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- Complementary Medicine -
Effects of Roselle and Ginger on cisplatin-induced reproductive toxicity in rats
Amr Amin, AlaaEldin A. Hamza
Department of Biology, United Arab Emirates University, Al-Ain 17551, United Arab Emirates
Aim: To evaluate the protective effects of
Hibiscus sabdariffa (Roselle) and Zingiber officinale
(Ginger) against cisplatin-induced reproductive toxicity in rats and to study the mechanisms underlying these effects.
Methods: Ethanol extracts of
H. sabdariffa or
Z. officinale [1 g/(kg·day)] were given p.o. to male albino rats for 26 days,
which began 21 days before a single cisplatin i.p. injection (10 mg/kg body
weight). Results: Extracts of
H. sabdariffa and
Z. officinale reduced the extent of cisplatin-induced sperm abnormality and enhanced sperm motility. Both
extracts restored the control level of malondialdehyde (MDA) (lipid peroxidation marker) in the cisplatin-treated testis.
The cisplatin injection induced decline in the levels of superoxide dismutase (SOD), reduced glutathione (GSH) and
catalase (CAT) were significantly reversed to control levels in groups where cisplatin was preceded by the
administration of either H. sabdariffa
or Z. officinale. Conclusion: Both
H. sabdariffa and
Z. officinale treatment increased the activities of testicular antioxidant enzymes and restored sperm motility of cisplatin-treated rats. The protective
effects of tested plants are, therefore, suggested to be mediated by their potent antioxidant
activities. (Asian J Androl 2006 Sep; 8: 607_612)
Keywords: cisplatin; testicular toxicity; Hibiscus sabdariffa; Zingiber officinale; herb; sperm motility; reproductive toxicity
Correspondence to: Dr Amr. Amin, Department of Biology, United Arab Emirates University, Al-Ain 17555, United Arab Emirates.
Tel: +971-3-7134-389, Fax: +971-3-7671-291
Accepted 2006-01-09 Received 2006-02-27
Because of the relative spermiotoxicity of cisplatin (cis-diamminedichloroplatinum [II]), almost all patients under
chemotherapy show temporary or permanent azoospermia. The damage to both spermatogenesis and testicular
endocrine function can be temporary or permanent based on the applied dose of cisplatin . Within days of cisplatin
injection, animals undergo severe testicular damage, which is characterized by spermatogenic damage, germ cell
apoptosis, Leydig cell dysfunction and testicular steroidogenic disorder [2, 3].
Oxidative stress mediates cisplatin-induced nephrotoxicity . The administration of antioxidants such as vitamin
C reverses different cisplatin-associated side effects . Many natural products of plant origin protect against
drug-induced toxicity [4, 5]. However, to date, virtually no medicinal plant has been described to relieve the
cisplatin-induced reproductive side effects accompanied by chemotherapy applied to cancer patients.
Roselle (Hibiscus sabdariffa L., family: Malvaceae), an annual shrub, is commonly used to make jellies, jams and
beverages. In folk medicine,
H. sabdariffa has been commonly used for its anti-hypertension properties .
The anthocyanin pigments that confer its color make it a valuable food product. Many biological activities of anthocyanin, such as
antioxidative , hypocholesterole-mic and hepatoprotective activities , have been investigated.
Ginger rhizome (Zingiber officinale R., family: Zingiberaceae), is used worldwide as a spice. Both antioxidative 
and androgenic activity  of Z.
officinale were reported in animal models. All major active ingredients of
Z.officinale, such as Zingerone, Gingerdiol, Zingibrene, gingerols and shogaols, have antioxidant activity .
This investigation was set to evaluate the protective effects of
H. sabdariffa and
Z. officinale against cisplatin-induced testicular toxicity in male rats and to study the mechanisms underlying these effects.
2 Materials and methods
2.1 Chemicals, plants and extraction procedure
Cisplatin was purchased from Bristol-Myers Squibb Company (New Jersey, USA). Thiobarbituric acid, reduced
glutathione, 5,5¡¯-dithiobis (2-nitrobenzoic acid), Folin¡¯s reagent, epinephrine, superoxide dismutase,
H2O2 and bovine albumin were obtained from Sigma Chemical (St. Louis, MO, USA). All other chemicals were obtained from local
commercial suppliers. Dried flowers of
H.sabdariffa and roots of
Z. officinale were purchased from a local herbal
store. Plant specimens were authenticated at the herbarium of the United Arab Emi-rates University. Plant specimens
were then grounded into powder, which was subjected to microwave-assisted extraction .
Adult male Wistar strain albino rats (150_200 g) were obtained from the Animal House, United Arab Emirates
University. They were maintained on standard pellet diet and tap water
ad libitum and were kept in polycarbonate cages with wood chip bedding under a 12 h light/dark cycle and room temperature of 22_24ºC. Rats were
acclimatized to the environment for 2 weeks prior to experimental use. This study was approved by the Animal Ethics
Committee, United Arab Emirates University.
Animals were divided into four groups of five rats each. Extracts of
H. sabdariffa or
Z. officinale was given p.o. (1 g/kg body weight) each day for 26 consecutive days using an oral tube. Control and cisplatin groups were
gavaged distilled water instead. After
21 days of extracts or distilled water administration, cisplatin (dissolved in a
vehicle of normal saline) was injected i.p. to
Hibiscus, Zingiber or cisplatin group at a single dose of 10 mg/kg body
weight. Animals in the corresponding control group were administered with vehicle alone. Five days after cisplatin
injection, rats were killed using an anesthetic diethyl ether. Blood, testes and epididymid of each animal were collected.
Testes and epididymid were weighed and the relative organ weight was calculated (relative organ weight = organ
weight/body weight × 100). Epididymal sperm count, motility and abnormality were assessed immediately thereafter.
The animals were weighed daily throughout the study.
2.4 Epididymal sperm motility, count and abnormality
Epididymal sperm were collected by cutting the one epididymis into small pieces in 5 mL of physiological saline at
32ºC. A sperm viability test was done using the method described by World Health Organization (WHO) .
Epididymal sperm count and motility were evaluated and the methods used have been detailed elsewhere . Epididimal
weights also vary because of individual variation of weights and ages among tested rats. Therefore, sperm numbers
were calculated relative to epididimal weights (sperm per gram) .
2.5 Biochemistry and histology
Testes were separated and homogenized separately in ice-cold KCl (150 mmol/L). Supernatants were collected
and assays of lipid peroxidation, superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH) were
done. Protein was estimated by the Lowry¡¯s method as modified by Peterson . The methods have been
standardized in our laboratory and have been detailed elsewhere .
For the histological examinations, pieces of testis were fixed in 10% neutral phosphate-buffered formalin and
hydrated tissue sections, 5 µm in thickness, were stained with Hematoxylin and Eosin. The sections were examined
under a Leica DMRB/E light microscope.
2.6 Statistical analysis
Data were expressed as mean ± SE. Statistical significance between the various groups was determined using
unpaired t-test and analysis of variance .
3.1 Effect on organ weight of male rats
The weights of testes and epididymid, expressed as relative to body weight, in rats after cisplatin administration
were found to be significantly decreased, compared with the control group (Table 1). No significant changes in the
weights of testes and epididymid were found in rats treated with
H. sabdariffa or
Z. officinale before and after cisplatin treatment. The administration of cisplatin alone or along with tested herbs did not alter the body weight of the
animals (data not shown).
3.2 Effect on epididymal sperm indices
After cisplatin was administered, the epididymal sperm count and motility decreased significantly
(P < 0.01), whereas sperm abnormality was increased
(P < 0.01). Administration of either
H. sabdariffa or
Z. officinale attenuated the cisplatin-induced decrease of sperm count and motility and protected against sperm abnormality changes
3.3 Biochemical parameters
Significant decline (P < 0.01) in testicular content of reduced GSH (Figure 1A) and of the activity of CAT and
SOD (Figure 2A, B) was shown in the cisplatin-treated group
5 days post-treatment. A significant increase
(P < 0.01) of MDA was recorded after cisplatin treatment (Figure 1B). These markers of oxidative stress did not differ
significantly from control levels when
H.sabdariffa or Z. officinale was administered before cisplatin treatment.
3.4 Histologic evaluation
Cisplatin treatment caused severe degeneration in some seminiferous tubules (Figures 3 and 4). The tubules were
also shrunken and greatly depleted of germ cells. The number of Leydig cells was clearly reduced in the
cisplatin-treated rats. Debris from the degeneration of cellular components was seen in the lumen. Congestion of blood vessels
was also observed between tubules. Animals pretreated with
H. sabdariffa or Z.officinale showed normal testicular
morphology and spermatogenesis with slight degeneration of spermatids and spermatozoa in some tubules (Figure
The cisplatin-induced testicular damage in animals is commonly associated with spermatogenic damage, germ cell
apoptosis, Leydig cell dysfunction and testicular steroidogenic disorder [2, 3]. Administration of testosterone has
protective effects on Leydig cells  and spermatogenesis  in rats.
The present investigation shows a significant weight reduction of testes and epididymid as well as a decrease
in the quality of epididymal sperm (sperm count, motility and morphology) after treatment with cisplatin. Cisplatin-induced
testicular damage was also confirmed by histopathological lesions. In the present study, cisplatin-treated animals showed
an elevation in testicular MDA level vs. the control
animals. Testicular activities of reduced GSH, CAT and SOD were
lower in the cisplatin-treated animals relative to those in the control animals. The concurrent decrease of antioxidants in
cisplatin-induced tissues might potentially explain the upregulation of MDA production . A correlation between
MDA and cisplatin-induced complications has also been reported . It has been demonstrated that cisplatin toxicity
in the kidney is mediated by the depletion of antioxidants and the elevation of lipid peroxidation . It has been
suggested that cisplatin generates free radicals by interacting with DNA . Cisplatin has enhances the production
of reactive oxygen species in kidneys , which interferes with antioxidant defense system and results in tissue
injury. Therefore, overproduction of free radicals and, hence, oxidative stress might account, at least in part, for The
testicular injury associated with cisplatin treatment .
Recently, much attention has been focused on the protective effects of antioxidants and naturally-occurring
substances against cisplatin-induced nephrotoxicity [5, 10]. However, little is known about herbal plants as protective
agents against cisplatin-induced testicular toxicity. Administration of
H. sabdariffa extract before cisplatin treatment
clearly restored the testicular damage and quality of sperm caused by cisplatin, in addition to retaining the control
values of oxidative stress markers. Accumulating evidence suggests that the protective effects of
H. sabdariffa against oxidative damage could be attributed to its antioxidative properties [8, 24]. The antioxidant activity of
H. sabdariffa could be attributed to its phenolic contents; namely, anthocyanins [7, 8].
prevention of cisplatin-induced oxidative stress damage in rats with
H. sabdariffa supports the hypothesis that the
mechanism of testicular damage could be attributed, at least in part, to the overproduction of free radicals.
Administration of Z. officinale before cisplatin treatment also attenuates testicular damage induced by cisplatin
treatment, as shown by the normal sperm count and morphology and by the histopathological recovery compared to
the cisplatin-treated group. The protective effect of
Z. officinale is reflected by the normalization of antioxidant
activities and the concurrent decrease of malondialdehyde in testes. The major active phenolic ingredients isolated
from Z. officinale (Zingerone, Gingerdiol, Zingibrene, gingerols and shogaols) have antioxidant activity [9,
11]. The protective effect of
Z.officinale against cisplatin toxicity could also be mediated by its androgenic activities. Kamtchouing
et al.  reported that
Z. officinale extracts have a potent androgenic activity in male rats. This activity is reflected
by the increase of both testis weight and serum testosterone levels.
In conclusion, the present study shows that
H.sabdariffa and Z. officinale have significant protective effects
against cisplatin-induced testicular damage and oxidative stress in rats. Further investigations are underway to
elucidate the molecular mechanism of protection mediated by Roselle and ginger.
We are grateful to Mr Sayel Daud (Twam Hospital) and to Ms Enas Idris for their valuable assistance. We also
thank Professor Michael Buratovich (Spring Arbor University, Michigan, USA) for proofreading the manuscript.
Authors are also indebted to Mr Hamdi Kandil for his professional help revising all the graphs.
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