Antioxidative protective effect of icariin on the FeSO4/H2O2-damaged human sperm based on confocal raman micro-spectroscopy

  • Zhan-sen Huang (黄展森)
  • Heng-jun Xiao (肖恒军)
  • Tao Qi (齐 涛)
  • Zhi-ming Hu (胡志明)
  • Hao Li (李 浩)
  • Di-ling Chen (陈地灵)Email author
  • Ya-lin Xu (徐亚林)Email author
  • Jun Chen (陈 俊)Email author


Oxidative stress is implicated in male infertility and significantly higher reactive oxygen species are detected in 25% of infertile males. Although different agents of various alternative medicines, including traditional Chinese medicine, have been tried with varying success, evidence remains limited on whether and how much herbs or supplements might help increase the anti-oxidant ability of the sperm. This study examined the anti-oxidative effects of icariin, a flavonoid isolated from Herba Epimedii, on the human sperm. We prepared the FeSO4/H2O2-damaged human sperms, which were co-cultured with icariin in vitro, and then observed the changes of the sperm by employing Raman micro-spectroscopy. The results showed that Raman mapping with a 514 nm excitation laser allowed clear differentiation of the nucleus, neck, and, in particular, the mitochondria-rich middle piece of a human sperm cell. The effect of icariin on different organelles of the sperm was quantified by localized spectral Raman signatures obtained within milli-seconds, and icariin could keep the “Raman fingerprint” of the human sperm the same as the control groups, suggesting that icariin could protect the human sperm from being damaged by FeSO4/H2O2. Icariin may serve as a tonifying and replenishing agent of herbal origin for enhancing reproductive functions.

Key words

anti-oxidative protection mechanism icariin human sperm Raman spectroscopy 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Tawadrous GA, Aziz AA, Mostafa T. Seminal soluble fas relationship with oxidative stress in infertile men with varicocele. Urology, 2013, 82(4):820–823PubMedCrossRefGoogle Scholar
  2. 2.
    Foksinski M, Gackowski D, Rozalski R, et al. Effects of basal level of antioxidants on oxidative DNA damage in humans. Eur J Nutr, 2007, 46(3):174–180PubMedCrossRefGoogle Scholar
  3. 3.
    Guz J, Gackowski D, Foksinski M, et al. Comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men. PLoS One, 2013, 8(7): e68490PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Pahune PP, Choudhari AR, Muley PA, The total antioxidant power of semen and its correlation with the fertility potential of human male subjects. J Clin Diagn Res, 2013, 7(6):991–995PubMedPubMedCentralGoogle Scholar
  5. 5.
    Kilarkaje N, Mousa AM, Al-Bader MM, et al. Antioxidants enhance the recovery of three cycles of bleomycin, etoposide, and cisplatin-induced testicular dysfunction, pituitary-testicular axis, and fertility in rats. Fertil Steril, 2013, 100(4):1151–1159PubMedCrossRefGoogle Scholar
  6. 6.
    Liang HR, Vuorela P, Vuorela H, et al. Isolation and immunomodulatory effect of flavonol glycosides from Epimedium hunanense. Planta Med, 1997, 63(4):316–319PubMedCrossRefGoogle Scholar
  7. 7.
    Du Q, Xia M, Ito Y. Purification of icariin from the extract of Epimedium segittatum using high-speed counter-current chromatography. J Chromatogr A, 2002, 962(1–2):239–241PubMedCrossRefGoogle Scholar
  8. 8.
    Liu WJ, Xin ZC, Xin H, et al. Effects of icariin on erectile function and expression of nitric oxide synthase isoforms in castrated rats. Asian J Androl, 2005, 7(4): 381–388PubMedCrossRefGoogle Scholar
  9. 9.
    Makarova MN, Pozharitskaya ON, Shikov AN, et al. Effect of lipid-based suspension of Epimedium koreanum Nakai extract on sexual behavior in rats. J Ethnopharmacol, 2007, 114(3):412–416PubMedCrossRefGoogle Scholar
  10. 10.
    Tian L, Xin ZC, Liu WJ, et al. Effects of icariin on the erectile function and expression of nitrogen oxide synthase isoforms in corpus cavernosum of arterigenic erectile dysfunction rat model. Zhonghua Yixue Zazhi (Chinese), 2004, 84(11): 954–957Google Scholar
  11. 11.
    Zhang ZB, Yang QT. The testosterone mimetic properties of icariin. Asian J Androl, 2006, 8(5):601–605PubMedCrossRefGoogle Scholar
  12. 12.
    Xin ZC, Kim EK, Lin CS, et al. Effects of icariin on cGMP-specific PDE5 and cAMP-specific PDE4 activities. Asian J Androl, 2003, 5(1):15–18PubMedGoogle Scholar
  13. 13.
    Lee MK, Choi YJ, Sung SH, et al. Antihepatotoxic activity of icariin, a major constituent of Epimedium koreanum, Planta Med, 1995, 61(6):523–526PubMedCrossRefGoogle Scholar
  14. 14.
    He W, Sun H, Yang B, et al. Immunoregulatory effects of the herba Epimediia glycoside icariin. Arzneimittelforschung, 1995, 45(8):910–913PubMedGoogle Scholar
  15. 15.
    Niu R. Action of the drug Herba Epimedii on testosterone of the mouse plasma and its accessory sexual organ before and after processing. Zhongguo Zhongyao Zazhi (Chinese), 1989, 14(9):530–532, 574Google Scholar
  16. 16.
    Xiong YB. The study on the relation between icariin and gonad. J Tradit Chin Med (Chinese), 1995, 26(S3):98–99Google Scholar
  17. 17.
    Xiong YB, Zhou CH. The effect of extracts from Herba Epimedii and Semen Cuscutae on the function of male reproduction. China J Chinese Materia Medica (Chinese), 1994, 29(2): 89–91Google Scholar
  18. 18.
    Qin DN, She BR, She YC, et al. Effects of flavonoids from semen cuscutae on the reproductive system in male rats. Asian J Androl, 2000, 2(2):99–102PubMedGoogle Scholar
  19. 19.
    Xie F, Wu CF, Lai WP, et al. The osteoprotective effect of Herba epimedii (HEP) extract in vivo and in vitro. Evid Based Complement Alternat Med, 2005, 2(3):353–361PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Yin XX, Chen ZQ, Dang GT, et al. Effects of Epimedium pubescens icariin on proliferation and differentiation of human osteoblasts. Zhongguo Zhongyao Zazhi (Chinese). 2005, 30(4): 289–291Google Scholar
  21. 21.
    Frost RL, Xi Y, Scholz R, et al. The molecular structure of the phosphate mineral chalcosiderite-avibrational spectroscopic study. Spectrochim Acta A Mol Biomol Spectrosc, 2013, 111:24–30PubMedCrossRefGoogle Scholar
  22. 22.
    Kotchey GP, Gaugler JA, Kapralov AA, et al. Effect of antioxidants on enzyme-catalysed biodegradation of carbon nanotubes, J Mater Chem B Mater Biol Med, 2013, 1(3):302–309PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    Richard-Lacroix M, Pellerin C. Novel method for quantifying molecular orientation by polarized Raman spectroscopy: a comparative simulations study. Appl Spectrosc, 2013, 67(4):409–419PubMedCrossRefGoogle Scholar
  24. 24.
    Zhuang Z, Li N, Gou Z, et al. Study of molecule variations in renal tumor based on confocal micro-Ranman spectroscopy. J Biomed Optics, 2013, 18(3):31103CrossRefGoogle Scholar
  25. 25.
    Pudlas M, Koch S, Bolwien C, et al. Raman spectroscopy as a tool for quality and sterility analysis for tissue engineering applications like cartilage transplants. Int J Artif Organs, 2010, 33(4):228–237PubMedGoogle Scholar
  26. 26.
    Won-in K, Thongkam Y, Pongkrapan S, et al. Raman spectroscopic study on archaeological glasses in Thailand: ancient Thai glass. Spectrochim Acta A Mol Biomol Spectrosc, 2011, 83(1):231–235PubMedCrossRefGoogle Scholar
  27. 27.
    Smith ZJ, Huser TR, Wachsmann-Hogiu S. Raman scattering in pathology. Stud Health Technol Inform, 2013, 185:207–234PubMedGoogle Scholar
  28. 28.
    Kubasek WL, Wang Y, Thomas GA, et al. Peticolas, Raman spectra of the model B-DNA oligomer d(CGCGAATTCGCG)2 and of the DNA in living salmon sperm show that both have very similar B-type conformations. Biochemistry, 1986, 25(23):7440–7445PubMedCrossRefGoogle Scholar
  29. 29.
    Huser T, Orme CA, Hollars CW, et al. Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells, J Biophotonics, 2009, 2(5):322–332PubMedCrossRefGoogle Scholar
  30. 30.
    Meister K, Schmidt DA, Bründermann E, et al. Confocal Raman microspectroscopy as an analytical tool to assess the mitochondrial status in human spermatozoa. Analyst, 2010, 135(6):1370–1374PubMedCrossRefGoogle Scholar
  31. 31.
    Sánchez V, Redmann K, Wistuba J, et al. Oxidative DNA damage in human sperm can be detected by Raman microspectroscopy, Fertil Steril, 2012, 98(5):1124–1129.e 1-3PubMedCrossRefGoogle Scholar
  32. 32.
    Huang Z, Chen G, Chen X, et al. Rapid and label-free identification of normal spermatozoa based on image analysis and micro-Raman spectroscopy. J Biophotonics, 2013, doi:10.1002/jbio.201300003Google Scholar
  33. 33.
    Sigman M. Refining the measurement of sperm DNA fragmentation. Fertil Steril, 2012, 98(5):1123PubMedCrossRefGoogle Scholar
  34. 34.
    Liu F, Zhu Y, Liu Y, et al. Real-time Raman microspectroscopy scanning of the single live sperm bound to human zona pellucid. Fertil Steril, 2013, 99(3):684–689.e4PubMedCrossRefGoogle Scholar
  35. 35.
    Thomas GJr., Kyogoku Y. Biological infrared and Raman spectroscopy part C. Bram EGJr, Grasselli JG. editors. Marcel Dekker. USA. 1977, 717–872Google Scholar
  36. 36.
    Goodwin DC, Brahms J. Form of DNA and the nature of interactions with proteins in chromatin. Nucleic Acids Res, 1978, 5(3):835–850PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    Xu Y, Zhang Z, Zhang H. Raman spectroscopic study of DNA after photosensitive damage caused by hypocrellins A and B. Sci China C Life Sci, 1998, 41(4):360–366PubMedCrossRefGoogle Scholar
  38. 38.
    Zhao HX, Xu YM, Zhang ZY. Ranman spectroscopic study of DNA photodamage sensitized by hypocrellin B and 5-brominated-hypocrellin B. Chinese Science Bulletin, 1998, 43(2):1128–1134CrossRefGoogle Scholar
  39. 39.
    Xu Y, Zhou Z, Yang H, et al. Raman spectroscopic study of microcosmic photodamage of the space structure of DNA sensitized by Yangzhou haematoporphyrin derivative and photofrin II. J Photochem Photobio B, 1999, 52(1–3):30–34Google Scholar
  40. 40.
    Kolodziejski N. Tip-enhanced Raman spectroscopy for the base interrogation of DNA. Methods Cell Biol, 2013, 114:611–628PubMedCrossRefGoogle Scholar
  41. 41.
    Wang L, He D, Zeng J, et al. Raman spectroscopy, a potential tool in diagnosis and prognosis of castration-resistant prostate cancer. J Biomed Opt, 2013, 18(8): 87001PubMedCrossRefGoogle Scholar
  42. 42.
    Schulze HG, Konorov SO, Piret JM, et al. Label-free imaging of mammalian cell nucleoli by Raman microspectroscopy. Analyst, 2013, 138(12):3416–3423PubMedCrossRefGoogle Scholar
  43. 43.
    Erfurth SC, Peticolas WL. Melting and premelting phenomenon in DNA by laser Raman scattering. Biopolymer, 1975, 14(2):247–264CrossRefGoogle Scholar
  44. 44.
    Bansal J, Singh I, Bhatnagar PK, et al. DNA sequence detection based on Raman spectroscopy using single walled carbon nanotube. J Biosci Bioeng, 2013, 115(4):438–441PubMedCrossRefGoogle Scholar
  45. 45.
    Ghaleno LR, Valojerdi MR, Janzamin E, et al. Evaluation of conventional semen parameters, intracellular reactive oxygen species, DNA fragmentation and dysfunction of mitochondrial membrane potential after semen preparation techniques: a flow cytometric study. Arch Gynecol Obstet, 2014, 289(1):173–180PubMedCrossRefGoogle Scholar
  46. 46.
    Smith TB, De Iuliis GN, Lord T, et al. The senescence-accelerated mouse prone 8 as a model for oxidative stress and impaired DNA repair in the male germ line. Reproduction, 2013, 146(3):253–262PubMedCrossRefGoogle Scholar
  47. 47.
    Bogliolo L, Murrone O, Di Emidio G, et al. Raman spectroscopy-based approach to detect aging-related oxidative damage in the mouse oocyte. J Assist Reprod Genet, 2013, 30(7):877–882PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Zhan-sen Huang (黄展森)
    • 1
  • Heng-jun Xiao (肖恒军)
    • 2
  • Tao Qi (齐 涛)
    • 1
  • Zhi-ming Hu (胡志明)
    • 1
  • Hao Li (李 浩)
    • 1
  • Di-ling Chen (陈地灵)
    • 3
    Email author
  • Ya-lin Xu (徐亚林)
    • 4
    Email author
  • Jun Chen (陈 俊)
    • 1
    Email author
  1. 1.Departerment of Infertility and Sexual Medicinethe Third Affiliated Hospital of Sun Yat-sen UniversityGuangzhouChina
  2. 2.Department of Urologythe Third Affiliated Hospital of Sun Yat-sen UniversityGuangzhouChina
  3. 3.Guangdong Institute of MicrobiologyGuangzhouChina
  4. 4.Kaiping Central HospitalKaiping, GuangzhouChina

Personalised recommendations