INTRODUCTION:

Nanotechnology has been a formed scope that takes action in different fields of medicine, involving cardiology, endocrinology, ophthalmology, immunology, Pulmonology and oncology. In addition, it’s extremely applied in specialized areas like tumor targeting, brain targeting and gene delivery.

Nanotechnology is well efficient to enhancing the bioavailability of water-indissoluble compounds, hold the therapeutic drugs from barriers 20 Literature Review such as blood-brain barrier, highly developed of a novel kind of bioactive polymer as well as load big payloads. Noxious ecological factors, infections, and inflammation lead to reactive oxygen species (ROS) generated by immature sperm cells and leucocytes. cell signaling was dysreulated by ROS, that result defect cellular functions, cells' proliferation, and eventually increase apoptosis¹. Oxidative stress and the inflammatory process can influence on sperm and oocyte, which is related with DNA damage or, viability of these cells thus disturbance in motility of sperms that may be aid in development of infertility, abortion, fetus anomaly and sterility^2,3^. Enzymatic and nonenzymatic antioxidant system save cells against oxidative stress⁴. Spermatogenesis is complex operation and an extremely concurrent, uniform, long, and that happened in testes at the area of germinal epithelium^5.The testes is highly susceptible exogenous and endogenous stress specially in seminiferous tubule. There are wide variety of ROS can influence on tissue testes at different stages of differentiation germinal cells or in somatic testicular cells resulting of temporary or permanent unappealable sterility⁶. The purification of the Silymarin from milk thistle extract as a hydro-alcoholic form, involving of a compound of the variant of flavonolignans such as silydianin, silychristin, isosilybin A isosilybin B, silybin A and silybin B. These active ingradient involve together around 60–85% of the content of milk thistle extract^7,8. Today, silymarin is commonly used as a supportive and alternative therapy in control and relive different medical problems. Awang⁹ reported that silimarine has a potent antioxidant activity as compared to vitamin E. In addition, Chen et al. notify that silymarin recover Thioacetamide-generate hepatotoxicity¹⁰. However, no clear studies have reported the influence of SM on sperm characteristics and fertility. In our project, the influence of silymarin and silymarin nanoparticles on reproductive damage induced by alloxan monohydrate was determined to evaluate sperms parameters.

Characterization and synthesis of Nanoparticles of Silymarin:

Preparation of silymarin -TPGS nanoparticles: Nanoparticles of silymarin of were prepared by the Nano precipitation method according to^11,12. Briefly,the organic solution included 100 mg of Silymarin was placed in a glass test tube and then transferred 1ml of acetone solvent, the dissolving Silymarin was incubated overnight, then vortex on high speed until all silymarin extract is entirely dissolved (~10 min). An aqueous phase was prepared by adding 100ml of 0.03% w/v Vitamin E-TPGS to a 200 ml glass beaker for overnight, after that put on a magnetic stir bar at stirring speed 500 rpm, and then 10ml of 0.03% w/v Vitamin E-TPGS was added to glass test tube of emulsified polymer solution. Then the test tube was stirred, then 100μl of silymarine extract was immediately added to the equeous phase of TPGS under high stirring by vortex at 900rpm for 10 min, until all quantity of extract added to TPGS. 5. The emulsified silymarine solution was put in the small beaker 50ml and then directly transferred to the ultrasonicator and immersed in the ice water and sonicate the emulsion for 9min, pulse on time 15 seconds with pulse off time 15 seconds and 50% amplitude. Finally emulsified nanoparticle was filtered (Whatman filter paper 1) to discard any precipitated and then centrifuged at 14,000rpm at 4°C. The supernatant distilled water.

Experimental animals:

The present study was conducted at the, College, of Veterinary Medicine,/Al-Qasim Green University. Adult male rats weighted (180±200g) with ages range between 60-65 days. A total fifty Mature Wister male albino rats with a mean weight of 170 to 190g, obtained from the animal house at the College of Veterinary Medicine, Al Qasim Green University. Diabetes was induced in rats by fasting for more than 16 hours by a intra-peritonealy injection of single dose of 150mg/kg from alloxan monohydrate¹³^.

The animal study groups were divided into four groups according to variation between groups, each group include 10 rats, group control group, group two diabetic rats, group three diabetic rats with silymarin at dosed of 100mg/kg.bw daily orally, for 60 day, group four diabetic rats with extract of silymarin nanoparticles at dose 100mg/kg.bw daily orally, for 60 day. Blood was collected from heart puncture for estimate serum FSH, L.H and testosterone , then rats were sacrificed by euthanized by dislocation of the cervical spine under anesthesia using a cocktail consist of Ketamine–Xylazine. In the subsequent, step, the tail of the epididymis was digested and macerated, and duct of vas deferens separated into many tiny smaller parts and placed in buffer solution at 37°C for sperm feature calculation.

Sperm motility and Sperm count:

There are different ways used to calculate the movement and the total number of sperm, but we chose the most common procedure has already been described by¹⁴ Briefly, for counting sperm, 30μL of the diluted sperm were transmitted to the hemocytometer with a plastic Pasteur pipette and held for 5 min until the sperm reached the counting site. Then, under 400 x magnification, the counting sperms were done in five squares of twenty-five squares the mean was studied. For motility of sperm , briefly, 20μl of the newly ready suspension was lay on the normal slide. Fully five microscopic domains of sperm motility were evaluated and were performed on 200 sperms from each sample. Sperm was counted as "mobile" when it exhibits either progressive movement or spontaneous strikes of the skin if the head of sperm is bind to the vitreous slide. Three separate experiments were outright on more than 100 sperm per case.

Sperm morphology A part of sperm suspension was utilized to make smears to evaluate the sperm shape abnormalities. The morphology of sperm was also planned using of A 20μl of 0.5% nigrosin and eosin then sperms morphology were determined according to assay prescribed by^15,16.

Statistical analysis:

The statistical analysis was done by utilizing Complete Randomized Design (CRD( method confirmed by AL-Rawi and Kalaf-Allah¹⁷^. The mean differences between the averages of the studied traits were fixed at the probability level of (0.01).

RESULT AND DISCUSSION:

Characterization of Silymarin nanoparticles:

The current data regarding polydispersity index (PDI) and particle size of Silymarin nanoparticles with TPGS are presented in figures 1 and 2; they were recorded a effective diameter of 144nm. The size of the NPs was augment by an raise of the by using of aqueous phase TPGS listed in table (1). PDI was employed to determine the distribution of the particle size of nano- suspensions. The results showed a lower PDI at 0.003, which give guide a more homogenous particle size distribution and a stable particle population of uniform distribution. It is a fact that natural products are a healthier match than synthetic drugs. Our result showed that acetone as organic phase and TPGS as aqueous phase reduce particle size and PDI as compared with dimethyl sulfoxide (DMSO) and polyvinyl alcohol (PVA) as surfactant aqueous phase. In addition to acetone is preferable on other organic phase DCM and DMSO , due to easily evaporation from solution due to low melting point^18,19^. Tween 80 significantly increased drug and Cmax after oral administration, also inhibition of P-gp in the gut improved absorption²⁰^.Therefore, Tween 80 is likely to improve systemic exposure of P-gp substrates after oral administration but due to high size 440 nm so that not used in final experiment. In addition to TPGS can act as an effective P-gp inhibitor to hasten uptake of drug by cell as well as increased solubility and stability of therapeutic agents, and their efficacy^21,22.

Table(1) Particle size and polydispersity index (PDI), of Silymarin nanoparticles.

Ref. No	Organic phase	Aqueous phase	Particle size nm	Polydispertion index
1	Acetone	TPGS	144	0.003
2	DMSO	PVA	207	0.004
3	DCM	Tween 80	440	0.277

Table (2): determination absorbance and UV-Visible Spectroscopy of silimarine nanoparticles .

Ref. No	ABS	λ_max	% T	λ_max
1	3.64	378	97.9	877.4
2	4.55	302	86.3	970.3
3	3.56	360	30.12	1100

λ_max (Lambda max), ABS (absorbance), T(transmittance).

Diagram (1) Refer to particles size nm of silimarine nanoparticles by zeta sizers with polydispersion index

Figure (1): SEM image of particle size and morphology of Silymarin nanoparticles.

Chemical constitute of Silimarine extract:

Table(3) shows that thirteen active ingredients were estimated in silimarine by GC/MS. The main active compounds were Pinene,Terpineol, Caryophyllene, Epiglobulol, Cadinol, Silchristin, Silydianin and Silbin.Kuki, Nagy ²³. that confirmed the silimarine active compound like silychristin A, silydianin, silychristin B, silybin A and silybin B, using HPLC chromatograms. Chromatograms of methanolic extracts of dried milk thistle WERE seen eight active compound include , Silychristin, Taxifolin Apigenin-7-glucoside, , Silybin B, Isosilybin A, Silydianin, Silybin A and Isosilybin B, respectively²⁴^.

Table 1 Chemical components of extracted clove determined by GC/MS:

Component retention time

Pinene 5

Terpineol 12

Caryophyllene 18

Epiglobulol 20

Cadinol 21

Silchristin 37

Silydianin 40

Silbin 63

Sperm concentration and sperm motility:

Compared to the results obtained from negative control group (47 X10⁶ ± 1.47and 81±1.68) respectively , the datum confirmed that the average hesitance of Sperm counting and sperm motility a significantly reduced in group 2 at probability, that received alloxan monohydrate at a dose of 150mg/kg to the recorded mean value(23.5 X10⁶±1.55 and 60.25±4.2). Additionally, the rate of group four uncommonly raised than rats in group 3rd. The concentration and motility of sperms appear that the rate of sperm immotile was significantly elevated in group 2 than the 3rd and fourth groups under probability is 0.01. In dissimilarity, a significant elevation in concentration of sperm was spotted when the silimarine nanoparticles when compared with untreated group at probability (0.01), to reach mean value ( 36 x10⁶± 1.58 and 42 x10⁶±2.19).

Figure (2): refer to effect of silimarine nanoparticles on sperm concentration and sperm motility after exposed rats to alloxan.

The sperm viability significantly reduced while the positive control group received alloxan was compared with the healthy rats at probability 0.01. In contrast, viability of sperm (70±2.4) elevated in the rats of groups 3 that received orally silimarine extract but was statistically significant when compared with fourth group that received silimarine nanoparticles. On the other aspect Sperm abnormality results appear that the mean recurrence of sperm with healthy morphology, a significantly elevated for positive group that received only alloxan when was compared with the negative control group to recorded mean value (45.7±3.37 and 14.25±1.10) respectively at probability 0.01, also, in comparison with the positive group, the mean was significantly minimize after received silimarine in 3th group to recorded mean value (25±3.85). In addition the animal in group fourth that received silimarine nanoparticles showed clear improvement in the percentages of sperm abnormality mimic to negative control group to recorded mean value was (15±1.08).

Effect of nano- silimarine on FSH, L.H and Testosterone:

The present study showed that serum testosterone were significantly reduced in rats received oxidative stress as alloxane monohydrate while the fourth group that received alloxan and treated with nanoparticles of silimarine showed clear improvement to recorded mean value (1±0.09) as compared with the postive control group at (p=0.01). In serum testosterone was still moderate elevation in the rats of groups 3 that received orally silimarine extract but was statistically not significant when compared with positive control group to recorded mean value (0.67±0.03 and 0.38±0.021) respectively. On the other aspect Sperm data were appear that serum FS H and L.H were, significantly elevated for the fourth group that received alloxan and treated with silimarine nanoparticles to recorded mean value (0.75±0.08 and 0.83±0.07) respectively at probability 0.01. In the present study, a significant elevation in concentration sperm was observed in rats received silimarine extract or silimarine nanoparticles. Additionally, alloxan is considered potent oxidative stress could damage testicular tissues such as Sertoli cells and tubules of seminiferous, which, spermatids created in the seminiferous tubule that may drive to developing of the synthesis of spermatogenesis), or produce a defect in the endogenous antioxidant enzymes of epididymis' spermn by the generation of free radical. The finding was in agreement with the study of²⁵^. The previous work and reported that rats exposed to alloxan resulted marked reduction in the count of the sperm. Similarly, ^[26] had noted a marked reduction in the sperm count after exposed to alloxan in rats. Alloxan can affect the expected death of cell by activating caspase 3 which has an essential role in death of cells, and as a consequence, could an important role in sperm reduction.

Figure(3) refer to effect of silimarine extract and silimarine nanoparticles on sperm abnormality and sperm viability after exposed rats to alloxan.

Figure(4) refer to effect of silimarine extract and silimarine nanoparticles on serum level FSH, L.H and Testosterone that exposed rats to alloxan.

The finding was in agreement with the study of^25. The previous work and reported that rats exposed to alloxan resulted marked reduction in the count of the sperm. Similarly,²⁶ had noted a marked reduction in the sperm count after exposed to alloxan in rats. Alloxan can affect the expected death of cell by activating caspase3 which has an essential role in death of cells, and as a consequence, could have an important role in sperm reduction. There are several postulated mechanism by which sperm concentration can be adversely affected by alloxan and these mechanisms include direct toxicity of sperm cell or prohibit cell growth thus our data showed clear reduction of testosterone level that effect on sperm concentration and motility. Fatehi²⁷ refer that silymarin at dose 50mg/kg for one week overcome the oxidative stress radiation exposed to rats, through reducing testicular parameters (sperm morphology, concentration, and motility near to normal rats as well as reduce sperm DNA damage with improvement of the testicular tissue. Moreover, administration rats with alloxan monohydrate may induce the mimic influence on the sperm cells and changing functionality of it such as sperm motility and viability, leading to clear defects in the conception operation. Many research reported milk thistle is an herbal potent antioxidant that results in saving cell membranes from lipid peroxidation via reducing the creation of free radicals²⁸^.

Regardless of the technique interested, milk thistle has been noted to conserve the genetic material of the sperms from damages, also stimulate cells within tissues to create protein synthesis, and elevate the generation of fresh cells to scavengers the damaged cell, in certain, augment an essential role in many tumors to minimize the adverse effects chemotherapy remedy for normal tissues adjacent tumors. Sperm motility and vitality is the most accurate indicator that assesses the integrity of the sperm's membrane and its ability to conceive²⁹.The mammalian sperm membrane was more susceptible to lipid oxidation in reactive oxygen species causing reduced intracellular ATP and sperm vitality So is sperm motility ^X Therefore, it is likely that the changes in sperm capacity and motility present in the present study were received by oxidative stress that induces lipid peroxidation in the sperm layer. Since SM contains many active compounds such as silychristin A, silydianin, silychristin B, silybin A and silybin B it has powerful antioxidant properties, which elevate the sperm defense system of antioxidants and support sperm vitality and capacity³¹^. In Additionally, SM has different minerals and vitamines such vitamin C, and E which prevent sperm against alloxan, that saving sperm motility³².

Oufi, Al-Shawi³³ reported that silibinin isolated from silymarin lead to a significant elevation in the serum level of testosterone in rapprochement with the negative control group. As well as that to, silibinin increases the diameter of spermatids and primary spermatocytes that improves some testicular functions. on the other aspect, a recent study confirmed that SMN shows a protective effect against methotrexate-induced testicular damage via elevating the activities of antioxidant enzymes³⁴^. The SMN remedy preserved from varicocele-induced testicular damage and these rats appears a significant elevation in tubular differentiation, repopulation, and spermiogenesis with the improvement of seminiferous tubules. moreover, SMN clear influence on the varicocele-induced carbohydrate reduction in germinal cells. that has a positive effect on the pathogenesis of varicocele³⁵^.The use of antioxidants such as SMN can help restore fertility by scavenging free radicals and blocking transcription factors such as nuclear factor kappa B, with downregulation of E2f1 mRNA.

Figure (5 ) A: rats of animal received alloxan only appear a single tubule with decreased spermatogenic cells and Sertoli cells , The seminiferous tubular atrophy and germinal epitheliums aplasia represent no spermatogenetic activity, the structure of the testes exhibited more disorganization , 40X( H and E). B: rats treated with silimarine extract showed interstitial edema with mild thickening of tunica albugina (H&E; magnification, ×10).

Figure (6) rats treated with nanoparticles of silimarine showed effectively increased the number of germ cells and spermatozoa revealing a typical fully normal appearing spermatogenesis activity in seminiferous tubules(H&E; magnification ×40).

ACKNOWLEDGMENT:

I would like to be grateful University of AL-qassim Green University - College of veterinary medicine – Iraq of its support in the current work.

CONCLUSIONS:

In conclusion, our findings show that TPGS-coated silymarin nanoparticles have an active phytochemical that can affect the preservation of sperm characteristics after exposure to alloxan which may work by blocking oxidative and inflammatory activity thus increasing the internal antioxidant system or regenerating animals Seminal cells. Accordingly, it may be suitable a complementary herbal medicine to prevent reproductive failure.

ETHICAL:

This research has been approved by Ethics Commission of Veterinary Medicin according NO:533FD2.

REFERENCE:

1. Tahereh, S. and R. Mansoor. Antioxidants and infertility treatment, the role of Satureja Khuzestanica:a mini-systematic review. 2011. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC421643.

2. Bisht S et al., Oxidative stress and male infertility. Nature Reviews Urology, 2017. 14(8): p. 470-485. https://www.nature.com/articles/nrurol.2017; 69

3. Sinha A. and S. Gupta. The Role of Antioxidant Supplementation in Male Infertility. Clinical Research in Obstetrics and Gynecology, 2018;1(1): p. 1-12. https://asclepiusopen.com/clinical-research-in-obstetrics-and-gynecology/volume-1-issue-1/6.php

4. Zarif-Yeganeh M. and M. Rastegarpanah. Clinical role of silymarin in oxidative stress and infertility: A short review for pharmacy practitioners. Journal of research in pharmacy practice. 2019; 8(4): p. 181. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952757

5. Holstein, A.-F., W. Schulze, and M. Davidoff, Understanding spermatogenesis is a prerequisite for treatment. Reproductive Biology and Endocrinology, 2003; 1(1): p. 1-16. https://rbej.biomedcentral.com/articles/10.1186/1477-7827-1-107

6. Take G et al. Effect of melatonin and time of administration on irradiation-induced damage to rat testes. Brazilian Journal of Medical and Biological Research, 2009. 42(7):p.621-628. https://www.scielo.br/scielo.php?pid=S0100-879X2009000700006&script=sci_abstract&tlng=pt

7. Surai, P.F., Silymarin as a natural antioxidant: an overview of the current evidence and perspectives. Antioxidants.2015; 4(1): p. 204-247. https://www.scielo.br/scielo.php?pid=S0100-879X2009000700006&script=sci_abstract&tlng=pt

8. Masoumi, H., et al., Clinical and pharmacological applications of silymarin components at cellular and molecular level: a review. Research Journal of Medical Sciences.2016; 10(3): p. 102-107. http://eprints.skums.ac.ir/949/

9. Awang D. Milk thistle. Can Pharm J. 1993; 126(4034): p. 422. https://journals.sagepub.com/doi/abs/10.1177/875512259501100510

10. Chen I.S., et al., Hepatoprotection of silymarin against thioacetamide‐induced chronic liver fibrosis. Journal of the Science of Food and Agriculture. 2012; 92(7): p. 1441-1447. https://onlinelibrary.wiley.com/doi/abs/10.1002/jsfa.4723

11. Jasim A M. H FHasan, and M J Awady. Preparation of Vorapaxar loaded with Vitamin E TPGS and PVA emulsified PLGA nanoparticles In vitro studies. Research Journal of Pharmacy and Technology. 2019; 12(9): p. 4503-4510. https://www.indianjournals.com/ijor.aspx?target=ijor:rjpt&volume=12&issue=9&article=076

12. Jasim A M et al., Characterization and Synthesis of Selenium-TPGS Nanoparticles for Target Delivery Clove to Minimize Cytogenic and Liver Damage Induced in Adult Male Rats. Nano Biomed. Eng, 2021. 13(2): p. 127-136. http://nanobe.org/Assets/userfiles/sys_eb538c1c-65ff-4e82-8e6a-a1ef01127fed/files/13(2)_p127-136%20(Adnan%20Mansour%20Jasim).pdf

13. Jasim A A Dirwal and AAl-Yassri. Role of zontivity loaded by PLGA coated by TPGS in ameliorating induced cardiovascular and pulmonary disorder in adult male. East African Medical Journal, 2019; 96(6). https://www.ajol.info/index.php/eamj/article/view/200791

14. CooperT G., et al. World Health Organization reference values for human semen characteristics. Human reproduction update, 2010; 16(3): p. 231-245. https://academic.oup.com/humupd/article-abstract/16/3/231/639175

15. Adnan, M., et al., Effect of long-term administration of Atonik compound in female rabbits on hematological and pathological changes in important functional body organ. Journal of Contemporary Medical Sciences, 2016. 2(7): p. 96-102. http://www.jocms.org/index.php/jcms/article/view/87

16. Ibraheem AK. efficacy of dq-tocopheryl polyethylene glycol succinate (tpgs) to minimize atonik toxicity in male rats. Plant Archives. 2020; 20(1): p. 1738-1342. http://www.plantarchives.org/SPECIAL%20ISSUE%2020-1/1738-1742%20(128).pdf

17. Al-Rawi, K. and A. Khalafalla, Analysis of Experimental Agriculture Disgen. Dar Al-Kutub for Printing and Publishing. Mosul Univ. 2000.

18. Sah, E. and H. Sah, Recent trends in preparation of poly (lactide-co-glycolide) nanoparticles by mixing polymeric organic solution with antisolvent. Journal of Nanomaterials, 2015; 2015. https://dl.acm.org/doi/abs/10.1155/2015/794601

19. Dibenedetto A., et al. Organic carbonates: efficient extraction solvents for the synthesis of HMF in aqueous media with cerium phosphates as catalysts. ChemSusChem, 2016; 9(1): p. 118-125. https://chemistry-europe.onlinelibrary.wiley.com/doi/abs/10.1002/cssc.201501181

20. Zhang H et al., Commonly used surfactant, Tween 80, improves absorption of P-glycoprotein substrate, digoxin, in rats. Archives of pharmacal research, 2003;26(9)https://link.springer.com/article/10.1007/BF02976689

21. Guo Y . The applications of Vitamin E TPGS in drug delivery. European journal of pharmaceutical sciences, 2013. 49(2): p. 175-186. https://www.sciencedirect.com/science/article/pii/S0928098713000651

22. Mohammed QA. protective role of vitamin–tpgs to overcome oxidative stress induced by dipping of sheep with cypermethrin. Plant Archives, 2020; 20(1): p. 1105-1109. http://plantarchives.org/20-1/1105-1109%20(5735).pdf

23. Kuki, A., et al., Identification of silymarin constituents: an improved HPLC–MS method. Chromatographia, 2012. 75(3-4): p. 175-180. https://link.springer.com/content/pdf/10.1007/s10337-011-2163-7.pdf

24. Tayoub G. H Sulaiman, and M ALorfi. Quantitative identification of total silymarin in wild Silybum marianum L. by using HPLC. International Journal of Herbal Medicine, 2018;6(2): p. 110-114. https://www.florajournal.com/archives/2018/vol6issue2/PartB/7-1-21-585.pdf

25. Serban C. Effect of sour tea (Hibiscus sabdariffa L.) on arterial hypertension: a systematic review and meta-analysis of randomized controlled trials. Journal of hypertension, 2015; 33(6): p. 1119-1127. https://link.springer.com/article/10.1007/s13205-020-02612-8

26. Elangovan A et al. Momordica cymbalaria improves reproductive parameters in alloxan-induced male diabetic rats. 3 Biotech, 2021;11(2): p. 1-14.

27. Fatehi D et al. Radioprotective effects of Silymarin on the sperm parameters of NMRI mice irradiated with γ-rays. Journal of Photochemistry and Photobiology B: Biology, 2018; 178: p. 489-495. https://www.tandfonline.com/doi/abs/10.3109/09553002.2013.741285

28. Adhikari M et al. In vitro studies on radioprotective efficacy of silymarin against γ-irradiation. International journal of radiation biology, 2013; 89(3): p. 200-211.

29. Wong A et al. Addition of eosin to the aniline blue assay to enhance detection of immature sperm histones. Fertility and sterility, 2008;90(5): p. 1999-2002. https://www.sciencedirect.com/science/article/pii/S0015028207036485

30. Fanoudi S et al. Milk thistle (Silybum Marianum) as an antidote or a protective agent against natural or chemical toxicities: a review. Drug and chemical toxicology, 2020;43(3): p. 240-254. https://www.tandfonline.com/doi/abs/10.1080/01480545.2018.1485687

31. Tuli H S et al. Path of Silibinin from diet to medicine: A dietary polyphenolic flavonoid having potential anti-cancer therapeutic significance. in Seminars in Cancer Biology. 2020; Elsevier. https://www.sciencedirect.com/science/article/pii/S1044579X20302066

32. Bansal, J., et al., Hepatoprotective Models and Various Natural Product Used in Hepatoprotective Agents: a Review. Pharmacognosy Communications, 2014; 4(3). https://www.researchgate.net/profile/Rishabha-Malviya/publication/276866455_Hepatoprotective_Models_and_Various_Natural_Product_Used_in_Hepatoprotective_Agents_a_Review/links/572dd3ff08aeb1c73d128fcc/Hepatoprotective-Models-and-Various-Natural-Product-Used-in-Hepatoprotective-Agents-a-Review.pdf

33. Oufi H G. N N. Al-Shawi, and S.A. Hussain, What are the effects of silibinin on testicular tissue of mice? Journal of Applied Pharmaceutical Science, 2012; 2(11): p. 9. http://www.japsonline.com/admin/php/uploads/688_pdf.pdf

34. Yaman T. Protective effects of silymarin on methotrexate-induced damages in rat testes. Brazilian Journal of Pharmaceutical Sciences, 2018; 54(1). https://www.scielo.br/j/bjps/a/xSVm9VZrWYjRxgv3yFVrGcT/abstract/?lang=en

35. Moshtaghion S M . Silymarin protects from varicocele-induced damages in testis and improves sperm quality: evidence for E2f1 involvement. Systems biology in reproductive medicine, 2013; 59(5): p. 270-280. https://www.tandfonline.com/doi/abs/10.3109/19396368.2013.794253

Received on 28.04.2021 Modified on 08.09.2021

Research J. Pharm.and Tech 2022; 15(4):1664-1670.

DOI: 10.52711/0974-360X.2022.00278