Toxicological implications of aqueous extract of Clematis brachiata Thunb. leaves in male Wistar rats

Abstract

Aqueous extract of Clematis brachiata Thunb. leaves at the doses of 100, 200 and 400 mg/kg body weight was investigated for its toxic effect in male Wistar rats on days 1, 7 and 21. The extract at all the doses did not significantly (P > 0.05) alter the liver-and kidney-body weight ratio, conjugated bilirubin, total protein, globulin, sodium, potassium, chloride, inorganic phosphorus, calcium ions, LDL-C, RBC, Hb, PCV, MCV, MCH, MCHC and RCDW levels throughout the exposure period. In contrast, albumin, urea, HDL-C, LUC, neutrophils, monocytes, eosinophils and basophils levels increased. The 100 mg/kg body weight of the extract significantly (P < 0.05) reduced the total bilirubin only at the end of the experimental period whereas the 200 and 400 mg/kg body weight increased the bilirubin content of the animals. Whereas the increase in serum GGT activity did not manifest until after the 21 daily doses of the extract, the activities of serum ALP and AST increased throughout the exposure period. In addition, serum ALT activity was not altered by the extract. WBC, platelet, lymphocytes, uric acid, triacylglycerol and the computed atherogenic index were reduced by the extract. The results have suggested that the aqueous extract of C. brachiata leaves may be clinically beneficial to the animals as it may not predispose to cardiovascular risk, but has selective toxic effect on the haematological parameters and the functional indices of the liver and kidney of male rats. Overall, the extract of C. brachiata leaves is not completely safe when repeatedly consumed for three weeks at the doses investigated for three weeks.

Full text

African Journal of Pharmacy and Pharmacology Vol. 3(11), pp.531-538, November 2009
Available online http://www.academicjournals.org/ajpp
ISSN 1996-0816 © 2009 Academic Journals
Full Length Research Paper
Toxicological implications of aqueous extract of
Clematis brachiata Thunb. leaves in male Wistar rats
A. J. Afolayan*, M. T. Yakubu, J. R. Appidi and M. Mostafa
Center for Phytomedicine Research, Department of Botany, University of Fort Hare, Alice 5700, South Africa.
Accepted 8 September, 2009
Aqueous extract of Clematis brachiata Thunb. leaves at the doses of 100, 200 and 400 mg/kg body
weight was investigated for its toxic effect in male Wistar rats on days 1, 7 and 21. The extract at all the
doses did not significantly (P > 0.05) alter the liver- and kidney-body weight ratio, conjugated bilirubin,
total protein, globulin, sodium, potassium, chloride, inorganic phosphorus, calcium ions, LDL-C, RBC,
Hb, PCV, MCV, MCH, MCHC and RCDW levels throughout the exposure period. In contrast, albumin,
urea, HDL-C, LUC, neutrophils, monocytes, eosinophils and basophils levels increased. The 100 mg/kg
body weight of the extract significantly (P < 0.05) reduced the total bilirubin only at the end of the
experimental period whereas the 200 and 400 mg/kg body weight increased the bilirubin content of the
animals. Whereas the increase in serum GGT activity did not manifest until after the 21 daily doses of
the extract, the activities of serum ALP and AST increased throughout the exposure period. In addition,
serum ALT activity was not altered by the extract. WBC, platelet, lymphocytes, uric acid, triacylglycerol
and the computed atherogenic index were reduced by the extract. The results have suggested that the
aqueous extract of C. brachiata leaves may be clinically beneficial to the animals as it may not
predispose to cardiovascular risk, but has selective toxic effect on the haematological parameters and
the functional indices of the liver and kidney of male rats. Overall, the extract of C. brachiata leaves is
not completely safe when repeatedly consumed for three weeks at the doses investigated for three
weeks.
Key words: Clematis brachiata, cardiovascular risk, functional indices, haematological parameters, selective
toxicity, serum lipids.
INTRODUCTION
Phytomedicines have been used as the mainstay in the
treatment of various diseases and preservation of human
health since early ages of civilization. For example,
Farnsworth et al. (1985) reported that 80% of the
population in the developing world still rely on traditional
medicine for their primary health care needs. Unfor-
tunately, most of these herbal remedies have not been
assessed for their safety as the traditional medicine
practitioners believe that herbs are safe simply because
they are natural in origin.
Therefore, safety or toxicity in herbal therapy is an
important challenge for traditional medicine. Clematis
brachiata Thunb. (Ranunculaceae), known as traveller’s
joy (English) is widely distributed in South Africa,
*Corresponding author. E-mail: Aafolayan@ufh.ac.za. Fax: +27
866282295.
Swaziland, Namibia and Botswana. It is a slender, twin-
ing, woody deciduous climber that grows up to 5m in
length. It bears sasses of small, sweetly scented, creamy
white flowers in the late summer and autumn. It is
claimed to possess wonderful pain relieving properties
and as such used by travellers that embarked on long
distance by foot.
The leaves are used to ease headaches, coughs,
chest ailments, abdominal upsets and schistomiasis
(Roberts, 1990; Spang et al., 2000). The hot decoction
of the leaves, stems and roots are separately used to
relief cold, malaria, sinus infections and asthma (Chhabra
et al., 1991; Koch et al., 2005; Pendota et al., 2008). Des-
pite the various uses of C. brachiata in traditional medi-
cine of South Africa, there is inadequate information on
the toxic implications of aqueous extract of the plant
leaves. Therefore, the aim of this study was to evaluate
the toxicity risk of the aqueous extract of the plant leaves
using male Wistar rats as a model.

532 Afr. J. Pharm. Pharmacol.
MATERIALS AND METHODS
Plant material
C. brachiata leaves collected in April, 2008 from a natural
population growing within the premises of University of Fort Hare,
Alice, South Africa, was identified by Prof. D. S. Grierson of the
Department of Botany, University of Fort Hare. A voucher specimen
(M. Mostafa med., 2008/1) was deposited at the Giffen Herbarium
of the University.
Animals
Male albino rats (Rattus norvegicus) of Wistar strain weighing
191.21 ± 5.92 g were obtained from the Animal House of the
Agriculture and Rural Development Research Institute, University of
Fort Hare, Alice. The animals were housed in clean alluminum
cages placed in well-ventilated house conditions (temperature 23 ±
1°C; photoperiod: 12 h natural light and 12 h dark: humidity: 45 -
50%). They were maintained on Balanced Trusty Chunks (Pioneer
Foods (Pty) Ltd, Huguenot, South Africa) and clean tap water ad
libitum.
Assay kits
The assay kits for creatinine, urea, calcium, sodium, potassium,
chloride, phosphorus, albumin, bilirubin, cholesterol, triacylglycerol,
high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein
cholesterol (LDL-C), alkaline phosphatase (ALP), gamma glutamyl
transferase (GGT), alanine aminotransferase (ALT) and aspartate
aminotransferase (AST) were obtained from Roche Diagnostic GmbH,
Mannhein, Germany. All other reagents used were of analytical grade
and were supplied by Merck Chemicals (Pty) Ltd., Bellville, South
Africa.
Preparation of extract
The leaves were washed in running tap water, oven-dried at 40°C for
48 h and thereafter pulverized. The powder (200 g) was boiled for 20
min in a litre of distilled water and allowed to cool for 2 h (boiling was
done in accordance with the way it is usually prepared in folklore
medicine of South Africa). The extract was then suction-filtered with a
Buchner funnel and Whatman no. 1 filter paper. The filtrate was later
freeze-dried (Savant Refrigerated Vapor Trap, RVT4104, USA) to give a
yield of 45 g. This was reconstituted in distilled water to give the desired
doses (100, 200 and 400 mg/kg body weight) used in this study.
Animal grouping and extract administration
Seventy-two, male Wistar rats were completely randomized into
four groups of eighteen and were orally administered on daily basis
using a metal oropharyngeal cannula as follows: Group A (control)
received 0.5 ml of distilled water (the vehicle) while groups B, C and
D were treated like the control except they received same volume
containing 100, 200 and 400 mg/kg body weight of the extract
respectively.
Six rats from each group were sacrificed 24 h after their respect-
tive 1, 7 and 21 daily doses. The study was carried out following
approval from the Ethical Committee on Animal Use and Care of
University of Fort Hare, South Africa.
Preparation of serum
The procedure described by Yakubu et al. (2005) was employed
in the preparation of serum. Briefly, under ether anaesthesia, rats
were allowed to bleed through their cut jugular veins which were
slightly displaced (to prevent contamination of the blood with
interstitial fluid) into clean, dry centrifuge tubes. An aliquot (2 ml) of
the blood was collected into sample bottles containing EDTA (BD
Diagnostics, Preanalytical Systems, Midrand, USA) for the
hematological analysis. Another 5 ml of the blood was allowed to
clot for 10 min at room temperature and then centrifuged at 1282 g
× 5 min using Hermle Bench Top Centrifuge (Model Hermle, Z300,
Hamburg, Germany). The sera were later aspirated with Pasteur
pipettes into sample bottles and used within 12 h of preparation for
the assay. The rats were thereafter quickly dissected in the cold;
the liver and kidney excised, transferred into ice-cold 0.25 M
sucrose solution, blotted with clean tissue paper and then weighed.
Determination of biochemical parameters
The levels of sodium, potassium, calcium, magnesium, chloride,
inorganic phosphorus, urea, creatinine, total and conjugated
bilirubin, albumin, globulin, total protein, ALP, GGT, ALT, AST,
cholesterol, triacylglycerol, HDL-C and LDL-C were determined in
the serum using assay kits from Roche Diagnostics on Roche
Modular System (model P800) Mannhein, Germany, by adopting
the standard procedures described by Tietz et al. (1994). The
Horiba ABX 80 Diagnostics (ABX Pentra Montpellier, France) was
used for the determination of haematological parameters: red blood
cells (RBC), haemoglobin (Hb), packed cell volume (PCV), mean
corpuscular volume (MCV), mean corpuscular haemoglobin (MCH),
mean corpuscular haemoglobin concentration (MCHC), large
unstained cell (LUC), red cell distribution width (RCDW), white
blood cell (WBC), neutrophils, monocytes, lymphocytes, eosino-
phils, basophils and platelets. Organ to body weight ratio was
computed using the expression;
Weight of organ / Total weight of the animal) × 100.
Statistical analysis
Data were expressed as means of six replicates ± SD. They were
subjected to one way analysis of variance (ANOVA) and means
were separated by Duncan Multiple Range Test. Percentage data
were arcsine-transformed before analysis. Significant levels were
tested at P < 0.05.
RESULTS
The extract at all the doses investigated did not signifi-
cantly (P > 0.05) alter the computed liver- and kidney-
body weight ratios throughout the exposure period (Table 1).
The total bilirubin content of the animals was not altered
significantly (P > 0.05) until after 21 daily administration
when the 100 mg/kg body weight reduced the level of total
bilirubin in the animals. In contrast the 200 and 400
mg/kg body weight of the extract increased the total
bilirubin concentration during this period (Table 2).
Whereas the levels of conjugated bilirubin, total protein
and globulin were not significantly altered by the extract,
the albumin level increased significantly. The serum GGT
activity of the animals only increased after 21 daily doses
of the extract. In addition, the activities of serum ALP and
AST of the animals increased throughout the period of
exposure, whereas the serum ALT activity was not signi-

Afolayan et al. 533
Table 1. Effect of aqueous extract of Clematis brachiata leaves on the organ- body weight ratios of male Wistar rats. (n = 6, x ± SD).
Day 1
Day 7
Day 21
Test samples
Doses (mg/kg
body weight)
Liver
-
body we
ight
ratio (%)
Kidney
-
body
weight ratio (%)
Liver
-
body weight
ratio (%)
Kidney
-
body
weight ratio (%)
Liver
-
body weight
ratio (%)
Kidney
-
body
weight ratio (%)
Distilled water 0.5 ml 3.37 ± 0.68 0.72 ± 0.08 3.40 ± 0.29 0.70 ± 0.05 3.42 ± 0.19 0.69 ± 0.08
Aqueous extract 100 3.40 ± 0.70 0.68 ± 0.01 3.47 ± 0.20 0.65 ± 0.02 3.42 ± 0.32 0.65 ± 0.05
Aqueous extract
200 3.40 ± 0.54 0.70 ± 0.10 3.46 ± 0.17 0.64 ± 0.04 3.40 ± 0.30 0.67 ± 0.05
Aqueous extract 400 3.45 ± 0.43 0.69 ± 0.05 3.47 ± 0.17 0.67 ± 0.06 3.44 ± 0.12 0.69 ± 0.03
Test values are not significantly different (P > 0.05) from their respective control values.
Table 2. Effect of aqueous extract of Clematis brachiata leaves on the liver function indices of male Wistar rats. (n = 6, x ± SD).
Parameters
Day 1 Day 7 Day 21
Doses (mg/kg body weight)
Control 100 200 400 Control 100 200 400 Control 100 200 400
Total protein (g/L) 67.00 ±
1.73 64.33 ±
5.50 60.66 ±
8.52 66.00 ±
5.64 67.08 ±
0.94 63.66 ±
6.57 64.33 ±
6.08 66.66 ±
4.15 67.00 ±
1.03 67.00 ±
1.06 68.66 ±
1.57 66.00 ±
3.76
Albumin (mmol/L) 15.66 ±
0.81 17.33 ±
1.52* 18.0 ±
1.73* 19.66 ±
0.57* 15.71 ±
0.63 17.94 ±
0.00* 17.99 ±
0.40* 18.33 ±
0.17* 15.48 ±
0.14 19.33 ±
0.15* 18.66 ±
0.15* 19.00 ±
0.00*
Globulin (g) 47.34 ±
1.02 48.00 ±
0.16 46.66 ±
1.41 47.34 ±
0.83 47.40 ±
0.46 46.66 ±
0.44 47.33 ±
0.23 48.33 ±
0.21 47.34 ±
1.01 45.67 ±
3.11 46.00 ±
2.08 47.00 ±
0.86
Total bilirubin (µmol/L) 7.00 ±
0.28 7.16 ±
0.08 7.16 ±
0.12 7.26 ±
0.17 7.07 ±
0.13 7.00 ±
0.23 7.13 ±
0.09 7.09 ±
0.17 7.00 ±
0.30 5.00 ±
0.08* 10.00 ±
0.07* 11.66 ±
0.08*
Conjugated bilirubin
(µmol/L) 1.33 ±
0.07 1.33 ±
0.05 1.33 ±
0.07 1.33 ±
0.07 1.34 ±
0.04 1.31 ±
0.08 1.33 ±
0.08 1.35 ±
0.04 1.33 ±
0.07 1.32 ±
0.06 1.29 ±
0.09 1.33 ±
0.07
Alkaline phosphatase
(U/L) 315.01 ±
12.23 361.33 ±
9.88* 371.33 ±
7.76* 364.33 ±
9.92 314.20 ±
13.71 398.0 ±
7.44* 394.33 ±
7.61* 342.66 ±
5.73* 313.66 ±
16.96 335.33 ±
8.03* 411.66 ±
19.50* 367.00 ±
8.30*
Gamma glutamyl
transferase(U/L) 8.28 ±
0.18 8.00 ±
0.43 8.26 ±
0.15 8.33 ±
0.07 8.30 ±
0.06 8.30 ±
0.02 8.35 ±
0.03 8.22 ±
0.15 8.33 ±
0.08 11.66 ±
0.18* 12.33 ±
0.57* 14.66 ±
0.57*
Alanine
aminotransaminase (U/L) 51.00 ±
5.88 52.33 ±
5.29 51.66 ±
6.02 49.00 ±
9.58 51.0 ±
8.88 51.66 ±
6.50 53.66 ±
6.50 50.33 ±
7.71 51.0 ±
8.88 49.00 ±
8.88 51.33 ±
8.11 53.00 ±
8.00
Asparate
aminotransaminase (U/L) 168.13 ±
9.13 188.66 ±
7.26* 192.33 ±
6.89* 184.00 ±
4.28* 165.77 ±
11.86 195.0 ±
3.64* 185.00 ±
4.58* 186.33 ±
3.61* 168.13 ±
9.13 182.33 ±
4.09* 197.00 ±
7.21* 195.66 ±
3.08*
Test values carrying asterisks for each parameter and day are significantly different from their controls (p < 0.05) while those without asterisk are not significantly different (P > 0.05).
ficantly altered by the extract throughout the same
period (Table 2). The extract did not significantly
alter the sodium, potassium, chloride, inorganic
phosphorus and calcium ions of the animals (Table
3). Again, the increase in creatinine levels did not
manifest until after seven daily doses. The extract at
all the doses investigated increased urea levels of
the animals throughout the experimental period. In
contrast, magnesium ions were reduced only at 200

534 Afr. J. Pharm. Pharmacol.
Table 3. Effect of aqueous extract of Clematis brachiata leaves on the kidney function indices of male Wistar rats. (n = 6, x ± SD).
Parameters
Day 1
Day 7
Day 21
Doses (mg/kg body weight)
Control
100
200
400
Control
100
200
400
Control
100
200
400
Sodium (mmol/L) 140.00 ±
1.00 140.33 ±
1.08 139.33 ±
2.08 138.66 ±
4.57 140 ±
1.00 139.66 ±
1.15 140.00 ±
0.00 139.00 ±
1.00 140.00 ±
1.00 137.00 ±
4.00 139.33 ±
0.57 138.66 ±
1.52
Potassium (mmol/L) 5.50 ±
0.26 5.10 ±
0.15 5.40 ±
0.16 5.50 ±
0.26 5.50 ±
0.26 5.60 ±
0.40 5.40 ±
0.36 5.53 ±
0.32 5.50 ±
0.26 5.36 ±
0.45 5.43 ±
0.15 5.36 ±
0.35
Calcium (mmol/L) 2.52 ±
0.05 2.46 ±
0.05 2.45 ±
0.03 2.43 ±
0.01 2.52 ±
0.05 2.48 ±
0.05 2.43 ±
0.54 2.49 ±
0.04 2.52 ±
0.05 2.48 ±
0.04 2.47
±0.04 2.47 ±
0.09
Magnesium (mmol/L) 1.24± 0.01 1.23 ±
0.03 0.93 ±
0.02* 0.90 ±
0.06* 1.24± 0.01 1.21 ±
0.02 0.81 ±
0.06* 0.93 ±
0.04* 1.24± 0.03 1.20 ±
0.04 0.74 ±
0.02* 0.76 ±
0.02*
Chloride (mmol/L) 103.00 ±
2.64 104.00 ±
1.73 105.33 ±
1.08 104.00 ±
2.00 103.20 ±
1.75 102.66 ±
4.57 105.66 ±
2.51 104.66 ±
1.52 103.00 ±
2.64 103.66 ±
2.08 102.33 ±
1.52 104.66 ±
0.57
Inorganic
Phosphorus (mmol/L) 3.20 ±
0.19 3.20±
0.17 3.03 ±
0.35 2.90 ±
0.46 3.20 ±
0.10 3.33 ±
0.06 2.96 ±
0.40 2.97 ±
0.37 3.20 ±
0.15 3.35 ±
0.04 3.10 ±
0.30 3.00 ±
0.37
Urea (mmol/L) 4.50 ±
0.37 6.13 ±
0.25* 6.36 ±
0.15* 6.60 ±
0.16* 4.53 ±
0.27 6.10 ±
0.10* 5.43 ±
0.10* 5.60 ±
0.19* 4.40 ±
0.24 6.80 ±
0.30* 6.83 ±
0.20* 7.33 ±
0.40*
Creatinine (mmol/L) 44.33 ±
5.77 40.33 ±
9.07 42.66 ±
4.04 46.66 ±
5.50 44.33 ±
5.77 42.00 ±
6.46 45.66 ±
3.78 45.33 ±
4.04 44.33 ±
5.77 52.00 ±
0.00* 51.66 ±
2.08* 58.00 ±
1.60*
Uric acid (mmol/L) 0.11 ±
0.01 0.12 ±
0.01 0.13 ±
0.01 0.11 ±
0.01 0.11 ±
0.01 0.07 ±
0.01* 0.07 ±
0.00* 0.08 ±
0.00* 0.11 ±
0.00 0.08 ±
0.00* 0.07 ±
0.00* 0.07 ±
0.00*
Test values carrying asterisks for each parameter and day are significantly different from their controls (p < 0.05) while those without asterisk are not significantly different (P > 0.05).
and 400 mg/kg body weight of the extract. The
reduction in serum uric acid became manifest only
after seven daily doses and was sustained
throughout the exposure period.
Administration of the extract at all the doses for
seven days significantly increased (P < 0.05) the
serum cholesterol concentration of the animals
(Table 4). The increase washowever not sustained
beyond this point as the cholesterol values of the
extract treated animals compared well with the
control values. The increase in triacylglycerol content
of the animals by single administration of the extract
was not sustained beyond the first day as the
concentration of the lipid was reduced there-after
by the extract. The HDL-C increased throughout
the exposure period, whereas the LDL-C levels
remained significantly unaltered. In contrast, the
extract reduced the computed atherogenic index
(Table 4).
Although, the extract did not significantly alter the
levels of RBC, Hb, PCV, MCV, MCH, MCHC and
RCDW, the levels of LUC, monocytes, eosinophils
and basophils increased significantly throughout the
exposure period (Table 5). Again, the neutrophils
also increased throughout the experimental period
except in the animals administered with the single
dose of 100 mg/kg body weight. In contrast, the
extract reduced the WBC, platelet and lym-
phocytes right from the commencement of the
administration and was sustained throughout the
experimental period (Table 5).
DISCUSSION
The assessment of biochemical parameters such
as hematological, liver and kidney function as well
as lipid profile of animals following the adminis-
tration of chemical compounds, including plant
extract, plays significant role in the evaluation of
toxicity risk or safety of such compounds
(Maaroufi et al., 1996; Amresh et al., 2008).
Biochemical indices of organ function if altered will
impair the normal functioning of the organs
(Afolayan and Yakubu, 2009). Organ-body weight
ratio can be used to indicate organ swelling,
atrophy or hypertophy (Amresh et al., 2008).
Therefore, the lack of an effect on the liver and
kidney body weight ratio in this study suggests
that the extract did not cause inflammation or
constriction at the cellular levels of the rat organs.
This agrees with the findings of Amresh et al. (2008)
where Cissampelos pareira did not produce any

Afolayan et al. 535
Table 4. Effect of aqueous extract of Clematis brachiata leaves on the serum lipid profile of Wistar rats. n = 6, x ± SD.
Day 1
Day 7
Day 21
Doses (mg/kg body weight)
Para
meters
Control
100
200
400
Control
100
200
400
Control
100
200
400
Cholesterol (mmol/L) 1.23 ±
0.15 1.80 ±
0.20* 1.56 ±
0.08* 1.66 ±
0.05* 1.20 ±
0.15 1.43 ±
0.07* 1.43 ±
0.05* 1.53 ±
0.05* 1.23 ±
0.15 1.26
±0.15 1.23 ±
0.11 1.20 ±
0.10
Triacylglycerol (mmol/L) 1.06 ±
0.04 1.30 ±
0.05* 1.80 ±
0.12* 1.43 ±
0.09* 1.00 ±
0.06 0.76 ±
0.05* 0.80 ±
0.00* 0.66 ±
0.04* 1.01 ±
0.03 0.73 ±
0.03* 0.80 ±
0.03* 0.68 ±
0.08*
High-density lipoprotein
(mmol/L) 0.83 ±
0.05 1.43 ±
0.04* 1.20 ±
0.03* 1.20 ±
0.06* 0.84 ±
0.05 1.30 ±
0.07* 1.30 ±
0.00* 1.17 ±
0.06* 0.80 ±
0.05 1.20 ±
0.07* 1.20 ±
0.03* 1.10 ±
0.06*
Low-density lipoprotein
(mmol/L) 0.51 ±
0.05 0.53 ±
0.02 0.52 ±
0.06 0.50 ±
0.08 0.51 ±
0.06 0.50 ±
0.03 0.53 ±
0.02 0.51 ±
0.06 0.52 ±
0.03 0.52 ±
0.06 0.51 ±
0.07 0.53 ±
0.04
Atherogenic index (LDL-
C/HDL-C) 0.61 ±
0.04 0.37 ±
0.06* 0.43 ±
0.02* 0.42 ±
0.02* 0.61 ±
0.07 0.38 ±
0.04* 0.41 ±
0.07* 0.51 ±
0.02* 0.65 ±
0.05 0.43 ±
0.03* 0.43 ±
0.05* 0.48 ±
0.01*
Test values carrying asterisks for each parameter and day are significantly different from their controls (p < 0.05) while those without asterisk are not significantly
different (P > 0.05).
Table 5. Effect of the aqueous extract of Clematis brachiata leaves on the haematological parameters of male Wistar rats. (n = 6, x ± SD).
Parameters
Day 1
Day 7
Day 21
Doses (mg/kg body weight)
Control
100
200
400
Control
100
200
400
Control
100
200
400
WBC (x 10
9
/l) 11.80 ±
0.05 8.13±
0.12* 6.89 ±
0.06* 8.60 ±
0.10* 11.72 ±
0.10 7.55 ±
0.06* 7.35 ±
0.07* 9.14 ±
0.01* 11.58 ±
0.10 8.18 ±
0.06* 7.25 ±
0.05* 7.50 ±
0.03*
RBC (x 10
12
/l) 7.50 ± 0.31 7.54 ±
0.18 7.68 ±
0.04 7.47 ±
0.29 7.57 ± 0.26 7.51 ±
0.27 7.38 ±
0.37 7.48 ±
0.28 7.50 ±
0.31 7.55 ±
0.20 7.41 ±
0.29 7.48 ±
0.22
Hb (g/dl) 13.83 ±
0.35 13.43 ±
0.70 13.5 ±
0.81 13.73 ±
0.47 13.83 ±
0.35 14.26 ±
0.41 13.9 ±
0.43 14.56±
0.45 13.83 ±
0.35 13.76 ±
0.46 14.33 ±
0.49 14.66±
1.15
PCV (l/l) 0.47 ±
0.005 0.45 ±
0.03 0.47 ±
0.02 0.47 ±
0.02 0.47 ±
0.005 0.49 ±
0.01 0.47 ±
0.01 0.48 ±
0.005 0.47 ±
0.01 0.46 ±
0.01 0.47 ±
0.02 0.48 ±
0.02
MCV (fl) 63.23 ±
2.98 63.30 ±
3.13 62.50 ±
2.04 62.73 ±
0.66 63.23 ±
2.98 63.36 ±
1.02 62.13 ±
1.33 61.46 ±
3.40 63.23 ±
2.98 63.60 ±
0.87 64.63 ±
0.40 62.90 ±
1.86
MCH (pg) 18.46 ±
1.10 18.86 ±
1.13 17.6 ±
0.43 18.36 ±
0.15 18.46 ±
1.10 18.5 ±
0.43 18.8 ±
0.43 18.46 ±
0.25 18.46 ±
1.10 19.06 ±
0.23 19.06 ±
1.2 18.56 ±
1.00
MCHC (g/dL) 29.20 ±
0.36 29.90 ±
0.43 28.60 ±
0.75 29.26 ±
0.40 29.20 ±
0.36 29.13 ±
0.28 29.30 ±
0.10 30.03 ±
0.17 29.20 ±
0.36 29.96 ±
0.27 30.03 ±
0.41 29.46 ±
0.43
RCDW (%) 14.03 ±
1.30 14.09 ±
1.01 14.03 ±
0.95 14.03 ±
1.22 14.03 ±
1.30 14.56 ±
0.76 14.26 ±
1.00 14.23 ±
0.80 14.03 ±
1.30 14.96 ±
0.58 14.76 ±
0.77 14.33 ±
0.45
Platelet (x
109/l) 829.33±
15.73 446.66 ±
4.77* 702.66 ±
5.61* 706.00 ±
4.34* 827.45±
15.73 709.00 ±
5.86* 673.66 ±
8.14* 729.00 ±
7.09* 836.25±
8.73 659.66 ±
8.85* 683.66 ±
7.79* 722.00 ±
4.53*

536 Afr. J. Pharm. Pharmacol.
Table 5 Contd.
Neutrophils (%) 9.26 ±
0.50 10.86 ±
0.05 15.40±
0.13* 13.36 ±
0.06* 9.26 ±
0.44 11.9 ±
0.09 * 13.33 ±
0.04* 12.13 ±
0.07* 9.26 ±
0.42 16.86 ±
0.18* 14.30 ±
0.18* 18.53 ±
0.15*
Monocytes (%) 14.36 ±
0.76 20.10 ±
1.67* 29.2 ±
1.32* 25.73 ±
1.80* 14.30 ±
0.69 22.76 ±
1.40* 26.33 ±
1.80* 20.20±
1.69* 14.46 ±
0.46 21.33 ±
2.60* 20.00 ±
1.42* 19.86 ±
1.15*
Lymphocytes
(%) 66.43 ±
2.41 50.63 ±
1.42* 48.4 ±
2.20* 45.5 ±
2.21* 63.43 ±
4.41 50.56 ±
3.51* 47.03 ±
2.33* 48.16 ±
1.17* 66.61 ±
1.41 52.0 ±
0.50* 47.13 ±
1.81* 48.26 ±
1.35*
LUC (%) 7.03 ±
0.26 10.96 ±
0.14* 14.40 ±
0.22* 12.43 ±
0.14* 7.05 ±
0.24 12.00 ±
0.09* 10.86 ±
0.07* 11.13 ±
0.11* 7.07 ±
0.21 11.76 ±
0.28* 10.76±
0.05* 10.10 ±
0.10*
Eosinophils (%) 0.60 ±
0.03 2.56 ±
0.08* 1.83 ±
0.03* 2.63 ±
0.07* 0.63 ±
0.03 2.33 ±
0.06* 1.53 ±
0.05* 1.76 ±
0.25* 0.61±
0.02 1.56 ±
0.15* 2.40 ±
0.02* 2.76 ±
0.08*
Basophils (%) 0.26 ±
0.02 0.90 ±
0.03* 0.36 ±
0.02* 0.36 ±
0.00* 0.22 ±
0.04 0.46 ±
0.03* 0.86 ±
0.08* 0.70±
0.02* 0.25 ±
0.02 0.43 ±
0.05* 0.40 ±
0.07* 0.46 ±
0.02*
Test values carrying asterisks for each parameter and day is significantly different from their controls (p < 0.05) while those without asterisk are not significantly different (P > 0.05).
effect on the organ-body weight ratio of the animals.
Albumin, total bilirubin, globulin and the totality of
the molecules can be used to assess the
secretory ability of the liver (Yakubu et al., 2003).
The extract exhibited selective toxicity on the liver
functional endpoints since only albumin and total
bilirubin were affected whereas total protein,
conjugated bilirubin and globulin were not altered
significantly. Bilirubin is an important catabolic
product of blood with biological and diagnostic
values (Chowdhury et al., 1989). The alterations in
the extract in the levels of total bilirubin prior to the
end of experimental period implies that adverse
effect have occurred on the normal functioning of
the liver. The elevated levels of serum albumin
also suggest adverse effect of the extract on the
secretory ability of the liver and hence the normal
functioning of the organ.
There are many enzymes found in the serum
that did not originate from the extracellular fluid.
During tissue damage, some of these enzymes
find their way into the serum through leakage
arising from altered membrane permeability (Wills,
1985). Serum enzyme measurements are there-
fore a valuable tool in clinical diagnosis, providing
information on the effect and nature of patho-
logical damage to any tissue. ALP and GGT
are ectoenzymes of the plasma membrane where-
as ALT and AST are of the cytosol (Shahjahan et
al., 2004). Damage to structural integrity of tissues
is always reflected by increase in some of these
enzymes in the serum. Therefore, the increase in
serum ALP and GGT activity in this study could
possibly imply damage to the plasma membrane.
This was however supported by similar increase
in the serum AST. The lack of an effect on the
activity of serum ALT implied selective toxicity of
the extract on the cytosolic enzyme. Alteration in
the level of ALP, GGT and AST will have cones-
quential effect on the adequate transportation of
required ions across the cell membrane, gluta-
thione metabolism as well as metabolism and
regulation of amino acids respectively.
Renal function indices such as serum electro-
lytes, urea, creatinine and uric acid can be used to
evaluate the functional capacity of the nephrons of
animals (Yakubu et al., 2003). In this study, the
levels of sodium, potassium, chloride, inorganic
phosphorus and calcium ions were not signify-
cantly altered. This may suggest selective and
and dose specific effect of the extract of C.
brachiata leaves. Creatinine, urea and uric acid
are major catabolic products of muscle, protein
and purine metabolism respectively. The increase
in the serum creatinine and urea contents of the
animals may be attributed to compromising the
renal functional capacity. The extract might have
interfered with creatinine and urea metabolism
leading to increased synthesis or the tissue might
have compromised all or part of its functional
capacity in tubular and glomerular excretion (Zilva et
al., 1991). Similarly, the reduction in the serum urea
content of the animals suggests abnormality in the
physiological excretion of urea.
Alterations in the concentration of major lipids of
animals such as cholesterol, high and low-density
lipoprotein cholesterols, and triglycerides can give
useful information on the lipid metabolism as well as
predisposition of the animals to cardiovascular risk
(Yakubu et al., 2008). The elevated level of serum
cholesterol following the administration of the
extract may be attributed to increase in the
concentration of acetyl CoA arising probably from
enhanced -oxidation of fatty acid (Rang et al.,
1995). High blood cholesterol concentration is one

one of the important risk factors of cardiovascular
disease (Treasure et al., 1995). In this study, the levels of
serum cholesterol which compared well with the control
by the end of the experimental period suggests that the
extract may not pose threat to the well being of the
animals notwithstanding the initial increase. Similarly, the
reduction in the levels of triacylglycerol may be adduced
to reduced lipolysis. HDL-C is considered to have anti-
atherogenic properties. Therefore, increase in HDL-C
may be clinically beneficial to the animals since it will
reduce cardiovascular risk. The lack of an effect on the
serum LDL-C of the animals further buttress the selective
effect of the extract. It has been reported that cardio-
vascular diseased patients had markedly elevated levels
of triacylglycerol but reduced HDL-C. Since the elevation
in triacylglycerol in this study was not accompanied by
reduction in HDL-C, it is unlikely that the extract may
potentate cardiovascular risk. In addition, the reduction in
the computed atherogenic index also lends support to the
non-cardiovascular risk of the extract.
The lack of an effect on the RBC suggests that the
extract did not alter the balance between the rate of
production and destruction of the blood corpuscles. Since
MCHC, MCH and MCV relate to individual red blood cells
while Hb, RBC, PCV and RCDW are associated with total
population of red blood cells, the lack of an effect on
these indices implies that neither the incorporation of
haemoglobin into red blood cells nor the morphology and
osmotic fragility of the red blood cells was affected
(Adebayo et al., 2005). Therefore, it is unlikely that the
extract will affect the oxygen-carrying capacity of each of
the RBC as well as the total population.
Interestingly, WBC and indices relating to it were
significantly altered during the experimental period. The
decrease in WBC may be due to impairment in the rate of
entrance of the haematological parameter into the blood
from the bone marrow and an enhanced rate of removal
from circulation. The elevated level of neutrophils may
stimulate phagocytosis in the animals. In contrast,
reduction in lymphocytes observed in this study suggests
adverse effect on the effector cells of the immune
system. Since monocytes, eosinophils and basophils are
necessary for normal functioning of the immune system,
the alterations in these blood indices has suggested
challenges on the immune system. Platelets when pre-
sent in sufficient size, number and function are involved
in the process of normal coagulation of the blood (William
and Levine, 1982). Therefore, the reduction in platelet
levels by the extract may be attributed to diminished
effect on thrombopoietin (Li et al., 1999). The altered
levels of WBC and factors relating to it by the extract,
accompanied with lack of effect on RBC and factors
relating to it suggest selective and localized stimulatory
effect on the bone marrow. This may be an indication of
localized systemic toxicity of the extract which may
adversely affect the normal functioning of the WBC and
its related indices. This agreed with the findings of
Afolayan et al. 537
Adebayo et al. (2005) on ethanolic extract of Bougain-
villea spectabilis leaves.
Our present findings have shown that aqueous extract
of C. brachiata is not completely ‘safe’ at the doses
investigated. While it appears to be clinically beneficial to
the animals as it may not predispose to cardiovascular
risk, it has the tendency to selectively affect the normal
functioning of the liver and kidney of the animals. In
addition, the heamatotoxic effect of the extract may be
localized to the WBC and factors relating to it.
ACKNOWLEDGEMENT
This research was supported with grants from Govan
Mbeki Research and Development Centre, University of
Fort Hare and the National Research Foundation, South
Africa. The authors are also grateful to BCSIR,
Bangladesh and University of Ilorin, Nigeria for
supporting the Postdoctoral Fellowship Programmes of
Drs. M. Mostafa and M. T. Yakubu respectively.
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