientific basis for
traditional herbal medicines claimed to possess antioxidant, anti-inflammatory and analgesic activities, is
on the increase, this study is designed to evaluate the possible therapeutic and antioxidative effects of A.
floribunda on crude oil oxidative stress in rats.
Study area
The experiment was carried out at the Department of Medical Biochemistry, School of Basic Medical
Sciences, University of Benin, Benin City, Edo State, Nigeria (Latitude 6° 23’ 44”; Longitude 5° 36’ 49”;
Altitude 360 feet).
68
Collection of Crude oil, Plant Materials, Feeds and Animals

The crude oil was obtained from Warri Refinery and Petrochemical Company, Nigeria. Albino rats
(Wistar strain) were obtained from the Department of Animal Science, Faculty of Agriculture, University
of Benin, Edo State, Nigeria and rat pellets was purchased from vital feeds Nigeria. The fresh stem bark of
A. floribunda was collected from a forest area in Edo State, Nigeria, by herbal practitioner. The plant was
identified and authenticated by a botanist in the Department of Plant Biology and Biotechnology, University
of Benin, Nigeria and a voucher specimen deposited in the Herbarium of the Department for reference.
Preparation of Crude oil, Plant Materials and Animals

The stem bark was washed, dried at room temperature, macerated and sieved through a micro pore
sieve. The macerated form of A. floribunda stem bark was soaked in ethanol for 72 hours with occasional
stirring. The extracts were filtered using double layered muslin cloth and the filtrate was concentrated to
dryness with a rotary evaporator at reduced pressure. The crude extract was stored in a refrigerator until
required.
Thirty albino rats with a mean weight 170±5 g were maintained in the Laboratory Animal Unit of the
Department of Biochemistry, Faculty of Life Sciences, University of Benin. They were fed with standard
diet and water ad libitum. They were acclimatize for two weeks. The study was approved by the ethics
committee of the University of Benin, Nigeria. The rats were handled in accordance with the guidelines on
the care and wellbeing of research animals.
Phytochemical Screening of the Ethanol Extract

The phytochemical screening of the A. floribunda stem bark extract was carried out using standard
procedures (17).
Experimental Conditions
Albino Wistar rats were randomly divided into five groups of six rats each (n=6).
Group I served as Control and received distilled water ad libitum.
Group II received Crude oil (5ml/kg BW) by gavage every other day for 28 days of treatment.
Group III received A. floribunda extract (200mg/kg BW) by gavage for 28 days of treatment.
Group IV and Group VI received Crude oil (5 ml/kg BW) every other day with 200mg/kg A. floribunda
and 400mg/kg A. floribunda extract combination respectively by gavage administration for 28 days.
Water was provided ad libitium to all groups. The experiment lasted for twenty eight (28) days and
rats sacrificed on the twenty ninth (29th) day. The blood and liver were recovered for analyses.
Collection of Blood and Liver Samples

After treatment, the rats were sacrificed by cervical decapitation on the 29th day, 24 hours after the last
treatment. The blood samples were collected into labeled sample tubes and allowed to clot for 30 min at
room temperature. Serum was separated from the blood by centrifuging at 3000 rpm for 15 min. The
supernatant (serum) was stored in sterile vial and kept in the freezer for biochemical analyses. The liver
was excised and homogenised with 5 ml (50 mM, pH 7.4) phosphate buffer to give a 20% (w/v) liver
homogenate. The homogenate was centrifuged at 3000 rpm for 20 min and the supernatant obtained for
biochemical analyses.
Biochemical Analyses
The serum sample prepared from the blood was used to determine alkaline phosphatase (ALP), alanine
aminotransferase (ALT), aspartate aminotransferase (AST), uric acid (UA) and lactate dehydrogenase
(LDH) activity. The liver function markers and LDH in the serum were measured using Randox test kit as
specified by the manufacturers, Randox laboratory ltd (Antrim, UK, BT29 4QY). While the supernatant
from liver homogenate was used to determine oxidative stress markers.
69
The total proteins was determined by the method described by (18) while superoxide dismutase (SOD)
activity was assayed according to the method described by (19) and was expressed as units/mg tissue
weight. One unit of enzyme was defined as the amount of the enzyme required for 50% inhibition of
oxidation of epinephrine to adrenochrome in one minute. Catalase (CAT) specific activity was determined
according to the method of (20) and was expressed as units/g wet tissue. Dichromate in acetic acid is
reduced to chromic acetate when heated in the presence of H2O2 with the formation of perchromic acid as
an unstable intermediate. The amount of perchromic acid formed was taken as an activity unit. Peroxidase
(POX) activity was determined using the method of (21) and was expressed as units/mg protein. The activity
unit of the enzyme was defined as amount of purpurogallin formed in the oxidation of pyrogallol to
purpurogallin by peroxidase at 200C. Malondialdehyde (MDA) levels was determined by reaction with
thiobarbituric acid (TBA) and used as a lipid peroxidation marker (22). Uric acid was determined by
enzymatic colorimetric method (23). All experiments were performed in five replicates.
Statistical evaluations of all data were done using one-way analysis of variance (ANOVA) to test for
differences in groups. All analysed results represented mean ± standard error of mean (SEM) and Duncan’s
multiple comparisons test was used to determine significant differences between means. Instat-Graphpad
software, San Diego, California, USA, was used for this analysis. A P value < 0.05 was considered
statistically significant.
Results and Discussion
The results of the phytochemical constituents of the ethanol stem bark extracts of A. floribunda is
shown in Table 1. The phytochemical screening of the stem bark extract of A. floribunda revealed the
presence of tannins, flavonoids, saponins, glycosides, alkaloids, steroids and polyphenols (Table 1). This
result is comparable to earlier reports of the phytochemistry of A. floribunda and other plant species by
various researchers (9, 24, 25 & 26) who indicated the presences of various phytochemicals. However, we
did not observe the presence of anthraquinone in our study. While Manikandan et al (27) reported that
alkaloids and flavonoids protect cells by acting as powerful antioxidants which prevent or repair damage
done to red cells by free radicals or highly reactive oxygen species, others reported that flavonoids possesses
antioxidant, anti-inflammatory, anticancer and antimicrobial activity (9, 12, 24, 25, 26).
Tannins are reported to have some interactions with protein to give an effect which makes it important
for management of inflamed or ulcerated cells. The phytochemical constituents present in the extract and
the quantity of these constituents may be responsible for the antioxidant activities of the extracts and may
also be responsible for their acclaimed medicinal uses.
The presence of these phytochemical constituents may have reduced the toxic effects of crude oil.
The presence of the phytochemical constituents in the extract of A. floribunda therefore, supports its
use and pharmacological importance in the treatment of ailments.
Crude oil and its products have become an essential constituent of human life due to their industrial
and domestic importance. However, clinical and experimental studies have shown that exposure to
petroleum hydrocarbon and other constituents is a risk factor for oxidative stress and degenerative diseases
(9, 28).
70
Table 1. Phytochemical constituents of the ethanol extracts of A. floribunda
Phytochemicals Glycosides Tannins Saponins Flavonoids Alkaloids Sterols Polyphenols
Qualitative +++ + ++ + ++ ++ ++
estimates
(++) = Present in moderate concentrations; (+) = Present in low concentrations
Table 2 shows the effects of crude oil and A. floribunda on body weight in rats treated by using oral
dosing needle with 5ml/kg crude oil and/or treatment with different doses of A. floribunda. While the rats
in groups 2 significantly decreased in the percentage change in body weight when compared with control
and the treatment groups, the rats in the other groups 4 (5ml/kg crude oil + 200mg/kg A. floribunda) and 5
(5ml/kg crude oil + 400mg/kg A. floribunda) recorded non-significant (P>0.05) increase in percentage
change in body weight when compared with the control (groups 1). While groups 2 rats recorded 7%
decrease in percentage change in body weight, groups 3, 4, and 5 recorded an increase of 25, 21 and 16%
respectively when compared with control. Body weight loss is an indication of an abnormal condition and
may arise from decreased food intake due to the immobility accompanying stress, or reduced absorption of
food (29). The body weight were significantly increased in the rats given the extract and extract with 5ml/kg
crude oil when compared to untreated crude oil rats. The extract may have inhibit the loss in body weight
in rats by increasing food intake through attenuation of abnormal condition of oxidative stress or improving
the absorption capacity of the intestine (29).
Since crude oil induces lipid peroxidation as a result of elevated production of reactive oxygen species
(ROS) (30, 31), the observed decrease (P<0.05) in body weight in group 2 (crude oil treated rats) may be
due to crude oil toxicity. Crude oil may have impaired normal growth and development through a variety
of mechanisms. This research is in agreement with other researchers who reported weight loss in animals
exposed to crude oil and other contaminants (9, 30 & 31). The non-significant alteration in body weight
with the combined treatment may be due to the antioxidant protective role of A. floribunda.
Table 2: Effects of ethanol extracts of A. floribunda stem bark and crude oil on the body weight in
albino Wistar rats
% Change in Body
Group/Assay (g) Initial Body Weight Final Body Weight
Weight (%∆BWt.)
Group 1: Control 165.52±2.17 205.40 ± 3.89a 24
Group 2: Co only 174.50±3.38 162.89.± 4.20b -7
Group 3: E only (200mg/kg) 166.20±5.00 207.46 ± 5.06a 25
Group 4: Treated(200mg/kg) 163.44±5.30 198.16 ± 3.17a 21
a
Group 5: Treated(400mg/kg) 172.46±3.90 200.52 ± 5.07 16
Results are expressed as means ± SEM of five (5) replicates. Body weight is expressed as g while change
in body weight is expressed as %.
The results of total protein concentration and the serum marker enzymes are shown in Table 3. The
concentration of total protein in group 2 decreased significantly (p<0.05) when compared with control and
the other groups (p<0.05). The result also recorded increased activity of ALP, AST, ALT and LDH in crude
oil exposed rats when compared with control and other groups (Table 3).
The administration of crude oil to the rats recorded significant increase in marker enzymes (ALT, AST,
ALP and LDH) in the serum after 28 days of alternate treatment with dosing needle. Administration of the
A. floribunda stem bark extract of two different doses, provided significant antioxidative protection,
71
resulting in decreased elevated serum activities of marker enzymes (Table 3 and Figure 1) when compared
to crude oil untreated rats. The toxicity induced by crude oil in the liver tissues was assessed by measuring
the antioxidant defense enzymes like SOD, POX, CAT and MDA levels (Table 4). Table 4 recorded
reduction in the activities of SOD and POX but increase in CAT and MDA when compared with the control
group. Treatment of the rats with A. floribunda stem bark extract to group 4 and group 5 reduced the toxicity
of crude oil.
Table 3: Effects of crude oil and A. floribunda extracts on marker enzymes and total proteins in albino
rats
Group Group 1 Group 2 Group 3 Group 4 Group 5

/Assay Control Co only E only Co+E200mg/kg Co+E400mg/kg
Protein 54.6±2.9a 46.0+1.6b 57.6+2.0a 54.7+2.3a 58.6+2.4a
AST 221.5±10.82 242.14±8.10 220.5±8.5 222.18±8.09 220.46±10
ALT 46.37±1.52 63.16±6.7 46.17±3.12 49.33±5.23 45.83±1.28
ALP 260.56±19.1a 332.4±15b 249.11±11.81a 272.14±19.7a 301.9±12.44a
LDH 143.64±6.5a 172.65±9.5b 143.77±6.7a 144.78±6.8a 142. 94±7.6a
Results are expressed as means ± SEM of determinations from five samples. ALT = Alanine aminotransferase,
AST=Aspartate aminotransferase, ALT=alkaline phosphatase and LDH = Lactate Dehydrogenase, were expressed as
U/L. Means carrying different notations are statistically different at p< 0.05.
400
b G ro u p 1
M a r k e r E n z y m e s ( U /L )
a G ro u p 2
300 a
a
b
G ro u p 3
a
G ro u p 4
200 b
G ro u p 5
100 b
0
P
H
T
L
L
S
D
A
F ig . 1 : E f f e c t s o f c r u d e o i l a n d A . f lo r ib u n d a e x t r a c t s o n l iv e r m a r k e r e n z y m e s i n r a t s .E a c h b a r r e p r e s e n t s t h e m e a n s ± S E M f r o m
f iv e s a m p l e s . A L T = A la n in e a m in o t r a n s f e r a s e , A S T = A s p a r t a t e a m in o t r a n s f e r a s e , A L T = a lk a lin e p h o s p h a t a s e a n d L D H = L a c t a t e
D e h y d r o g e n a s e , w e r e e x p r e s s e d a s U /L . B a r s c a r r y i n g d i f f e r e n t n o t a t i o n s a r e s t a t i s t ic a l ly d if f e r e n t a t p < 0 .0 5 u s in g I n s t a t
G ra ph pa d.
The increase recorded in serum ALT, AST, ALP and LDH may be an indication of liver injury
resulting from a loss of hepatocytes membrane integrity (26). This increase in serum marker parameters
may be due to the liver damage resulting from the liver inflammation and disruption induced by crude oil
toxicity (9). In this study, the plant extract prevented crude oil-induced increase of ALT, AST, ALP and
LDH. It is possible, that the extract protects cell membranes or counteracts the deleterious effects of crude
oil. These results could suggest the presence of some compounds (tannins, alkaloids) which may inhibit the
cytochrome P450 responsible for metabolism or boost the gene responsible for the regeneration of liver cells,
or scavenged free radicals. The administration of the extract inhibited the increase in serum markers at both
doses though non-significantly when compared with the control values. These results suggest that the plant
extract could protect the liver against inflammation and disorders.
Crude oil, A. floribunda stem bark extract and a combination of both, recorded significant (p<0.05)
decrease and non-significant increases in total protein respectively. This could be due to the oxidation of
proteins by crude oil-induced free radicals. The oxidation of these proteins could cause protein denaturation
72
thus reduced their liver and blood concentrations. The treatment with the extract maintained total protein
to within the normal control values. This may be due to its antioxidant character.
Table 4 recorded the effects of crude oil and A. floribunda on oxidative stress parameters in rats treated
by using oral dosing needle with 5ml/kg crude oil and/or treatment with different doses of A. floribunda
stem bark extract for 28 days. Significant variations were observed in oxidative stress parameters
[superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), peroxidase (POX)] and Uric acid
(UA) (Table 4). The rats exposed to crude oil induced significant (p<0.05) reduction in the activity of SOD
and POX but activated significant (p<0.05) increase in CAT, MDA and UA, the groups treated with A.
floribunda stem bark extract relatively maintained a non-significant alteration from the control values in a
dose dependent manner.
Table 4: Effects of crude oil and A. floribunda extracts on oxidative stress markers in albino rats
Group Group 1 Group 2 Group 3 Group 4 Group 5

/Assay Control Co only E only Co+E25mg/kg Co+E50mg/kg
POX 3.50±0.35a 1.38±0.33b 2.86±0.15a 2. 94±0.45a 3. 01±0.25a
Catalase 3.54 ±0.47a 5.84±0.38b 3.23±0.21a 3.32±0.37a 4.15± 0.36a
SOD 4.94±0.25a 2.75±1.09b 4.04±0.50a 4.38 ±0.58a 4.60±0.86a
MDA 8.02±1.05a 15.82±1.25b 7. 94±1.02a 7.76±1.04a 7.89±0.58a
UA 3.61±0.27a 4.98±0.83b 3.86±0.47a 3.94±0.36a 4.01±0.84a
Results are expressed as means ± SEM of determinations from five samples. Catalase activity is expressed as unit/g
wet tissue. SOD = Superoxide dismutase activity is expressed as unit/mg protein. POX = Peroxidase activity is
expressed as unit/mg protein. MDA= Malondialdehyde level is presented in mole MDA/g tissue.
Antioxidant defense systems under physiological condition may induce a slight oxidative stress as a
compensatory response against ROS and thus protect the organisms from oxidative damage. The activity
of antioxidant may be increased or inhibited under chemical stress depending on intensity and duration of
the applied stress as well as susceptibility of the exposed species (32).
Catalase (CAT), SOD and POX are important in protecting cells against oxidative stress and damage.
The observed significant decrease in the activities of SOD and POX could be due to their involvement in
antioxidative functions which may have resulted in induced formation of pro-oxidants and relative decrease
in antioxidants status of cells, while the increase in CAT may be a physiological adaptation for the
elimination of generated ROS. Other researchers also reported decrease in antioxidant enzymes (1, 5, 32 &
33). This result is in agreement with the report of other researchers who used other types of treatments (34,
35). Since oxidative stress due to the toxicants is usually indicated by increased levels of products of
oxidative damage (MDA) and subsequent increase in defence enzymes (POX, SOD and CAT) in response
to the stress (36) or decrease due to overwhelming effect of the pollutants (37), we may say that the decrease
in the activity of SOD is due to the overwhelming effect of the toxicants from the crude oil where the system
used the SOD to detoxify the resulting superoxide radicals. The non-significant reduction or relative
maintenance of POX activity in the A. floribunda treated group is suggestive of antioxidative, and hepato-
protective ability of the stem bark extract (38).
The increase observed in the levels of malondialdehyde (MDA) [lipid peroxidation product] confirms
the induction of oxidative stress in the rats exposed to crude oil only (32, 33).
The significant increase recorded in uric acid level of group 2 rats compared to the control and the
other groups treated with the plant extracts, shows that crude oil have toxic effect on uric acid level in
blood. The reason may be over production by liver, poor elimination by the kidneys or as non-enzymatic
antioxidant defense against excess ROS produced as a result of oxidative stress (8, 39). A. floribunda
recorded good results in combating crude oil-induced toxicity in albino rats by reducing lipid peroxidation
and normalize the levels of uric acid in the serum of rats treated with the extracts. Other researchers also
73
recorded increased uric acid levels during intoxication and ameliorative effects of other plant species (8,
39)
When hepatocytes membranes are allowed to be damaged by ROS, as observed by significant variation
in the oxidative stress parameters in this study, a variety of liver transaminases are released into the blood
from the cytosol (11). This indicates hepatocytes damage even without evident hepatic impairment. The
elevated levels of AST and ALT are indicative of cellular leakage and loss of functional integrity of cellular
membrane and liver. Increased activity of AST has been reported in CCl4-intoxicated experimental animals
(40). This increase may be due to abnormal dynamic properties of cellular membranes following exposure
to hydrocarbon fractions present in crude oil. Metabolisms of aliphatic and aromatic hydrocarbons which
are major constituents of crude oil as well as other environmental contaminants have resulted in changes in
cell membrane due to ROS (32, 40).
Uboh et al (40) also reported that serum ALT activity increase as a result of liver injury in patients
developing severe hepatotoxicity. The ALP activities on the other hand are related to functioning of
hepatocytes, its increase in serum is due to increased synthesis in the presence of increased biliary pressure
(1). This could be attributed to damage to the structural integrity of the liver and possible necrotic lesions
in the hepatocytes (1). ALT might have leaked from damaged cells, due to increased permeability of the
hepatocellular membrane, or due to necrosis, indicating organ dysfunction (40). However, the close to
normal control levels of the marker enzymes in the treated groups affirm the antioxidant protective role of
the stem bark extracts on the hepatocytes. This is in agreement with the reports of Ujowundu et al (33) who
recorded the hepato-protective and antioxidant potentials of O. gratissimum and G. latifolium respectively
against petroleum-based products-induced hepatotoxicity in albino rats. The antioxidant role of A.
floribunda stem bark extract (26) may have prevented the induction of oxidative stress in the liver (1).
The significantly higher (p < 0.05) alkaline phosphatase (ALP) activity in the rats exposed to crude oil
when compared with control may imply damage in the liver cells, since the activity of this enzyme in the
serum is reported to be increased in liver damage (32, 40). Alkaline phosphatase is involved in the transport
of metabolites across the cell membranes, protein synthesis and synthesis of certain enzymes, secretory
activities and glycogen metabolism. The increase in ALP activity may not be unconnected with a
disturbance in the transport of metabolites or alteration in the synthesis of certain enzymes as in other
hepatotoxic conditions (32, 40).
The significant decrease in total protein in crude oil group may be attributed to decrease in synthetic
function of liver due to crude oil exposure. However, the non-significant decrease or near control values of
total protein concentrations in the treatment group may indicate ameliorative effects of the stem bark
extracts. This observation is in agreement with the studies of George et al (41), which recorded hepato-
protective potential of O. gratissimum and G. latifolium against ethanol-induced and CCl4-induced
hepatoxicity in albino rats respectively (11, 33).
Conclusion
Administration of A. floribunda to rats exposed to crude oil can prevent severe alterations of serum
marker enzymes and oxidative status. This study has demonstrated that treatment with A. floribunda stem
bark extract significantly attenuated the alterations induced by crude oil. Crude oil significantly increased
the levels of some serum marker enzymes (AST, ALP, ALT and LDH), and oxidative stress markers (CAT,
SOD, POX, UA) and increased the level of total protein. In the case of using the extract alone, there was
little alteration in the activity of these parameters. On the other hand, co-administration of crude oil with A.
floribunda stem bark extract-treated rats improved most of the biochemical parameters to within normal
levels. This may be due to the antioxidant properties of the stem bark. We can deduce that crude oil-induced
lipid peroxidation and oxidative stress observed in the albino rats, and treatment with A. floribunda stem
bark extract resulted in significant ameliorating effect. The antioxidant properties of the plant species
support the bioactive roles of its protective effects on crude oil toxicity. Further study should be undertaken
to strengthen the evidence for dietary antioxidants as modulators of the adverse effects caused by increased
chemical or environmental contamination.
74
Acknowledgments
Special thanks to laboratory staff of Medical Biochemistry, University of Benin, for their technical
assistance.
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76
R. A. Oyegoke & A. T. Oladiji
0795-8080/2021 $10.00 + 0.00

Biokemistri
BKR 2020093/33107
Safety evaluation of Dialium guineense fresh fruit pulp meal-

based diet
R. A. Oyegoke* and A. T. Oladiji
Department of Biochemistry, Faculty of Life Sciences, University of Ilorin, Ilorin, Kwara State, Nigeria
*Author to who correspondence should be addressed: rukayat.oyegoke@gmail.com; +2348062075089.
(Received December 18, 2020; Revised version received March 8, 2021; Accepted March 12, 2021)
ABSTRACT: Toxicological investigation of plant species used as food/drug requires the examination of a wide
array of assays including antioxidant parameters and tissues functional indices. This study investigated the safety of
the fruit pulp of Dialium guineense in rats by assaying for liver and kidney antioxidant parameters as well as some
selected functional indices. Thirty-six rats were assigned into 6 groups, A – F and maintained on basal diet, 5, 10,
20, 30 and 40% inclusion of Dialium guineense fruit pulp respectively for five weeks. The study revealed a non-
significant difference (p > 0.05) in the antioxidant parameters except for catalase in which a significant decrease (p
< 0.05) was observed at 30 and 40% in both tissues. For the aminotransferases, gamma glutamyl transferase,
globulin, total and conjugated bilirubin as well as the five electrolytes (Na+, K +, HCO32-, PO42- ,Cl-) assayed for, no
significant difference (p > 0.05) was observed. However, rats maintained on the diet at all supplementation levels
had a significant increase (p < 0.05) in the total protein, albumin and urea level except for creatinine. The
insignificant effects in most parameters suggest that the pulp might not interfere with normal functioning of the
biomolecules at the percentage inclusions.
Keywords: Dialium guineense, Antioxidant , Liver, Kidney, Inclusion
Introduction
Traditional medicine has a long history (Devi et al., 2016). It is the sum total of the knowledge, skills
and practices based on the theories, beliefs and experiences indigenous to different cultures. This can be
explicable or not and it is used in the maintenance of health, as well as in the prevention, diagnosis,
improvement or treatment of physical and mental illnesses (Devi et al., 2016). Medicinal plants form an
important component of the natural wealth of any country. The tropical rainforest of which Nigeria is a
part has been described by Sofowora (1982) as a reservoir of phytomedicines. These plants have
traditionally been used by Nigerians because, they are natural products, environmentally friendly, easily
available, cheap and curative than many orthodox medicines (Murray, 1995). They are preferred because
of the ills of conventional medications which include toxic effects on humans, resistance by man and
animals, Non availability/ Non accessibility, High cost of most of the drugs (Egharavba and Ikhatua,
2008).
77
In line with the interaction of food in management of diseases, phytomedicines are also now
preferred to be taken as food supplement instead of concoctions, decoctions etc. which goes in tandem
with the use of orthodox drugs. However, the use of it in either ways as food or as drug still requires
thorough safety evaluation because the toxicological level of most of them have not been established.
Toxicology is the study of the adverse physicochemical effects of chemical, physical or biological agents
on living organisms and the ecosystem. This includes the prevention and amelioration of such adverse
effects.
The effects on organisms can occur at multiple levels, including the molecular and the organ levels.
One of the most important assay required to determine the level of toxicity of a food or drug in the
biological system is the assay of the antioxidant defense system. Reactive oxygen species are highly
reactive, oxygen-containing molecules including the free radicals which cause biological damage known
as oxidative stress to the tissues and cells of the body (Ridnour et al., 2005). These molecules when
generated in the body system by introduction of toxins from the food or drug taken in are mopped by the
antioxidant molecules through the antioxidant defense system therefore, the need to certify the integrity of
this system.
Enzymatic defense system include the superoxide dismutase (SOD) and catalase (CAT) while the
gluthathione is non-enzymatic. SOD catalyze the dismutation of superoxide anions (O2-) generated in the
gastrointestinal tract to hydrogen peroxide (H2O2) and oxygen (O2) rendering the potentially harmful
superoxide anion less hazardous thereby protecting the mucosal and the epithelial cells. Catalase catalyzes
the reduction of H2O2 generated as a result of oxidative stress in the GI tract to water and oxygen using
either an iron or manganese cofactor (Chelikani et al., 2004). Gluthathione (GSH) is a major thiol based
non-enzymic antioxidant in living organism, which performs a key role in co-ordinating the innate
antioxidant defence mechanisms. GSH acts as ROS scavenger in the stomach and duodenum (Koevary,
2012).
Another in the series antioxidant defense system assay is the lipid peroxidation, which is a process
induced by free radicals leading to oxidative deterioration of polyunsaturated fatty acids. The lipid
peroxidation product, malondialdehyde, is commonly used as a measure of the oxidative stress in cells.
Lipid peroxidation, being a free radical reaction, occurs when the hydroxyl radicals, possibly oxygen,
react with the unsaturated lipids of the bio-membranes, resulting in the generation of lipid peroxide
radicals (ROO-), lipid hydroperoxide (ROOH) and fragmentation products such as malondialdehyde. This
aldehyde is a highly toxic molecule and is a marker of lipid peroxidation. Its interaction with the DNA
and proteins has often been referred to as potentially mutagenic and atherogenic (Marnett, 1999). Also of
toxicological relevance are the aminotransferases and the gamma glutamyl transferase enzymes. Alanine
aminotransferase (ALT) catalyzes the transamination reaction between L-alanine and α-ketoglutarate
(George et al., 1994), to form pyruvate and glutamate while aspartate aminotransferase (AST) catalyses
the transamination reaction between L-aspartate and α-ketoglutarate to form glutamate and oxaloacetate
at optimum pH of 7.4.
ALT is less abundant than AST in human tissues (Wilkinson, 1963), it is native to the cytosol,
though small amount have been found in the mitochondria of liver cells and also in the heart muscles
(Wilkinson, 1963; Bhargavan and Sreenivasan, 1965). AST is widely distributed in animal tissues with a
relatively high concentration in the heart muscle (Bhargava and Screenivasan, 1965).
The liver, kidney, pancreas, lung and spleen also contain considerable quantities of the enzyme
(Miller and Luthrade, 1990). It is found both in the cytosol and in the mitochondria of the cells (Boyd,
1960). Both enzymes are very important in the diagnosis of liver and kidney diseases caused by drug
toxicity or infection (Nelson and Cox, 2005). Another in this series is the gamma glutamyl transferase
enzyme which is a membrane localized enzyme. It catalyzes the transfer of amino acid from one peptide
to another amino acid or peptide (i.e. it acts as amino acid transferase) (Burtis and Ashwood, 2001).
Gamma glutamyl transferase plays a major role in glutathione metabolism and reabsorption of amino
acids from the glomerular filterate and from intestinal lumen (Kaplan and Pesce, 1996). The enzyme is
found in a number of tissues. It occurs mainly in the cells of the liver, kidney, pancreas and prostate. It is
78
also present in the plasma membrane of renal tubular cells, endoplasmic reticulum of the hepatocytes
(Murray et al., 2000) and seminal vesicles (Kohdaira et al., 1986).
Serum levels of gamma glutamyl transferase are commonly elevated in response to many drugs and
toxins (Ruppin et al., 1982). In the same vein, liver and kidney function tests are also very important
guage in assessing the function of these organs as well as determining if there are signs of toxicity. The
concentration of proteins, bilirubin and albumin in the serum can be used to ascertain the state of the liver
and different types of liver damage while the kidney function parameters include urea, creatinine, uric
acid and serum electrolytes like K+, Na+, PO42-, HCO32-, Cl- (Yakubu et al., 2003).
Dialium guineense (Wild) belongs to the family of Fabaceae. It is known as Velvet or Black
tamarind (English), Awin (Yoruba), Icheku (Igbo), Tsamiyar kurmi (Hausa) (Aiyeloja and Bello, 2006). It
is a woody plant that grows up to 15 m high in the rain forest region of West Africa (Okegbile and Taiwo,
1990). D. guineense is used as chewing stick (indigenous tooth brush) among Nigerian populace
(Akinpelu et al., 2011), also the stem bark as well as the leaves are also used as folklore remedies for the
treatment of infections such as diarrhoea, severe cough, bronchitis, wound, stomach aches, malaria fever,
jaundice, ulcer and haemorrhoids (Bero et al., 2009). The fruit pulp of the plant is claimed locally to have
anti-ulcerogenic potential (Aiyeloja and Bello, 2006) and this have been scientifically proven by Oyegoke
and Oladiji (2014). Some of the other scientifically validated activities of the plant leaves, stem bark and
fruit pulp include its analgesic and antibacterial activities, antioxidant and antimicrobial activities (Gideon
et al., 2013).
The upsurge in the use of this plant in the flora as herbal remedy for anti-ulcer efficacy and others
necessitate thorough scientific investigation in order to provide information on its safety and toxicity risk.
In the same vein, there is dearth of information on this plant especially the fruit pulp on its toxicological
implications in open scientific literature; therefore the need to provide information to fill this lacuna.
Therefore, the main objective of the study was to investigate the safety /toxicity risk of Dialium guineense
fruit pulp meal-based diet while the specific objectives evaluate the safety of the diet in rats as it relates to
antioxidant enzymes, protein and non- antioxidant enzymes as well as liver and kidney function indices.
Plant Material
The fruit pulp of Dialium guineense was purchased from Dawanu Market in Kano City, Nigeria. It
was authenticated in the Department of Plant Biology, University of Ilorin, Ilorin, Nigeria. A voucher
specimen (UIH1064) was deposited in the Herbarium of the Department.
Laboratory Animals
Thirty six albino rats (Rattus novergicus) of both sexes (167.09 ± 6.78g, 5-7weeks old) were
obtained from the Animal Holding Unit of the Department of Biochemistry, University of Ilorin, Ilorin,
Nigeria. They were kept in well-ventilated house conditions with free access to rat pellets (Premier Feed
Mills Company Limited, Ibadan) and tap water before the commencement of the experiment.
Ingredients for Feed formulation and their sources

Yellow maize seeds, cellulose (corn cob) and soybean were purchased from Oja Oba Market, Ilorin,
Nigeria. Sunola Refined soybean oil is a product of Kewalrams Nigeria Limited while the
vitamin/mineral mix, lysine and D-methionine were purchased from Rofiat Feeds Nigeria Limited.
Chemicals and Assay Kits

The chemicals and assay kits used in this study were of the finest qualities commercially available.
Albumin, bilirubin, urea, creatinine, alanine aminotransferase, aspartate aminotransferase and gamma
glutamyl transferase were purchased from Randox Laboratories Limited, United Kingdom. Other reagents
were purchased from British Drug Houses (BDH), Poole, England.
79
Preparation of Plant Material

Fresh fruit pulps of Dialium guineense were manually removed from the seed after expunging it
from the seed coat. They were then oven-dried at 40oC and pulverized in an electric blender (Super
Master Electrical Appliance Industries Co., Kyoto, Japan) and then stored for later use.
Composition of Diet
The Composition of diet is presented in Table 1.
Table 1: Composition of the diet (g/kg)
Ingredient Control Dialium guineense fruit pulp meal-based diet

(Basal diet)
5% 10% 20% 30% 40%
Corn Starch 506 480.70 455.40 404.80 354.20 303.60
D. guineense fruit pulp 25.30 50.60 101.20 151.80 202.40
Cellulose 40 40 40 40 40 40
Sucrose 100 100 100 100 100 100
Soybean 250 250 250 250 250 250
Soybean Oil 50 50 50 50 50 50
Vitamin/Mineral Mix 50 50 50 50 50 50
D-Methionine 4 4 4 4 4 4
* Vitamin/Mineral mix: Vitamin A 4,000,000 i.u; Vitamin D3, 800,000 i.u.; Tocopherols, 400 i.u; Vitamin K3 800mg , Folacin,
200mg; Thiamine, 600mg; Riboflavin 1,800mg; Niacin, 6000mg; Calcium pathothenate, 4 mg; Biotin, 8 mg; Manganese,
30,000mg, Zinc, 20,000mg; 8,000mg; Choline chloride 80,000mg; Copper, 2,000mg; Iodine, 480mg; Cobalt, 80 mg; Selenium,
40mg; BHT, 2,500mg. Anticaking agent, 6000mg.
Unit of diet composed – g/ kg.
Animal Grouping
36 rats were acclimatized for one week. They were kept in well-ventilated house conditions
Temperature: 22 ±30C; photoperiod: 12 hrs light and 12 h dark; humidity: 40–45%) with free access to rat
pellets (Premier feed mills Company Limited, Ibadan) and tap water before the commencement of the
experiment. After a week of acclimatization, the animals were grouped into six as follows:
A – Control group fed on Control (Basal) Diet

B - Group fed on 5% Dialium guineense fruit pulp meal-based diet
C - Group fed on 10% Dialium guineense fruit pulp meal-based diet
D - Group fed on 20% Dialium guineense fruit pulp meal-based diet
E - Group fed on 30% Dialium guineense fruit pulp meal-based diet
F - Group fed on 40% Dialium guineense fruit pulp meal-based diet
The animals were maintained on their respective diets for period of five weeks before sacrifice.
Preparation of serum and tissue homogenates

The procedures described by Akanji and Ngaha (1989) were used to collect the blood while the
tissues (Liver and kidney) homogenates as well as serum were prepared according to the procedure
described by Yakubu et al (2003).
Procedures for determination of various biochemical parameters for safety evaluation study
Determination of the activities of antioxidant enzymes and protein concentration
Superoxide dismutase activity was determined by the method described by Mistra and Fridovich
(1972). Catalase was assayed according to the procedure described by Beers and Sizer (1952). Reduced
80
glutathione concentration was determined using the procedure of Ellman (1959) while the concentration
of malondialdehyde (MDA) was determined using the method of Buege and Aust (1978).
Determination of the activities of the Aminotransferases and Gamma glutamyl transferases

The procedure described by Reitman and Frankel (1957) was used to assay for the activity of
aspartate aminotransferase and alanine aminotransferase while the method described by Szasz (1969) was
used to assay for the activity of gamma glutamyl transferase.
Determination of liver and kidney function tests

The protein concentration in the serum of the animals was assayed, using Biuret reagent as described
by Gornall et al (1949) while the procedure described by Doumas et al (1971) was used for the
determination of albumin in the serum of the animals. The determination of serum globulin content was
done using the method described by Tietz (1995) by subtracting the concentration of serum albumin from
the total serum protein content. Bilirubin was determined using the method described by Sherlock
(1951). The method used for the determination of urea in the serum was that described by Veniamin and
Vakirtzi (1970). Serum creatinine was carried out using the procedure described by Bartels and Bohmer
(1972). For the determination of the electrolytes, sodium ion, potassium ion, bicarbonate ion, phosphate
ion and chloride ion were determined by the method of Tietz (1995), Tietz (1990), Tietz (1995), Fiske and
Subbarrow (1925) and Skeggs and Hochstrasser (1964) respectively.
Data was expressed as mean values ± SEM of six replicates. All results were statistically analyzed
using one-way ANOVA and Duncan’s Multiple Range Test. Differences between group means were
considered significant at P < 0.05.
Results
Effect of Dialium guineense fruit pulp meal-based diet on the activities of liver and kidney
antioxidant enzymes and protein concentration
The effect of Dialium guineense fruit pulp meal-based diet on the liver and kidney superoxide
dismutase and catalase activities, reduced glutathione and malondialdehyde concentrations of rats is
shown in Table 2. Feeding of rats on Dialium guineense fruit pulp meal-based diet at all inclusion levels
did not significantly alter (p > 0.05) the liver and kidney superoxide dismutase activity, reduced
glutathione and malondialdehyde concentrations when compared with the control values. However, for
the catalase activity in the liver and kidney, there was a significant reduction (p < 0.05) in rats maintained
on 30 and 40% of the diet whereas the activity of the enzyme was not altered (p > 0.05) in rats maintained
on 5%, 10% and 20% of the diet when compared with the control values.
Effect of Dialium guineense fruit pulp meal-based diet on the liver and kidney aminotransferases
and liver gamma glutamyl transferase activities of rats
The effect of Dialium guineense fruit pulp meal-based diet on the liver and kidney aminotransferases
and liver gamma glutamyl transferase activities of rats is presented in Tables 3 and 4 respectively.
Dialium guineense fruit pulp meal-based diet at 5% to 40% inclusion levels did not produce any
significant change (p > 0.05) in the activities of alanine aminotransferase and aspartate aminotransferase
in the liver and kidney as well as liver gamma glutamyl transferase when compared with the animals
maintained on the basal (control) diet (Tables 3 and 4). Similarly, there was no significant difference (p >
0.05) in the activities of the three enzymes in the serum of rats when compared with the control values.
81
Table 2: Effect of Dialium guineense fruit pulp meal-based diet on the activities of liver and kidney antioxidant enzymes and protein
concentration
Treatment Superoxide Dismutase Catalase Reduced Glutathione Malondialdehyde

Groups
Liver Kidney Liver Kidney Liver Kidney Liver Kidney

Control 39.62 ± 0.47a 25.40 ± 1.01a 26.97 ± 1.08a 17.28 ± 0.99a 18.45 ± 1.44a 11.56 ± 1.02a 0.22 ± 0.02a 0.25 ± 0.02a
5% D.g 38.84 ± 0.67a 24.92 ± 0.95a 26.78 ± 1.96a 17.02 ± 0.56a 18.58 ± 1.23a 11.77 ± 0.78a 0.26 ± 0.01a 0.26 ± 0.01a
10% D.g 39.13 ± 0.98a 25.21 ± 1.03a 26.88 ± 1.01a 16.98 ± 1.02a 18.49 ± 0.98a 11.98 ± 0.89a 0.24 ± 0.03a 0.26 ± 0.04a
20% D.g 38.79 ± 0.68a 24.98 ± 1.44a 26.62 ± 0.98a 16.99 ± 0.78a 18.02 ± 1.06a 11.03 ± 0.85a 0.24 ± 0.01a 0.25 ± 0.03a
30% D.g 40.01 ± 0.02a 24.98 ± 0.89a 24.43 ± 0.46b 15.44 ± 0.56b 18.39 ± 0.95a 11.40 ± 0.56a 0.21 ± 0.03a 0.24 ± 0.01a
40% D.g 39.12 ± 0.78a 25.78 ± 0.40a 24.41 ± 1.07b 15.56 ± 0.78b 18.24 ± 1.41a 11.05 ± 1.02a 0.20 ± 0.04a 0.25 ± 0.07a
Data are means of six determinations ± SEM. Values with superscripts different from the control for each parameter down each column are significantly
different (p < 0.05). D.g – Dialium guineense fruit pulp meal-based diet.
82
Table 3: Effect of Dialium guineense fruit pulp meal-based diet on the liver, kidney and serum
aminotransferase activities of rats
Treatment Alanine aminotransferase (U/L) Aspartate aminotransferase (U/L)

Groups
Liver Kidney Serum Liver Kidney Serum

a a a a a
Control 58.51 ± 2.01 28.65 ± 1.02 1.52 ± 0.09 202.15 ± 4.32 131.40 ± 3.64 1.03 ± 0.04a
5% D.g 57.29 ± 1.09a 27.95 ± 0.98a 1.50 ± 0.05a 203.01 ± 3.12a 132.10 ± 2.46a 1.05 ± 0.03a
10% D.g 57.32 ± 0.56a 28.45 ± 1.03a 1.46 ± 0.03a 203.43 ± 0.14a 132.37 ± 1.69a 1.07 ± 0.06a
20% D.g 57.44 ± 1.08a 27.75 ± 1.32a 1.56 ± 0.04a 202.14 ± 2.11a 131.65 ± 1.30a 1.06 ± 0.08a
30% D.g 58.03 ± 0.23a 28.52 ± 0.98a 1.50 ± 0.07a 203.15 ± 2.13a 132.16 ± 3.59a 1.01 ± 0.09a
40% D.g 58.62 ± 1.02a 28.44 ± 1.32a 1.54 ± 0.02a 203.98 ± 2.14a 131.38 ± 2.59a 1.05 ± 0.04a
Data are means of six determinations ± SEM. Values with the same superscript a, across the same colum for each
parameter are not significant different (p>0.05). D.g – Dialium guineense fruit pulp meal-based diet.
Table 4: Effect of Dialium guineense fruit pulp meal-based diet on liver gamma glutamyl
transferase activity of rats
Gamma glutamyl transferase (U/L)

Treatments Liver Serum
a
Control 256.12 ± 5.12 3.95 ± 0.12a
a
5% D.g. 255.45 ± 4.98 4.02 ± 0.65a
a
10% D.g. 254.16 ± 3.87 4.34 ± 0.18a
a
20% D.g. 254.47 ± 2.98 4.04 ± 0.68a
a
30% D.g. 253.11 ± 3.25 3.87 ± 0.45a
a
40% D.g. 253.23 ± 2.29 3.98 ± 0.67a
Data are means of six determinations ± SEM. Values with the same superscript a, across the same column for each
parameter are not significant different (p>0.05). D.g – Dialium guineense fruit pulp meal-based diet.
Effect of Dialium guineense fruit pulp meal-based diet on liver and kidney function indices of rats
The effect of Dialium guineense fruit pulp meal-based diet on liver function indices is shown in
Table 5. Dialium guineense fruit pulp meal-based diet significantly increased (p < 0.05) the serum total
protein and serum total albumin concentrations in rats at all inclusion levels (5% - 40%) when compared
with the control values. For the protein and albumin concentration, there was also a significant decrease
(p < 0.05) in rats fed on 5%, 10% and 20% of the diet when compared with those fed on 30% and 40% of
the diet. However, the rats maintained on 30% and 40% supplementation compared favourably (p > 0.05)
with each other. There was no significant difference (p > 0.05) in the serum globulin, total bilirubin and
conjugated bilirubin levels of rats fed on the diet at all inclusion levels when compared with the control
values. The effect of Dialium guineense fruit pulp meal-based diet on kidney function indices of rats is
presented in Table 6. Feeding of rats with Dialium guineense fruit pulp meal-based diet significantly
increased (p < 0.05) the urea and creatinine concentration in the serum of rats when compared with the
control values. However, for the creatinine concentration, there was a significant decrease (p < 0.05) in
rats fed on 20%, 30% and 40% of the diet was compared with those fed on 5% and 10% of the diet. For
83
all the electrolytes, Na+, K+, HCO32-, PO42- and Cl-, there was no significant difference (p > 0.05) in all the
rats fed on the diet when compared with the control value.
Table 5: Effect of Dialium guineense fruit pulp meal-based diet on liver function indices of rats
Treatment Groups
Parameters Control 5% D.g 10% D.g 20% D.g 30% D.g 40% D.g
Total protein 43.57 ± 2.11a 45.29 ± 1.04b 45.32 ± 2.45b 46.16 ± 1.07c 47.69 ± 1.45d 47.36 ± 1.04d
(mg/ml)
Albumin (g/l) 25.08 ± 1.02a 27.42 ± 0.45b 27.11 ± 1.45b 27.93 ± 1.39b 28.83 ± 0.03c 28.30 ± 1.00c
Globulin (g/l) 18.13 ± 0.34a 18.21 ± 0.35a 18.89 ± 0.44a 18.84 ± 1.14a 18.34 ± 1.19a 18.51 ± 1.04a
Total 12.45 ± 1.06a 12.29 ± 1.11a 12.31 ± 0.08a 12.37 ± 1.01a 12.39 ± 0.98a 12.38 ± 0.69a
bilirubin
(µmole/l)
Conjugated 5.12 ± 0.23a 4.99 ± 0.78a 5.01 ± 0.92a 5.08 ± 0.29a 5.03 ± 0.61a 5.08 ± 0.56a
bilirubin
(µmole/l)
Data are means of six determinations ± SEM. Values with superscripts different from the control down each column
for each parameter are significantly different (p < 0.05). Key :- D.g – Dialium guineense fruit pulp meal-based diet.
Table 6: Effect of Dialium guineense fruit pulp meal-based diet on kidney function indices of rats
Treatment Groups
Parameters Control 5% D.g 10% D.g 20% D.g 30% D.g 40% D.g
Urea (mmol/l) 28.62 ± 1.03a 31.43 ± 2.01b 32.04 ± 1.07b 32.92 ± 1.05b 33.56 ± 0.43b 32.99 ± 1.01b
Creatinine 90.12 ± 2.16a 85.43 ± 1.01b 85.62 ± 0.32b 83.32 ± 1.41c 83.21 ± 0.33c 82.82 ± 0.56c
(µmole/l)
Na+ 22.14 ± 1.12a 22.22 ± 1.21a 21.61 ± 0.22a 22.11 ± 1.06a 21.83 ± 1.45a 21.78 ± 1.98a
K+ 2.13 ± 0.23a 2.49 ± 0.33a 2.74 ± 0.45a 2.18 ± 0.09a 2.08 ± 0.16a 2.14 ± 0.08a
HCO3- 3.89 ± 0.68a 3.88 ± 0.84a 3.79 ± 0.45a 3.90 ± 0.11a 3.92 ± 10.68a 3.52 ± 0.23a
PO43- 0.78 ± 0.03a 0.68 ± 0.05a 0.69 ± 0.01a 0.72 ± 0.06a 0.77 ± 0.04a 0.71 ± 0.06a
Cl- 28.68 ± 1.69a 28.22 ± 1.68a 27.45 ± 0.98a 28.42 ± 1.69a 28.44 ± 0.98a 27.46 ± 2.11a
Data are means of six determinations ± SEM. Values with superscripts different from the control down each column
for each parameter are significantly different (p < 0.05). Key :- D.g – Dialium guineense fruit pulp meal-based diet.
Discussion
Dialium guineense fruit pulp has been scientifically investigated and proven for various nutritional
and therapeutic uses. However, a holistic investigation of its possible toxic effects is needed for proper
pharmaceutical and nutritional use. Evaluation of antioxidant parameters as well as liver and kidney
function indices are some of the pointers that have been used to predict the effects of foreign compounds
(such as plant products) on body systemic functions (Anderson, 2001).
Free radicals scavenging enzymes and protein such as superoxide dismutase (SOD) and catalase
(CAT), reduced glutathione are known to be the first line of cellular defense against oxidative damage,
disposing O2 and H2O2 before their interaction to form the more harmful hydroxyl (OH·) radical (Lil et
al., 1988). SOD is an important defense enzyme that catalyzes the dismutation of superoxide anions into
O2 and H2O2 (Bannister and Bannister, 1987). CAT is a hemeprotein that catalyzes the reduction of H2O2
to H2O and O2 and protects the tissue from highly reactive oxygen free radicals and hydroxyl radicals
(Zamocky and Koller, 1999).
In the present study, the activities of SOD and CAT in liver and kidney were not significantly
affected which suggests that there was apparently a reduced formation of superoxide anions which might
84
have inactivated these enzymes and decreased their activities. Reduced glutathione is one of the most
abundant non-enzymatic antioxidant bio-molecules present in the tissues (Meister, 1984). Its functions
include removal of free oxygen species such as H2O2, superoxide anions and alkoxy radicals, maintenance
of membrane protein thiols and to act as a substrate for GSH-Px (Townsend, 2003).
The absence of significant effect on the reduced glutathione concentration of rats maintained on the
fruit pulp meal-based diet also supports the findings obtained earlier from superoxide dismutase and
catalase activities and this might be due to the non-interference of the fruit pulp with the synthesis of this
biomolecule or by the presence of various bioactive molecueles like tannins and/ or flavonoids in the pulp
(Oyegoke and Oladiji, 2014) which might enhance the activity of this biomolecule. The absence of
significant effect in both the liver and kidney malondialdehyde levels corroborate with earlier results on
the antioxidant enzymes and protein and suggest that there was no peroxidation that might lead to tissue
damage. This implies that the antioxidant defense mechanisms might still be intact. Aminotransferase
which include alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are enzymes located
in the cytosol and mitochondria where they are involved in the transfer of amino group from α-amino to
α-keto acids. They are also involved in the biochemical regulation of intracellular amino acid pool
(Chapatwala et al., 1982). These aminotransferases belong to the plasma non-functional enzymes which
are normally localized within the cells of liver, heart, kidney and muscles. Their presence in serum may
give information on tissue injury or organ dysfunction (Wells et al., 1986).
Tissue and serum levels of ALT and AST can be used to assess the toxic impact of chemical
compound including plant derived substances. The absence of effect on the activities of both enzymes in
the organs and serum suggests that the function of the organs was not compromised by the diet (Wells et
al., 1986). γ-Glutamyl transferase (GGT) is the most sensitive enzymatic indicator of hepatobiliary
disease (Mayne, 1998). It is a membrane localized enzyme that plays a major role in the glutathione
metabolism and reabsorption of amino acid from the glomerular filterate and intestinal lumen (Kaplan and
Pesce, 1996). The absence of significant effect in the activities of GGT in the liver and serum of rats fed
on the fruit pulp meal-based diet may suggest that the activities of the liver had not been compromised.
The concentrations of albumin, globulin and bilirubin in the serum of the animals can indicate the
secretory and synthetic functioning of the liver and can be used to ascertain types of liver damage
(Yakubu et al., 2003). Albumin is the major protein present within the blood and represents a reliable test
to assess the degree of liver damage in animals. Albumin which is manufactured by the liver, is a major
protein that circulates in the blood stream (Tietz, 1986). The increase in albumin concentration of rats fed
on fruit pulp meal-based diet is an indication of dehydration (Naganna, 1989). This might also be due to
increased rate of hepatic synthesis of albumin without proportionate increase in the rate of its elimination
(Yakubu et al., 2003). Globulins are a larger protein than albumins and are important for its
immunological responses (Tietz, 1986). They are also an heterogenous complex mixture of protein
molecule whose role is to regulate osmotic pressure (homeostatis) (Ganong, 2001). The absence of
significant effect on serum globulin concentration of rats maintained on fruit pulp meal-based diet of
Dialium guineense suggest that the rate of transportation of nutrients, defence, coagulation processes,
buffering capacity of the blood and haemaostasis was not altered (Ganong, 2001).
Bilirubin is the major breakdown product that results from the destruction of old red blood cells. It is
removed from the blood by the liver, hence it is a good indication of the function of liver (Murray et al.,
2000). Bilirubin concentration is elevated in the blood either by increased production of bilirubin or
decreased liver uptake (as a result of liver disease).
The findings in the present study showed that there was no significant effect on the total and
conjugated bilirubin of rats fed with the fruit pulp meal-based diet of Dialium guineense at all
supplementation (5% to 40%) when compared to the control. This observation might suggest that the
experimental diet had no adverse effect on the liver (Chebeseborough, 1992). Renal function indices such
as serum electrolytes (sodium, potassium, phosphate, chloride and bicarbonate), urea and creatinine can
be used to evaluate the functional capacity of the nephrons of animals at the glomerular and tubular levels
(Yakubu et al., 2003).
85
Although, creatinine, urea and uric acids are major catabolic products of muscle, protein and purine
metabolism, creatinine is regarded as the most endogenous marker in the diagnosis and treatment of
kidney disease and its clearance is estimated as an indication of glomerular filtration rate (Chawla, 1999).
The significant reduction in the concentration of creatinine at all inclusion levels of Dialium guineense
fruit pulp meal-based diet when compared with the control group may be an indication of glomerular or
tubular dysfunction (Saad et al., 2006).
Urea plays a key role in the countercurrent exchange system of the nephrons, which allows for
reabsorption of water and critical ions. The significant increase in the serum urea concentration of all the
groups of animals fed on the fruit pulp meal-based diet may be an indication of glomerular dysfunction of
the nephron (Chawla, 1999). Electrolytes are present in the body and the balance of the electrolytes in the
body is essential for normal function of cells and organs. The concentrations of electrolytes in the serum
of animals could give an insight into the effect of chemical compound including plant/food substances on
tubular or glomerular part of the kidney (Ashafa et al., 2009). The absence of significant effect of the
levels of all the electrolytes investigated in the animals fed on the diet at the various inclusion levels
implies that the fruit pulp might not interfere with the metabolism of these electrolytes.
Conclusion
The absence of significant effects on the activity of most enzymes and concentrations of metabolites
investigated for the safety evaluation studies suggest that the fruit pulp supplementation might not
interfere with the normal functioning of the enzymes and metabolites. This implies that the fruit pulp
may be safe for consumption at the percentage inclusion evaluated.
Acknowledgment
The authors acknowledge the technical assistance rendered by the technologists of the Department of
Biochemistry Laboratory, Faculty of Life Sciences, University of Ilorin, Ilorin, Kwara State, Nigeria.
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ISSN 0795-8080

VOLUME 33, NUMBER 1

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0795-8080/2021 $10.00 + 0.00

Selected liver and kidney function indices of Wistar rats fed

*Corresponding author E-mail: lolasojiomoniwa@gmail.com; Telephone: 07034205898

(Received November 24, 2020; Accepted February 4, 2021)

Materials and Methods

Plants collection and authentication

Feed preparation and administration to experimental rats

C - Control rats fed diet without Ficus exasperata leaves (FEL)

Table 1: Feed Formulation of Ficus exasperata leaf-based diet

Ingredients (g/kg) Control 10% 30% 50%

Corn starch 512 412 212 12

Animal sacrifice and tissue collection

Liver function indices

Kidney function indices

Liver function indices

Kidney function indices

Day 7 Day14 Day21

Kidney function indices

Urea (mg/dl) Creatinine (mg/dl )

F1 109.63±2.40a 109.24±2.00a 109.20±1.00a 3.81±0.27a 3.80±0.24a 3.81±0.26a 2.51±0.10a 2.52±0.02a 2.51±0.02a

F2 108.34±2.32a 109.62±1.00a 108.22±2.02a 3.53±0.02a 3.58±0.18a 3.56±0.17a 2.49±0.02a 2.48±0.12a 2.50±0.02a

F3 109.28±1.74a 108.00±1.84a 108.24±2.24a 3.61±0.27a 3.61±0.28a 3.60±0.26a 2.51±0.02a 2.51±0.10a 2.51±0.01a

Groups PO43- (mg/dl) Cl- (mmol/l) HCO3- (mmol/l)

C 7.69±0.39a 7.68±0.38a 7.68±0.39a 79.26±1.18a 78.26±1.15a 78.30±1.20a 32.16±3.43a 33.23±2.24a 32.20±2.20a

F1 7.66±0.40b 7.64±0.80a 7.66.0.41a` 80.36±1.59a 78.40±1.08a 79.00±1.89a 33.32±1.57a 33.20±2.00a 33.00±1.18a

F2 7.72±0.47ab 7.70±0.45a 7.70±0.44a 81.11±0.81a 79.05±0.80a 78.42±1.15a 35.37±4.11a 34.02±1.18a 32.00±2.00a

F3 7.69±0.39a 7.70±0.36a 7.70±0.42a 79.26±1.18a 79.08±1.18a 77.02±2.28a 32.16±3.43a 33.20±2.02a 33.34±1.00a

0795-8080/2020 $10.00 + 0.00

Hypolipidemic potentials of methanol extracts of Vernonia

*Author for correspondence: Tel: 07032869195; E-mails: anthonynnamudi@gmail.com; annamudi@pums.edu.ng

(Received November 26, 2020; Accepted February 4, 2021)

Keywords: Vernonia colorata, high fat diet, obesity, dyslipidemia

atherosclerosis, cardiovascular diseases, cerebrovascular diseases, hypertension and type 2 diabetes

Materials and Methods

Collection and Identification of Plant

Preparation of Methanol Extracts of Vernonia colorata

Figure 1: Vernonia colorata showing its leaves and fruits.

Table 1: Composition of Diets

Concentration (g/1000 g of feed)

Group I: Basal Diet

Body Weight Measurement

Effect on Total Cholesterol concentration:

Effect on Triacylglycerol concentration:

Effect on HDL cholesterol concentration:

Effect on LDL cholesterol concentration:

0795-8080/2021 $10.00 + 0.00

Antigenotoxic and hepatoprotective activities of ethanol

*Correspondences to: ronodunola@yahoo.com and aygoke@yahoo.com, Tel.: +234 8023387152

(Received November 27, 2020; Accepted February 4, 2021)

Key words: Arsenite, Genotoxicity, Hepatotoxicity, Herbal, Micronucleus, Transaminases.

Materials and Methods

Chemicals and Reagents for Analysis

Preparation of Ethanol leaf extract of Eclipta alba (ELEA)

Experimental Protocols and Treatments

Liver function enzymes assays

p-nitrophenylphosphate + H20 ALP -→ phosphate + p-nitrophenol

Micronucleus (MN) assay

Liver and Kidney histological analysis

Hepatic Cell Analysis

Effect of ELEA on SA-induced hepatotoxicity in male albino Wister rats