The aim of our study was to reveal the in vitro effects of Salvia officinalis L. (37.5, 75, 150, 200, 250, 300 and 600 µg/ml) extract on the TM3 Leydig cell viability, membrane integrity, steroidogenesis and reactive oxygen species production after 24 h and 48 h cultivation. For the present study, the extract prepared from Salvia officinalis L. leaves was analysed by high performance liquid chromatography (HPLC) for selected flavonoids and phenolic acids followed by a determination of its free radicals scavenging activity (DPPH). Furthermore, Leydig cell viability was assessed by the mitochondrial toxicity assay (MTT), while the membrane integrity was evaluated by 5- carboxyfluorescein diacetate-acetoxymethyl ester (5-CFDA-AM). The level of steroid hormones was performed by enzyme-linked immunosorbent assay (ELISA) from the culture media, while the superoxide radical generation was measured by the nitroblue tetrazolium chloride (NBT) assay. The results show that experimental concentrations did not damage the cell membrane integrity and viability when present at below 300 µg/ml, it was only at 600 µg/ml that a significant (P<0.05) cell viability decline was observed after a 48 h cultivation. A significant (P<0.05) stimulation of testosterone secretion was recorded at 250 µg/ml for 24 h, while the prolonged cultivation time significantly (P<0.05) increased the testosterone and progesterone production at 150, 200, 250 and 300 µg/ml. Furthermore, none of the selected doses exhibited significant ROS-promoting effects however, the highest dose of Salvia initiated the free radical scavenging activity in cultured mice Leydig cells.

Department of Animal Physiology Faculty of Biotechnology and Food Sciences Slovak University of Agriculture in Nitra itra Slovak Republic

Zobrazit více v PubMed

ABDELKADER M, AHCEN B, RACHID D, HAKIN H. Phytochemical study and biological activity of sage (Salvia officinalis L.) Int J Life Sci Biotechnol. 2014;8:1253–1257. doi: 10.5281/zenodo.1326832. DOI

AHERNE SA, KERRY JP, O’BREIN NM. Effects of plant extracts on antioxidant status and oxidant-induced strass in Caco-2 cells. Br J Nutr. 2007;97:321–328. doi: 10.1017/S0007114507250469. PubMed DOI

ALZWEIRI M, AL SARHAN A, MANSI K, HUDAIB M, ABURJAI T. Ethnopharmacological survey of medicinal herbs in Jordan, the northern badia region. J Ethnopharmacol. 2011;137:27–35. doi: 10.1016/j.jep.2011.02.007. PubMed DOI

BAHR HI, IBRAHIM AE. Phytopreventive effect of Salvia officinalis L. on infertility induced by hypothyroidism in male albino rats. Biomedical. 2015;4:40–44.

CHEN GW, CHEN TY, YANG PM. Differential effect of herbal tea extracts on free fatty acids-, ethanol- and acetaminophen-induced hepatotoxicity in FL83B hepatocytes. Drug Chem Toxicol. 2019;17:1–6. doi: 10.1080/01480545.2019.1692026. PubMed DOI

CHEN YCH, NAGPAL ML, STOCCO DM, LIN T. Effects of genistein, resveratrol, and quercetin o steroidogenesis and proliferation of MA-10 mouse Leydig tumor cells. J Endocrinol. 2007;192:527–537. doi: 10.1677/JOE-06-0087. PubMed DOI

CHOI HS, KIM JQ, CHA YN, KIM C. A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells. J Immunoassay Immunochem. 2006;27:31–44. doi: 10.1080/15321810500403722. PubMed DOI

ELIDA B, DANIEL Z, PAYAL P, VISHAL J, TEJAS L, INNA K, SIDNEY J, SIDHARTHA D. A novel dietary supplement containing multiple phytochemicals and vitamins elevates hepatorenal and cardiac antioxidant enzymes in the absence of significant serum chemistry and genomic changes. Oxid Med Cell Longev. 2010;3:129–144. doi: 10.4161/oxim.3.2.11157. PubMed DOI PMC

ESMAEILI MA, SONBOLI A, KANNANI MR, SADEGHI H. Salvia sahendica prevents tissue damages induced by alcohol in oxidative stress conditions. Effect on liver and kidney oxidative parameters. J Med Plants Res. 2009;3:276–283.

FATTAHI E, VASEGHI M. Protective effect of salvia officinalis on testes tissue damages of rats intoxicated by diazinon. JMPB. 2015;1:39–43.

FREI B, HIGDON JV. Antioxidant activity of tea polyphenols in vivo: evidence from animal studies. J Nutr. 2003;133:3275–3284. doi: 10.1093/jn/133.10.3275S. PubMed DOI

HALO M, JR, MASSANYI P, GREN A, LASAK A, SLANINA T, ONDRUSKA L, MUCHACKA R, GALBAVY D, IVANIC P, SCHNEIR R, FORMICKI G. Time and dose-dependent effect of Viscum album quercus on rabbit spermatozoa motility and viability in vitro. Physiol Res. 2019;68:955–972. doi: 10.33549/physiolres.934223. PubMed DOI

ISMAIL BH, HAMMED SM. Effect of Salvia officinalis on the histological parameters and physiological criteria of male reproductive system in mice. J Vet Sci. 2013;6:157–162.

JAMBOR T, KOVACIKOVA E, GREIFOVA H, KOVACIK A, LIBOVA L, LUKAC N. Assessment of the effective impact of bisphenols on mitochondrial activity and steroidogenesis a dose-dependency in mice TM3 Leydig cells. Physiol Res. 2019;4:689–693. doi: 10.33549/physiolres.934200. PubMed DOI

JASEM E, NASIM J, GHOLAMREZA M, NASER S, NABER M, MARYAM SL, ABBAS N, VAHID R. Evaluation of the effects of Salvia hypoleuca on the cAMP-responsive element modulator (CREM) gene expression and spermatogenesis in rat. Mid East Fertility Society J. 2010;15:274–277. doi: 10.1016/j.mefs.2010.08.002. DOI

KOSAR M, DORMAN HJ, BASER KH, HILLTUNEN R. Salvia officinalis L.: composition and antioxidant-related activities of a crude extract and selected sub-fractions. Natur Product Com. 2010;5:1453–1456. doi: 10.1177/1934578X1000500921. PubMed DOI

KOVACIK A, TIRPAK F, TOMKA M, MISKEJE M, TVRDA E, ARVAY J, ANDREJI J, SLANINA T, GABOR M, HLEBA L, FIK M, JAMBOR T, CISAROVA M, MASSANYI P. Trace elements content in semen and their interactions with sperm quality and Red Ox status in freshwater fish Cyprinus carpio: A correlation study. J Trace Elem Med Biol. 2018;50:399–407. doi: 10.1016/j.jtemb.2018.08.005. PubMed DOI

LIMA CF, CARVALHO F, FARNANDES E, BASTOS ML, SANTOS-GOMES PC, FARNANDES-FERREIRA M, PEREIRA-WILSON C. Evaluation of toxic/protective effects of t essential oil of Salvia officinalis on freshly isolated rat hepatocytes. Toxicol In Vitro. 2004;18:457–465. doi: 10.1016/j.tiv.2004.01.001. PubMed DOI

LUKŠIČ L, ÁRVAY J, VOLLMANNOVÁ A, TÓTH T, SKRABANIA V, TRČEK J, GERM M, KREFT I. Hydrothermal treatment of Tartary buckwheat grain hinders the transudation of rutin to quercetin. J Cereal Sci. 2016;72:131–134. doi: 10.1016/j.jcs.2016.10.009. DOI

MOSMANN T. Rapid colorimetric assay for cellular growth and survival. Application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;16:55–63. doi: 10.1016/0022-1759(83)90303-4. PubMed DOI

PAPIEZ M, GANCARCZYK M, BILINSKA B. The compounds form the hollyhock extract (Althaea rosea Cav. Var. nigra) affect the aromatization in rat testicular cells in vivo and in vitro. Folia Histochem Cytobiol. 2002;40:353–359. PubMed

RAHTE S, EVANS R, EUGSTER PJ, MARCOURT L, WOLFENDER JL, KORTENKAMP A, TASDEMIR D. Salvia officinalis for hot flushes. Towards determination of mechanism of activity and active principles. Planta Med. 2013;79:753–760. doi: 10.1055/s-0032-1328552. PubMed DOI

SAHA R, ROYCHODHURY S, KAR K, VARGHESE AC, NANDI P, SHARMA GD, FORMICKI G, SLAMA P, KOLESAROVA A. Coenzyme Q10 ameliorates cadmium induced reproductive toxicity in male rats. Physiol Res. 2019;68:141–145. doi: 10.33549/physiolres.934000. PubMed DOI

SALAH MM, HUSSEIN MS, MAHMOOD R, KHALID LB. Effect of Salvia officinalis L (sage) aqueous extract on liver and testicular function of diabetic albino male rats. J Univ Babylon. 2016;24:390–399.

SÁNCHÉZ-MORENO C, LARRAURI A, SAURA-CALIXTO F. A procedure to measure the antioxidant efficiency of polyphenols. J Sci Food Agricul. 1998;76:270–276. doi: 10.1002/(SICI)1097-0010(199802)76:2<270::AID-JSFA945>3.0.CO,2-9. DOI

SCHEER A, TINSON C, SHERRY JP, SHCHRIMER K. Application of alamar blue/5carboxylfluorescein diacetate acetoxymethyl ester as a noninvasive cell viability assay in primary hepatocytes from rainbow trout. Anal Biochem. 2005;344:76–85. doi: 10.1016/j.ab.2005.06.009. PubMed DOI

SINGH S, LI SS. Epigenetic effects of environmental chemicals bisphenol A and phthalates. Int J Mol Sci. 2012;13:10143–10153. doi: 10.3390/ijms130810143. PubMed DOI PMC

SINGLEOTN VL, ROSSI JA. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Viticul. 1965;6:1444–1458.

SULTAN C, BALAGUER P, TEROUANNE B, GEORGET V, PARIS F, JEANDEL C, NICOLAS J. Environmental xenoestrogens, antiandrogens and disorders of male sexual differentiation. Mol Cell Endocrinol. 2001;178:99–105. doi: 10.1016/S0303-7207(01)00430-0. PubMed DOI

SUN J, WANG H, LIU B, SHI W, SHI J, ZHANG Z, XING J. Rutin attenuates H2O2 - induced oxidation damage and apoptosis in Leydig cells by activating PI3K/Akt signal pathways. Biomed Pharmacother. 2017;88:500–506. doi: 10.1016/j.biopha.2017.01.066. PubMed DOI

TOHAMY AA, IBRAHIM SR, MONEIM AEA. Studies on the effect of Salvia aegyptiaca and Trigonella foenum graecum extracts on adult male mice. JAPS. 2012;2:36–43. doi: 10.7324/JAPS.2012.2501. DOI

TVRDÁ E, BOTMAN B, HALENÁR M, SLANINA T, LUKÁČ N. In vitro effects of Salvia officinalis on bovine spermatozoa. Int J Biol Biomol Agricul Food Biotechnol Engineering. 2017;11:89–95.

WANG Y, CHEN F, YE L, ZIRKIN B, CHEN H. Steroidogenesis in Leydig cells: effects of aging and environmental factors. Reproduction. 2017;154:111–122. doi: 10.1530/REP-17-0064. PubMed DOI PMC

ZUPKO I, HOHMANN J, REDEI D, FALKAY G, JANISCAK G, MATHE I. Antioxidant activity of leaves of Salvia species in enzyme-dependent and enzyme-independent systems of lipid peroxidation and their phenolic constituents. Planta Medica. 2001;67:366–368. doi: 10.1055/s-2001-14327. PubMed DOI

The Mechanisms and Management of Age-Related Oxidative Stress in Male Hypogonadism Associated with Non-communicable Chronic Disease

The effect of Apium graveolens L., Levisticum officinale and Calendula officinalis L. on cell viability, membrane integrity, steroidogenesis, and intercellular communication in mice Leydig cells in vitro