Antioxidant effects of virgin coconut oil and corn oil on histomorphology of seminiferous tubules in phenytoin induced toxicity in rats

Antioxidant effects of virgin coconut oil and corn oil on histomorphology of seminiferous tubules in phenytoin induced toxicity in rats

Khalique-ur-Rehman¹, Hina Khan²,                                 , Sadia Sundus³,  Raja Faisal², Muhammad Sajid Khan⁴, Devi kumarai²

1. Department of Anatomy, Chandka Medical College, Larkana, Sindh
2. Department of Anatomy, Al-Tibri Medical College and Hospital, Isra University, Karachi Campus
3. Department of Anatomy, Fazaia Ruth PFau Medical College, Karachi
4. Department of Physiology, Shahida Islam Medical College, Multan-Bahawalpur Road, Lodhran

Corresponding Author:

Dr Hina Khan

Associate Professor Anatomy

Deputy Director of Research and PG Affairs

Al-Tibri Medical College and Hospital

Isra Unversity. Karachi Campus

Cell # 0346-3318553

e-mail: drhinasalman@gmail.com

Introduction:

It is an anticonvulsant, non-sedative drug which is used in the treatment of epilepsy. Male patients after the use of this drug complained of impotence. Chemically it is regarded as NO3 group name Antiepileptic. Its generic name is 5, 5 diphenyl-substituted hydantoin Trade Name; Epigran, Dilantin, Dantoino (1). Phenytoin is excreted in human semen in small quantities, and this may possibly affect the testosterone levels. Reduce the plasma concentration of free testosterone has been detected in male epileptic patients receiving phenytoin. Those patients who are receiving phenytoin, the serum level of testosterone is reduced. (41-42) It has been observed that phenytoin has a mutagenic effect on human sperm cells. It is observed that the low fertility rate is greater in epileptic patients rather than the general population. There are reports that they reduce testicular weight, spermatic count, and gives abnormal morphology of spermatozoa. They reduce the motility of spermatozoa interfere with normal HPG (hypophyseal gonadal) pathway (4). Its biological name is Cocos Nucifera. The coconut oil is abstracted by mechanical means from a mature kernel of the coconut with or without the use of heat, and chemical refining is called Virgin coconut oil. Virgin coconut oil is derived from COPRA. Myristic acid and Lauric acid are related to the average serum cholesterol concentration in humans. The lauric acid level in coconut is controlled by environment and genetics. Lauric acid is a fatty acid derived from coconut for developing monolaurin. Monolaurin is an antimicrobial agent for killing bacteria’s, yeasts and viruses. Coconut oil contains flavonox and polyphenol that have potent antioxidant effects, particularly in male fertility. The corn oil is composed of Triacylglycerol 99% and Poly Unsaturated fatty acids (PUFA). Sperm cells contain a high proportion of (PUFA), and normal spermatozoa possess high % of PUFA. These PUFA are formed from linoleic acid (LA), and they give fluidity to the sperm plasma membrane, which helps in the fusion events of fertilization. More recently, antioxidants (AOX) and Phytosterols (PST), commonly found in substantial amounts in special oils, such as Pecan nut (PNO) Corn (CO) and grape seed (GSO) oils. These oils are associated with a lower risk of inflammation, dyslipidemia and low risk of oxidative stress, which is required for the maintenance of endothelial integrity.

Material and Method:

The experimental study was carried out at Al-Tibri Medical College and Hospital, with a duration of six months from June 2019 to November 2019. After taken ethical consideration from the ethical review committee of the concerned institute and the animals were taken from the animal house of Al-Tibri Medical College and Hospital. Total 48 male Wister albino rats were taken through a randomized sampling technique with a weight of 150-250gms and equally divided into four different groups and kept them in separate cages for six weeks.  Group A (control group) given normal diet and1 unit normal saline intraperitoneal daily once a day, Group B (Experimental group) given an inj. Phenytoin 10mg/kg/body wt intra-peritoneal once daily. Group C (Virgin coconut oil) 6.7ml orally and the same dose of phenytoin once daily intra-peritoneal. Group D received 2.5 ml once daily along with the similar application of inj. Phenytoin. The entire treatment plan was given for six weeks, and the sample was taken on 4^th, 5^th and 6^th week of the study. Throughout the period 12hours day and light cycle were maintained.

Sampling:

Before starting the study, the weight of the animals was taken ad randomly divided into four groups. At 4^th, 5^th and 6^th week of study, the weight of the animal was taken, and the animals were anaesthetized with ethanol containing jar. After given anaesthesia, the animals were sacrificed, and through dissection, the testis was removed and stored in formalin, the tissue of the testis were sent for preservation, embedding and staining process for the histomorphological examination of the tissue.  The tissue was stained with H&E.

Histomorphology:

The tubular dimension was taken from different field area of the slide (choose five field area), and tubular diameter was measured through micrometre and then meant of five different field area at 400x were taken for the comparison among different groups. The reading was recorded in um.

Tubular Dimension= length x Breath/2

Photomicrograph was taken from a DSLR camera for the comparison among the different groups.

Statistical Analysis:

The Mean diameter of seminiferous tubules was recorded and to compare the mean difference among the groups, the one way ANOVA followed by post hoc Tukey’s was applied. The level of significance was considered p=<0.05.

Results:

Table: 1 shows Mean values of tubular dimension among different therapeutic groups in comparison with group B (experimental group). P-value <0.05 was considered significant.

Photomicrograph: 1.1 shows the histomorphology of seminiferous tubules among different groups on 4^th week of sampling. Significant reduction of tubular dimension was seen in Group B (phenytoin induced group). In contrast, significant restoration of tubules was found in Group A (control) and Group C (Virgin coconut oil) as compared with Group D (corn oil).

Photomicrograph: 1.2 shows the histomorphology of seminiferous tubules among different groups on 5^th week of sampling. Significant reduction of tubular dimension was seen in Group B (phenytoin induced group). In contrast, significant restoration of tubules was found in Group A (control) and Group C (Virgin coconut oil) as compared with Group D (corn oil).

Photomicrograph: 1.3 shows the histomorphology of seminiferous tubules among different groups on 6^th week of sampling. Significant reduction of tubular dimension was seen in Group B (phenytoin induced group). In contrast, significant restoration of tubules was found in Group A (control) and Group C (Virgin coconut oil) as compared with Group D (corn oil).

Discussion:

In accordance with the study, that was observed testicular toxicity induced with the administration of antiretroviral therapy that leads to potent effects on male fertility same as in our study induced with phenytoin. In a similar study, the virgin coconut oil was used to observe the antioxidant effects on male fertility and sperm morphology. There was a significant reduction of sperm motility p<0.01, in animals, after treated with HAART and on another hand in a group of virgin coconut oil HAART the significant restoration of numbers sperm and maintain the sperm motility almost near to the normal value. A similar finding was found in our study in a group of phenytoin and virgin coconut oil, and the readings were taken at the interval of 4, 5 and 6 weeks (11, 12). The ratio of poly-unsaturated fatty acids had potent inhibitory action that influences on lipid.

Peroxidation and Virgin coconut oil are highly rich in poly-unsaturated fatty acids, so similar in our study the virgin coconut oil showed their antioxidant effects in male fertility as similar in this study. The virgin coconut oil evidence more significant antioxidant role in comparison with copra oil and groundnut oil. At the same time, the same results were found in our study (13). In the same study, the virgin coconut oil and HARRT showed a significant reduction in the diameter of seminiferous tubules moreover there was no effect on the germinal layer. In accordance with the results of our study, the virgin coconut oil along with phenytoin had a potent role in the restoration of tubular diameter and thickness of germinal epithelium in the animal taken long term phenytoin intra-peritoneal once daily. The effects were measure with the interval of 4, 5 and 6 weeks.

Additionally, virgin coconut oil mixed with groundnut oil or olive oil was observed to be more effective in inhibiting LDL oxidation, and stimulate hepatic antioxidant enzyme activity. Antioxidant activity of virgin coconut oil was linked to the presence of high levels of polyphenol compounds in the oil. Polyphenols were reported to be a stronger antioxidant than vitamin C and E in vitro on a molecular basis (14, 15).

Conclusion: Virgin coconut oils showed significant restoration of seminiferous tubules dimension when used along with phenytoin for six weeks in comparison of corn oil. Virgin coconut oil showed significant antioxidative effects and altered the toxic effects of drugs if administered simultaneously.

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Table I: Showing the comparison of the tubular dimension of seminiferous tubules among the different groups (um)

Group A                             Group B                       Group C                            Group D

Photomicrograph 1.1 represents the tissue of seminiferous tubules with H&E stain and showing the tubular dimension at 4th week among different groups (400x). (GL) germinal layer, (TD) tubular dimension

Group A                             Group B                       Group C                            Group D

Photomicrograph 1.2 represents the tissue of seminiferous tubules with H&E stain and showing the tubular dimension at 5th week among different groups (400x). (GL) germinal layer, (TD) tubular dimension

Group A                             Group B                       Group C                            Group D

Photomicrograph 1.3 represents the tissue of seminiferous tubules with H&E stain and showing the tubular dimension at 6^th week among different groups (400x). (GL) germinal layer, (TD) tubular dimension