Ameliorative Effect of Moringa oleifera Fruit Extract on Reproductive Parameters in Diabetic-induced Male Rats

Background: Diabetes mellitus negatively impact the male reproductive parameters. The ability of Moringa oleifera fruit to improve the reproductive parameters in diabetic-induced male has yet to be documented. Aim: To investigate the anti-hyperglycaemic potential of M. oleifera fruit aqueous extract and its effect on reproductive parameters in diabetic-induced male rats. Materials and Methods: A total of 20 male Sprague Dawley rats were divided into four groups; normal control (without diabetic induction), negative control (diabetes without treatment), positive control (diabetes with metformin) and treatment group (diabetes with 500 mg/kg M. oleifera fruit aqueous extract). Treatments were given via oral gavage for 21 consecutive days. Analysis of fasting blood glucose level, sperm quality, testicular histology and relative testis weight were performed. Results: The administration of M. oleifera fruit aqueous extract exhibited a significant reduction in fasting blood glucose level and a pronounced increase in sperm quality (sperm count, motility, viability and morphology) compared with the negative control group. M. oleifera fruit extract restored the histology of the testes as they were seen to be packed with sperms and showed an organised spermatogenesis. The relative testis weight showed no significant difference between all groups. Conclusion: The results obtained suggested that M. oleifera fruit aqueous extract possessed an antihyperglycaemic property and improved the reproductive parameters in diabetic-induced rats.

significant increase of reactive oxygen species (ROS). 8ROS damages the plasma membrane of the sperm which negatively affects the sperm motility and its ability to fertilise the oocyte.Besides, diabetes mellitus was found to induce the loss of sperm motility via the mitochondrial DNA damage associated with the activation of mitochondrial ROS generation.Numerous reports have demonstrated a higher sperm DNA fragmentation in diabetic men compared to normal control. 9In addition, Shrilatha 10 found out that the streptozotocin-induced diabetic rats manifested a significant increase of oxidative stress in the testis.The onset of diabetes mellitus is also known to alter the production of reproductive hormones by interrupting the hypothalamuspituitary-testicular axis.The expression of insulin has been detected in the hypothalamus and pituitary gland. 11The absence of insulin signalling in the hypothalamus of knock-out mice inhibits the development of Leydig cells, thus disrupts the biosynthesis of testosterone by the Leydig cells. 12umerous anti-diabetic drugs are available in the market such as metformin and glibenclamide but those drugs do not completely address the reproductive S55 problems commonly faced by diabetic men.Unlike modern synthetic drugs, herbs provide holistic treatment as it can tackle multiple diseases simultaneously.Herbs play a significant role in the discovery and development of medicinal drugs in which 25% of modern medicines were derived from natural plants. 13ew herbs have been documented as highly potential anti-diabetic agents.One of such plant is Lunasia amara.It has been documented that L. amara extract inihibits the carbohydrate-metabolizing enzyme in the intestine in an in vitro study, hence forbidding the glucose uptake into the blood stream. 14Nevertheless, the most effective anti-hyperglycemic dose of L. amara (500 mg/kg) was found to be toxic to the reproductive parameters in diabetic-induced rats as it significantly damages the sperm quality. 15The diabetic-induced sperm damage may be worsen by the anti-diabetic mechanism of L. amara.Therefore, there is an urgent need to study the potential of medicinal herbs in regulating diabetes mellitus and male infertility comprehensively in order to treat both of the pathological conditions without rising any negative side effects.Throughout the years, traditional herbs have been widely used to treat diabetes and infertility in many communities around the world. 16One of them is Moringa oleifera Lam. which is a native plant in subtropical and tropical countries. 17M. oleifera is also known as the Drumstick tree because of the long slender shape of its fruit.] Many works reported the pro-fertility effects of M. oleifera fruit among normal rats however, reports on the effect of M. oleifera fruit on reproductive parameters among diabetic models are scarce.Therefore, this study aimed to investigate the anti-hyperglycaemic effect of M. oleifera fruit aqueous extract and its ability to improve reproductive parameters in diabetic-induced male rats.

Chemicals
All the reagents were procured from Sigma-Aldrich Inc. (USA), unless stated otherwise.Commercial assay kit for testosterone was purchased from Cayman Chemical Company (USA).

Experimental animals
A total of 20 male Sprague Dawley rats (300-500 g) at age between 10-12 weeks were provided by the Animal House, Universiti Kebangsaan Malaysia (UKM), Malaysia.The rats were acclimatized for three days prior to the experiment and were kept at 28°C under 12h light/dark cycle.The rats received standard pellet diet and water ad libitum.Animal maintenance was strictly performed according to the guidelines approved by the Animal Ethics Committee of Faculty of Medicine, Universiti Kebangsaan Malaysia, Malaysia (FST/2017/MAHANEM/29-MARCH/833-MARCH-2017-FEB.-2019).

Preparation of M. oleifera fruit aqueous extract
Fresh fruits of M. oleifera were collected from Tasek Gelugor, Penang, Malaysia.The fruits were authenticated by Mr. Damanhuri, a botanist from the Herbarium of Faculty of Science and Technology, UKM, Malaysia.Voucher specimen (40344) was deposited in the aforementioned Herbarium.The M. oleifera fruits were weighed, washed and dried at 50°C for 6 h.A total of 450 g dried powdered fruits were extracted with distilled water at 80°C for three hours using reflux apparatus.The ratio of the dried powdered fruits to the distilled water was 1:10.The extract was filtered and freeze-dried for five days to yield a total of 68.57g yellow powdered extract.

Induction of experimental diabetes
The rats were fasted for 16h prior to the diabetic induction.Diabetes was induced by a single intravenous injection of streptozotocin (STZ) at a dose of 50 mg/kg body weight, dissolved in 0.1 M citrate buffer at pH 4.5.Rats with fasting blood glucose (FBG) level elevated above 13 mmol/l were considered diabetic and were used in the experiment. 22

Experimental design
The animals were randomly divided into four groups with five rats in each group; normal control (without diabetic induction), negative control (diabetes without treatment), positive control (diabetes with metformin) and treatment group (diabetes with 500 mg/kg of M. oleifera fruit aqueous extract).Treatments were given for 21 consecutive days via oral gavage after five days of STZ injection.At the end of the treatment, FBG level was determined and the rats were euthanised through a single intravenous injection of 130 mg/kg sodium pentobarbital.Finally, sperms were collected and reproductive organs were dissected out, weighed and preserved.

Anti-hyperglycemic study
Blood was drew out from the tail vein of 16-h fasted rats.The fasting blood glucose (FBG) level was determined prior to the treatment and at the end of the treatment using Accu-Chek® Performa glucometer (Roche Diabetes Care GmbH, Manheim, German).

Determination of relative testis weight
Rats were sacrificed and the testes were dissected out, washed with 0.9% sodium chloride and weighed.The testes of each rat were measured individually.The average value of both testes obtained from each rat was taken as one observation.The relative testes weight was calculated based on the following equation: 24  Microscope Carl Zeiss, USA) with the help of image analyser (iSolution Lite, IMT i-Solution Inc., USA). 25

Statistical analysis
Statistical analysis was carried out using the IBM SPSS Statistics version 22 software (SPSS Inc., USA).Data were analysed by one-way analysis of variance (ANOVA) followed by Tukey's test for post hoc analysis.Correlations were assessed using Pearson's correlation coefficient.P value < 0.05 was considered statistically significant.Results were expressed as mean ± standard error mean (SEM).

Fasting blood glucose level
The effects of M. oleifera fruit extract on FBG level are displayed in Table 1.M. oleifera extract at 500 mg/kg significantly (p < 0.05) produced an anti-hyperglycemic effect in STZ-induced rats (11.1 ± 0.81 mmol/L) compared with negative control (23.7 ± 0.89 mmol/L).M. oleifera fruit extract exhibited the glucose-lowering effect similar to that of metformin where FBG level was reduced by 43.94% (in M. oleifera) and 45.90% (in metformin) after the 21-day treatment.

Sperm quality (sperm count, motility, viability and morphology)
The effects of M. oleifera fruit extract on reproductive parameters are displayed in Table 1.The negative control group revealed a marked reduction (p < 0.05) in sperm counts which is (2.80 ± 0.58) × 10 6 than the normal control, (88.80 ± 5.24) × 10 6 .Whereas, oral administration of M. oleifera fruit extract significantly (p < 0.05) increased the sperm count and the percentage of viable sperms compared with negative control group.In addition, a strong negative correlation was observed between FBG level and sperm count in M. oleifera-treated group (r = -0.83;p < 0.05).Sperm motility was restored in the M. oleifera-treated rats in which the percentage of progressive (PR) sperms increased while the percentage of non-progressive (NP) and immotile (IM) sperms decreased significantly (p < 0.05), in contrast to the negative control group.Moreover, a pronounced increase (p < 0.05) in the percentage of viable sperms was observed in M. oleifera-treated group (69.17 ± 2.34 %) compared  with negative control (6.67 ± 0.91 %) and positive control (11.44 ± 1.44 %).The percentages of sperms with normal morphology were similar in normal control (33.76 ± 1.98 %) and M. oleifera-treated group (30.00 ± 2.36 %).These values were significantly (p < 0.05) higher than that of the negative control (10.00 ± 0.63 %).Even though metformin significantly (p < 0.05) improved the percentage of sperms with normal morphology compared with the negative control group, the effect was not as good as recorded by the M. oleifera-treated group.The M. oleifera-treated group displayed normal sperm morphology with intact hooked head, midpiece and tail, similar to that of normal control group.On the contrary, the negative control group revealed various abnormal sperm morphologies such as detached head and tail, bent midpiece, short tail and coiled tail (Figure 1).

Relative testes weight
No significant changes were observed in the relative testes weight between all groups after 21 days of treatment (Table 1).

Testicular histology
Histological testis section of normal control group (Figure 2A) shows that the seminiferous tubules are in normal shape with Leydig cells in between them.The tubule is lined with many layers of spermatogenic cells representing different stages of development; spermatogonia, spermatocyte and spermatids, while the lumen is highly packed with spermatozoa.Sertoli cells are seen at interval in between the spermatogenic cells.On the other hand, negative control group (Figure 2B) shows abnormal shape of seminiferous tubules with no interstitial space in between.The stratification of spermatogenic cells seems to be mixed and cannot be distinguished.Furthermore, the lumen of the tubule is devoid of spermatozoa.Meanwhile, the positive control group (Figure 2C) shows the presence of vacuoles in the seminiferous tubules.The tubules were round in shape yet contained depleted spermatogenic cells and a very few spermatozoa in the lumen.The scarcity of spermatozoa in the lumen suggested an impaired spermatogenesis.Interestingly, histological examination of the testicular tissue of M. oleifera-treated group (Figure 2D) did not exhibit any microscopic lesion and disorganisation of the tubular elements.The lumen is tremendously packed with spermatozoa and the tubules returned to its round shape with adequate interstitial space in between them.M. oleifera-treated group shows the histological structure of testis similar to the normal control group in which the presence of different stages of spermatogenic cells and the highly packed spermatozoa in the lumen indicating normal spermatogenesis.The quantitative analysis of the testes revealed a significant (p < 0.05) increase in the diameter of the seminiferous tubules of M. oleifera-treated group compared to the negative control group (Table 1).Additionally, the seminiferous tubules diameter showed a strong inverse correlation with the FBG level in M. oleifera-treated group (r = -0.89;p < 0.05).

DISCUSSION
Diabetes mellitus causes disruption of sperms quality, disturbance in hypothalamus-pituitary-gonadal axis and erectile dysfunction. 26One of the underlying mechanisms of infertility in diabetic subjects is due to the increase of oxidative stress in light of the fact that it is one of the main contributing factors that leads to various diabetic complications. 9his hypothesis was further supported by Shrilatha 10 who found out that there was a significant increase of oxidative stress in the testes of diabetic-induced rats after 6 weeks of STZ injection.STZ damages the pancreatic β-cells and consequently inhibits insulin production. 22Insufficient insulin production prevents the uptake of glucose into the body cells, thus causes a striking rise of blood glucose level. 27. oleifera pods and seeds are known to be rich in antioxidants. 28The M. oleifera fruit aqueous extract displayed a higher antioxidant activity compared to ethanolic extract.Phytochemical screening of M. oleifera fruit aqueous extract revealed the presence of alkaloids, glycosides, flavonoids, triterpenes and tannins. 29Hence, the reason to why aqueous extraction was applied in the present study.This present study showed that the M. oleifera fruit aqueous extract is able to significantly reduce the FBG level similar to that of metformin.Related finding has been reported by Gupta et al. 19 where M. oleifera fruit methanol extract was discovered to possess an anti-hyperglycaemic effect without causing hypoglycaemic in non-diabetic rats.M. oleifera fruit inhibits the enzymatic activity of α-amylase and α-glucosidase in the intestine which hinders the breakdown of complex carbohydrate into simple sugar to be absorbed into the blood vessels, thus preventing hyperglycaemic state. 18The antihyperglycaemic effect of M. oleifera fruit might also be due to the presence of bioactive compounds such as niazinin, niazirin A, and 4-[(6-deoxyα-l-mannopyranosyl) oxy]-benzaldehyde. 30Meanwhile, positive control group demonstrated a decreased FBG level due to the role of metformin as an established anti-diabetic drug that is capable to inhibit gluconeogenesis which in turn reduce glucose production by the liver. 31he results obtained also showed that the administration of M. oleifera fruit extract significantly enhanced the reproductive parameters of diabetic-induced male rats in addition to its anti-hyperglycemic effect.The improvement of sperm quality might be due to the antioxidant activity of flavonoids which strengthen the sperm antioxidant defence system, thereby preventing lipid peroxidation reaction on the sperm membrane. 21,32The improvement in both the histology of testes and the diameter of the seminiferous tubules might also be due to the reduction of oxidative stress in the testes and the pronounced elevation of testosterone which is crucial for the regulation of spermatogenesis.Sharma and Paliwal 33 reported that the presence of antioxidants, especially saponins, in M. oleifera fruit extract reduce the oxidative stress in the testis which in turn increase the production of testosterone.
In contrast, administration of metformin into the diabetic-induced rats did not significantly improve the sperm quality and testosterone level compared to negative control.Experimental studies suggested that metformin inhibits the in vitro proliferation of Leydig cells, thus reducing the production of testosterone.Meanwhile, the treatment of metformin in the in vivo setting was reported to be associated with a significant decrease in the size of the testis and the population of the Sertoli cells that affects the spermatogenesis efficiency. 34

CONCLUSION
M. oleifera fruit aqueous extract was able to ameliorate the reproductive parameters in diabetic-induced rats.The daily administration of 500 mg/kg M. oleifera fruit aqueous extract for 21 days reduced the diabetic-associated reproductive problems which could be due to the antioxidant and androgen-increasing properties of M. oleifera fruit extract.The current data provide an evidence on the efficacy of M. oleifera fruit aqueous extract as a complementary agent to improve male reproductive parameters specifically in diabetic subjects.However, detailed mechanism on how the reproductive parameters in diabetic-induced rats were improved is yet to be elucidated.
Testes were fixed overnight in Bouin's solution.Next, the testes were dehydrated in a series of graded alcohol and embedded in paraffin wax.The epidydimal tissues were then sectioned at 5 µm, stained with haematoxylin and eosin (H and E) and mounted with DPX.The stained sections were examined qualitatively.The mean diameter of round seminiferous tubules was measured under a light microscope (Digital Pharmacognosy Journal, Vol 10, Issue 6 (Suppl), Nov-Dec, 2018