Sodium benzoate is a salt of benzoic acid and frequently used as food additive to prevent the microbial growth including bacteria and fungi. It is extensively used in a variety of food stuff including carbonated drinks, fruit juices, pickles, sauces, tooth paste’, pharmaceuticals² and cosmetics. Sodium benzoate forms benzene as result of reaction of benzoic acid and ascorbic acid in soft drinks and fruit juices, which is a carcinogen³. It is able to induce neurotoxicity, nephrotoxicity and teratogenecities during pregnancy⁴. Many authors reported the effects of sodium benzoate on male reproductive system, where as information regarding female reproductive system is inadequate.

Natural antioxidants have become very popular for medical and food applications and are preferred by consumers than synthesized antioxidants⁵. Pomegranate (Punica granatum) plant extract from its different parts possess antioxidant activity, which is highest among many foods⁶. Peel of pomegranate contains high content of polyphenols such as condensed tannins and pro-anthocyanidins, anthocyanins (delphinidin, cyanidin and pelargonidin 3-glucosides and 3, 5-diglucosides) and flavonoids which are referred to as antioxidants⁷. The present work was undertaken to study pathomorphological changes in ovary and uterus of rats induced by sodium benzoate toxicity and its amelioration with Punica granatum (pomegranate) peel extract.

Healthy female albino Wistar rats weighing between 150 and 200 g were obtained from Sri Raghavendra Agencies, Bangalore and maintained in clean polypropylene cages in the laboratory animal room having temperature 25 ± 1°C. On standard pellet diet, tap water adlibitum, and daily dark - light cycle (12/12 hrs), the rats were acclimatized for one week prior to start of the experiment. The experimental work on rats was performed with approval of the Institutional Animal Ethical Committee (IAEC) of Sri Venkateswara Veterinary University.

Sodium benzoate was procured from the Loba Chemie Pvt. Ltd., Mumbai with 99% purity. The Punica granatum(pomegranate) peel extract powder product code No. C/VP/C/PUGR-01 was procured from Chemiloids Company, Vijayawada, Andhra Pradesh State.

The rats were randomly divided into four groups each containing 6 animals and sodium benzoate and Punica granatum peel extract were administered by oral gavage for 8 weeks. Rats in Group I served as control and allowed feed and water adlibitum. Rats in Group II were given sodium benzoate in distilled water at a dose of 200 mg/kg b.wt. daily. Rats in Group III were treated orally with Punica granatum peel extract (200 mg/kg b.wt. dissolved in distilled water) and Group IV rats were co-treated with sodium benzoate (200 mg/kg b.wt.) and Punica granatum peel extract (200 mg/kg b.wt.). During the experimental period, pooled serum samples were collected from all groups at fortnight intervals. All rats were maintained for 8 weeks and sacrificed at the end of research study.

There was significant (P< 0.05) increase in mean serum thiobarbituric acid reactive substance levels in sodium benzoate treated rats (group II) when compared to the control rats (group I). A significant (P< 0.05) decrease in mean serum TBARS was noticed in Punica granatum ameliorated rats (group IV), when compared to the sodium benzoate treated rats (group II) (Fig. 1).

Mean serum progesterone concentration of sodium benzoate treated rats (group II) significantly (P< 0.05) decreased when compared to the control rats (group I). A significant increase in mean serum progesterone was noticed in Punica granatum ameliorated rats (Group IV) when compared to the sodium benzoate treated rats (group II) (Fig. 2). A significant (P< 0.05) reduction was observed in mean serum estrogen concentration in sodium benzoate treated rats (group II) when compared to the control rats (group I). Punica granatum ameliorated rats (group IV) showed significant increase in mean serum estrogen concentration when compared to the sodium benzoate treated rats (group II) (Fig. 3).

Grossly, there was slight reduction in size of uterus was observed in sodium benzoate treated rats (group II). Whereas in ameliorated group (group IV), there was no changes in uterus. Small and atrophied ovaries were seen in sodium benzoate treated rats (group II), whereas in Punica granatum ameliorated rats showed near to normal sized ovaries (Fig. 4).

Histopathology of uterus of sodium benzoate treated (group II) rats showed degenerated and desquamated columnar lining epithelium with focal areas of atrophied changes (Fig: 5) in majority of rats. Areas of squamous metaplasia of lining epithelium were also observed in some treated rats. Endometrial stroma showed moderate to severe fibroblast proliferation, periglandular fibrosis (Figs: 6 & 7), severe infiltration of eosinophils and along with few mononuclear cells, congested blood vessels, severely degenerated and desquamated glandular epithelium (Fig: 8) with atrophied and distorted glands (Fig: 9) were more evident in all treated rats. Hyperplasia of glandular epithelium with reduced lumen was also noticed in focal areas. Atrophied lining columnar epithelium with reduced endometrial stromal thickness and reduced number of glands (Fig: 10) were prominently noticed in some sodium benzoate treated rats. Myometrium revealed thickened and congested blood vessels, areas of haemorrhages, perivascular eosinophilic infiltration, highly distorted smooth muscles architecture with eosinophilic infiltration in between muscle fibres in all treated rats (Fig: 11)

The changes noticed in the Punica granatum amelioration rats (group IV) were less severe like near to normal lining epithelium, mild degenerative changes in endometrial glands, mild periglandular fibrous tissue proliferation and mild infiltration of eosinophils. The thickness of endometrium was coming to normal size with increased number of glands (Fig: 12). Myometrium revealed, almost regularly arranged smooth muscle layer with mild infiltration of eosinophils. Uterus regained its near to normal appearance when compared to sodium benzoate treated rats (group II) (Fig: 13).

The uterus of Punica granatum treated rats showed normal in appearance and similar to the uterus of control rats.

Microscopically ovaries of sodium benzoate treated rats revealed thickened and wrinkled tunica albuginea with fibrous tissue proliferation. Thickened and congested blood vessels, pockets of hemorrhages (Fig: 14) and moderate proliferation of fibroblast in the medullary part. Hypertrophy and hyperplasia of interstitial stromal cellsand areas of necrosis (Fig: 15) were also noticed in medulla. Primary and secondary follicles showed degenerated and atrophic changes with severely degenerated granulosa cells, pyknotic nuclei in theca interna and theca externa cells (Fig: 16) with completely disintegrated oocyte and its nucleus leaving the cystic space in all treated rats. Degenerated and loss of luteal cells in corpus luteum (Fig: 17) and reduced number of primordial follicleswere also evident in majority of treated rats.

Histopathology of ovaries of group IV rats were similar to that of group II with very mild changes like mild degenerative changes in primary and secondary follicles, near to normal thickness of tunica albuginea, normal number of primordial follicles and ovary regained its near to normal appearance (Fig: 18) when compared to sodium benzoate treated rats (group II) by the end of 8^th week of experiment.

The ovary of Punica granatum treated rats showed normal in appearance and similar to the ovary of control rats.

In the present study, significant increase in lipid peroxidation (MDA) was noticed in sodium benzoate treated rats (group II) when compared to control rats (group I). This result was in line with the observation of ^11,12,13. The elevated levels of MDA might be attributed with the action of reactive oxygen species on cell membrane lipids¹⁴, development of products of lipid peroxidation and inhibition to the activity of antioxidants¹⁵.

In Punica granatum ameliorated group (Group IV), significant reduction in serum MDA values compared to sodium benzoate treated group was observed and it might be due to its antioxidant defence activity^16,17,18 and lipid peroxidation inhibition activity of Punica granatum^19,17,20.

These decreased levels of progesterone and estrogen might be atributed with the action of reactive oxygen species on cell membrane lipids²¹, development of lipid peroxidation and inhibition to the activity of antioxidant enzymes¹⁵. Sodium benzoate induced oxidative stress might suppress the sensitivity of the gonadotrophic cells to gonadotropin-releasing hormone and prevent gonadotropin secretion²². Inhibition of follicular stimulating hormone and leutinizing hormone by sodium benzoate might be a result of its negative effect on central nervous system that could inhibit the neural stimulus essential for the release of pituitary gonadotrophins ²³.

In Punica granatum ameliorated rats (Group IV), significant improvement was noticed in mean serum P and E₂ values compared to sodium benzoate treated group and this might be due to its antioxidant defence activity^16,17,18 and lipid peroxidation inhibition activity of Punica granatum^19,17,20.

This toxic effect of SB on reproduction might be due to SB induced reactive oxygen species and effect on biological membranes. The peroxidation of unsaturated fatty acids in biological membranes led to decreased membrane fluidity and disruption of membrane integrity and function which might lead to serious pathological changes in organs²⁴ and due to inhibition on the release of reproductive hormones^21,23.

The changes noticed in the Punica granatum amelioration rats (group IV) were very mild the uterus and ovary regained its near to normal appearance when compared to sodium benzoate treated rats (group II) which might be due to antioxidant property and free radical scavenging activity of Punica granatum activity^16,17,18.

In conclusion, the results from present study demonstrated that administration of sodium benzoate resulted in the reproductive organs damage. Amelioration with Punica granatum peel extract followed by the administration of SB mitigated the toxic effects induced by SB and has provided the positive impact on the therapeutic implementation of the Punica granatum peel extract in animals exposed to sodium benzoate.

The results described in this paper were part of MVSc thesis. The authors’ wishes to acknowledge the faculty and staff of Department of Veterinary Pathology, College of Veterinary Science, Tirupati for their cooperation through-out the period of research study. Funds and facilities of department were utilized for conducting present study.

References