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Biokemistri
Nigerian Society for Experimental Biology
ISSN: 0795-8080
Vol. 20, Num. 2, 2008, pp. 47-52
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Biokemistri, Vol. 20, No. 2, Dec, 2008, pp. 47-52
The effect of Hibiscus
sabdariffa calyx extract on cisplatin-induced tissue damage in rats
Tebekeme Okoko1* and Ibiba F. Oruambo2
1Biochemistry Division, Department of Chemical
Sciences, Niger Delta University, P.M.B 71, Wilberforce Island, Bayelsa State,
Nigeria
2Department of Chemistry, Rivers State
University of Science and Technology, P.M.B 5080, Port Harcourt, Rivers State,
Nigeria
*To whom correspondence
should be addressed. E-mail: tebebuddy@yahoo.com , Tel:
+2348051069342
Received 14 December 2008
Code Number: bk08008
Abstract
The effect of Hibiscus
sabdariffa calyx extract on cisplatin-induced tissue damage was studied. A
total of twenty rats were used for the study and split into four groups of five
rats per group-group I, II, III, and IV. Tissue damage was induced in rats of
groups II, III, and IV by a single intraperitoneal administration of cisplatin
(5 mg/kg b.w.). Four days later, rats in groups III and IV were given 200 mg/kg
b.w. and 300 mg/kg b.w respectively of the Hibiscus sabdariffa calyx
extract once daily for another five days. Rats in group I were untreated
controls. Tissue damage was later assessed in sera by measuring the levels of
alanine aminotransferase, aspartate aminotransferase, urea, and creatinine.
Thiobarbituric acid reactive species, catalase, and reduced glutathione levels
were also measured in the livers and kidneys. The results reveal that the
administration of cisplatin alone resulted to a significant increase in the
levels of the serum markers over controls (p< 0.05). Cisplatin also caused a
significant decrease in catalase activity, and also caused a significant
reduction in the levels of reduced glutathione in the liver and kidney over
controls (p< 0.05). Cisplatin also caused a significant increase in the
levels of thiobarbituric acid reactive species in the liver (p<
0.05). All the markers assessed were brought to near control levels when the Hibiscus
sabdariffa extract was given to the rats with the dose of 300 mg/kg b.w.
possessing a higher activity. The results show that the Hibiscus sabdariffa
extract ameliorated cisplatin-induced tissue damage in rats which may be dose-dependent.
Keywords:Hibiscus sabdariffa, urea, creatinine,
cisplatin, glutathione
INTRODUCTION
The
consumption of botanicals as complementary/ alternative medicine has been
encouraged because they are relatively cheap and coupled with the belief that
they could significantly contribute to the improvement of human health in terms
of cure and the prevention of various human disorders in addition to the less frequent
side effects reported when compared to modern medicine1. Among these
botanicals of focus is Hibiscus sabdariffa Linn which is known as the
Red Sorrel in English and the Zobo plant in Nigeria. Hibiscus sabdariffa
Linn is an annual dicotyledonous herbaceous tropical plant that belongs to the
family Malvaceae. The tender leaves of the plant are eaten as salad and curries
traditionally and as a remedy for various ailments such as liver disorders,
hypertension, pyrexia2, and as a diuretic, emollient, and purgative3.
The infusions of the calyces have been shown to reduce intestinal transit,
anti-inflammatory and anti-mutagenic effects4-6 among others. Duke
and Atchley7 revealed that the calyces of the plant (the principally
utilized part) contain 14 mg ascorbate, 300 μg β-carotene, 1.9 g protein, 12.3 g total carbohydrate, 2.7 mg Iron per
100 g dry weight. In addition to these nutrients, various phytochemicals have
also been identified in the calyces which include saponins, tannins,
anthocyanins, cyanogenic glycosides, flavones, and polyphenols8,9.
Despite these established bioactivities, it is a common belief that the plant
is still underutilised as it is obvious that it could also possess other known
activities due to the phytochemicals identified. Based of these, this work
focused on the effect of the methanolic extract of the calyces of Hibiscus
sabdariffa on cisplatin-induced oxidative damage in rat. Cisplatin (also
called cisplatinum and carboplatin) is an anticancer drug but its cytotoxic
potential is a major limiting factor of its chemotherapy as its use causes
various tissue injuries notable among them is dose related kidney damage10.
MATERIALS
AND METHODS
Animals
Twenty
albino rats (Rattus novergicus) of both sexes (100-115 g) were purchased
from the Nigerian Institute of Medical Research, Yaba, Lagos and kept in the
animal housing facility of the Department of Biological Sciences of the Niger Delta University. They were allowed unlimited access to tap water and growers mash
ad libitum housed in standard rat cages.
Plant
material and preparation of extract
The
calyces of matured Hibiscus sabdariffa were collected fresh from a
garden and sun-dried. The dried calyces were subsequently ground using a
blender to fine powder. Five hundred grams of the powder was extracted using
light petroleum in a soxhlet extractor for 24 hours. A second extraction was
made using 80 % methanol and concentrated using a rotary evaporator at 400C
and finally dried in a vacuum dessicator at 400C. The resulting
residue which weighed 8.13 g (recovery 1.63%) was later stored under -40C
until required. However, a 10 mg/ml solution of the extract was prepared in
distilled water before administration to the rats.
Treatment
of animals
The
rats were split into four groups of five rats each after matching the weights.
They were acclimatized to laboratory conditions for five days before
commencement of treatment. Tissue damage was induced in rats of groups II, III,
and IV by a single intraperitoneal-administration of cisplatin (5 mg/kg body
weight). Four days later, doses of 200 mg/kg b.w., and 300 mg/kg b.w. of the Hibiscus
sabdariffa calyx extract was administered to rats in group III, and group
IV respectively through the oral route using a gavage needle once daily for
five days. Rats in group II were given sterile water in place of the Hibiscus
sabdariffa extract. Rats in group I were untreated controls. They were all
allowed unlimited access to tap water and growers mash.
Collection
of tissues
Twenty
four hours after the last administration of the extract, each rat was subjected
to light anaesthesia in a urethane saturated chamber, and dissected. The
thoracic region was exposed and blood was drawn through direct cardiac
punctures and delivered into sample containers having no anticoagulant. Blood
samples were allowed to clot for 20 minutes and centrifuged at 4000 rpm for 15
minutes. Sera were collected using micropipettes and analysed. The livers and
kidneys were also excised immediately and washed in cold saline. Ten percent tissue
homogenates were prepared in phosphate buffer (0.05M, pH 7.25). Perinuclear
fractions were obtained after centrifuging homogenates at 1500 rpm for 20
minutes using a centrifuge.
Biochemical
analysis
Alanine
aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in
sera as described11. Creatinine and urea were determined in serum by
the modified Jaffe, and the modified Berthelot-Searcy enzymatic methods
respectively using assay kits obtained from QCA Spain. Catalase in liver and
kidney was measured in tissue homogenates following the method of Aebi12
with slight modifications. Briefly, 0.1 ml of perinuclear fraction was added to
1.5 ml of phosphate buffer (0.05M, pH 7.25), and 1.4 ml of distilled water. The
reaction was initiated by the addition of 0.1 ml of 0.01M H2O2.
Amount of H2O2 consumed was determined by measuring
absorbance at 240 nm after 30 s. The enzyme activity was expressed as k/g
protein. Thiobarbituric acid reactive species (TBARS) were measured in the
perinuclear fractions as reported13. Reduced glutathione (GSH) was
also measured in the perinuclear fractions as previously described14.
Statistical
analysis
Results
of representative values were expressed as mean ± standard error of the mean
(S.E.M). Comparisons between two groups were made using a two-tailed Students t-test.
P values below 0.05 were considered statistically significant.
RESULTS
The
levels of ALT, AST, creatinine and urea levels in sera are shown in Table 1.
When the rats were treated with cisplatin alone, there was a significant
increase in the levels of ALT, AST, creatinine and urea in group II over
control rats (p < 0.05), while the levels of these markers in groups
III, and IV were significantly reduced close to control levels after
administration of the extract (p < 0.05), with group IV having the
lowest values. Table 2 shows the levels of GSH, catalase, and TBARS in the
livers and kidneys of the rats.
Table
1: Effect of pre-treatment with or
without extract on some serum enzymes of hepatotoxicity, and non-enzymatic
markers of kidney dysfunction
Group |
ALT (U/L) |
AST (U/L) |
Creatinine(mg/L) |
Urea (mg/L) |
I |
15.6±0.02 |
28.8±0.03 |
16.4±0.54 |
126.7±1.65 |
II |
24.8±0.17a |
52.4±2.82a |
27.3±1.43a |
293.6±7.32a |
III |
18.3±0.44b |
33.5±0.57ab |
17.8b ±
0.21 |
137.8±11.21ab |
IV |
15.8±1.22b |
29.2±1.45bc |
16.2 ± 1.04b |
128.2±9.21abc |
Values
presented as the means ± S.E.M of readings from 5 individual rat sera (a
significantly different from Group I; b significantly
different from group II; c significantly different from group
III, p < 0.05)
Table
2: Effect of pre-treatment with or
without extract on the glutathione, catalase, and TBARS levels in liver and
kidney
Tissues and
Groups |
TBARS
(μg/mg protein) |
GSH
(mg/g protein) |
Catalase
(k/g protein) |
Liver I |
1.13±0.00 |
8.93±0.54 |
31.72±1.42 |
II |
2.67±0.12a |
6.26±0.07a |
17.44±0.23a |
III |
1.25±0.01b |
7.43±0.13ab |
23.17±0.65ab |
IV |
1.23±0.14b |
9.53±0.48bc |
27.56±1.21abc |
Kidney I |
0.50±0.01 |
2.73±0.06 |
105.76±8.55 |
II |
0.49±0.05 |
1.63±0.03a |
68.12±3.28a |
III |
0.49±0.03 |
2.74±0.01b |
87.35± 9.71ab |
IV |
0.51±0.03 |
2.87±0.22b |
93.58±7.76abc |
Values presented as the means ± S.E.M of readings from
perinuclear fractions from homogenates from 5 individual rat tissue (a
significantly different from Group I; b significantly
different from group II, c significantly different from group
III, p < 0.05)
When
the rats were treated with only cisplatin, there was a significant reduction in
the levels of GSH and catalase in both liver and kidney over controls (p < 0.05), while these reduced levels were increased close to control levels
after the extract was given to the rats (p < 0.05) with the group
given the dose of 300 mg/kg b.w. having the highest elevations. The level of
TBARS in liver was significantly higher when the rats were treated with
cisplatin alone (p < 0.05). However, the level of the marker was
significantly reduced when the extract was administered to the rats (p <
0.05). The levels of TBARS in the kidneys did not show any significant
difference between the groups (p > 0.05).
DISCUSSION
Cisplatin
is a cell cycle phase non-specific cytotoxic agent15 and it is this
property that has been exploited in the treatment of various cancers. As with
other drugs, its lack of specificity for neoplastic cells makes it seriously
limiting. A major consequence is the reported nephrotoxicity and hepatotoxicity16.
Though the toxicokinetics of cisplatin has not been fully known, lipid
peroxidation may be involved in its adverse effects. The principal active
metabolite is free filterable platinum17 which has the propensity of
adding to double bonds of membrane lipids, form adducts with proteins, DNA, and
can also form stable products with other macromolecules.
Chemotherapeutic
levels known to induce mild kidney dysfunction in rats is thought to be a
single dose of 5 mg/kg body weight which peaks about 3 5days10,18
thus the choice of a single dose of 5 mg/kg body weight, and the four days
exposure before the administration of the extract for the present study.
The
increase in serum creatinine and urea levels in the rats after the
intraperitoneal administration of cisplatin (group II) shows that cisplatin
induced mild kidney dysfunction which is in accordance with previous studies19.
This damage to the kidney was further experienced with the depletion in the
levels of GSH and catalase in the kidney.
The
increase in the levels of ALT and AST in the sera of rats in group II also show
that at that dose (5 mg/kg b.w.), cisplatin also damages the liver which was
further evidenced by the elevated level of TBARS, and the reduction in the
levels of GSH and catalase in the perinuclear fraction of the rat livers. The
antioxidant enzyme catalase and GSH with other peroxidases constitute a
supportive team against reactive oxygen species20,21 hence the
depletion of GSH and catalase could enhance lipid peroxidation22.
The
reduction in the levels of TBARS, ALT, AST, and the elevation of catalase and
GSH in the liver close to control levels in rats of groups III and IV clearly
show that the Hibiscus sabdariffa extract reduced the liver damage
induced by cisplatin. Also the reduction in the levels of serum creatinine,
urea and the elevation of the levels of kidney GSH and catalase in rats of
group III, and IV also indicate that the extract also reduced cisplatin induced
kidney dysfunction. From the results, it was obvious that the bioactivity of
the extract is dependent on the dose administered. The mechanism of this action
is not fully understood. However, it could be suggested that the extract
reduced the lipid peroxidation induced by cisplatin. It could also be suggested
that the various phytochemicals inherent in the extract acted synergistically
to sequester the free filterable platinum hence making it less available for
cellular damage. The calyces of Hibiscus sabdariffa are rich in vitamin
C, hence the reduction in the liver and kidney damages may be ascribed partly
to this natural antioxidant which also functions in the conversion of
α-tocopheroxy radical to α-tocopherol23. The extract may
also reduce the involvement of Ca2+ dependent permeabilisation of
renal cortex mitochondria as it has been shown that there is a possible
involvement of Ca2+ dependent permeabilisation of renal cortex
mitochondria in cisplatin nephrotoxicity19. Anthocyanins are
ubiquitous in the calyces of the plant thus this property of the extract may be
attributed partly to these flavonoids which are known to be potent antioxidants
and free radical scavengers25.
Further
work should focus on elucidating the actual protective mode of the extract.
Since there are various phytochemicals in the calyces, proper fractionation of
the extract should be done and each fraction subjected to both in vitro and in
vivo studies since some notable bioactivities of terpenes have also been
reported25 in addition to the fact that flavonoids also possess
pro-oxidant activities 26. Progress in this area will also extend
the borders of even cisplatin chemotherapy.
The
occurrence of Hibiscus sabdariffa Linn in the biosphere is natures generosity
in providing mankind with cheap and natural bioactive materials. Thus the
exploitation of this natural gift in the production of neutraceuticals is
recommended.
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© 2008 Nigerian Society for
Experimental Biology
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