Evaluation of the effect of ethanolic extract of Croton zambesicus on the testes of Swiss albino mice

Department of Anatomy, School of Basic Medical Sciences, Igbinedion University, Okada, P.M.B. 0006, Benin City, Edo State, Nigeria. Department of Anatomy and Cell Biology, Faculty of Basic Medical Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria. Department of Chemistry, Faculty of Science, University of Lagos, Akoka, Nigeria. Department of Physiology, School of Basic Medical Sciences, Igbinedion University, Okada, P.M.B 0006, Benin City, Edo State. Nigeria. Department of Physiological sciences, Faculty of Basic Medical Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria.

The genus Croton is well known for its diterpenoid content and a lot of different types of diterpenes (phorbol esters, clerodane, labdane, kaurane, trachylobane, pimarane, etc.) have been isolated from this genus (Block et al., 2004).Ngadjui et al. (2002) revealed that compounds isolated from C. zambesicus which are used in traditional medicine for the treatment of a number of diseases including malaria, were characterized and screened for anti-plasmodial activity using inhibition of growth of Plasmodium berghei in mice.The compounds include abiatane diterpenoids, quinines, triterpenoids and flavonoid.It was clear from the results that flavonoid exhibited strong anti-malarial activity against the multidrug resistant K1 strain of Plasmodium falciparum.Labdane, clerodane and trachylobane diterpenes have been identified in the stem bark of C. zambesicus (Ngadjui et al., 2002).Recently Block et al. (2002) identi-fied a new cytotoxic trachylobane diterpene from the leaves of C. zambesicus.In order to continue their investigations on the composition of the cytotoxic dichloromethane extract of the leaves, Block et al. (2004) have isolated and characterised two new trachylobane and one isopimarane diterpenes together with transphytol, a-amyrin and sterols.Okokon et al. (2005) concluded that the alkanoid fractions of the leaf and stem of C. zambesicus is active against microorganism and that the essential oil found in the leaves also contain Pcymene, linalool and beta-caryophyllene.
Despite these huge achievements on the successful isolation of some important phytochemicals, there is very little literature concerning the study of the effect of C. zambesicus on vital organs such as the testes.We therefore set to elucidate the possible effects of ethanolic extract of C. zambesicus on the function of the testes.This will in a way guide the usage and dosage of this important plant.

Plant materials
The leaves of C. zambesicus were procured from a local market in Ile-Ife, Osun-State, Nigeria.It was identified by the Department of Botany, Obafemi Awolowo University, Ile-Ife, Nigeria, were a voucher was deposited at the Herbarium.The leaves were oven dried at 40 o C for 6 days and then grounded to a fine powder.

Preparation of extract
The powdered material (100 g) was percolated with ethanol.The extract obtained yield (26.27%) was partitioned between dichloromethane and water.The aqueous fraction was concentrated in vacuum at 20 o C (yield 3.98%).The fraction was dissolved in normal saline and administered orally at a dose of 5 and 10 mg/kg as the plant extract for a period of five consecutive days.

Animal treatment
Fifteen Swiss male albino mice (27 -30 g) were used for the experiment.They were maintained under standard laboratory conditions in the Animal Holdings of Igbinedion University, Okada, Nigeria.They were fed with standard pelleted diet and water adlibitum.The animals were randomly assigned into groups A, B and C (n = 5).Groups B and C were administered with 5 and 10 mg/kg doses of the extract respectively.An equivalent volume of normal saline was given to group A (control group) for five consecutive days.The care and handling of the animals is in line with the rules and guidelines of animal right committee of the Igbinedion University, Okada, Nigeria.

Testes weight, volume and density estimation
On the day six, the mice were decapitated and the testes collected and weighed using a Metler (153) balance.The volume was also measured by water displacement method.The density was calculated arithmetically by dividing the mass by the volume.

Sperm characteristics
The testes from each mouse were carefully exposed and removed.They were trimmed free of the epididymis and adjoining tissues.
From each separated epididymis, the caudal part was removed and placed in a beaker containing 1 mL physiological saline solution.
Each section was quickly macerated with a pair of sharp scissors and left for a few minutes to liberate its spermatozoa into the saline solution.Sperm motility, concentration and progressive motility were determined as earlier described (Monica, 2000).Semen drops were placed on the slide and two drops of warm 2.9% sodium citrate were added.The slide was covered with a cover slip and examined under the microscope using X40 objective for sperm motility.Sperm count was done under the microscope using improved Neubauer haemocytometer.

Assay for Malondialdehyde (MDA)
The testes were removed and blotted dried on a filter paper and weighed.A 10% homogenates of each testis in chilled phosphate buffer was immediately prepared with a Potter-Elvhjem homogenizer.The homogenates were centrifuged (3000 rpm for 10 min) and the supernatant immediately stored in the freezer (-20EC) and assayed within 48 h.MDA an index of lipid peroxidation was determined using the method of Bueger and Aust (1978).1.0 ml of the supernatant was added to 2 ml of the tricarboxylic acidthiobarbituric acid-hydrochloric acid reagent (TCA-TBA-HCl) boiled at 100 o C for 15 min, and allowed to cool.Flocculent materials were removed by centrifuging at 3000 rpm for 10 min.The supernatant was removed and the absorbance read at 532 nm against a blank.MDA was calculated using the molar extinction coefficient for MDA-TBA-complex of 1.56 × 105 M -1 CM -1 .

Determination of catalase activity
This was determined adopting the methods of Aksenes and Njaa (1981).Hydrogen peroxide was prepared with phosphate buffer; 0.2 ml of sample was added to 1.8 ml of 30 mM of hydrogen peroxide (H202) substrate in a 2 ml curvette.The phosphate buffers were used as a blank.The absorbance for the test sample, blank and standard was read against a blank at 240 nm at 30s interval for 1 min.The enzyme activity was calculated using the molar extinction coefficient of 40.00 per M per CM expressed as unit per ml.

Determination of total protein
This was determined using Biuret method (Gonall et al., 1949).5.0 ml of blank Biuret reagent prepared by dissolving CuSO4 5H20 crystal in 500 ml of distilled water was added to sample blank.These were mixed well and allowed to stand for 20 min at room temperature 25 -27 o C. Absorbance was read for one test and standard against a blank at 540 nm.The concentration of protein was calculated using: optical density for standard × concentration of standard.

Statistical analysis
Data were expressed as mean ± SEM.The data were analysed by student's t-test using statistical software STATISTICA VERSION 5. Values of P < 0.05 were considered significant.

RESULTS AND DISCUSSION
Close examination of the experimental animals showed no pathological symptoms throughout the experimental period.The consumption of food was also appreciably okay.This explains the reason for a slight increase observed in the weight difference even though it was not significantly different (P > 0.05) when the weight of the treated animals were compared with the control (Table 1).The results showed nonsignificant increase P > 0.05 when the volume, weight and density of the testes in the treated group were compared with the control (Table 2).Assay for MDA (an index of lipid peroxidation) and catalase activity showed a significant reduction (P < 0.05) when the treated group was compared with the control group (Table 3).There was, however, a significant increase (P < 0.05) in the sperm concentration, sperm motility, and progressivity in the treated group in contrast to the control.This significant increase was observed to be dose dependent (Table 4).This present study demonstrated that C. zambesicus, a very important ornamental plant in Africa, is a good booster of testicular functions in Swiss mice.The increment (P > 0.05) in the (body) weight difference, when the control group was compared with the treated (Table 1) implies an equal continuous progressivity of the body cells through the cell circle to accommodate tissue growth or cell turn over (Heath et al., 1999).The increment in the weight, volume and density of the testes, as compared with the control group, might be related to the proliferative and differentiating changes in the surface epithelium of the seminiferous tubules which enhance the active role of the extract in spermatogenesis (Heath et al., 1999;Senger, 1999).The increment observed in the measured parameters (Tables 1 and 2), clearly demonstrates the non interference of the extract in the normal metabolic activities (Ezeasor, 1985) which was reflected in the slight increase in the weight of the treated group even though it was not statistically significant.
The leaf extract of C. zambesicus demonstrates an antioxidant activity in the testes as it inhibits lipid peroxidation which was measured by the concentration of MDA (Table 3).The significant reduction in the concentration of MDA in the treated group implies its role in scavenging natural free radicals by donating electron to it (Mira et al., 2000).Akpantah et al. (2006) in a related work with Garcinia kola seed concluded that the flavonoid in G. kola has antioxidant activity which acts in synergy with natural antioxidant.Flavonoids have been associated with antimicrobial effects in various studies using plant extracts (Nweze et al., 2004;Abo et al., 1999;Corthout et al., 1991).However, more research is required to determine other roles of flavonoids in C. zambesicus.The flavonoid isolated in C. zambesicus (Ngadjui et al., 2002;Okokon et al., 2005) may have possessed the antioxidant activity which is responsible for the reduction in MDA (an index of lipid peroxidation) even at high dosage (Table 4).The activity of this antioxidant must have acted in synergy with the catalase which is also an antioxidant.This suggests that the extract prevent lipid peroxidation.The little literature concerning the phytochemical study of C. zambesicus, made Block et al. (2002) to identify a new cytotoxic trachylobane diterpene from the leaves of C. zambesicus and just very recently, Block et al. (2004) isolated and characterised two new trachylobane and one isopimarane diterpenes together with trans-phytol, aamyrin and sterols.
This study which revealed the antioxidant properties of C. zambesicus in testes, further confirm its non toxic effect and its importance in African traditional medicine.The observed increase in sperm concentration in the treated group was dose dependent (68.24 ± 7.35 and 79 ± 7.61 x 106 mL for 5 and 10 mg/kg respectively).This is a significant improvement over the control (56.71± 6.19 x 106 mL).This is a confirmation of the importance of C. zambesicus as a potent antioxidant and free radical scavenger (Ngadjui et al., 2002;Okokon et al., 2005) which ameliorated the increased free radicals generated by the natural and experimental stress.Although the mechanism of action of the extract for the increased sperm concen-tration is yet to be elucidated, it may however be connected with the induction of the germinal Ofusori et al. 2437 epithelial cells called spermatogonia to continuously proliferate to replenish themselves and to differentiate through definite changes of development (Guyton and Hall, 2000).Data for sperm motility and progressivity (Table 4) showed a significant increase (P < 0.05) when the treated group was compared with the control group.This could be due to the influence of the extract on the mitochondria in the body of the tail of the spermatozoon to synthesize energy in the form of adenosine triphosphate (Guyton and Hall, 2000).In conclusion, this plant has been demonstrated to have a promising profertility property which can be exploited in fertility therapy.

Table 1 .
Weights of the animals before and after treatment (mean ± SEM).

Table 2 .
Weight, volume and density of the testes of treated and control animals (mean ± SEM).

Table 4 .
Characteristic of sperm in control and experimental animal (mean ± SEM)