Protective effects of Tribulus Terrestris extract on cisplatin-induced ovarian damage: Antioxidants and anti-inflammatory insights

Abstract

Background

Cisplatin (CIS) is a widely used chemotherapeutic agent; however, it is associated with ovarian toxicity. Tribulus Terrestris (TT) is recognized for its antioxidant and anti-inflammatory properties. This study aims to evaluate the effects of TT extract on ovarian tissue damage induced by cisplatin.

Material and Method

Twenty-five female BALB/c mice were divided into five groups (n = 5): Control, CIS (Cisplatin only), CIS + TT100 (100 mg/kg TT extract daily + CIS), CIS + TT300 (300 mg/kg TT + CIS), and CIS + TT500 (500 mg/kg TT daily + CIS). After 15 days, blood samples were collected for hormonal analysis, and ovaries were harvested for histopathological, immunohistochemical, and biochemical assessments.

Results

The CIS group exhibited a significant decline in follicle count compared to the control group (P < 0.001). In contrast, the CIS + TT groups showed a notable increase in follicle count (P < 0.05). TT treatment also resulted in significant improvements in antioxidant markers (SOD, CAT) and a reduction in oxidative stress (MDA) compared to the CIS group. Moreover, E2, AMH, and progesterone concentrations were decreased in the CIS group, while these levels were restored in the TT-treated groups (P < 0.001). The expression of inflammatory markers TNF-α and IL-1β was higher in the CIS group and decreased in the TT-treated groups.

Conclusion

Tribulus Terrestris extract effectively mitigates cisplatin-induced ovarian toxicity by enhancing follicular count, improving antioxidant activity, and reducing oxidative stress. TT treatment also elevated AMH and progesterone levels while decreasing inflammatory markers, underscoring its potential as a protective agent against cisplatin-induced ovarian damage.

Keywords

premature ovarian failure Tribulus Terrestris antioxidant inflammation

Introduction

One of the primary health concerns stemming from the administration of antineoplastic medications in female recipients is ovarian toxicity. Research has substantiated that chemotherapy serves as a significant catalyst for the onset of premature ovarian failure (POF), subsequently leading to infertility and ovarian endocrine dysfunction.¹ POF is defined as the cessation of ovarian functionality before the age of 40 in women and pertains to a condition in which the quantity of ovarian follicles rapidly diminishes before menopause. Clinical indicators of POF encompass irregular menstrual cycles or abrupt pause of menstruation, vaginal dryness, excessive perspiration, and hair loss, among others.^2,3

Cis-diamminedichloroplatinum II, commonly referred to as cisplatin, is a chemotherapeutic agent widely employed in the treatment of various cancer types, and substantial tumors.^4,5 While cisplatin is highly effective in combating cancer, it is associated with significant adverse effects, notably hepatotoxicity and nephrotoxicity. Cisplatin falls into the category of moderate gonadal risk drugs, and its administration to women of reproductive age can result in the loss of primordial follicles and the development of POF, leading to female infertility and early menopause. The precise mechanism underlying cisplatin-induced side effects remains multifactorial.^6–8

Reproductive toxicity induced by cisplatin can be attributed to several factors, including the generation of reactive oxygen species (ROS), disruption of steroid production, inflammatory responses, and activation of apoptosis pathways.^5,9,10 Inflammation is a prominent component of cisplatin toxicity, with cisplatin stimulating extracellular signal-regulated kinases, leading to the release of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6, along with enhanced nuclear factor kappa B (NF-κB).^11,12 Translocation of these events results in increased poly (ADP-ribose) polymerase (PARP) activity, a marker of DNA damage due to inflammation, ultimately culminating in cell death.^13,14 Therefore, the preservation of fertility in women undergoing cisplatin treatment is a critical concern. Phytochemicals, such as Tribulus Terrestris (TT), present a promising adjuvant treatment option due to their considerable benefits over conventional therapies.¹⁵

Tribulus Terrestris is a perennial plant from the Zygophyllaceae family, found in regions such as the Mediterranean, western Asia, China, Korea, and Europe. Its fruits and seeds have been traditionally utilized for various medicinal purposes. TT is rich in chemical compounds, including flavonoids, tannins, and phenolic acids, which exhibit antioxidant, antimicrobial, and anti-inflammatory properties.¹⁶ Numerous studies have highlighted the beneficial effects of TT extract in mitigating male reproductive toxicity caused by chemotherapy agents like cyclophosphamide and cisplatin, primarily through its ability to reduce oxidative stress and inflammation—two critical factors in reproductive damage.^17,18 While the anti-inflammatory and antioxidant effects of Tribulus Terrestris extract on female reproductive health have been well-documented, particularly in conditions such as polycystic ovary syndrome, research on its potential to prevent reproductive toxicity induced by antineoplastic drugs in women remains scarce.^19,20 This gap underscores the necessity for further investigation into its protective role in maintaining female reproductive health during cancer treatment.

This study aims to examine the preventive effects of a dry extract of Tribulus Terrestris on the ovaries of female mice. It investigates the mechanisms underlying these protective effects by assessing the impact of TT co-administration with cisplatin on ovarian tissue levels of IL-1β and TNF-α, as well as markers of oxidative stress (MDA, SOD, and CAT). Additionally, the study evaluated serum hormonal profiles (AMH, 17β-estradiol, and progesterone) and examined histopathological and immunohistochemical changes in ovarian tissues.

Materials and methods

Animal

A total of 25 female BALB/c mice, weighing 20–25 g and aged 8–12 weeks, were obtained from the Animal House of Tabriz Medical Science University for this experiment. The study adhered to the ARRIVE guidelines and complied with the Guide for the Care and Use of Laboratory Animals, ensuring regular health checks, weight monitoring, and appropriate anesthesia. The sample size was determined using the resource equation method. Ethical approval for all procedures was granted by the Ethical Committee of Tabriz Medical Science University (IR.TBZMED.AEC.1402.050). Mice were individually housed in standard cages under controlled conditions: temperature of 22°C–24 °C, humidity at 45%, and a 12/12-h light/dark cycle. They had unrestricted access to food and water and underwent a 7-day acclimatization period before the study commenced.

Preparation of plant extract

Tribulus Terrestris was sourced from a traditional medicine research center (herbal code 14,019, Agriculture Faculty, Tabriz University) and verified by a botanist. Initially, 250 g of TT was ground into a powder. The seeds were then subjected to extraction using the soxhlet method, with two rounds of defatting conducted. In the first round, n-hexane was employed as a defatting agent, followed by a second round using 70% ethanol for the final extraction. The resultant extract was subjected to lyophilization, resulting in a lyophilized extract weighing 19 g. This extract was subsequently dissolved in distilled water and administered intraperitoneally (IP) to the mice, with dosages specified in milligrams of extract per kilogram of body weight.²¹

Experimental design

Adult mice were housed under controlled conditions. After an adaptation phase, they were monitored for estrous cycle assessment via vaginal smears. Following this, the mice were randomly assigned to five groups, each consisting of five mice:

(1) Group 1 (Control): Mice received an equivalent volume of normal saline via IP injection.

(2) Group 2 (Cisplatin): Mice received a single dose of cisplatin (5.5 mg/kg) (Sigma-Aldrich).²²

(3) Group 3 (G1, Cisplatin + TT 100 mg/kg): Mice received cisplatin (5.5 mg/kg) and 100 mg/kg of Tribulus Terrestris (TT) extract^23,24

(4) Group 4 (G2, Cisplatin + TT 300 mg/kg): Mice received cisplatin (5.5 mg/kg) and 300 mg/kg of TT extract.²⁵

(5) Group 5 (G3, Cisplatin + TT 500 mg/kg): Mice received cisplatin (5.5 mg/kg) and 500 mg/kg of TT extract.²⁵

This treatment regimen continued for 14 consecutive days, guided by prior research.^17,26,27 Twenty-four hours after the final TT administration, the estrous cycle was reassessed. Mice were then anesthetized using ketamine (50 mg/kg) and xylazine (10 mg/kg), and blood samples were collected via cardiac puncture. These samples were centrifuged at 3000 rpm for 15 min to obtain serum, which was frozen at −80°C for subsequent analysis. Following blood collection, euthanasia was performed via cervical dislocation, and the left ovarian tissues were extracted for histological and immunohistochemical (IHC) staining, while the right ovaries were designated for biochemical assessments. Ovarian tissue samples were stored at −70°C for further biochemical examination.

Histological process

To achieve this objective, Samples underwent examination under a light microscope. The microscope was utilized to observe and assess various aspects of the tissue samples, including the hemorrhage, tissue bleeding, and any signs of tissue disruption. Additionally, the morphological evaluation of follicles was conducted, explicitly focusing on follicle diameter and any signs of degeneration. As well as the count of follicles was performed by light microscope. These assessments were conducted to gain insights into the histopathological changes and alterations within the ovarian tissue.

Biochemical assays in ovaries

After the experiment, ovarian tissues were homogenized in 10 ml of phosphate-buffered saline (PBS) and centrifuged at 5000×g for 1 h at 4°C. The resulting supernatants were used to analyze Malondialdehyde (MDA), Superoxide Dismutase (SOD), and Catalase (CAT).

Malondialdehyde (MDA)

Lipid peroxidation was evaluated using MDA as a biomarker via the thiobarbituric acid reactive species (TBARS) assay. The product was quantified colorimetrically at 532 nm to determine MDA levels.²⁸

Superoxide dismutase (SOD)

SOD, an essential antioxidant enzyme, catalyzes the dismutation of superoxide anions into hydrogen peroxide and molecular oxygen. The SOD activity was assessed by monitoring the rate of auto-oxidation of a chromogen. Inhibition of this rate correlated linearly with SOD concentration, measured colorimetrically at 480 nm.²⁸

Catalase (CAT)

CAT activity was measured by monitoring the consumption of H₂O₂ colorimetrically, with readings taken at 240 nm.

Hormone assays

The hormonal profile in mouse serum samples was quantitatively assessed using ELISA kits, and the results were expressed in milligrams per milliliter (mg/mL). Specific ELISA kits designed for mice were employed to determine the serum levels of progesterone (Novus Biologicals, NBP2-60125), 17β-estradiol (CUSABIO, CSB-E05109m), and Anti-Müllerian hormone (AMH) (AVIV SYSTEM BIOLOGY, OKEH00320). All procedures followed the manufacturer’s instructions.

Inflammatory markers

IL-1β and TNF-α were evaluated as inflammatory markers. Immunohistochemical expression of the mentioned markers in the ovarian tissues was accomplished using a ready-to-use rabbit polyclonal antibody (D9542, Sigma-Aldrich). The staining of IL-1β and TNF-α was assessed according to the intensity of IHC-positive cells.

Statistical analysis

The statistical analysis was conducted using SPSS software, version 19, a commonly used tool for data analysis in scientific research. All the obtained results were presented as mean ± standard deviation (SD), measuring the variability within the data. To evaluate the differences among the five groups, One-way analysis of variance (ANOVA) was employed. The ANOVA test is suitable for comparing means across multiple groups or conditions. Following the ANOVA analysis, a Tukey test was applied as a post hoc test to further examine pairwise differences between the groups. In this study, a significance level of P < 0.05 was chosen as the threshold for statistical significance.

Figure 1.

Histological finding.

Results

Histopathological evaluation

Figure 1 shows the histopathology of ovaries in all groups of the study. In the control group, the histological structure and morphology of ovaries showed no signs of hemorrhage or tissue degeneration. However, in the CIS group, examination of ovarian tissue histopathology indicated evident signs of tissue degeneration, hemorrhage, and disruption. Moreover, the number of primary, secondary, and tertiary follicles notably decreased in the CIS group compared to the control group, while the higher count of atretic follicles was seen in the CIS group relative to the control group (P < 0.01). In groups treated with Tribulus Terrestris extract (100, 300, and 500 mg/kg), there was a substantial increase in the number of primary, secondary, and tertiary follicles compared to the CIS group (P < 0.05). Furthermore, the number of atretic follicles in therapeutic groups exhibited a significant reduction compared to the CIS group (P < 0.05) (Table 1).

Figure 2.

The immunohistochemistry of IL-1β. Control, Cisplatin: received a single-dose injection of Cisplatin, CIS + TT100: Cisplatin + 100 mg/kg of Tribulus Terrestris, CIS + TT300: Cisplatin + 300 mg/kg of Tribulus Terrestris, CIS + TT500: Cisplatin + 500 mg/kg of Tribulus Terrestris.

Table 1.

The number of follicles, primary, secondary, tertiary, and atretic follicles, in all groups of the study.

Groups	Primary follicles (Mean ± SD)	Secondary follicles (Mean ± SD)	Tertiary follicles (Mean ± SD)	Atretic follicles (Mean ± SD)
Control	45.32 ± 6.08	21.67 ± 2.12	10.32 ± 0.24	7.11 ± 1.37
Cisplatin	23.25 ± 2.64*	8.32 ± 1.02*	3.24 ± 0.08*	21.72 ± 2.45*
Cisplatin + 100 mg/kg of Tribulus Terrestris (G1)	29.67 ± 4.52**	12.54 ± 1.05**	5.14 ± 0.04**	16.41 ± 2.11**
Cisplatin + 300 mg/kg of Tribulus Terrestris (G2)	32.33 ± 2.38**	14.12 ± 1.25**	6.14 ± 0.06**	14.08 ± 2.31**
Cisplatin + 500 mg/kg of Tribulus Terrestris (G3)	38.33 ± 5.52**	17.45 ± 1.92**	7.24 ± 0.14**	11.46 ± 1.66**

(*) shows a significant difference compared to the control group (P < 0.01). (**) shows a significant difference compared to the cisplatin group (P < 0.05).

Control, Cisplatin: received a single-dose injection of Cisplatin, G1: Cisplatin + 100 mg/kg of Tribulus Terrestris, G2: Cisplatin + 300 mg/kg of Tribulus Terrestris, G3: Cisplatin + 500 mg/kg of Tribulus Terrestris. Yellow Circles, Red Arrows, Blue Arrows, and Green Arrows show Primary, Secondary, Tertiary, and Atretic Follicles, respectively. Scale Bar: 100X.

Oxidative stress markers in ovarian tissue

Table 2 shows the ovaries tissues levels of oxidative stress markers, MDA, SOD, and CAT. In mice treated with cisplatin, the level of MDA, an indication of lipid peroxidation, remarkably enhanced compared to the control group (P < 0.01), while levels of SOD and CAT significantly decreased compared to the control group. Conversely, in all groups that received cisplatin + TT extract (100, 300, and 500 mg/kg), a noteworthy decline in MDA levels and a remarkable increase in the levels of SOD and CAT were observed (P < 0.05).

Table 2.

Oxidative stress markers, SOD, MDA, and CAT, in mice ovaries tissues.

Groups	SOD (U/mg) (Mean ± SD)	CAT (U/mg) (Mean ± SD)	MDA (nmol/mg) (Mean ± SD)
Control	36.8 ± 4.15	98.11 ± 4.50	2.3 ± 0.25
Cisplatin	13.6 ± 1.22*	62.1 ± 3.25*	4.13 ± 0.30*
Cisplatin + 100 mg/kg of Tribulus Terrestris (G1)	22.4 ± 3.17**	63.70 ± 4.35**	3.21 ± 0.40**
Cisplatin + 300 mg/kg of Tribulus Terrestris (G2)	23.2 ± 1.21**	72.3 ± 3.45**	3.01 ± 0.45**
Cisplatin + 500 mg/kg of Tribulus Terrestris (G3)	24.5 ± 2.35**	82.8 ± 4.50**	2.97 ± 0.50**

(*) shows a significant difference compared to the control group (p < 0.01). (**) shows a significant difference compared to the cisplatin group (p < 0.05).

Hormone profile

Serum levels of E2, AMH, and progesterone significantly decreased in mice treated with cisplatin compared to the control group (P < 0.01). On the other hand, in all groups that received Tribulus Terrestris, G1, G2, and G3, serum levels of all mentioned hormones remarkably increased in comparison with the cisplatin group (P < 0.05). These findings are summarized in Table 3.

Table 3.

Hormone profile findings.

Groups	Progesterone (ng/mL) (Mean ± SD)	E2 (pg/mL) (Mean ± SD)	AMH (ng/mL) (Mean ± SD)
Control	6.13 ± 1.15	29.48±3.20	3.13 ± 0.11
Cisplatin	2.26 ± 0.22*	16.15±2.45*	1.73 ± 0.29*
Cisplatin + 100 mg/kg of Tribulus Terrestris (G1)	3.73 ± 0.17**	25.31 ± 2.15**	2.2 ± 011**
Cisplatin + 300 mg/kg of Tribulus Terrestris (G2)	3.86 ± 0.21**	24.30 ± 2.16**	2.6 ± 0.20**
Cisplatin + 500 mg/kg of Tribulus Terrestris (G3)	4.5 ± 0.35**	22.08 ± 1.76**	2.9 ± 0.11**

(*) shows a significant difference compared to the control group (P < 0.01). (**) shows a significant difference compared to the cisplatin group (P < 0.05).

IHC result

Figures 2 and 3 show the IHC staining of two inflammatory markers, IL-1β and TNF-α. According to these figures, strong protein expression intensity in the tissue sections of the cisplatin group was observed. In contrast, the control group did not exhibit expression intensity of TNF-α and IL-1β proteins. However, in groups treated with Tribulus Terrestris extract (G1, G2, and G3), the expression of IL-1β and TNF-α proteins was weaker than in the cisplatin group.

Figure 3.

The IHC of TNF-α. Control, Cisplatin: received a single-dose injection of Cisplatin, CIS + TT100: Cisplatin + 100 mg/kg of Tribulus Terrestris, CIS + TT300: Cisplatin + 300 mg/kg of Tribulus Terrestris, CIS + TT500: Cisplatin + 500 mg/kg of Tribulus Terrestris.

Discussion

The findings of this study highlight the potential protective effects of Tribulus Terrestris extract against ovarian damage induced by cisplatin, a widely used chemotherapeutic agent. Through a multidimensional assessment that includes histopathological evaluation, oxidative stress markers, hormone profiles, and immunohistochemistry, we gain a comprehensive understanding of how Tribulus Terrestris extract impacts ovarian health.

Histopathological examination revealed significant damage to ovarian tissue in the cisplatin-treated group. Observations included tissue degeneration, hemorrhage, and structural disruption, alongside a notable reduction in the number of follicles—primary, secondary, and tertiary. Additionally, there was a marked increase in atretic follicles, suggesting a degenerative process within the ovarian tissue. These alterations align with the well-documented adverse effects of cisplatin on reproductive organs.^29,30 For instance, Algandaby et al. reported that cisplatin treatment resulted in oxidative stress, ovarian degeneration, and apoptosis.²⁹ Numerous studies have shown that cisplatin negatively affects sexual and reproductive functions in both males and females, likely due to its activation of ROS and free radicals.^31,32 This leads to damage in the inner mitochondrial membrane, resulting in the release of cytochrome c and subsequent activation of caspases, culminating in apoptosis.³³ Such conditions contribute to ovarian tissue damage and a decrease in estrogen production, ultimately impairing sexual function in females.²² The clinical implications of cisplatin-induced oxidative stress on ovarian tissue are significant. ROS-induced damage to ovarian follicles and oocytes can lead to ovarian toxicity, resulting in ovarian dysfunction, impaired folliculogenesis, and reduced ovarian reserve.^34,35 Clinically, this may manifest as ovarian failure, infertility, or premature menopause in women undergoing cisplatin-based chemotherapy. Moreover, cisplatin-induced oxidative stress can promote drug resistance by activating survival pathways and DNA repair mechanisms in cancer cells. This compromise in efficacy can lead to treatment failure and disease recurrence in patients with ovarian cancer.^11,36

In contrast, the treatment groups that received Tribulus Terrestris extract demonstrated a significant preservation of ovarian tissue architecture. The counts of various follicles were substantially higher in these groups compared to the cisplatin group. Furthermore, the number of atretic follicles was notably reduced, suggesting a protective effect of Tribulus Terrestris extract against degenerative changes in ovarian follicles. This histopathological evidence indicates that Tribulus Terrestris extract may mitigate the structural damage caused by cisplatin in the ovaries. These results are consistent with the antioxidant and anti-inflammatory properties of Tribulus Terrestris extract, which protects ovarian tissue from oxidative damage.^37,38 For instance, Dehghan et al. found that Tribulus Terrestris extract can safeguard ovarian tissue from oxidative damage related to polycystic ovary syndrome.³⁷ Additionally, a study by Figueiredo et al. highlighted the antioxidant and anticancer effects of Tribulus Terrestris extract.³⁹

Oxidative stress is a well-documented mechanism responsible for the toxic effects of cisplatin on various tissues, including the ovaries. The assessment of oxidative stress markers—specifically MDA, SOD, and CAT—offered valuable insights into the antioxidative potential of Tribulus Terrestris extract. Cisplatin is a widely used chemotherapeutic agent for various cancers, including ovarian cancer. It induces oxidative stress in ovarian tissue through several mechanisms. Upon entering the cell, cisplatin interacts with intracellular components, leading to the generation of reactive oxygen species (ROS) such as superoxide anion (O₂^-), hydrogen peroxide (H₂O₂), and hydroxyl radicals (•OH). These ROS can cause direct damage to cellular structures, including lipids, proteins, and DNA, resulting in cell dysfunction and death.³⁶ Additionally, cisplatin-induced ROS can disrupt mitochondrial function, leading to mitochondrial membrane depolarization, impaired ATP production, and the release of cytochrome-c. This cascade can activate apoptotic pathways, contributing to ovarian cell death. Furthermore, cisplatin can form DNA adducts, causing DNA damage that activates repair mechanisms. However, excessive DNA damage can overwhelm the cell’s repair capacity, resulting in persistent lesions and the activation of apoptotic pathways.^40,41

In the cisplatin group, there was a significant increase in MDA levels, indicative of elevated lipid peroxidation and oxidative damage. Concurrently, the activities of SOD and CAT were notably decreased, suggesting a compromised antioxidant defense system in response to cisplatin-induced oxidative stress.⁴¹ In contrast, the groups treated with Tribulus Terrestris extract displayed a significant reduction in MDA levels, indicating a potential antioxidant effect. Additionally, the activities of SOD and CAT were significantly higher in these groups compared to the CIS group, reflecting an enhancement of the antioxidant defense system.¹⁹ These findings are consistent with previous studies highlighting the antioxidant properties of Tribulus Terrestris extract. The active compounds in Tribulus Terrestris, such as flavonoids and saponins, are recognized for their free radical-scavenging capabilities. The ability of Tribulus Terrestris extract to mitigate oxidative stress in ovarian tissue suggests a protective mechanism against cisplatin-induced damage.^19,38

Cisplatin-induced changes in hormone levels, particularly progesterone and estrogen, are linked to reproductive toxicity.^42,43 The CIS group exhibited a significant decrease in AMH and progesterone levels, along with a reduction in estrogen, indicating a hormonal imbalance due to cisplatin exposure. In contrast, the treatment groups receiving Tribulus Terrestris extract showed a significant reversal of these hormonal disturbances, reflected in increased hormone levels. Tribulus Terrestris is recognized for its potential role in hormone regulation. The extract is thought to modulate hormone levels by influencing the activity of enzymes involved in steroidogenesis. The restoration of hormonal balance in the Tribulus Terrestris-treated groups suggests that it plays a regulatory role in maintaining hormonal homeostasis. This modulation may contribute to its protective effects against cisplatin-induced reproductive toxicity.^38,44

Cisplatin-induced oxidative stress can initiate inflammatory responses in ovarian tissue, leading to the production of pro-inflammatory cytokines and chemokines. This inflammatory microenvironment may further exacerbate oxidative damage and tissue injury. Although ovarian tissue possesses antioxidant defense mechanisms to counteract ROS and mitigate oxidative stress, cisplatin can severely compromise these defenses, resulting in oxidative damage that surpasses the tissue’s capacity for repair and regeneration.^40,45 Inflammation is a critical component of cisplatin-induced toxicity.⁴⁶ Cytokines such as IL-1β and TNF-α are pivotal in mediating the inflammatory response. Immunohistochemical analysis revealed a marked expression of IL-1β and TNF-α proteins in the ovarian tissue of the cisplatin-treated group, indicating heightened inflammatory activity.¹¹ In contrast, the groups treated with Tribulus Terrestris extract demonstrated reduced expression of IL-1β and TNF-α proteins, suggesting an anti-inflammatory effect. The anti-inflammatory properties of Tribulus Terrestris are attributed to its bioactive compounds. The observed downregulation of inflammatory markers in the treated groups further supports the extract’s potential as an anti-inflammatory agent.⁴⁴

In summary, the comprehensive analysis of histopathological, oxidative stress, hormone profiles, and immunohistochemical parameters provides compelling evidence for the protective effects of Tribulus Terrestris extract against cisplatin-induced ovarian damage. The findings indicate that Tribulus Terrestris significantly mitigates ovarian toxicity, as evidenced by increased follicular count, improved antioxidant markers (SOD, CAT), reduced oxidative stress (MDA), and elevated levels of AMH and progesterone. Furthermore, treatment resulted in decreased expression of pro-inflammatory cytokines TNF-α and IL-1β, highlighting its potential as a therapeutic agent to safeguard ovarian function during chemotherapy. While these results show promise for future clinical applications, further studies are necessary to confirm efficacy and safety in humans.

Study limitations

While the findings are promising, several limitations must be acknowledged. This research utilized a mouse model, necessitating careful consideration when extrapolating results to humans. The treatment period was limited, and the study lacked healthy control groups for Tribulus Terrestris extract administration. Furthermore, the optimal dosage and duration for maximum efficacy require further investigation. Also, clinical trials involving human subjects are essential to validate the translational potential of Tribulus Terrestris extract in addressing cisplatin-induced reproductive toxicity.

Footnotes

Acknowledgments

The research protocol was approved and supported by the Student Research Committee, Tabriz University of Medical Sciences.

ORCID iD

Morteza Abdi

Ethical considerations

This study adhered to the ARRIVE guidelines for reporting animal research. All experimental procedures were conducted in compliance with the Guide for the Care and Use of Laboratory Animals, ensuring animal welfare monitoring, including regular health checks, weight tracking, and use of appropriate anesthesia. The sample size was determined based on prior research to minimize animal use while ensuring statistical validity. The research protocol received ethical approval from the Ethical Committee of Tabriz University of Medical Sciences, under the ethical code IR.TBZMED.AEC.1402.050.

Author Contributions

Morteza Abdi, Amirreza Jourabchi and Hadi Karimzadeh designed and implemented the present study and also wrote the manuscript. The extraction process is done under the supervision of Laleh Khodaiee. Abbas Majdi Reviewed the written content. All authors have read and approved the final manuscript.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Students Research Committee, Tabriz University of Medical Science, Tabriz, Iran (No: 70919).

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.*

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