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Male infertility is a global health issue, with pharmaceutical agents such as cimetidine contributing significantly to gonadotoxicity through antiandrogenic and oxidative mechanisms. The search for natural protective agents has highlighted
This study evaluated the protective effects of
Thirty-five rats were divided into seven groups: control, cimetidine (120 mg/kg), ethanol extract (200 mg/kg), and cimetidine + fractions (aqueous, butanol, and hexane). After 8 weeks oral administration of extracts/fractions, sperm parameters, serum hormones (LH, FSH, and testosterone), oxidative stress markers (catalase, SOD, and MDA), and histopathology were assessed.
Cimetidine significantly reduce follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone levels (
Male infertility is a growing global public health concern, accounting for nearly 50% of all infertility cases and affecting over 17% of the reproductive age population worldwide, according to the World Health Organization (
Collard green (
Despite its ethnobotanical relevance, the potential protective effects of
This study therefore aims to investigate the therapeutic efficacy of ethanol extracts and solvent fractions of
Thirty-five adult male Wistar rats (8–10 weeks old, weighing 180–220 g) were utilized in this study. The animals were obtained from the animal house of Kampala International University, Western Campus, Ishaka, Uganda. The rats were maintained in standard laboratory cages under controlled environmental conditions, with ad libitum access to water and a standard pellet diet. They were acclimatized to the laboratory environment for 1 week before the start of the experiment.
Cimetidine tablets (400 mg) used in this study was purchased from Cosmos Ltd., Nairobi, Kenya. For the extraction and fractionation process, a total of 5.5 L of 99% ethanol and 400 mL each of n-hexane and n-butanol were used. These solvents were purchased from God’s Grace Biomed Supply Ltd., Kampala, Uganda. The chemical-grade solvents were sourced from established manufacturers and used without further purification. Specifically, the n-hexane was obtained from Sigma-Aldrich (Cat. No. 296090), n-butanol from Merck (Cat. No. 101990), and 99% ethanol from Sigma-Aldrich (Cat. No. 24103). All reagents used in the study were of analytical grade and handled according to standard laboratory procedures.
An ethical clearance from the Research Ethics Committee (REC), Kampala International University Western Campus, Ishaka, Uganda was obtained. Also, REC number (KIU-2024-389) Uganda National for Science and Technology with registration number (HSF5192ES) was also obtained.
Fresh leaves of
Showing taxonomic and morphological identification details of
| Items | |
|---|---|
| Date of collection | 03-09-2024 |
| Collection number | IOE-24-001 |
| Local name | Sukuma Wiki |
| Family name | Cruciferous family/Acephala group |
| Generic name | Chepkilumnda |
| Scientific name | |
| Growth form | Rosette growth |
| Color | Dark green |
| Type of inflorescence | Raceme |
| Leaf shape | Ovate shape |
| Type of margin | Smooth or slightly undulating margin |
| Leaf venation | Palmate venation pattern |
| Pubescence | Glabrous (smooth and hairless) leaves |
| Leaf arrangement | Alternate leaf arrangement |
| Common plants around | Tomatoes, onions, garlic, beans, carrots |
| Type of ecosystem | Subtropical and tropical gardens |
| Locality | Bwejuragye, Ishaka town |
| Habitat | Subtropical and tropical areas |
Fresh leaves of
For solvent partitioning, 100 g of the ethanol extract was reconstituted in 400 mL of distilled water and transferred into a separating funnel. An equal volume (400 mL) of n-hexane was added, and the mixture was vigorously shaken to allow for phase separation. The upper n-hexane layer was collected and labeled as the “n-hexane fraction.” The remaining aqueous phase was subsequently extracted with 400 mL of n-butanol using the same procedure. The resulting n-butanol layer was collected and labeled as the “n-butanol fraction.” The residual aqueous layer was lyophilized to obtain the “aqueous fraction.” The n-hexane and n-butanol fractions were concentrated by evaporation in an oven at 40 °C (
A stock solution was prepared by dissolving 5.2 g of cimetidine in 100 mL of distilled water. From this stock, a dose of 120 mg/kg of cimetidine was administered orally to each rat using an oral cannula. The dosing regimen for cimetidine administration was adapted from Liu et al. (
Group 1 received 2 mL/kg of distilled water for 8 weeks, group 2 received 200 mg/kg of ethanol extract for 8 weeks, group 3 received 120 mg/kg of cimetidine for 8 weeks, group 4 received 120 mg/kg of cimetidine + 200 mg/kg of aqueous extract for 8 weeks, group 5 received 120 mg/kg of cimetidine + 200 mg/kg of ethanol extract for 8 weeks, group 6 received 120 mg/kg of cimetidine + 200 mg/kg of n-butanol fraction for 8 weeks, and group 7 received 120 mg/kg of cimetidine + 200 mg/kg of n-hexane fraction for 8 weeks. The detailed study design is shown in
Experimental design.
| Groups | Number of rats | Treatments | Number of days |
|---|---|---|---|
| Group 1 (control) | 5 | 2 mL/kg of distilled water | 8 weeks treated |
| Group 2 (ELEBO) | 5 | 200 mg/kg of ethanol extract | 8 weeks treated |
| Group 3 (CTD) | 5 | 120 mg/kg cimetidine | 8 weeks treated |
| Group 4 (AFBO + CTD) | 5 | 120 mg/kg cimetidine + 200 mg/kg of aqueous extract | 8 weeks treated |
| Group 5 (ELEBO + CTD) | 5 | 120 mg/kg cimetidine + 200-mg/kg ethanol extract | 8 weeks treated |
| Group 6 (BFBO + CTD) | 5 | 120 mg/kg cimetidine + 200 mg/kg n-butanol fraction | 8 weeks treated |
| Group 7 (HFBO + CTD) | 5 | 120 mg/kg cimetidine + 200 mg/kg n-hexane fraction | 8 weeks treated |
Cimetidine was administered orally (daily) for 8 weeks. 1, Control; 2, ELEBO (ethanol leaf extract of
A young adult male Wistar rat was selected for the study and acclimatized under standard laboratory conditions, with unrestricted access to water and a standard pellet diet. Prior to the initiation of dosing, the animal underwent a fasting period of 3–4 h, during which water was made freely available. At the commencement of the acute oral toxicity evaluation, the rat, weighing 204 g, received a single oral dose of the test substance at the limit dose of 2,000 mg/kg body weight, administered via oral gavage using an appropriate cannula. The corresponding volume administered was 2 mL/kg body weight.
Post-administration, the animal was subjected to intensive monitoring for clinical signs of toxicity during the first 30 min, followed by periodic observations over the subsequent 24 h, and thereafter daily monitoring for a total of 14 days. Parameters assessed included signs of toxicity, behavioral changes, alterations in body weight, and mortality. Throughout the observation period, no clinical signs of toxicity or mortality were recorded.
Following the initial assessment and in accordance with the OECD guideline 425 (up-and-down procedure) (
The body weights of the rats were measured periodically throughout the study using a digital weighing scale. Following euthanasia, the testes, epididymis, and anterior pituitary gland were carefully excised, cleared of adherent adipose tissue, and weighed using a computerized analytical balance. The following was used to calculate relative organ weight (Rw) and percentage weight change (%W) (
At the end of the 8-week treatment period, all experimental animals were anesthetized with a combination of ketamine (80 mg/kg) and xylazine (10 mg/kg) (
Using the enzyme-linked immunosorbent assay (ELISA) method and in accordance with the manufacturer’s instructions, the levels of luteinizing hormone, follicle-stimulating hormone, and testosterone in the serum was assessed (
The rat luteinizing hormone (LH) Elisa kit (Catalogue CSB-E12654r Cusabio Wuhan, Hubei Province, China) was obtained and used for the analysis of LH according to manufacturer’s instruction. Briefly, all reagents were equilibrated to room temperature (approximately 22 °C) prior to use. The required number of wells was secured in a well holder. Subsequently, 50 μL each of standards, specimens, and controls were dispensed into the designated wells. To each well, 100 μL of enzyme conjugate reagent was added. The contents were thoroughly mixed using a microplate mixer for 30 s and incubated at 22 °C for 60 min in the dark. Following incubation, the plate contents were discarded by inverting the microplate over a sink. The wells were then washed five times using a microplate washer with the provided washing buffer. After the final wash, the plates were tapped onto absorbent tissue paper to remove residual liquid. Next, 100 μL of tetramethylbenzidine (TMB) substrate solution was added to each well, and the plate was gently mixed and incubated for 15 min. The enzymatic reaction was terminated by adding 50 μL of stop solution to each well, followed by gentle mixing for 30 s. A color change from blue to yellow indicated successful reaction termination. The optical density (OD) was measured using a microplate reader at a wavelength of 450/630 nm (
The rat follicle-stimulating hormone, ELISA Kit (Catalogue CSB-E06869r Cusabio Wuhan, Hubei Province, China) was obtained and used for the analysis of FSH according to manufacturer’s instruction. Briefly, all reagents were brought to room temperature (approximately 22 °C) prior to use. The required number of wells was secured in a microplate well holder. Subsequently, 50 μL of standard, specimen, and control solutions were added to the designated wells. An enzyme conjugate reagent (100 μL) was then dispensed into each well. The contents of the wells were thoroughly mixed using a microplate mixer for 30 s, followed by incubation at 22 °C for 60 min. After incubation, the reaction mixture was discarded by inverting the plate over a sink. The microtiter wells were washed five times with the provided washing buffer using a microplate washer. After washing, the plate was gently tapped onto absorbent tissue paper to remove any residual liquid. Next, 100 μL of tetramethylbenzidine (TMB) substrate solution was added to each well, and the plate was gently mixed and incubated for 15 min. The reaction was terminated by adding 50 μL of stop solution to each well and mixing for 30 s. A color change from blue to yellow indicated the enzymatic reaction had occurred. The optical density (OD) was measured at 550/630 nm using a microplate reader (
The Rat Free Testosterone, F-TESTO ELISA Kit hormone Elisa kit (Catalogue CSB-E05097r Cusabio Wuhan, Hubei Province, China) was obtained and used for the analysis of Testosterone according to manufacturer’s instruction. Briefly, a volume of 0.01 mL of serum was pipetted into the designated wells of the microplate. Subsequently, 0.05 mL of the working testosterone enzyme reagent was added to each well, and the plate was gently swirled for 20–30 s to ensure thorough mixing. Thereafter, 0.05 mL of the testosterone biotin reagent was added to all wells, followed by another gentle swirling for 20–30 s. The plate was then covered and incubated at room temperature (approximately 22 °C) for 60 min. Following incubation, the contents of the wells were discarded by decantation, and residual liquid was blotted using absorbent paper. The wells were washed three times with 0.35 mL of wash buffer per well, decanting and blotting the plate dry after each wash cycle. After washing, 0.1 mL of the working substrate solution was added to each well, and the plate was incubated at room temperature for 15 min. The enzymatic reaction was terminated by adding 0.05 mL of stop solution to each well, followed by gentle mixing for 15–20 s. All reagents were added in the same sequence across all wells to minimize variability in reaction timing. The optical density was measured at 450 nm using a microplate reader (Model SM600, Shanghai Yongchuang Medical Instrument Co., China) within 30 min of adding the stop solution. A standard dose–response curve was used to determine the concentration of testosterone in each sample (
The estimation of the following antioxidants was assessed according to the following methods.
Catalase (CAT) activity was assessed using the method described by Sinha (
Malondialdehyde (MDA), a marker of lipid peroxidation, reacts with thiobarbituric acid (TBA) to form a pink chromogen with a maximum absorbance at 532 nm. The concentration of MDA in the tissue supernatant was determined using the method described by Prabhakar et al. (
Superoxide dismutase (SOD) catalyzes the dismutation of superoxide radicals into molecular oxygen and hydrogen peroxide. The enzymatic activity was determined based on SOD’s ability to inhibit the autoxidation of epinephrine (adrenaline) to adrenochrome, as described by Misra and Fridovich (
To initiate the assay, 2.0 mL of tissue supernatant was mixed with 2.5 mL of 0.05 M sodium carbonate buffer (pH 10.2) in a cuvette. The reaction was initiated by the rapid addition of 0.3 mL of freshly prepared 0.3 mM adrenaline solution, followed by thorough mixing. A reference (blank) cuvette contained 2.5 mL of the same carbonate buffer, 0.3 mL of adrenaline, and 0.2 mL of distilled water (in place of the sample). The formation of adrenochrome was monitored by measuring the increase in absorbance at 480 nm using a spectrophotometer. Absorbance readings were recorded at 30 s and again at 150 s. SOD activity was calculated based on the degree of inhibition of adrenaline autoxidation, using the appropriate equation as described in the original method.
The testes and epididymis were dissected out carefully and fixed in 10% neutral buffered formalin for 48 h. They were processed for histological examination following standard histological protocols (
One-way analysis of variance (ANOVA) was used for data analysis with the aid of GraphPad software version 8. Comparison was done using Tukey test. Results were expressed as mean ± S.E.M.
Effects of ethanol extract and fractions of
Effects of ethanol extract and solvent fractions of
Effects of ethanol leaf extract and solvent fractions of
Effects of ethanol leaf extract and fractions of
Effects of ethanol leaf extract and fractions of
Effects of ethanol extract and fractions of
Effects of ethanol extract and fractions of
Effects of ethanol extract and solvent fractions of
Effect of ethanol extract and solvent fractions of
Effect of ethanol extract and fractions of
Effect of ethanol extract and fractions of
This study evaluated the protective effects of the ethanol extract and solvent fractions of
This study evaluated the effects of
Importantly, co-treatment with ELEBO, both alone and combined with cimetidine (ELEBO + CTD), significantly attenuated the suppression of FSH induced by cimetidine. This restorative effect likely stems from the antioxidant-rich phytochemicals in
These results emphasize the critical role of phytochemical synergy in
The study also demonstrated that luteinizing hormone (LH) levels, which are essential for stimulating Leydig cells to produce testosterone and maintain spermatogenesis, were significantly reduced by cimetidine. ELEBO and ELEBO + CTD co-treatment effectively restored LH levels, suggesting a positive effect on pituitary-gonadal axis function and Leydig cell activity. Although partial LH restoration was observed with the solvent fractions (AFBO, BFBO, and HFBO), the whole ethanol extract produced the most pronounced effect, further supporting the hypothesis of synergistic interactions among bioactive compounds in the full extract.
Consistent with these hormonal improvements, testosterone levels suppressed by cimetidine were significantly restored in both the full extract and solvent fraction groups. Cimetidine’s inhibition of Leydig cell function and androgen biosynthesis is well documented (
These findings concur with previous studies on natural interventions for drug-induced reproductive toxicity. For example, Kolawole et al. (
Increased malondialdehyde (MDA) levels in the cimetidine-only group further confirm elevated lipid peroxidation, reinforcing the link between oxidative stress and cimetidine-induced testicular toxicity. Notably, co-treatment with
Although some reports suggest that cimetidine-induced DNA damage may not always result in overt infertility in rats, oxidative stress remains a recognized contributor to male reproductive dysfunction (
This study reported a significant decline in superoxide dismutase (SOD) activity following reproductive toxicity with cimetidine which was reversed upon administration of
Microscopic examination of testicular tissue sections provides critical insights into the protective and restorative effects of
The groups co-administered cimetidine with the ethanol extract (ELEBO), aqueous (AFBO), n-butanol (BFBO), and n-hexane (HFBO) fractions of
Protecting the integrity of seminiferous tubules is crucial in male reproduction. Damage to these structures often results in poor sperm quality and reduced fertility. The enhancement observed in the treated groups demonstrates that
The epididymal tissue sections from all treatment groups, including cimetidine-treated animals, demonstrated normal structural organization with unaltered epithelial tissue integrity. The columnar epithelial structure and presence of spermatozoa in the lumen indicate that the extract and its fractions protected the epididymis’ functional capacity during cimetidine exposure. This observation is significant because the epididymis plays a vital role in sperm maturation, storage, and transport. Impairment of sperm maturation due to structural damage to the epididymis can reduce fertilizing capacity (
Cimetidine, a histamine H₂-receptor antagonist, is well-documented to exert reproductive toxicity in males, primarily through its antiandrogenic effects, without significantly affecting body mass. This aligns with findings from Liu et al. (
The absence of significant changes in body weight among rats treated with
Further analysis of the relative weight of the epididymis revealed no significant differences among the experimental groups (
Similarly, no significant differences were observed in the relative weights of the testes across all groups, indicating that cimetidine and
The relative weight of the anterior pituitary gland remained unchanged across all treatment groups. As the anterior pituitary regulates male reproductive function by secreting luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which, respectively, stimulate testosterone production and support spermatogenesis, functional disruptions at this level may impair fertility even in the absence of morphological alterations. The absence of pituitary weight changes suggests that
This interpretation is supported by findings from Adelakun et al. (
This study demonstrates that
Given the promising findings of this study, it is recommended that future research focus on further elucidating the mechanisms underlying the protective effects of
In addition, long-term toxicity studies and pharmacokinetic profiling are essential to evaluate the safety, optimal dosage, and bioavailability of the extract when used chronically. This will be crucial in advancing the extract from experimental use to clinical application. Furthermore, controlled clinical trials involving human participants are necessary to validate the efficacy of
Finally, the development of standardized nutraceutical formulations containing
While this study provides valuable insights into the biological effects and phytochemical potential of
Future research should include broader extraction techniques, extended biochemical profiling, and translational studies to validate these findings in human subjects.
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.
The animal study was approved by Kampala International University Western Campus, Ishaka, Uganda and National for Science and Technology. The study was conducted in accordance with the local legislation and institutional requirements.
EI: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Writing – original draft, Writing – review & editing. DO: Data curation, Supervision, Writing – review & editing. VO: Supervision, Validation, Conceptualization, Writing – review & editing. OO: Methodology, Supervision, Writing – review & editing. FU: Software, Supervision, Writing – review & editing. PA: Conceptualization, Investigation, Methodology, Resources, Supervision, Validation, Writing – review & editing.
The author(s) declare that no financial support was received for the research and/or publication of this article.
The technical assistance of Mrs. Ikuomola Odunayo Ibukun is greatly acknowledged.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors declare that no Gen AI was used in the creation of this manuscript.
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IOE, Ikuomola Orire Emmanuel; WHO, World Health Organization; STIs, Sexually transmitted infections; DHT, Dihydrotestosterone; GCMS, Gas chromatography-mass spectrometry; LH, Luteinizing hormone; FSH, Follicle-stimulating hormone; SOD, Superoxide dismutase; CAT, Catalase; MDA, Malondialdehyde; ELISA, Enzyme-linked immunosorbent assay; OECD, Organisation for Economic Co-operation and Development; REC, Research Ethics Committee; KIU, Kampala International University; MOH, Ministry of Health; HPG, Hypothalamic-pituitary-gonadal; GnRH, Gonadotropin-releasing hormone.