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Plant Science Today (PST)

Research Articles

Early Access

Pharmacological evaluation of Dactylorhiza Hatagirea against cyclophosphamide-induced reproductive toxicity in male wistar rats

DOI
https://doi.org/10.14719/pst.11857
Submitted
19 September 2025
Published
24-12-2025

Abstract

D. hatagirea (DH) is a species belonging to the Orchidaceae family predominantly found in higher altitude regions such as the Himalayas,, which is reported to possess several biological properties such as anti-oxidant, anti-viral and anti-inflammatory potential. Cyclophosphamide (CP) is a commonly used chemotherapeutic drug that is reported to cause severe reproductive toxicity due to oxidative stress induction. The current study aims at evaluating the effect of DH root extract on mitigating reproductive toxicity induced via CP administration using male wistar rats owing to its previously reported antioxidant potential. Briefly, sequential extraction of DH root was performed using petroleum ether, chloroform and ethanol, followed by its phytochemical evaluation. Biochemical evaluation of the ethanolic DH extract showed optimum antioxidant potential compared to other extracts. Further, in vivo evaluation of the effect of DH extract on CP-induced reproductive toxicity was conducted using varying
dosages of 100 mg/kg and 200 mg/kg for 4 weeks and 8 weeks time periods. Our results indicate the efficacy of DH extract by the restoration of tissue in CP-induced damage post 8 weeks of administration with 200 mg/kg DH treatment. Moreover, an elevated level of testosterone with respect to vehicle control in 4 and 8 weeks of 200 mg/kg DH treatment was also observed, which suggests the ability of DH root extract to elevate reproductive capacity in animals. An increase of 163.37 % and 116.25 % w.r.t. control was observed for testosterone and follicle-stimulating hormone (FSH) was obtained. An up-regulation of antioxidant enzyme catalase (CAT) and down-regulation of malondialdehyde (MDA) levels suggest the possible mode of action of DH extract towards mitigating the toxic effects of CP administration in the test groups. Thus, our study proposes DH root extract as a potential therapeutic agent to mitigate CP-induced reproductive damage.

References

  1. 1. Toyin YM, Olakunle AT, Adewunmi AM. Toxicity and beneficial effects of some african plants on the reproductive system. Toxicological survey of African medicinal plants. 2014 Jan 1:445-92. https://doi.org/10.1016/B978-0-12-800018-2.00015-7
  2. 2. Jalali AS, Hasanzadeh S. Crataegus monogyna fruit aqueous extract as a protective agent against doxorubicin-induced reproductive toxicity in male rats. Avicenna J. Phytomed. 2013;3(2):159. PMCID: 25050270
  3. 3. Amin A, et. al. A standardized extract of Ginkgo biloba neutralizes cisplatin-mediated reproductive toxicity in rats. Biomed Res. Int. 2012;2012(1):362049. 10.1155/2012/362049. https://doi.org/10.1155/2012/362049
  4. 4. Ahmed HA, Ali HA, Mutar TF. Protective effects of olive leaf extract against reproductive toxicity of the lead acetate in rats. Environ. Sci. Pollut. Res. Int. 2021 Nov;28(44):63102-10. 10.1007/s11356-021-15240-3. https://doi.org/10.1007/s11356-021-15240-3
  5. 5. Ara C, Arshad A, Faheem M, Khan M, Shakir HA. Protective Potential of Aqueous Extract of Allium cepa against Tartrazine Induced Reproductive Toxicity. Pak. Vet. J. 2022 Jul 1;42(3). 10.29261/pakvetj/2022.029
  6. 6. Akbari A, Nasiri K, Heydari M, Mosavat SH, Iraji A. The protective effect of hydroalcoholic extract of Zingiber officinale Roscoe (Ginger) on ethanol-induced reproductive toxicity in male rats. J. evid.-based complement. 2017;22(4):609-17. 10.1177/2156587216687696 https://doi.org/10.1177/2156587216687696
  7. 7. Yousaf B, Amina, Liu G, Wang R, Qadir A, Ali MU, Kanwal Q, Munir B, Asmatullah, Abbas Z. Bisphenol A exposure and healing effects of Adiantum capillus-veneris L. plant extract (APE) in bisphenol A-induced reproductive toxicity in albino rats. Environ. Sci. Pollut. Res. Int. 2016 Jun;23(12):11645-57. 10.1007/s11356-016-6330-0 https://doi.org/10.1007/s11356-016-6330-0
  8. 8. Rezvanfar MA et al., Protection of cyclophosphamide-induced toxicity in reproductive tract histology, sperm characteristics and DNA damage by an herbal source; evidence for role of free-radical toxic stress. Hum. Exp. Toxicol. 2008;27(12):901-10. 10.1177/0960327108102046. https://doi.org/10.1177/0960327108102046
  9. 9. Oh MS et al., Yukmijihwang-tang protects against cyclophosphamide-induced reproductive toxicity. Reprod. Toxicol. 2007 Nov 1;24(3-4):365-70. 10.1016/j.reprotox.2007.05.007 https://doi.org/10.1016/j.reprotox.2007.05.007
  10. 10. Abdi M, Fadaee M, Jourabchi A, Karimzadeh H, Kazemi T. Cyclophosphamide-Induced Infertility and the Impact of Antioxidants. Am J Reprod Immunol. 2024 Dec;92(6):e70014. 10.1111/aji.70014. https://doi.org/10.1111/aji.70014
  11. 11. Nazar N, Hussain AI, Hussain SM, Nigam PS. Natural Products as Economical Agents for Antioxidant Activity. Phytochemicals from Medicinal Plants. 2019 Nov 15:201-32. https://doi.org/10.1201/9780429203220-11
  12. 12. Sharma S, Kumar V, Seth CA, Sourirajan A, El-Shazly M, Dev K. A comprehensive review on the phytochemistry, pharmacological properties and in vitro propagation of an endemic medicinal orchid, Dactylorhiza hatagirea. Naunyn Schmiedebergs Arch. Pharmacol. 2024 May;397(5):2621-35. 10.1007/s00210-023-02827-5. https://doi.org/10.1007/s00210-023-02827-5
  13. 13. Wani IA, Kumar V, Verma S, Tasleem JA, Rather IA. Dactylorhiza hatagirea (D. Don) Soo: A Critically Endangered Perennial Orchid from the North-West Himalayas. Plants 2020, 9(12), 1-17. https://doi.org/10.3390/plants9121644
  14. 14. Ben IO, Woode E, Abotsi WK, Boakye-Gyasi E. Preliminary phytochemical screening and in vitro antioxidant properties of Trichilia monadelpha (Thonn.) JJ De Wilde (Meliaceae). J. med. biomedical sci. 2013 Aug 8;2(2):6-15.
  15. 15. Trease G, Evans SM. Pharmacognosy. 15th ed. London: Bailer Tindal; 2002. pp. 23-67.
  16. 16. Vijay DT, Rajendra SB. Estimation of Total Phenol, Tannin, Alkaloid and Flavonoid in Hibiscus Tiliaceus Linn. Wood Extracts. J Pharmacogn Phytochem. 2014 Sep 7;2(4):41-47..
  17. 17. Sultana M, Verma PK, Raina R, Prawez S, Dar MA. Quantitative analysis of total phenolic, flavonoids and tannin contents in acetone and n-hexane extracts of Ageratum conyzoides. Int. J. Chemtech Res. 2012 Jul;3:996-9.
  18. 18. Saeed N, Khan MR, Shabbir M. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complement. Altern. Med. 2012 Nov 16;12(1):221.10.1186/1472-6882-12-221. https://doi.org/10.1186/1472-6882-12-221
  19. 19. Govindappa M, Naga Sravya S, Poojashri MN, Sadananda TS, Chandrappa CP. Antimicrobial, Antioxidant and in virto Anti-inflammatory activity of Ethanol extract and Active Phytochemical screening of Wedeliatrilobata (L.) Hitchc. J Pharmacogn Phytochem. 2011; 3(3):43-51. https://doi.org/10.5530/pj.2011.25.15
  20. 20. Aliyu AB, Ibrahim MA, Musa AM, Musa AO, Kiplimo JJ, Oyewale AO. Free radical scavenging and total antioxidant capacity of root extracts of Anchomanes difformis Engl.(Araceae). Acta Pol Pharm. 2013 Jan 1;70(1):115-21.PMID: 23610966.
  21. 21. Har LeeWei HL, Intan Safinar Ismail IS. Antioxidant activity, total phenolics and total flavonoids of Syzygium polyanthum (Wight) Walp leaves. Int J Med Arom Plants. 2012;2(2):219-28.
  22. 22. Chen YF, Yang CH, Chang MS, Ciou YP, Huang YC. Foam properties and detergent abilities of the saponins from Camellia oleifera. Int J Mol Sci. 2010;11(11):4417-25. https://doi.org/10.3390/ijms11114417
  23. 23. Shamsa F, Monsef H, Ghamooshi R, Verdian-Rizi M. Spectrophotometric determination of total alkaloids in some Iranian medicinal plants. Thai J Pharm Sci. 2008;32(1):17-20. https://doi.org/10.56808/3027-7922.2196
  24. 24. Winterbourn CC, Hawkins RE, Brian M, Carrell RW. Estimation of red cell superoxide dismutase activity. J Lab Clin Med. 1975;85(2):337-41.
  25. 25. Garratt DC. The quantitative analysis of drugs. Berlin: Springer Science & Business Media; 2012.
  26. 26. Brand-Williams W, Cuvelier ME, Berset CL. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci Technol. 1995;28(1):25-30. https://doi.org/10.1016/S0023-6438(95)80008-5
  27. 27. Kunchandy E, Rao MN. Oxygen radical scavenging activity of curcumin. Int J Pharm. 1990;58(3):237-40. https://doi.org/10.1016/0378-5173(90)90201-E
  28. 28. Thakur M, Thompson D, Connellan P, Deseo MA, Morris C, Dixit VK. Improvement of penile erection, sperm count and seminal fructose levels in vivo and nitric oxide release in vitro by ayurvedic herbs. Andrologia. 2011;43(4):273-7. https://doi.org/10.1111/j.1439-0272.2010.01068.x
  29. 29. Cellat M, Yavaş İ, Uyar A, Etyemez M, Güvenç M. Safranal alleviates cyclophosphamide-induced testicular toxicity in rats. Food Sci Nutr. 2025;13(6):e70452. https://doi.org/10.1002/fsn3.70452
  30. 30. Rezaei S, Hosseinimehr SJ, Zargari M, Karimpour Malekshah A, Mirzaei M, Talebpour Amiri F. Protective effects of sinapic acid against cyclophosphamide-induced testicular toxicity in BALB/c mice. Andrologia. 2021;53(10):e14196. https://doi.org/10.1111/and.14196
  31. 31. Hari Priya B, et al. Mitigating cyclophosphamide-associated gonadotoxicity in male Wistar rats: therapeutic potential of hesperidin. Front Vet Sci. 2024;11:1376225. https://doi.org/10.3389/fvets.2024.1376225
  32. 32. Potnuri AG, Allakonda L, Lahkar M. Crocin attenuates cyclophosphamide-induced testicular toxicity by preserving glutathione redox system. Biomed Pharmacother. 2018;101:174-80 https://doi.org/10.1016/j.biopha.2018.02.068
  33. 33. Zheng HX, et al. Potential protective role of chlorogenic acid against cyclophosphamide-induced reproductive damage in male mice. Toxicol Res (Camb). 2024;13(5):tfae176. https://doi.org/10.1093/toxres/tfae176
  34. 34. Wang H, Gao XD, Zhou GC, Cai L, Yao WB. In vitro and in vivo antioxidant activity of aqueous extract from Choerospondias axillaris fruit. Food Chem. 2008;106(3):888-95. https://doi.org/10.1016/j.foodchem.2007.05.068
  35. 35. Cushnie TT, Cushnie B, Lamb AJ. Alkaloids: overview of antibacterial, antibiotic-enhancing and antivirulence activities. Int J Antimicrob Agents. 2014;44(5):377-86. https://doi.org/10.1016/j.ijantimicag.2014.06.001
  36. 36. Fu S, Wu D, Jiang W, Li J, Long J, Jia C, Zhou T. Molecular biomarkers in drug-induced liver injury: challenges and future perspectives. Front Pharmacol. 2020;10:1667. https://doi.org/10.3389/fphar.2019.01667
  37. 37. Olayinka ET, Ore A, Ola OS, Adeyemo OA. Ameliorative effect of gallic acid on cyclophosphamide-induced oxidative injury and hepatic dysfunction in rats. Med Sci. 2015;3(3):78-92. https://doi.org/10.3390/medsci3030078
  38. 38. Jalali AS, Hasanzadeh S, Malekinejad H. Achillea millefolium inflorescence aqueous extract ameliorates cyclophosphamide-induced toxicity in rat testis. Chin J Nat Med. 2012;10(4):247-54. https://doi.org/10.3724/SP.J.1009.2012.00247
  39. 39. Zhao Y, et al. Protective effect of Huangqi-Guizhi-Wuwutang against cyclophosphamide-induced spermatogenesis dysfunction in mice. J Ethnopharmacol. 2024;319:117260. https://doi.org/10.1016/j.jep.2023.117260
  40. 40. Liao S, et al. Cyclophosphamide activates ferroptosis-induced dysfunction of Leydig cells via the SMAD2 pathway. Biol Reprod. 2024;110(5):1012-24. https://doi.org/10.1093/biolre/ioae020
  41. 41. Song Z, Wang J, Zhu P, Wang Z, Liu X, Liu F. Melatonin ameliorates cyclophosphamide-induced spermatogenesis disorder by reducing pyroptosis. Andrologia. 2023;2023(1):2186029. https://doi.org/10.1155/2023/2186029
  42. 42. Sakr S, Mahran HA, Basily NS, Saif ME. Effect of Ginkgo biloba extract on cyclophosphamide-induced reproductive toxicity and oxidative stress in male albino mice. World J Pharm Sci. 2015;:16-25.
  43. 43. Sakr SA, Mahran HA, Abo-El-Yazid SM. Effect of fenugreek seeds extract on cyclophosphamide-induced histomorphometrical, ultrastructural and biochemical changes in testes of albino mice. Toxicol Ind Health. 2012;28(3):276-88. https://doi.org/10.1177/0748233711412427
  44. 44. Oyagbemi AA, et al. Gallic acid protects against cyclophosphamide-induced toxicity in testis and epididymis of rats. Andrologia. 2016;48(4):393-401. https://doi.org/10.1111/and.12459
  45. 45. Abarikwu SO, Otuechere CA, Ekor M, Monwuba K, Osobu D. Rutin ameliorates cyclophosphamide-induced reproductive toxicity in male rats. Tox Int. 2012;19(2):207. https://doi.org/10.4103/0971-6580.97224
  46. 46. Chen J, et al. Cynomorium songaricum Rupr. flavonoids improve cyclophosphamide-induced reproductive function damage by regulating the testosterone synthesis pathway. Front Pharmacol. 2024;15:1457780. https://doi.org/10.3389/fphar.2024.1457780
  47. 47. Sushma C, Devi KR. Antigenotoxic effects of Aegle marmelos fruit extract in cyclophosphamide-induced chromosomal aberrations and aberrant sperms in germ cells of Swiss albino mice. Int J Pure Appl Biosci. 2015;3(5):178-83. https://doi.org/10.18782/2320-7051.2119
  48. 48. Shalizar Jalali A, Hassanzadeh S, Malekinejad H. Chemoprotective effect of Crataegus monogyna aqueous extract against cyclophosphamide-induced reproductive toxicity. Vet Res Forum. 2011;2(4):266-73.
  49. 49. Raju G, Senthil KB, Ranjith S, Deepti S. Chemoprotective action of Dactylorhiza hatagirea (D. Don) Soo ethanol extract against cyclophosphamide-induced reproductive toxicity and oxidative stress on TM3 cells. Plant Sci Today. 2025;12(4). https://doi.org/10.14719/pst.10523
  50. 50. Jafari M, Akbari A, Esmailpour Z, Nadi Z, Baazm M. Protective effects of Withania somnifera against cyclophosphamide-induced testicular damage in rats. Clin Exp Reprod Med. 2024;51(3):205. https://doi.org/10.5653/cerm.2023.06415
  51. 51. Hosseini A, Zare S, Borzouei Z, Pakdel FG. Cyclophosphamide-induced testicular toxicity ameliorated by American ginseng treatment: an experimental study. Int J Reprod Biomed. 2018;16(11):711.
  52. 52. Ghaffarie T, Johari H, Najafian M, Kargar H. Effect of hydroalcoholic extract of cinnamon on the pituitary-gonadal axis in adult male rats under chemotherapy by cyclophosphamide. Zahedan J Res Med Sci. 2013;16(3):16-20.
  53. 53. Aghaei S, Nikzad H, Taghizadeh M, Tameh AA, Taherian A, Moravveji A. Protective effect of pumpkin seed extract on sperm characteristics, biochemical parameters and epididymal histology in adult male rats treated with cyclophosphamide. Andrologia. 2014;46(8):927-35. https://doi.org/10.1111/and.12175

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