Volume 11, Issue 9 (12-2013)                   IJRM 2013, 11(9): 711-0 | Back to browse issues page

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Walvekar M, Shaikh N, Sarvalkar P. Effects of glycowithanolides on lipid peroxidation and lipofuscinogenesis in male reproductive organs of mice. IJRM. 2013; 11 (9) :711-0
URL: http://journals.ssu.ac.ir/ijrmnew/article-1-468-en.html
1- Department of Zoology, Shivaji University, Kolhapur, India, India
2- Department of Zoology, Shivaji University, Kolhapur, India, India , nilofarshaikh20@rediffmail.com
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Free radicals are highly reactive due to presence of unpaired electron. To nullify the adverse effect of the free radicals protective system is present. Super oxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) is the enzymatic antioxidants and non-enzymatic antioxidants are glutathione, vitamin E (Tocopherol), ascorbic acid centrophenoxine, and curotenoides (1-4).
Cross linked product of oxidation damage are resistant to digestion by lysosomal enzyme. Lysosomes become unable to digest the phagocytosed material resulting into lipofuscinogenesis (5-7). Lipofuscinogenesis often exists in post mitotic cells of different animals (8, 9). Lipofuscin granules are of autofluroscent material which accumulates progressively with age in secondary lysosomes and linked with hydrolytic activity within lysosomes (10-14). In aging, mitochondria become enlarged, engulfed by lysosomes and contributes to formation of lipofuscin granules (15). Oxidative stress may result in unfavourable, physiological changes in the reproductive organs, including the epididymis and accessory glands (16).
Damage localized to the epididymis may affect normal sperm maturation processes (17, 18). Therefore, oxidative stress coupled with aging correlates with decreased semen quality and causes infertility. The Reactive Oxygen Species (ROS) originating from spermatozoa are of significant patho-physiological importance in the etiology of male fertility (19-26). Mammalian sperm cells possesses highly specific lipidic composition and high content of polyunsaturated fatty acids and because of their capacity to generate ROS, human spermatozoa are very sensitive to oxidative stress (27, 19). The excessive production of ROS results in destruction of natural antioxidants capacity of reproductive tract (28). Stress is one of the important factors that induce infertility in normozoosprmic individuals (29).
Withania somnifera also called as ‘Ashwagandha’ belonging to Solanaceae family. It is one of the important herbs used in Ayurvedic medicine. It is used as general tonic to increase energy, improve all over health and longevity and prevent the diseases in athletes, the elderly, and during pregnancy. It may prevent tumour growth patient with cancer (30-32).
Glycowithanolides (Withaferin A) chemically characterized as 4b, 27-dihydroxy 5b-6b-epoxy-1 oxawitha-2, 24-dienolide, is one of the main withanolides active principles isolated from plant. Withania somnifera showed chemogenetic variation and so for three chemotype I, II, III had been reported (33). Therefore the aim of the present investigation was to study the protective effects of glycowithanolides on oxidative stress of male reproductive system to reduce infertility during aging. Oxidative stress was induced in adult male mice by injecting low dose of D-galactose (34).
Materials and methods
Plant material
In this experimental study, fresh leaves of Withania somnifera were collected from Town Hall Garden Kolhapur. The plant was identified by Taxonomist from Botany Department, Shivaji University Kolhapur.
Plant extraction
Glycowithanolides were extracted from leaves of Withania somnifera plant as described by Bhattacharya et al (35). Fresh leaves of Withania somnifera were separated, washed with distilled water, blotted properly and kept for shade drying. Dried leaves were crushed, powdered and sieved. Then soaked in chloroform for 72 hrs to remove fatty material and separates withanolides. The solution was filtered and chloroform was evaporated by evaporator and thick paste was obtained. With the help of HPTLC the active principal glycowithanolides was confirmed. It was stored in glass bottle at 40C and used as active ingredient for dose preparation.
Swiss albino male Mus musculus (Linn) of age six month old weighing 50-55 gr were used for present investigation. They were bred and reared in departmental animal house approved by Committee for the Purpose of Control and Supervision on Experiments on Animals. (CPCSEA/233) in separate cages under proper condition of light, temperature and humidity. They were supplied with Amrut mice feed (Pranav, Agro Industries, and Sangli) and water ad libitum. In total 20 animals were divided into 4 groups of 5 animals each. All animals were treated in accordance with the (CPCSEA), New Delhi, India.
1) Control group: Mice were injected subcutaneously with 0.5 ml sterile water/day/animal for 20 days.
2) D-galactose treated group: 5% D-galactose 0.5 ml/day/animal were injected subcutaneously for 20 days (34).
3) Protective group: Mice were injected subcutaneously with 0.5 ml of 5% D-galactose/day along with glycowithanolides 20 mg/kg body wt for 20 days. This dose was selected according to Bhattacharya et al (35).
4) Curative group: Mice were injected with 0.5 ml of 5% D-galactose for 20 days, and then to study the recovery, glycowithanolides were injected 20 mg/kg body wt for next 20 days.
Determination of total lipid peroxidation (LPO)
After the completion of doses the animals were sacrificed by cervical dislocation, Testes, epididymis and seminal vesicle were dissected out, blotted and weighed. The tissues were homogenized in reaction mixture (2 mg/ml) containing 75mM phosphate buffer (pH= 7.04), 1 mM ascorbic acids and 1mM ferric chloride with 20% Trichloroacetic acid (TCA) and 0.67% Thiobarbituric acid (TBA).The mixture were heated in boiling water bath. The Thiobarbituric acid reacting substance TBARS in the form MDA was measured on spectrophotometer (Miltons Roy company) at 532 nm.
Determination of mitochondrial lipid peroxidation
For the mitochondrial fraction tissue was homogenized in 0.25 M sucrose and 1mM EDTA (2 mg/ml) and centrifugation was carried out at 3000 rpm for 10 min at 4oC (Cooling microfuge, Remi). The supernatant were again centrifuged at 10,000 rpm for 10 min at 4oC. The supernatant thus obtained were discarded, the pellete were resuspended 
in 0.2 ml 20% Triton X-100 and 0.8 ml distilled water and centrifuged at high speed 10.000 rpm for 10 min 4oC.
The pellete obtained after high centrifugation were suspended in reaction mixture and used as sample for estimation of MDA in mitochondrial fraction. The total and mitochondrial lipid peroxidation was studied by Wills methods, in which thiobarbituric acid reactive substance (TBARS) i.e. Malondialdehyde (MDA) was measured in to form of red colored malondialdehyde- TBA spectrophotometer (Miltons Roy Co.) at 532 nm against blank (36). Lipid peroxidation was measured in the form of n mole MDA/mg wet tissue.
Measurement of fluorescence product
Lipofuscinogenesis was studied Dillard and Tapple method (37). The testes, epididymis, and seminal vesicle were homogenized by using the mixture prepared earlier for lipid peroxidation. The extraction was carried out by addition of chloroform: methanol (2:1 v/v) to 0.5 ml of homogenized tissue sample. It was mixed well on vortex mixer and then 3ml of double distilled water was added and centrifuged at 300 g for 2 min. To 1ml of upper layer 0.1 ml of methanol was added and the fluorescence was measured on photoflurometer calibrated with Quinine sulphate.
Statistical Analysis
The statistical analysis was performed using One way Analysis of Variance (ANOVA) followed by Tukey’s Post Hoc test. A value of p<0.01 was considered statistically significant.
 The lipid peroxidation (both total and mitochondrial lipid peroxidation) and fluorescence product in the testes, seminal vesicle and epididymis were increased in mice with D-galactose induced aging group (group II) as compared to control (group I) and this increase was highly significant (p<0.0001); while there was decrease in total as well as mitochondrial lipid peroxidation and fluorescence product in protective group (group III) and curative group (group IV) mice as compared to aging induced mice. In Withania somnifera treated groups significant results were observed in curative groups as compared to protective group (Table I, II and III).

Table I. Lipid peroxidation (n moles MDA /mg wet weight of tissue) and fluorescence product in testes of aging induced mice and effect of glycowithanolides on the same (mean±SD)

Table II. Lipid peroxidation (n moles MDA /mg wet weight of tissue) and fluorescence product in seminal vesicle of aging induced mice and effect of glycowithanolides on the same (mean ± SD)

Table III. Lipid peroxidation (n moles MDA /mg wet weight of tissue) and fluorescence product in epididymis of aging induced mice and effect of glycowithanolides on the same (mean ± SD)

Free radicals oxidative stress has been implicated in the pathogenesis of a variety of diseases resulting usually from defective natural antioxidant defences. Potential antioxidants therapy should therefore, include either natural antioxidant enzymes or agents which are capable of augmenting the function of these oxidative free radical scavenging enzymes (38).
In the present study the active principle glycowithanolides of Withania somnifera were found to decreases the lipid peroxidation and fluorescence product in testes, epididymis and seminal vesicle. D-galactose is a reducing sugar, which react non-enzymatically with amino group in protein, lipid, and nucleic acids and form advanced glycation end products (AGES). AGES are responsible for production of free radicals thus they may accelerate the aging process (34). AGES accumulation in cell increases generation of ROS. These ROS cause the LPO of biomembrane through a chain reaction. The first step is initiation reaction, which begins by taking out “H” in unsaturated fatty acid by oxygen radicals. The second is the propagation and the final step is termination.
The extent of LPO has often been determined by the thiobarbituratic acid (TBA) test, which has also been considered for the detection of malondialdehyde (MDA). A significant increase in (p<0.0001) MDA level from control to D-galactose induced mice in testes, epididymis and seminal vesicle indicates increases in LPO. In protective group this level was decreased significantly as compared to D-galactose induced group. While in curative group the LPO was still decreased and comes near to control group, indicating that instead of simultaneous treatment of glycowithanolides with D-galactose; the later treatment will be definitely beneficial. The increase in LPO leads to the damage of cell membrane. The membrane wastes are not digested properly due to insufficiency of lysosomal enzymes (39). These wastes get accumulated in the lysosomes and called lipofuscin granules (40). These lipofuscin granules are autofluroscent and that fluorescence we measured by Spectroflurometer (Spectroflurometer-ELICO). Increase in fluorescence in testes, epididymis and seminal vesicle in D-galactose treated mice indicates lysosomes are unable to digested wastes and increases in lipofuscin granules takes place. This fluorescence product decreased in curative and protective group indicating antioxidative effect of glycowithanolides.
The increase in LPO damage spermatozoon and increase male infertility, decrease sperm-egg interaction and reduces invivo fertility (27, 41, 42). Sukcharoen et al demonstrated the association of LPO with mid piece abnormality decreased sperm count, motility and loss of the capacity of the spermatozoan to undergo the acrosome reaction and fertilize (43). The present finding indicates that glycowithanolides offer protection against D-galactose induced oxidative stress in testes, epididymis and seminal vesicle.
Conflict of interests
Authors do not have any conflict of interest.
Type of Study: Original Article |
Received: 2017/10/1 | Accepted: 2018/03/10 | Published: 2018/03/10

1. Beauchamp C, Fridovich I. Superoxide dismutase: improved assay and assay applicable to acrylamide gels. Anal Biochem 1971; 44: 276-278. [DOI:10.1016/0003-2697(71)90370-8]
2. Luck H. In: Methods in Enzymatic Analysis, 2nd English Ed Translated from 3rd German Ed. New York, Acad Press; 1974.
3. Beers R, Sizer I. A spectrophotometeric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 1952; 195: 133-134.
4. Nagy K, Zs-Nagy I. Alterations in the molecular weight distribution of proteins in rat brain synaptosomes during aging and Centrophenoxine treatment of old rats. Mech Ageing Dev 1984; 28: 171-176. [DOI:10.1016/0047-6374(84)90017-4]
5. Patro IK, Patro N. Lipofuscin in aging brain-A selective reappraisal. Ind Rev Life Sci 1992; 12: 133-144.
6. Tomake BA, Pillai MM. Age related changes in amylase and tryspin activity in the salivary glands of male mice. Indian J Gerontol 1996; 10: 1-6.
7. Pillai MM, Ashokan KV, Jadhav SJ, Pawar BK. Protective effect of Lactucasativa on the brain of mouse during aging. Indian J Gerontol 2002; 16: 199-121.
8. Patro IK, Sharma SP, Patro N. Formation and maturation of neuronal lipofuscin. Proc Nat Acad Sci India 1987; 59: 287-293.
9. Patro IK, Sharma SP, Patro N. Influence of crowding stress on neuronal aging. Age 1987; 10: 114.
10. Donato H, Sohal RS. Age related change in lipofuscin associated flurescent substance in the adult male housefly, Musca domestica. Exp Gerontol 1978; 13: 171-179. [DOI:10.1016/0531-5565(78)90010-4]
11. Sohal RS, Donato Hr. Effect of experimental prolongation of lifespan on lipofuscin content and lysosomal enzyme activity in brain of the housefly. J Gerontol 1979; 34: 489-496. [DOI:10.1093/geronj/34.4.489]
12. Ivy, GO, Schottler, F, Wenzel, J, Boudry, M, Lynch, G. Inhibitors of lysosomalenzymes: Accumulation of lipofuscin like dense bodies in the brain. Science 1984; 226: 985-987. [DOI:10.1126/science.6505679]
13. Sharma SP, Gupta SK, Patrol K. Influence of centrophenoxine on the anterior horn of protein maturation in wistar rats. Proc Nat Acad Sci India 1987; 57: 247-249.
14. Thakkar BK, Dastur DA, Munghani DK. Neuropathology & pathogenesis of experimental phenyluramine toxicity in young rodents. Indian J Med Res 1990; 92: 54-65.
15. Brunk UT, Terman A. Lipofuscin: mechanisms of age related accumulation and influence on cell function. Free Radic Biol Med 2002; 33: 611-619. [DOI:10.1016/S0891-5849(02)00959-0]
16. Sloter E, Schmid TE, Marchetti F, Eskenazi B, Nath J, Wyrobek AJ. Quantitative effects of male age on sperm motion. Hum Reprod 2006; 21: 2868-2875. [DOI:10.1093/humrep/del250]
17. Kidd SA, Eskenazi B, Wyrobek AJ. Effects of male age on semen quality and fertility: a review of the literature. Fertil Steril 2001; 75: 237-248. [DOI:10.1016/S0015-0282(00)01679-4]
18. Levitas E, Lunenfeld E, Weisz N, Friger M, Potashnik G. Relationship between age and semen parameters in men with normal sperm concentration analysis of 6022 semen sample. Andrologia 2007; 39: 45-50. [DOI:10.1111/j.1439-0272.2007.00761.x]
19. Aitken RJ, Irvine MD, Wu FC. Prospective analysis of sperm- oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am J obstet Gynecol 1989; 164: 542-551. [DOI:10.1016/S0002-9378(11)80017-7]
20. Aitken RJ, Clarkson JS, Hargreave TB, Irvine DS, Wu FC. Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia. J Androl 1989; 10: 214-220. [DOI:10.1002/j.1939-4640.1989.tb00091.x]
21. Aitken RJ, Clarkson, JS, Fishel C. Generation of reactive oxygen species, lipid peroxidation and human sperm function. Biol Reprod 1989; 41: 183-197. [DOI:10.1095/biolreprod41.1.183]
22. Iwasaki, A, Gagnon, C. Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil Steril 1992; 57: 404-416.
23. Zini A, de Lamirande E, Gagnon C. Reactive oxygen species in semen of infertile patient's level of superoxide dismutase and Catalase like activities in seminal plasma and spermatozoa. Int J Androl 1993; 16: 183-188. [DOI:10.1111/j.1365-2605.1993.tb01177.x]
24. Aitken RJ. A Free radical theory of male infertility. Reprod Fertil Rev 1994; 6: 19-24. [DOI:10.1071/RD9940019]
25. Zalata A, Hafez T, Comair F. Evalution of the role of reactive oxygen species in male infertility. Hum Reprod 1995; 10: 1444-1451. [DOI:10.1093/HUMREP/10.6.1444]
26. Zalata A, Hafex T, Mahmoud A, Comair F. Relationship between resazurine reduction test, reactive oxygen species generation, and gamma-glutamyltransferase. Hum Reprod 1995; 10: 1136-1140. [DOI:10.1093/oxfordjournals.humrep.a136106]
27. Aitken RJ, Clarkson JS. Cellular basis of defective sperm function its association with genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil 1987; 81: 459-469. [DOI:10.1530/jrf.0.0810459]
28. Lewis SE, Boyle PM, McKinney KA, Young IS, Thompson W. Total antioxidant capacity of seminal plasma in fertile and infertile men. Fertil Steril 1995; 64: 868-870. [DOI:10.1016/S0015-0282(16)57870-4]
29. McGrady AV. Effects of psychological stress on male reproduction. Arch Androl 1984; 13: 1-7. [DOI:10.3109/01485018408987495]
30. Chatterjee A, Pakrashi SC. The Treatise on Indian Medical Plants. Publications & Information Directorate; 1995: 208-212.
31. Bone K. Clinical Application of Ayurvedic and Chinese Herbs. Monographs for the Western Herbal practitioner. Aust phytother Press; 1996: 137-141.
32. Jayaprakasam B, Zhang Y, Seeram NP, Nair MG. Growth inhibition of tumor cell lines by withanolides from Withania somnifera leaves. Life Sci 2003; 74: 125-132. [DOI:10.1016/j.lfs.2003.07.007]
33. Abraham A, Kirson I, Glotter E, Lavie D. A chemotaxonomical study of Withania somnifera (L) Dunal. Phytochemistry 1968; 7: 957-962. [DOI:10.1016/S0031-9422(00)82182-2]
34. Song X, Bao M, LI D, Li YM. Advanced glycation in D-galactose induced mouse model. Mech Ageing Dev 1999; 108: 241-251. [DOI:10.1016/S0047-6374(99)00022-6]
35. Bhattacharya SK, Kalkunte SS, Ghosal S. Antioxident activity of glycowithanolides from Withania Somnifera. Indian J Exp Biol 1997; 35: 236-239.
36. Wills E D. Mechanism of lipid peroxidation in animal tissues. Biochem J 1966; 99: 667. [DOI:10.1042/bj0990667]
37. Dillard CJ, Tappel AL. Fluorescence product of lipid peroxidation of mitochondria and microsomes. Lipid 1971; 6: 715. [DOI:10.1007/BF02531296]
38. Bast A, Haenen GR, Doleman GJ. Oxidants and antioxidants: state of the art. AM J Med 1991; 91 : 2S-13S. [DOI:10.1016/0002-9343(91)90278-6]
39. Nakamura Y,Taleda M, Suzuki H, Morita H,Toda K, Haniguchi S, Nishimura T. Age dependent change in activities of lysosomal enzymes in rat brain. Neurosci Lett 1997; 97: 215-220. [DOI:10.1016/0304-3940(89)90166-3]
40. Hammer C, Braum F. Quantification of age pigment (lipofuscin). Comp Biochem Physiol B 1988; 90B:7-17. [DOI:10.1016/0305-0491(88)90030-2]
41. Aitken RJ, Buckingham D, West K, Wu FC, Zikopoulos K, Richardson DW. Differetial contribution of leukocytes and spermatozoa to the generation of reactive oxygen species in ejaculates of oligozoospermic patients and fertile donors. J Reprod Fertil 1992; 94: 451-462. [DOI:10.1530/jrf.0.0940451]
42. Mazzilli F, Rossi T, Marchesini M, Ronconi C, Dondero F. Superoxide anion in human semen related to semen parameters and clinical aspects. Fertil Steril 1994; 62: 862-869. [DOI:10.1016/S0015-0282(16)57017-4]
43. Sukcharoen N, Keith J, Irvine DS, Aitken RJ. Prediction of the in vitro fertilization (IVF) potential of human spermatozoa using sperm function tests: the effect of delay between testing and IVF. Hum Reprod 1996; 11: 1030-1040. [DOI:10.1093/oxfordjournals.humrep.a019291]

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