The protective effect of hydroalcoholic extract of Ephedra pachyclada leaves on ovarian damage induced by cyclophosphamide in rat: An experimental study


Background: Cyclophosphamide (CP) is an anticancer drug that acts as an alkylation agent after metabolism in the liver. CP has toxic effects on the body’s cells, especially the reproductive system’s function, and causes infertility. Moreover, medicinal plants have few side effects and are psychologically acceptable to patients.

Objective: This study aimed to investigate the impact of Ephedra pachyclada hydroalcoholic extract (EPHE) on ovarian tissue and hypothalamic-pituitary-gonadal axis in rats treated with CP.

Materials and Methods: In this experimental study, 48 adult female Wistar rats (180-200 gr, 9-10 wk) were randomly assigned to 6 experimental groups (n = 8/each): (a) control; (b) sham; (c) CP; (d) CP+250 mg/kg EPHE; (e) CP+500 mg/kg EPHE; (f) CP+1000 mg/kg EPHE. On the 29th day of the experiment, serum was collected; serum concentration of the luteinizing hormone, follicle-stimulating hormone, estrogen, progesterone, and antioxidant activity were measured. The number of ovarian follicles were also counted.

Results: In the CP groups, serum concentrations of follicle-stimulating hormone and luteinizing hormone significantly increased, and estrogen and progesterone significantly decreased (p ≤ 0.05). EPHE significantly compensated for the complications caused by CP and 1000 mg/kg had the greatest effect. Antioxidant reduction by CP was significantly enhanced by EPHE, especially at higher doses (p ≤ 0.05). The number of primordial, primary, secondary, and Graafian follicles showed a significant decrease in CP groups and EPHE groups showed a significant increase compared to the CP. EPHE showed that the concentration of 1000 mg/kg was more effective than other doses (p ≤ 0.05).

Conclusion: In addition to proving the effect of EPHE on the hypothalamic-pituitary-gonadal axis, our investigation showed antioxidant properties, which can be an effective factor in CP-treated rats.

Key words: Ephedra, Cyclophosphamide, Ovary, Follicles, Hydroalcoholic extract.

[1] More GS, Thomas AB, Chitlange SS, Nanda RK, Gajbhiye RL. Nitrogen mustards as alkylating agents: A review on chemistry, mechanism of action and current USFDA status of drugs. Anti-Cancer Agents Med Chem 2019; 19: 1080–1102.

[2] Gagelmann N, Bacigalupo A, Rambaldi A, Hoelzer D, Halter J, Sanz J, et al. Haploidentical stem cell transplantation with posttransplant cyclophosphamide therapy vs other donor transplantations in adults with hematologic cancers: A systematic review and metaanalysis. JAMA Oncol 2019; 5: 1739–1748.

[3] Shrief AI, Hamed WHE, Mazroa SA, Moustafa AM. Histological study of the role of CD34+ stem cells and mast cells in cyclophosphamide-induced thymic injury in rats and the possible attenuating role of melatonin. Histochem Cell Biol 2023; 59: 501–512.

[4] Sadeghi Dousari A, Satarzadeh N, Amirheidari B, Forootanfar H. Medicinal and therapeutic properties of ephedra. Revista Brasileira de Farmacognosia 2022; 32: 883–899.

[5] Gonzalez-Juarez DE, Escobedo-Moratilla A, Flores J, Hidalgo-Figueroa S, Martinez-Taguena N, Morales- Jimenez J, et al. A review of the Ephedra genus: Distribution, ecology, ethnobotany, phytochemistry and pharmacological properties. Molecules 2020; 25: 3283.

[6] Bouafia W, Hamdi A, Mouffouk S, Arcos RR, Araujo AJ, Bejarano RG, et al. Phenolic composition, in vitro alpha-amylase and pancreatic lipase inhibitory effects, anti-inflammatory and antioxidant activities of ephedra altissima. Indian J Pharm Sci 2022; 84: 890–901.

[7] Costa VM, Rossato Grando LG, Milandri E, Nardi J, Teixeira P, Mladenka P, et al. Natural sympathomimetic drugs: From pharmacology to toxicology. Biomolecules 2022; 12: 1793.

[8] Zhang BM, Wang ZB, Xin P, Wang QH, Bu H, Kuang HX. Phytochemistry and pharmacology of genus Ephedra. Chin J Nat Med 2018; 16: 811–828.

[9] Kallassy H, Fayyad-Kazan M, Makki R, El-Makhour Y, Rammal H, Leger DY, et al. Chemical composition and antioxidant, anti-inflammatory, and antiproliferative activities of lebanese ephedra campylopoda plant. Med Sci Monit Basic Res 2017; 23: 313–325.

[10] Biswas S, Das R, Banerjee ER. Role of free radicals in human inflammatory diseases. AIMS Biophysics 2017; 4: 596–614.

[11] Ghasemi Pirbalouti A, Amirmohammadi M, Azizi Sh, Craker L. Healing effect of hydro-alcoholic extract of Ephedra pachyclada Boiss in experimental gastric ulcer in rat. Acta Pol Pharm 2013; 70: 1003–1009.

[12] Kalhori Z, Soleimani Mehranjani M, Azadbakht M, Shariaatzadeh MA. Ovary stereological features and serum biochemical factors following induction of polycystic ovary syndrome with testosterone enanthate in mice: An experimental study. Int J Reprod BioMed 2018; 16: 267–274.

[13] Delkhosh A, Delashoub M, Tehrani AA, Bahrami AM, Niazi V, Shoorei H, et al. Upregulation of FSHR and PCNA by administration of coenzyme Q10 on cyclophosphamide-induced premature ovarian failure in a mouse model. J Biochem Mol Toxicol 2019; 33: e22398.

[14] Hamzeh M, Hosseinimehr SJ, Mohammadi HR, Yaghubi Beklar S, Dashti A, Talebpour Amiri F. Atorvastatin attenuates the ovarian damage induced by cyclophosphamide in rat: An experimental study. Int J Reprod BioMed 2018; 16: 323–334.

[15] Rostamzadeh A, Amini-Khoei H, Mardani Korani MJ, Rahimi-Madiseh M. Comparison effects of olive leaf extract and oleuropein compounds on male reproductive function in cyclophosphamide exposed mice. Heliyon 2020; 6: e03785.

[16] Hamidpour R, Hamidpour S, Hamidpour M, Shahlari M, Sohraby M, Shahlari N, et al. Russian olive (Elaeagnus angustifolia L.): From a variety of traditional medicinal applications to its novel roles as active antioxidant, antiinflammatory, anti-mutagenic and analgesic agent. J Tradit Complement Med 2016; 7: 24–29.

[17] Khamis T, Hegazy AA, El-Fatah SSA, Abdelfattah ER, Abdelfattah MMM, Fericean LM, et al. Hesperidin mitigates cyclophosphamide-induced testicular dysfunction via altering the hypothalamic pituitary gonadal axis and testicular steroidogenesis, inflammation, and apoptosis in male rats. Pharmaceuticals 2023; 16: 301.

[18] Sun Y, Liu Y, Ma X, Hu H. The influence of cell cycle regulation on chemotherapy. Int J Mol Sci 2021; 22: 6923.

[19] Spears N, Lopes F, Stefansdottir A, Rossi V, De Felici M, Anderson RA, et al. Ovarian damage from chemotherapy and current approaches to its protection. Hum Reprod Update 2019; 25: 673–693.

[20] Steinbrecht S, Kiebist J, Konig R, Thiessen M, Schmidtke K-U, Kammerer S, et al. Synthesis of cyclophosphamide metabolites by a peroxygenase from Marasmius rotula for toxicological studies on human cancer cells. AMB Express 2020; 10: 128.

[21] Jeelani R, Khan SN, Shaeib F, Kohan-Ghadr HR, Aldhaheri SR, Najafi T, et al. Cyclophosphamide and acrolein induced oxidative stress leading to deterioration of metaphase II mouse oocyte quality. Free Radic Biol Med 2017; 110: 11–18.

[22] Chi YN, Yang JM, Liu N, Cui YH, Ma L, Lan XB, et al. Development of protective agents against ovarian injury caused by chemotherapeutic drugs. Biomed Pharmacother 2022; 155: 113731.

[23] Barberino RS, Lins TLB, Monte APO, Gouveia BB, Campinho DSP, Palheta Jr RC, et al. Melatonin attenuates cyclophosphamide-induced primordial follicle loss by interaction with MT1 receptor and modulation of PTEN/Akt/FOXO3a proteins in the mouse ovary. Reprod Sci 2022; 29: 2505–2514.

[24] Mobasher MA, Hassen MT, Ebiya RA, Alturki NA, Alzamami A, Mohamed HK, et al. Ameliorative effect of citrus lemon peel extract and resveratrol on premature ovarian failure rat model: Role of iNOS/Caspase-3 pathway. Molecules 2022; 28: 122.

[25] Yılmaz TE, Taşdemir M, Kaya M, Arıcan N, Ahıshalı B. The effects of magnesium sulfate on cyclophosphamide-induced ovarian damage: Folliculogenesis. Acta Histochem 2020; 122: 151470.

[26] Kalich-Philosoph L, Roness H, Carmely A, Fishel-Bartal M, Ligumsky H, Paglin S, et al. Cyclophosphamide triggers follicle activation and “burnout”; AS101 prevents follicle loss and preserves fertility. Sci Transl Med 2013; 5: 185ra62.

[27] Huang C, Song K, Ma W, Ding J, Chen Z, Zhang M. Immunomodulatory mechanism of Bushen Huoxue Recipe alleviates cyclophosphamideinduced diminished ovarian reserve in mouse model. J Ethnopharmacol 2017; 208: 44–56.

[28] Rocamora-Reverte L, Melzer FL, Wurzner R, Weinberger B. The complex role of regulatory T cells in immunity and aging. Front Immunol 2021; 11: 616949.

[29] Ghiringhelli F, Larmonier N, Schmitt E, Parcellier A, Cathelin D, Garrido C, et al. CD4+ CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative. Eur J Immunol 2004; 34: 336–344.

[30] Tang S, Ren J, Kong L, Yan G, Liu C, Han Y, et al. Ephedrae herba: A review of its phytochemistry, pharmacology, clinical application, and alkaloid toxicity. Molecules 2023; 28: 663.