Pre and Post-Treatment Effects : Estimation of Serum Testosterone and Lipid Peroxidation Levels on Moringa olifera Extract Induced Cadmium Exposed Rats

Background: Cadmium (Cd), is a toxic metal which affects various organs including testis. It produces oxidative stress leading to male infertility. Moringa tree, is a natural plant with a great therapeutic value and hence it is found to be effective both in prevention and treatment of various conditions including reducing toxicity of hazardous materials. The aim of the present study was to examine the effects of Pre-and Post-treatment with Moringa oliefera leaf extract (MoE) on testis in cadmium exposed rats. Materials and Methods: The present study was conducted at the Department of Physiology, Kasturba Medical College (KMC), Mangalore, Manipal University (MU), Karnataka, India, between (2011-2013). This prospective study consisted a total of 30 rats. These were divided into 5 groups with group I being the control. Data were presented as mean ±SD. student’s t test was used as statistical tool, p<0.05 considered statistically significant. Group IV and V were pre-and post-MoE treated groups respectively. Serum testosterone and tissue lipid peroxidation levels were estimated. Results: Treatment with MoE prior and after administration of cadmium, respectively showed an increase significantly in the testosterone levels and a decrease in the tissue lipid peroxidation as compared to the group treated with cadmium. However, the pre-treatment showed better results in combatting the toxic effects of cadmium. Conclusion: This study shows that Moringa olifera leaf extract altered the testosterone and tissue lipid peroxidation levels. Also, pre-treatment showed better outcome.

phytochemicals with antioxidant property. 7Hence, extracts of this plant is indicated as an anticancer, antiinflammatory and hepatoprotective agent. 9,10ts antidiabetic actions also has been demonstrated.Till recent times, there are ongoing scientific researches being carried on some of the reported medicinal properties of this plant.
Male infertility is one of the important problems nowadays and affects almost about 10% of cou ples in the society.Recently there are few studies reported on Moringa oleifera in improving the fertil ity rate.A study done by the Bureau of Plant Indus try (BPI) revealed that the sperm count in men can be boosted by a steady diet of Moringa fruit. 12,13But till date there are no studies reported the compara tive results Moringa olifera prior and after the treat ment on serum testosterone and tissue lipid per oxidation levels in cadmium exposed rats.So, the present study was designed to elucidate the effect of extract of Moringa oliefera leaf on damage of testicles after the exposure of cadmium in wistar rats.

MATERIALS AND METHODS
The chemicals required for the biochemical assay were obtained from Durga Laboratories, India.Experiments were carried out after approval from the Institutional Animal Ethics Committee (IAEC).

Preparation of Extract
The leaves were collected and validated by a taxonomist.After drying the leaves at 37°C, powdered form of extract was prepared.20 gm of powder was soaked in 100 ml of water and chloroform in a shaker for three days.It was further concentrated Filtrate of this was concentrated to 10 % of its original.We concentrated the filtrate in rotary evaporator and dried in water bath. 14e procured male Wistar Albino rats (4 months old) from the institu tional animal house.They were provided food and water adlibitum.Rats were maintained and procedures were conducted according to the insti tutional ethical committee guidelines.

Experimental Design
The present study was conducted at the Department of Physiology, Kasturba Medical College (KMC), Mangalore, Manipal University (MU), Karnataka, India.A total of 30 male Wistar rats were divided into five (5) groups as summarized in the Table below. 4rmonal Assay Tesosterone was estimated in 2ml of blood which was collected from rats by cardiac puncture.Chemiluminescence Immuno Assay (CLIA) kit (Chen et al,1991) was used for the estimation of serum testosterone levels. 15issue lipid peroxidation (MDA) was analysed by using testis tissue hemosylate.Malondialdehyde was estimated by the standard procedure described by Kartha and Krishnamurthy. 16atistical Analysis Data were presented as mean ±SD.Student's t test was used as statistical tool, p<0.05 considered statistically significant.

RESULTS
Results depicted in Figure 1, show prior and post treatment with aqueous Moringa olifera leaf extract on tissue lipid peroxidation level and serum testosterone level.A significant decrease in testosterone and increase lipid peroxidation (MDA) was observed (p < 0.0001) in cadmium treated group when compared to the control and preand posttreated M. oliefera leaf extract (MoE).Highly significant decrease in lipid peroxidation of testis and significant increase in testosterone levels were observed when group IV and group V was compared to group III.

DISCUSSION
Effect of heavy metal exposure on testes (biology of reproduction) has become one of the major concern for the society as it is directly related to infertility. 17,18,19Recently, antidiabetic actions of Moringa olifera have also been reported. 20Cadmium is a toxic metal, implicated in oxidative stress 10,11 and embryonic tissue. 21,22Oxida tive stress arises from a variance between the production of oxi dants and physiological antioxidant capability. 23Lipid peroxidation is a measure of oxidative stress.Hence in the present experiment measured tissue lipid peroxidation and serum testosterone lev els. 23Our results showed significant increase in the testicular MDA of the cadmium alone treated group (oral 10mg/kg/bw, single dose).Testosterone levels were found to be decreased significantly in these rats.These findings indicate testicular toxicity.Our previous study reports on Pre treatment of Moringa olifera in rats also showed the similar results on cadmium induced rats. 24Imbalance of steroidogenic activity observed in this study are in accordance with the previous reports. 25,26Increased MDA level suggests that CdCl3 triggered testicular oxidative damage by enhancing the production of reactive oxygen species. 27Treatment with M. oleifera (pre and post 100 mg/kg oral for 10 days ) showed decreased toxic effect caused due to exposure of cadmium ,which is evidenced by an increase in the serum testosterone level coupled with decrease in the tissue MDA levels.However, the pretreated group showed a better effect in reducing the toxicity than the treated group after exposure to cad mium M. oleifera leaf extract (proved of having bio flavonoid and other effective antioxidants) administration prior to cadmium exposure and post to cadmium exposure caused in significant improvement of tox icity in the testis. 9,10Studies have also reported that pretreatment with M. oleifera leaf extract (containing fundamental antioxidant and phe nolic compounds) protects the testes against various lethal substances and the oxidative damage caused by them 26 Our study suggest that Moringa oleifera extract could diminish cadmium induced toxicity which owe to their antioxidant property.This may explain the fact that, the experimental group treated cadmium and M. oliefera leaf extract (MoE) showed an increase in testosterone concentration and subsequent reduc  tion in testicular MDA.Studies have shown antioxidant activities of M. oleifera leaves. 28Active components of this plant scavenge free radicals.It offers protection against oxidative damage on cellular macro mol ecules.Free radical scavengers may include polyphenols, flavonoids, and phenolic compounds. 29Moringa oleifera could enhance the anti oxidant system present in the epididymis which in turn preserved and facilitated the process of spermatogenesis.Apparently, it may have reversed concentrations of gonadotropins (FSH and LH) and ultimately testosterone. 30Therefore the present study suggests that M. oleifera leaf extract (MoE) can be used as preventive and convalescent agent to atten uate the deleterious effects of cadmium on the testis

CONCLUSION
Plant extract used in the present study offers a cheap alternative thera peutic potential in treating cadmium induced testicular damage and infertility.

Limitation of the Study
Further studies are warranted in isolating the active components respon sible for the reversal of cadmium induced decrease in sex hormone and testicular damage.

Figure 1 :
Figure 1: Changes in the MDA & testosterone level with prior & posttreatment with MoE in Cadmium exposed rats.Values are expressed in mean ± SD.