Veterinary Research Forum

Protective effects of royal jelly on dibutyl phthalate-induced oxidative and inflammatory ovarian damage in female Wistar rats

Document Type : Original Article

Authors

Mahdieh Nezami Majd 1
Goudarz Sadeghi Hashjin 2, 3
Hassan Malekinejad 4
Ali Rassouli 2

1 Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

2 Department of Comparative Biosciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

3 Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Near East University, Nicosia, Northern Cyprus

4 Department of Pharmacology and Toxicology, School of Pharmacy, Urmia University of Medical Sciences, Urmia, Iran

https://doi.org/10.30466/vrf.2026.2082953.5048
Abstract
Phthalates are endocrine-disrupting chemicals widely used in numerous consumer products, making human and animal exposure a significant public health concern. This study evaluated the protective effects of royal jelly (RJ) against dibutyl phthalate (DBP)-induced ovarian damage. Forty female Wistar albino rats were randomly allocated into eight groups (n = 5 per group): Control (corn oil), DBP (500 mg kg-1), RJ (200 mg kg-1), Quercetin (QCN; 50.00 mg kg-1), DBP + RJ (100, 200 and 300 mg kg-1) and DBP + QCN (50.00 mg kg-1) for 28 consecutive days by gavage. QCN was used as a positive antioxidant control. The RJ was dissolved in normal saline, whereas, QCN and DBP were dissolved in corn oil. The levels of testosterone, dehydroepiandrosterone and tumor necrosis factor alpha in the serum were measured. Oxidative and nitrosative stress biomarkers (malondialdehyde, nitric oxide, total thiol molecules) were assessed in the ovarian tissue. The histopathological alterations of ovarian tissue and ovarian index were also evaluated. The RJ significantly reduced DBP-induced elevations in serum testosterone and dehydro-epiandrosterone levels. Compared to the DBP group, RJ treatment significantly restored the ovarian index and reduced serum tumor necrosis factor alpha level. The RJ treatment significantly attenuated DBP-induced oxidative and nitrosative stress in ovarian tissue. The DBP disrupted normal ovarian architecture, characterized by increased atretic and cystic follicles and reduced corpora lutea formation. These alterations were markedly ameliorated by RJ. In conclusion, RJ attenuated DBP-induced ovarian toxicity as evidenced by reduced oxidative/nitrosative stress, decreased serum tumor necrosis factor alpha and lowered serum testosterone as well as dehydroepiandrosterone levels.

Keywords

Dehydroepiandrosterone
Dibutyl phthalate
Ovarian toxicity
Oxidative stress
Royal jelly
Subjects
1.     Hannon PR, Flaws JA. The effects of phthalates on the ovary. Front Endocrinol (Lausanne) 2015; 6: 8. doi: 10.3389/fendo.2015.00008.
2.     Radha MJ, Basha PM. Hepatotoxic evaluation of Di-n-butyl phthalate in Wistar rats upon sub-chronic exposure: a multigenerational assessment. Toxicol Rep 2020; 7(2): 772-778.
3.  Ore A, Adewale AA, Kehinde SA, et al. Potential roles of oxidative stress and insulin resistance in diisononyl phthalate induced dyslipidemia and hepatosteatosis in BALB/c mice. Adv Redox Res 2022; 5: 100038. doi: 10.1016/j.arres.2022.100038.
4.     Asghari MH, Saeidnia S, Abdollahi M. A review on the biochemical and molecular mechanisms of phthalate-induced toxicity in various organs with a focus on the reproductive system. Int J Pharmacol 2015; 11(2):
95-105.
5.     Hirshfield AN. Development of follicles in the mammalian ovary. Int Rev Cytol 1991; 124: 43-101.
Wang W, Craig ZR, Basavarajappa MS, et al. Di (2- ethylhexyl) phthalate inhibits growth of mouse ovarian antral follicles through an oxidative stress pathway. Toxicol Appl Pharmacol 2012; 258(2): 288-295.
6.     Hoyer PB, Keating AF. Xenobiotic effects in the ovary: temporary versus permanent infertility. Expert Opin Drug Metab Toxicol 2014; 10(4): 511-523.
7.     Zeng X, Xie YJ, Liu YT, et al. Polycystic ovarian syndrome: correlation between hyperandrogenism, insulin resistance and obesity. Clin Chim Acta 2020; 502: 214-221.
8.     Abdelsalam HM, Diab AA, Hussien AG, et al. Ginseng and royal jelly diminish diabetic hepato-and nephron-toxicity in STZ-induced diabetic male rats. Nutrire 2023; 48: 8. doi: 10.1186/s41110-023-00191-1.
9.     Ahmad S, Campos MG, Fratini F, et al. New insights into the biological and pharmaceutical properties of royal jelly. Int J Mol Sci 2020; 21(2): 382. doi: 10.3390/ ijms21020382.
10. Ab Hamid N, Abu Bakar AB, Mat Zain AA, et al. Composition of Royal Jelly (RJ) and its anti-androgenic effect on reproductive parameters in a polycystic ovarian syndrome (PCOS) animal model. Antioxidants (Basel) 2020; 9(6): 499. doi: 10.3390/antiox9060499.
11. Wang G, Wang Y, Yao L, et al. Pharmacological activity of quercetin: an updated review. Evid Based Complement Alternat Med 2022; 2022: 3997190. doi: 10.1155/2022/3997190.
12. Shah MZ, Shrivastava VK, Mir MA, et al. Effect of quercetin on steroidogenesis and folliculogenesis in ovary of mice with experimentally-induced polycystic ovarian syndrome. Front Endocrinol (Lausanne) 2023; 14: 1153289. doi: 10.3389/fendo.2023.1153289.
13. Ashari S, Karami M, Shokrzadeh M, et al. Quercetin ameliorates Di (2-ethylhexyl) phthalate-induced nephrotoxicity by inhibiting NF-κB signaling pathway. Toxicol Res (Camb) 2022; 11(2): 272-285.
14. Abd-Ellah MF, Aly HA, Mokhlis HA, et al. Quercetin attenuates di-(2-ethylhexyl) phthalate-induced testicular toxicity in adult rats. Hum Exp Toxicol 2016; 35(3): 232-243.
15. Nezami Majd M, Sadeghi-Hashjin G, Malekinejad H, et al. Hepatoprotective effect of royal jelly on dibutyl phthalate-induced liver injury in rats. Vet Res Forum 2025; 16(2): 97-105.
16. Ghanbari E, Khazaei MR, Khazaei M, et al. Royal jelly promotes ovarian follicles growth and increases steroid hormones in immature rats. Int J Fertil Steril 2018; 11(4): 263-269.
17. Niehaus Jr WG, Samuelsson B. Formation of malonaldehyde from phospholipid arachidonate during microsomal lipid peroxidation. Eur J Biochem 1968; 6(1): 126-130.
Green LC, Wagner DA, Glogowski J, et al. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 1982; 126(1): 131-138.
18. Hu ML. Measurement of protein thiol groups and glutathione in plasma. In: Packer L (Ed). Methods in enzymology. San Diego, USA: Academic Press 1994; 380-385.
19. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193(1): 265-275.
20. Brehm E, Zhou C, Gao L, et al. Prenatal exposure to an environmentally relevant phthalate mixture accelerates biomarkers of reproductive aging in a multiple and transgenerational manner in female mice. Reprod Toxicol 2020; 98: 260-268.
21. Zhou C, Gao L, Flaws JA. Exposure to an environmentally relevant phthalate mixture causes transgenerational effects on female reproduction in mice. Endocrinology 2017; 158(6): 1739-1754.
22. Zhou C, Gao L, Flaws JA. Prenatal exposure to an environmentally relevant phthalate mixture disrupts reproduction in F1 female mice. Toxicol Appl Pharmacol 2017; 318: 49-57.
23. Betteridge DJ. What is oxidative stress? Metabolism 2000; 49(2 Suppl 1): 3-8.
24. Agarwal A, Aponte-Mellado A, Premkumar BJ, et al. The effects of oxidative stress on female reproduction: a review. Reprod Biol Endocrinol 2012; 10: 49. doi: 10.1186/1477-7827-10-49.
25. Singh S, Lata S. Effect of exposure to di-butyl phthalate on reproductive physiology in adult female mouse. Int J Pharm Sci Res 2015; 6(11): 4788-4795.
26. Chen J, Yang S, Ma B, et al. Di-isononyl phthalate induces apoptosis and autophagy of mouse ovarian granulosa cells via oxidative stress. Ecotoxicol Environ Saf 2022; 242: 113898. doi: 10.1016/j.ecoenv. 2022.113898.
27. Kang JC, Jee JH, Koo JG, et al. Anti-oxidative status and hepatic enzymes following acute administration of diethyl phthalate in olive flounder Paralichthys olivaceus, a marine culture fish. Ecotoxicol Environ Saf 2010; 73(6): 1449-1455.
28. Khazaei F, Ghanbari E, Khazaei M. Improved hormonal and oxidative changes by Royal Jelly in the rat model of PCOS: An experimental study. Int J Reprod Biomed 2021; 19(6): 515-524.
29. Oršolić N, Jazvinšćak Jembrek M. Royal Jelly: biological action and health benefits. Int J Mol Sci 2024; 25(11): 6023. doi: 10.3390/ijms25116023.
30. Chi X, Liu Z, Wang H, et al. Royal jelly enhanced the antioxidant activities and modulated the gut microbiota in healthy mice. J Food Biochem 2021; 45(5): e13701. doi: 10.1111/jfbc.13701.
31. Ma P, Liu X, Wu J, et al. Cognitive deficits and anxiety induced by diisononyl phthalate in mice and the neuroprotective effects of melatonin. Sci Rep 2015; 5: 14676. doi: 10.1038/srep14676.
Biswas SK. Does the interdependence between oxidative stress and inflammation explain the antioxidant paradox? Oxid Med Cell Longev 2016; 2016(1): 5698931. doi: 10.1155/2016/5698931.
32. Metwally Ibrahim SEL, Kosba AA. Royal jelly supplementation reduces skeletal muscle lipotoxicity and insulin resistance in aged obese rats. Pathophysiology 2018; 25(4): 307-315.
33. Davis BJ, Weaver R, Gaines LJ, et al. Mono-(2-ethylhexyl) phthalate suppresses estradiol production independent of FSH-cAMP stimulation in rat granulosa cells. Toxicol Appl Pharmacol 1994; 128(2): 224-228.
34. Lovekamp TN, Davis BJ. Mono-(2-ethylhexyl) phthalate suppresses aromatase transcript levels and estradiol production in cultured rat granulosa cells. Toxicol Appl Pharmacol 2001; 172(3): 217-224.
Lovekamp-Swan T, Jetten AM, Davis BJ. Dual activation of PPARα and PPARγ by mono-(2-ethylhexyl) phthalate in rat ovarian granulosa cells. Mol Cell Endocrinol 2003; 201(1-2): 133-141.
35. Watkins DJ, Téllez-Rojo MM, Ferguson KK, et al. In utero and peripubertal exposure to phthalates and BPA in relation to female sexual maturation. Environ Res 2014; 134: 233-241.
36. Sen N, Liu X, Craig ZR. Short term exposure to di-n-butyl phthalate (DBP) disrupts ovarian function in young CD-1 mice. Reprod Toxicol 2015; 53: 15-22.
37. Xia LZ, Jiang MZ, Liu LL, et al. Protective effect of quercetin against phthalates induced hepatotoxicity in rats. Toxicol Res (Camb) 2022; 11(5): 863-871.
38. Mahmoud AA, Elfiky AM, Abo-Zeid FS. The anti-androgenic effect of quercetin on hyperandrogenism and ovarian dysfunction induced in a dehydro-epiandrosterone rat model of polycystic ovary syndrome. Steroids 2022; 177: 108936. doi: 10.1016/ j.steroids.2021.108936.
Veterinary Research Forum
Volume 17, Issue 10
October 2026
Pages 775-784

  • Receive Date 04 January 2026
  • Revise Date 29 August 2026
  • Accept Date 31 August 2026

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