Evaluation of antioxidant effects of neem based  constituent azadirachtin-A in rat spermatozoa

Ravindranath H Aladakatti; Umadevi C Jadaramkunti; Hiremath M B

doi:10.48165/jlas.2020.3.2.5

Authors

Ravindranath H Aladakatti Central Animal Facility, Indian Institute of Science, Bengaluru Karnataka, India Author
Umadevi C Jadaramkunti Department of Zoology, Government First Grade College, Rajnagar, Hubli, Karnataka, India Author
Hiremath M B Department of Biotechnology and Microbiology, Karnatak University, Dharwad, Karnataka, India Author

DOI:

https://doi.org/10.48165/jlas.2020.3.2.5

Keywords:

Azadirachtin-A, Antioxidant enzymes, Oxidative stress, Lipid peroxidation, Cauda epididymal spermatozoa and Rat

Abstract

Technical azadirachtin, a major component of neem, has low acute/subchronic toxicity and non-mutagenic/teratogenic in mammalian species along with minimal disruption to the ecosystem. The present in vitro study aims to address the potential evaluation of azadirachtin-A, a tetranortritarpinoid of neem seed kernel, on the antioxidant system of rat spermatozoa to promote oxidative stress in a dose-dependent manner. To assess the effect of the azadirachtin-A on activities of superoxide dismutase (SOD), catalase, Glutathione reductase (GR), Glutathione peroxidase (GPx), α-Glucosidase, production of Hydrogen peroxide (H2O2) and level of lipid peroxidation (LPO) in rat spermatozoa, increasing quantities 0.5 mM, 1 mM, 1.5 mM and 2.0 mM per ml of the azadirachtin-A was added to the cultured medium prior to the addition of cauda epididymal spermatozoa. The spermatozoa were observed at 6th h post-culture and the expressions of enzyme activities and production of H2O2, level of LPO were recorded. The activities of antioxidant enzymes decreased significantly while the levels of H2O2 generation and LPO increased significantly in azadirachtin-A treated spermatozoa in a dose dependent manner when expressed in terms of milligram protein and milligram DNA. The activity of α- glucosidase, a negative control against antioxidant enzymes, did not show any significant change at any of the doses. The results suggest that graded doses of azadirachtin-A elicits depletion of antioxidant defense system in sperm, indicating azadirachtin-A induced oxidative stress in the epididymal sperm of rats.

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References

Aitken RJ, Clarkson JS, Fishel S (1989). Generation of reactive oxygen species, lipid peroxidation and human sperm function. Biol. Reprod.40:183-197.

Aitken RJ (1989). The role of free oxygen radicals and sperm function. Int. J Androl. 12:95-97.

Aladakatti RH, Jadaramkunti UC (2015). In vitro effect of azadirachtin-A, a tetranortritarpinoid of neem seed kernel, on rat spermatozoa. Int. J. Gen. Chem. 1: 15-18.

Aladakatti RH, Sukesh B, Jadaramkunti UC, Hiremath MB (2011).Effect of graded doses of nimbolide (a major component of Azadirachta indica leaves) on biochemical and sperm functional parameters in male albino rats. J. Laborat. Anim. Sci.1: 24-30.

Alvarez JG, Storey BT (1989). Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gamete. Res. 23:77–90.

Alvarez JG, Touchstone JC, Blasco L, Storey BT (1987). Spontaneous lipid peroxidation and the production of hydrogen peroxide and superoxide in human spermatozoa. J. Androl. 8:338–348.

Arivazhagan S, Velmurugan B, Bhuvaneswari V, Nagini S (2004). Effects of aqueous extracts of garlic (Allium sativum) and neem (Azadirachta indica) leaf on hepatic and blood oxidant–antioxidant status during experimental gastric carcinogenesis. J Med. Food. 7: 334–339.

Atawodi SE, Atawodi JC (2009). Azadirachta indica (neem): a plant of multiple biological and pharmacological activities. Phytochem Rev.8:601-620.

Baligar NS, Aladakatti RH, Mukhtar Ahmed, Hiremath MB (2014). Hepatoprotective activity of the neem based constituent azadirachtin-A in carbon tetrachloride intoxicated Wistar rats. Can. J. Physiol. Pharmacol.92: 267–277.

Biswas K, Chattopadhyay I, Banerjee RK (2002). Biological activities and medicinal properties of neem (Azadirachta indica). Curr. Sci.82:1336–1345.

Burton K (1956). A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. J. Biochem. 62: 316–320.

Carlberg I, Mannervik BJ (1985). Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J.Biol.Chem. 250:5474–5480.

Chance B, Sies H, Boveris A (1979). Hydroperoxide metabolism in mammalian organs. Physiol.Rev. 59:527– 534.

Science

Chitra KC, Latchoumycandane C, Mathur PP (2002). Effect of nonylphenol on the antioxidant system in epididymal sperm of rats. Arch. Toxicol. 76: 545–551

Claiborne A (1985). Catalase activity. In: Greenwald R (Ed.) CRC Handbook of methods for oxygen radical research. (pp 283–284). CRC Press, Boca Raton, Fla.

Cooper TG, Yeung CH, Nashan D, Nieschlag E (1988). Epididymal markers in human infertility. J. Androl.9:91–101.

Das S, Das S, Pal S, Mujib A, Dey, S. (1999). Biotechnology of medical plants – Recent advances and potential. I Ed. Vol – II, UK 992 Publications (pp126 – 139), Hyderabad, India.

Glinsukon T, Somjaree R, Piyachaturawat P, Thebtaranonth Y(1986). Acute toxicity of nimbolide and nimbic acid in mice, rats and hamsters. Toxicol. Lett. 30: 159–166.

Grieveau JF, Lannou DL (1997). Reactive oxygen species and human spermatozoa: physiology and pathophysiology. Int. J. Androl. 20:61–69.

Gupta S, Kataria M, Gupta PK, Murganandan S, Yashroy RC (2004). Protective role of extracts of neem seeds in diabetes caused by streptozotocin in rats. J. Ethnopharmacol.90:185–189.

Gupta SS (1994). Prospects and perspectives of normal plants products in medicine. Ind.J.Pharmacol. 26:1-12.

Holloway AJ, Moore HDM, Foster PMD (1990). The use of in vitro fertilization to detect reductions in the fertility of spermatozoa from males exposed to ethylene glycol monomethyl ether. Reprod. Toxicol.4:21-27.

Ichikawa T, Oeda T, Ohmori H, Schill WB (1999). Reactive oxygen species influence the acrosome reaction but not acrosin activity in human spermatozoa. Int. J. Androl.22:37–42.

Jedlinska-Krakowska M, Bomba G, Jakubowski K, Rotkiewicz T, Jana B, Penkowski A (2006). Impact of oxidative stress and supplementation with vitamins E and C on testes morphology in rats. J. Reprod. Dev.52: 203-209.

Kessopoulou E, Tomlinson MJ, Barratt CLR, Bolton AE, Cooke ID (1992). Origin of reactive oxygen species in semen: spermatozoa or leucocytes? J. Reprod. Fertil.94:463–470.

Kono Y, Fridovich I (1982). Superoxide radical inhibits catalase. J.Biol.Chem. 257:5751–5754.

Kumbar SB, Jadaramkunti UC, Aladakatti RH (2012). In vitro effect of nimbolide, an isoprenoid of neem leaf, on antioxidant system of rat cauda epididymal spermatozoa: A dose dependent study. J. Appl. Pharm. Sci. 2: 84-93.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem.193:265–275.

Mann T (1964). The biochemistry of semen and of the male reproductive tract. Methuen, London.

Marklund S, Marklund G (1974). Involvement of superoxide anion radical in antioxidation of pyrogallol and a constituent assay for superoxide dismutase. Eur. J Biochem.47:469–474.

Mohandas J, Marshall JJ, Duggin GG, Horvath JS, Tiller DJ (1984). Low activities of glutathione-related enzymes as factors in the genesis of urinary bladder cancer. Cancer. Res.44:5086–5091.

Mordue (Luntz) AJ, Blackwell A (1993). Azadirachtin: an update. J. Insect Physiol.39: 903–924.

Ochsendorf FR, Buhl R, Bastlein A, Beschmann H (1998). Glutathione in spermatozoa and seminal plasma of infertile men. Hum. Reprod.13:353–359.

Ohkawa H, Ohishi N, Yagi K (1979). Assay for lipid peroxidation in animal tissues by thiobarbituric acid reaction. Anal. Biochem.95:351–358.

Pick E, Keisari Y (1981). Superoxide anion and H2O2 production by chemically elicited peritoneal macrophages induced by multiple nonphagocytic stimuli. Cell. Immunol.59:301–318.

Pigeolet E, Corbisier P, Houbion A, Lambert D, Michiels DC, Raes M, Zachary D, Ramacle J (1990). Glutathione peroxidase, superoxide dismutase and catalase inactivation by peroxides and oxygen derived free radicals. Mech. Ageing. Dev.51:283–97.

Raizada RB, Srivastava MK, Kaushal RA, Singh RP (2001). Azadirachtin, a neem biopesticide: subchronic toxicity assessment in rats. Food. Chem. Toxicol. 39: 477–483.

Rajeev K, Gagan G, Narmada P (2006). Drug Therapy for Idiopathic Male Infertility: Rationale Versus Evidence. J.Urology.176: 1307-1312.

Raji Y, Salami SA, Adisa RA (2009).Ameliorative Role of Vitamin E on Adverse Impact of Azadirachta indica in Reproductive Indices of Male Rats. J. Comp. Integ. Med. 6: Article 10: 1-11.

Sikka SC (2001). Relative impact of oxidative stress on male reproductive function. Curr. Med. Chem. 8:851–862.

Sinet PM, Garber P (1981). Inactivation of human Cu-Zn superoxide dismutase during exposure to O2 and H2O2. Arch. Biochem. Biophys.212:411–416.

Sundaram KMS (1996). Azadirachtin biopesticide a review of studies conducted on its analytical chemistry, environmental behavior and biological effects. J. Environ. Sci. Health. 31(4): 913–948.

Toyoda Y, Chang MC (1974). Capacitation of epididymal spermatozoa in a medium with high K/Na ratio and cyclic AMP for the fertilization of rat eggs in vitro. J Reprod. Fertil.36:125-134.

Umapathy E, Simbini T, Chipata T, Mbizvo M (2001). Sperm characteristics and accessory sex gland functions in HIV infected men. Arch. Androl.46:153–158.

Woods J, Garside DA (1996). An in vivo and in vitro investigation into the effects of alpha- chlorohydrin on sperm motility and correlation with fertility in the Han Wistar rat. Reprod. Toxicol. 103:199-207.

Zini A, Schlegel PN (1997). Identification and characterization of antioxidant enzyme mRNAs in the rat epididymis. Int. J Androl. 20:86–91.