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British Biotechnology Journal
4(12): 1253-1262, 2014
ISSN: 2231–2927
SCIENCEDOMAIN international
www.sciencedomain.org
Effects of Methanolic Extract of Dioclea reflexa
Hook F Seed on some Haematological and
Kidney Function Parameters in Rats following
Single or Repeated Carbon Tetrachloride
Intoxication
Sunday Ene-Ojo Atawodi
1*
and Uju Dorothy Iliemene
1
1
Biochemistry Department, Ahmadu Bello University, Zaria, Nigeria.
Authors’ contributions
This work was carried out in collaboration between both authors. Author SEA designed the
study wrote the protocol and the manuscript, while author UDI managed the analyses and
the literature searches and performed the statistical analysis. Both authors read and
approved the final manuscript.
Article Information
DOI: 10.9734/BBJ/2014/13836
Editor(s):
(1)
Samuel Peña-Llopis, Developmental Biology and Internal Medicine, Oncology Division, University of Texas
Southwestern Medical Center, USA.
Reviewers:
(1)
Anonymous, Helwan University, Egypt.
(2)
Yousif Y. Bilto, Dept. Biol and Biomed Sciences, The University of Jordan, Amman, Jordan.
Complete Peer review History:
http://www.sciencedomain.org/review-history.php?iid=800&id=11&aid=6726
Received 6
th
September 2014
Accepted 7
th
October 2014
Published 30
th
October 2014
ABSTRACT
Objective:
To investigate the effect of methanolic extract of Dioclea reflexa Hook F seed
on male rats following single (acute) or repeated (chronic) carbon tetrachloride
intoxication.
Methods: Male albino rats were divided into groups of six each consisting of plant extract
only, plant extract + carbon tetrachloride, solvent only, solvent + carbon tetrachloride,
vitamin E only, vitamin E + carbon tetrachloride and untreated control. The rats in the
Original Research Article
British Biotechnology Journal, 4(12): 1253-1262, 2014
1254
acute experiment received the extract (5mg/kg) by intraperitoneal injection for two days,
and 1 hour after administration of CCl
4
(0.6ml/kg body weight) on the third day, while
those in the chronic experimental model received 2.5mg/kg for 10 consecutive days with
72 hourly administration of CCl
4
at 0.3ml/kg.
Results: In the acute model, the level of total bilirubin and conjugated bilirubin were
significantly (P<0.05) reduced in D. reflexa seed extract pre-treated rats compared to the
CCl
4
control, while in the chronic model, the level of packed cell volume (PCV) and
hemoglobin were significantly (P<0.05) boosted in extract treated group compared to the
CCl
4
control group with concomitant reduction in the levels of bilirubin.
Conclusion: These results indicate that the seed of D. reflexa possess capacity to boost
haematological parameters and protect the kidney against acute and chronic toxicological
challenges.
Keywords: Dioclea reflexa; haematological effect; kidney damage; CCl
4
intoxication; acute
toxicity; chronic toxicity.
1. INTRODUCTION
Spices, condiments and herbs are recognized as sources of natural antioxidants and
phytochemicals that could play an important role in the chemoprevention of diseases that
have their etiology and pathophysiology in reactive oxygen species [1-3]. Thus, it has been
recommended that adequate intake of antioxidant through the consumption of antioxidant
rich foods can prevent the development of oxidative stress [4-6]. In line with our earlier work
on the assessment of the beneficial effects of some Nigerian foodstuffs and herbs [7-9,5,10],
the effects of dried seeds of Dioclea reflexa, an Eastern Nigeria condiment on some
haematological and kidney function parameters were investigated following single or
repeated intoxication with carbon tetrachloride.
Also known as “Ukpo” in Igbo and “Agbaarin” in Yoruba, Dioclea reflexa Hook F.
(Papilionaceae) is an annual climber crop that can be cultivated more than once a year. The
plant is high yielding, bearing pods that contain between three and four seeds [11], which
are usually dark brown to black, sometimes speckled, depending on variety. The
endosperm, which is rich in gum is pulverized and used as thickener in many traditional food
preparations, while there are several reports on its suitability in processed foods, including
use as a rheology modifier [12]. Both its seed and leaf have high economic values in culinary
and pharmaceutical industries [13].
Dioclea reflexa seed oil has acid value, saponification value, iodine value, ester value and
iodine number to be 8.69mg KOH/g, 251mg KOH/g, 72.8mg I/g, 242 and 27.9, respectively.
The fatty acid composition include unsaturated fatty acid, especially oleic acid, while the
saturated fatty acids include palmitic acid and stearic acid. The cake and seed flour of
Dioclea reflexa has appreciable levels of protein that could serve as important protein source
in livestock production and in human foods [14].
2. MATERIALS AND METHODS
2.1 Chemicals and Reagents
Methanol, petroleum ether, vitamin E as tocopherol acetate carbon tetrachloride, corn oil,
and assay kits were purchased from sigma chemical Co, Ltd (USA) and are of analytical
grades.
British Biotechnology Journal, 4(12): 1253-1262, 2014
1255
2.2 Plant Collection and Authentication
Dioclea reflexa seeds and plant parts were collected from Achalla village in Awka North
Local Government of Anambra State, Nigeria in June of 2009. It was authenticated at the
Herbarium Unit of the Department of Biological Sciences, Ahmadu Bello University, Zaria,
Nigeria where voucher number 1286 was assigned.
2.3 Sample Preparation and Extraction
The D. reflexa seeds were dried in the laboratory at room temperature and pulverized using
laboratory mortar and pestle. Pulverized material (35g) was placed in the thimble of soxhlex
extractor and extracted first, using petroleum ether (300ml) for 8 hours each and then
methanol (300ml), three times for 5 hours each. The methanol extracts were combined and
dried in vacuo at 45°C using a rotary evaporator (Büchi Labortechnik AG, Switzerland). The
methanol extracts were combined and dried in vacuo at 45°C using a rotary evaporator.
2.4 Management of Experimental Animals
Male albino rats (7-8weeks old and weighing about 120-150kg) were purchased from the
animal house of National Research Institute for Chemical Technology, Zaria, Nigeria. They
were acclimatized for two weeks prior to commencement of experiment. They were kept at
room temperature and were maintained ad libitum on tap water and growers mash (Vital
feeds, Jos, Plateau State Nigeria) except in the last 15 hours before termination of the
experiment. They were weighed prior to commencement and termination of the experiment.
2.5 Grouping of Animals
In the chronic experimental model, rats were divided into 7 groups of six rats each. Group 1
was administered the plant extract only, group 2 was administered plant extract and carbon
tetrachloride, group 3 was administered solvent only, group 4 was administered solvent and
carbon tetrachloride, group 5 was administered vitamin E only, group 6 was administered
vitamin E and carbon tetrachloride, group 7 was untreated control. The CCl
4
was
administered at a dose of 0.6ml/kg body weight before the administration of the first extract
or vitamin E, and subsequently every 72 hours until 10 days before termination of the
experiment. After the first day, extract and vitamin E were administered daily at a dose of
2.5mg/kg body weight for 10 days. The animals were sacrificed 24 hours after the last
administration of extract [9,10].
In the acute experimental model, rats were divided into 7 groups with each group containing
six rats. Group 1 was administered the plant extract only, group 2 was administered plant
extract and carbon tetrachloride, group 3 was administered solvent only, group 4 was
administered solvent and carbon tetrachloride, group 5 was administered vitamin E only,
group 6 was administered vitamin E and carbon tetrachloride and group 7 was the untreated
control. The rats received 24 hourly administration of the extract at 5mg/kg for three days by
intraperitoneal injection. On the third day, CCl
4
(0.6ml/kg) was administered 1hr after the
extract administration, and the animals sacrificed 24 hr later [9,10].
2.6 Collection and Storage Blood
At the point of sacrifice, blood from each rat was withdrawn from carotid artery at the neck
and collected in previously labeled test tubes and allowed to stand for 3 hours. Clear serum
British Biotechnology Journal, 4(12): 1253-1262, 2014
1256
were collected from the blood in eppendoff tubes and stored under - 20°C for biological
assay.
2.7 Determination of Packed Cell Volume (PCV) and Hemoglobin
Concentration
In determining the packed cell volume and hemoglobin concentration, whole blood samples
were collected into heparinized capillary tubes, filled up to 2/3 the length during animal
sacrifice, sealed with plasticine and centrifuged at 3000 × g for 10 minutes. Packed cell
volume was determined using hematocrit reader and expressed as percentage erythrocytes
content of the blood, while the hemoglobin concentration was calculated using the formula:
packed cell volume level divided by 3 and expressed as percentage (%) hemoglobin content
of the blood.
2.8 Determination of Bilirubin Concentration
Conjugated, unconjugated and total bilirubins were estimated colorimetrically at 560nm
following the principle described by Sherine and Safinaz [15] using Randox assay kit, while
the value of the total bilirubin (mg/dl) was obtained by multiplying the absorbance by a factor
of 10.8. In the case of direct bilirubin, the absorbance was read at 530 nm, and the value of
direct bilirubin (mg/dl) was obtained by multiplying the absorbance by a factor of 14.4, while
indirect bilirubin was estimated by difference.
2.9 Determination of Urea
Urea in serum is hydrolysed to ammonia in the presence of urease, and the ammonia is then
measured spectrophotometrically at 540nm by Berthelot’s reaction utilizing analytical kits
based on the principle described by Stephen and coworkers [16]. Urea level was calculated
from sample absorbance relative to the absorbance of standard of known concentration
(mg/dl).
2.10 Assessment of Creatinine Levels
Creatinine level was assayed colorimetrically at 510nm following 30 sec and 2 min
incubation with picric acid reagent by utilizing Randox reagent kit based on the principle
described by Stephen and co-workers [16]. Creatinine level was calculated as change in the
sample absorbance divided by the change in the absorbance of the standard multiplied by
the concentration of the standard.
2.11 Statistical Analysis
Results are expressed as mean ± SD. Analysis of variance(ANOVA) was used for statistical
analysis using the Statistical Package for Social Sciences (SPSS) version 20 software. A
value of P<0.05 was used to denote statistical significance.
3. RESULTS
3.1 Serum Total, Conjugated and Unconjugated Bilirubin Concentration
In the model of chronic injury, pre-treatment with methanolic extract of D. reflexa seed
significantly (P<0.05) prevented the CCl
4
induced elevation in the levels of total, conjugated
and unconjugated bilirubin (Fig
volume and hemoglobin concentration (Fig
levels of total, conjugate
d and unconjugated bilirubin (Fig
and hemoglobin concentration (
Fig. 1.
Mean bilirubin concentration (total, conjugated and unconjugated) in serum
after 10 days daily
intraperitoneal administration of
hourly ca
Fig. 2.
Mean bilirubin concentration (total, conjugated and unconjugated) in serum
rats on
intraperitoneal injection
methanolic extract of
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
D.
reflexa
only
D.
reflexa
+ CCl4
Bilirubin Conc. (mg/dl)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
D.
reflexa
only
D.
reflexa
+ CCl4
Bilirubin Conc. (mg/dl)
British Biotechnology Journal, 4(12):
1253
and unconjugated bilirubin (Fig
.
1) with concomitant increase in the levels of packed cell
volume and hemoglobin concentration (Fig
.
3). Similar observations were made for the
d and unconjugated bilirubin (Fig
.
2) as well as packed cell volume
and hemoglobin concentration (
Fig. 4) in the model of acute damage.
Mean bilirubin concentration (total, conjugated and unconjugated) in serum
intraperitoneal administration of
D. reflexa
extract (2.5mg) with 72
hourly ca
rbon tetrachloride intoxication
Mean bilirubin concentration (total, conjugated and unconjugated) in serum
intraperitoneal injection
of CCl
4
following 2 days pre-
treatment
methanolic extract of
D. reflexa (5mg/kg)
reflexa
+ CCl4
Solvent
only
CCl4
only
Vit E
only
Vit E +
CCl4
Normal
control
Treatments
Total
Conjugated
Unconjugated
reflexa
+ CCl4
Solvent
only
CCl4
only
Vit E
only
Vit E +
CCl4
Normal
control
Treatments
Total
Conjugated
Unconjugated
1253
-1262, 2014
1257
1) with concomitant increase in the levels of packed cell
3). Similar observations were made for the
2) as well as packed cell volume
Mean bilirubin concentration (total, conjugated and unconjugated) in serum
extract (2.5mg) with 72
Mean bilirubin concentration (total, conjugated and unconjugated) in serum
of
treatment
with
Conjugated
Unconjugated
Conjugated
Unconjugated
Fig. 3.
Mean packed cell volume (PCV) and haemoglobin concentration of rats after 10
days daily
administration of
Fig. 4.
Mean packed cell volume (PCV)
injection of CCl
4
following 2 days
0
10
20
30
40
50
60
D.
reflexa
only
D.
reflexa
+ CCl4
PCV & Haemoglobin level (% Avg)
0
10
20
30
40
50
60
D.
reflexa
only
D.
reflexa
+ CCl4
PCV & Haemoglobin level (% Avg)
British Biotechnology Journal, 4(12):
1253
Mean packed cell volume (PCV) and haemoglobin concentration of rats after 10
administration of
D. reflexa
extract (2.5mg) with 72 hourly ca
tetrachloride intoxication
Mean packed cell volume (PCV)
of rats in serum of rats on
intraperitoneal
following 2 days
pre-treatment
with methanolic extract of
5mg/kg
D.
reflexa
+ CCl4
Solvent
only
CCl4
only
Vit E
only
Vit E +
CCl4
Normal
control
Treatments
PCV
Haemoglobin
D.
reflexa
+ CCl4
Solvent
only
Solvent
+ CCl4
Vit E
only
Vit E +
CCl4
Normal
control
Treatments
PCV
Hemoglobin
1253
-1262, 2014
1258
Mean packed cell volume (PCV) and haemoglobin concentration of rats after 10
extract (2.5mg) with 72 hourly ca
rbon
intraperitoneal
with methanolic extract of
D. reflexa
Haemoglobin
PCV
Hemoglobin
British Biotechnology Journal, 4(12): 1253-1262, 2014
1259
3.2 Effect of D. reflexa Extract on Levels of Urea and Creatinine
To evaluate the effect of D. reflexa extract pre-treatment on kidney function in rats
chronically and acutely intoxicated with CCl
4
, the serum levels of urea and creatinine were
measured. In both models, extract pre-treatment greatly prevented the CCl
4
induced
elevation in the level of both urea (Tables 1 and 2) and creatinine (Tables 1 and 2) while no
such significant difference occurred between the vitamin E control, D. reflexa extract only
and untreated control groups (Table 1).
Table 1. Mean creatinine and urea level in serum of rats after 10 days daily
administration of D. reflexa methanolic extract (2.5mg) with 72 hourly carbon
tetrachloride intoxication
Urea and
creatinine
concentration mg/dl
Group
Treatment
Urea
Creatinine
1 D. reflexa only 67±5
a
1.0±0.0
a
2 D. reflexa + CCl
4
69±5
a
1.2±0.2
a
3 Solvent only 87±6
b
1.2±0.1
a
4 CCl
4
only 130±6
c
2.4±0.2
b
5 Vit E only 70±6
a
1.2±0.1
a
6 Vit E + CCl
4
82±6
b
1.2±0.2
a
7 Normal control 85±5
b
1.0±0.0
a
Values are Mean±SD; Values having different letters across the column are significantly different
(p<0.05)
Table 2. Mean creatinine and urea concentration in serum of rats on intraperitoneal
injection of CCl
4
following 2 days pre-treatment with methanolic extract of D. reflexa
(5mg/kg)
Urea and creatinine levels mg/dL
Group
Treatment
Urea
Creatinine
1 D. reflexa only 46±4
ab
1.0±0.0
a
2 D. reflexa + CCl
4
55±6
b
1.8±0.2
b
3 Solvent only 32±3
a
1.2±0.1
a
4 CCl
4
only 92±7
c
2.2±0.2
b
5 Vit E only 46±5
ab
1.2±0.1
a
6 Vit E + CCl
4
54±4
b
1.2±0.2
a
7 Normal control 37±3
a
1.0±0.0
a
Values are Mean ± SD; Values having different letters across the column are significantly different
(p<0.05)
4. DISCUSSION
The levels of packed cell volume, hemoglobin (Figs. 3 and 4) were significantly lower
(P<0.05) in the CCl
4
control group than in the extract or vitamin E-treated groups. Also the
CCl
4
– induced significant elevation in the levels of bilirubin (Figs. 1 and 2). Elevated levels
of urea and creatinine (Tables 1 and 2) were prevented by treatment with the methanolic
extract of D. reflexa seed or vitamin E.
Pre-treatment with the methanolic extract of D. reflexa significantly (P<0.05) prevented the
CCl
4
mediated elevation in the levels of bilirubin (P<0.05). That serum bilirubin (total, direct
British Biotechnology Journal, 4(12): 1253-1262, 2014
1260
and indirect) levels which were also significantly (P<0.05) elevated by CCl
4
treatment, was
remarkably reduced by pre-treatment or administration of the extract and vitamin E (Fig. 1),
strongly suggest that the seed methanolic extract of Dioclea reflexa protected the rat liver
against hepatotoxicity as much as vitamin E, since high level of bilirubin which is excreted by
the liver is an important index of liver dysfunction. Thus, the consumption of this popular
condiment may have wide ranging implication in preventing and ameliorating oxidative stress
related illnesses in the population where it is popularly consumed.
The decrease in the packed cell volume (Fig. 3) and hemoglobin concentration (Fig. 3) of the
CCl
4
control group compared with the extract or vitamin E-treated groups is consistent with
the bilirubin result (Fig. 1), since haem degrade following red blood cell destruction
consequent on CCl
4
intoxication [17]. Hence, while the lowered level of PCV and hemoglobin
suggest oxidative damage and red blood cell membrane destruction in the CCl
4
group, the
higher levels of PCV and hemoglobin in the extract treated group is a demonstration of the
capacity of D. reflexa to protect red blood cell membrane, and possibly boost the
haemopeitic system [18].
From Tables 1 to 2, it can be observed that there were high levels of urea and creatinine in
the CCl
4
control group which was significantly different (P<0.05) from the groups treated with
extract, vitamin E and normal control group. This is due to the fact that direct or indirect
exposure of kidney to nephrotoxic agents may result in ultra-structural damage to any of the
principal component of the nephrons [19]. This results in elevated level of blood urea which
is derived from normal metabolism of protein and is usually excreted in the urine and hence
elevated blood urea usually indicates glomerular damage. Similarly, creatinine, a metabolite
of creatine, is also excreted completely in the urine via glomerular filtration. Thus, an
elevation of its level in the blood is an indication of impaired kidney function [20]. That the
extract treated groups showed significantly (P<0.05) lowered concentrations of creatinine
and urea, confirms the potency of Dioclea reflexa seed extract in protecting and ameliorating
the kidney from oxidative stress-related damages. The ability of the D. reflexa seed
methanol extract to protect and ameliorate the organs of the rat against oxidative stress-
related damage should be as a result of its polyphenols and flavonoids content, since
polyphenols, especially flavonoids have been reported to exert positive effects on human
health through their antioxidant properties [2, 3, 5, 6, 21]. However, studies are underway to
establish the specific polyphenols in Dioclea reflexa seeds that are responsible for these
desirable health effects.
5. CONCLUSION
Dioclea reflexa seeds contain substances with potent capacity to protect the kidney and
blood from oxidative and related injuries under acute and chronic toxicological conditions.
COMPETING INTERESTS
Authors have declared that no competing interests exist.
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_________________________________________________________________________
© 2014 Atawodi and Iliemene; This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Peer-review history:
The peer review history for this paper can be accessed here:
http://www.sciencedomain.org/review-history.php?iid=800&id=11&aid=6726