Abstract
Chromolaena odorata is a medicinal plant widely used in traditional medicine. Despite its therapeutic applications, there are concerns regarding its potential toxic effects. This study investigates the dose-dependent effects of C. odorata on the fertility of adult male Wistar rats. A total of 25 male rats were randomly assigned into five groups (n = 5). Group A (Control), received distilled water (2 mL/kg), while groups B, C, D and E were administered 120, 200, 500 and 700 mg/kg of the aqueous extract of C. odorata daily for 28 days, respectively, after which they were sacrificed. Testicular weights, hormone levels, total protein and oxidative stress markers were evaluated, and the testes were evaluated histologically. GraphPad Prism version 10.2 was used to analyze the data. Results were described as the mean ± standard error of means (S.E.M.). One-way analysis of variance (at a 95% confidence level) was used to compare the means of the control group and experimental groups using GraphPad Prism (version 10.2.0). There was a statistically significant reduction (P < 0.05) in testicular weights, hormone levels (luteinizing hormone, follicle-stimulating hormone and testosterone) and total protein concentration, and a significant increase in catalase and malondialdehyde in groups D and E. Histomorphological analysis of groups D and E revealed severe structural distortions in the testes, presence of inflammatory cells, collapsed lumen with reduced luminal content, degenerating epithelium, loss of stereocilia cells and poorly arranged connective tissue fibers. High-dose administration of C. odorata extract induces significant histomorphological damage to the testes, disrupts reproductive hormone levels and increases oxidative stress, resulting in impaired male fertility in Wistar rats.
Lay summary
This study explored how Chromolaena odorata (Siam weed), a plant used in traditional medicine, affects male fertility. Using adult male Wistar rats, the research focused on the impact of high doses of the plant’s extract on reproductive health. After 28 days of treatment, rats given high doses showed a significant drop in the size of their reproductive organs and lower levels of key reproductive hormones, suggesting potential fertility issues. In addition, oxidative stress, which can damage cells, was higher in these rats, and their reproductive organs showed signs of serious damage, including inflammation and structural breakdown. The findings suggest that while Chromolaena odorata may have medicinal uses, consuming it in large amounts could harm male fertility by disrupting hormone balance and damaging reproductive tissues.
Introduction
Male fertility is a crucial aspect of reproductive health, and any disruption in the normal functioning of the male reproductive system can have profound implications on the ability to produce viable offspring. One of the factors that can affect male fertility is the use of medicinal plants and herbs, which are often consumed for their therapeutic benefits without thorough consideration of their potential side effects (Boroujeni et al. 2022). Chromolaena odorata, commonly known as Siam weed, is one such plant that has garnered attention due to its wide range of ethnomedicinal applications, particularly in Africa and Asia. It is traditionally used for its antimicrobial, anti-inflammatory and wound-healing properties (Vijayaraghavan et al. 2017, Zahara 2019). However, there are concerns regarding its potential toxic effects, particularly on reproductive health, when consumed in large doses.
Despite its widespread use, the effects of C. odorata on male reproductive function remain inadequately studied, especially concerning its impact on fertility. The bioactive compounds present in the plant, such as flavonoids, alkaloids and saponins, possess various pharmacological properties, but at high concentrations, they have been associated with toxicity, including oxidative stress and tissue damage (Phan et al. 2001, Olawale et al. 2022). This raises important questions about the safety of prolonged or high-dose consumption of C. odorata, particularly with respect to male reproductive health.
Given the growing use of C. odorata in traditional medicine, it is essential to understand its potential side effects on male fertility. This study will contribute to the body of knowledge on the toxicological profile of the plant and provide insights into the mechanisms by which it affects male reproductive health. Understanding these effects is particularly important for individuals who consume C. odorata in large quantities or over extended periods, as it may pose a risk to their reproductive health.
The present study aims to investigate the effects of C. odorata on male fertility using adult male Wistar rats as a model. Wistar rats are commonly used in biomedical research due to their physiological and anatomical similarities to humans, making them an ideal choice for studying reproductive toxicity (Bryda 2013). The focus of the study is to assess the impact of C. odorata on key reproductive parameters, including testicular and epididymal weights, levels of reproductive hormones, such as luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone, and markers of oxidative stress, such as catalase and malondialdehyde (MDA).
Materials and methods
Experimental animals
A total of 25 healthy adult male albino Wistar rats weighing an average of 180–220 g were used for this study. The animals were acclimatized to the animal house environment of the University of Medical Sciences, Ondo State, for two weeks before the administration. The rats were housed in a propylene cage with a wire mesh top for good aeration; with the macroenvironment temperature ranging between 27 and 31°C, the thermoneutral zone for laboratory rats. The rats were fed with pelleted feed from Olam International’s Chicken Feeds, Nigeria, and water ad libitum.
Plant source, and identification
Fresh leaves of C. odorata were collected from the premises of the University of Medical Sciences, Ondo, Ondo State, Nigeria, and it was properly identified and authenticated by a botanist.
Preparation of the extract
The aqueous extract of C. odorata was prepared using the method of extraction by Dahiru et al. (2006), with some modifications from Yakubu et al. (2007). Accordingly, about 3.6 kg of the wet C. odorata leaves were air-dried at 38°C for 72 h. The dried leaves were blended into powder form using an electric blender, which yielded about 600 g of C. odorata in powder. Six liters of distilled water was added to the powder in a plastic container in a ratio of 1 g to 10 mL. The mixture was then kept for 48 h at room temperature between 26 and 30°C, after which the extract was filtered using two doubled muslin clothes. The filtrate left was then concentrated in a steam bath to give about 100 g of brown to black fine semiliquid residue. The residue was then reconstituted in distilled water using the same dilution ratio to give the full dose in ml and the sample was stored in the refrigerator at 4°C for later use.
Experimental design
The animals were randomly divided into five groups (n = 5): A, B, C, D and E. Group A served as control and received distilled water (2 mL/kg) and experimental groups B, C, D and E were administered graded doses of 120, 200, 500 and 700 mg/kg, respectively, of the aqueous extract of C. odorata daily for 28 consecutive days. The extract was administered orally with the aid of an oropharyngeal cannula.
Sample collection and analysis
At the end of the treatment period, the rats were sacrificed (anesthetized using sodium pentobarbital, 50 mg/kg, intraperitoneally and euthanized by cervical dislocation) and blood samples were collected via cardiac puncture for hormonal and biochemical analyses. The testes were excised, weighed and processed for histological examination.
Biochemical assays
Serum levels of LH, FSH and testosterone (Testo) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Cat# LH: LH231F, Calbiotech Inc., USA. FSH: FS046F, Calbiotech Inc., USA. Testosterone: E-EL-0155). Procedures followed the instructions provided in the manufacturer’s manual. Changes in absorbance due to the release of p-nitroanilide (p-NA) were measured at 550/540 nm using a microplate reader. Total protein concentration (RTU BXC0173, Fortress Diagnostics Limited, Unit 2C Antrim Technology Park, United Kingdom) was determined using the biuret method, while oxidative stress markers, catalase and malondialdehyde (MDA) (Catalase: E-BC-K031-S, Elabscience, China. Colorimetric TBARS Microplate Assay Kit, FR40.130619, Oxford Biomedical Research, Inc, USA) were measured using spectrophotometric methods (Oyovwi et al. 2021, Uwejigho et al. 2023).
Histological analysis
The testes tissues were fixed in Bouin’s fluid and the epididymis tissues in 10% formalin. The tissues were processed and stained with hematoxylin and eosin (H&E) for light microscopy. Histological sections were examined for structural integrity, inflammation and tissue organization. The tissue processing involved dehydration using ascending grades of ethanol (70, 80 and 95%). Dehydrated tissue samples were then cleared in xylene, followed by the embedding of tissue samples in paraffin wax for about 8–10 h using automated tissue processing due to the large number of samples. Embedded samples were then sectioned using a laboratory microtone. Sample sections were afterward placed in a water bath to remove wrinkles and then placed on a slide for viewing. Slides were then passed through an ascending graded bath of alcohol and then cleared once again with xylene to remove wax. Tissue staining was performed, where tissue samples were stained on the slide using hematoxylin and eosin stain and dried in a hot air oven for a few minutes. After staining, tissue sections were covered using a glass coverslip and allowed to dry for a few minutes before being mounted on a microscope (Adjene et al. 2014). To ensure objectivity and avoid bias, randomized sectioning, systematic sampling, blinded analysis and standardized criteria were employed in the histophotomicrographs of selected portions.
Ethical approval
Prior to the commencement of this research, ethical clearance was obtained from the University of Medical Sciences Ethics Committee, Ondo, Ondo State, Nigeria.
Statistical analysis
The data procured were analyzed using descriptive statistics and inferential statistics. Values were presented as the mean ± standard error of means (SEM) on figures. All statistical analysis was done with the aid of the Prism 10 for Windows (version 10.2.0) software manufactured by the GraphPad Software LLC. The significance of the difference in the means of all parameters was determined using one-way analysis of variance (95% confidence interval) for more than two sets of data comparison, and Paired t-test was used for body wight comparison. A post-hoc test was carried out using Dunnett’s multiple comparisons for all groups and compared with control. A P-value of <0.05 was considered statistically significant.
Results
The results of this study are presented in bars and micrographs. Values are represented as the mean ± SEM for each group; alphabets “*” indicate a significant difference at P < 0.05 compared with control.
Effect of Chromolaena odorata on body weight and organ weight
Figure 1 shows the initial and final body weights of the experimental animals, and Fig. 2 shows the testicular and epididymis weights of the experimental animals. The result shows no statistically significant differences in the body weights. There was a statistically significant reduction in the testicular weight (Fig. 2) in groups treated with C. odorata at doses of 500 mg/kg (P = 0.0103) and 700 mg/kg (P = 0.0018). The result also shows a significant reduction (P = 0.0108) in the weights of the epididymis in the group treated with 700 mg/kg of the aqueous extract of C. odorata.
Effect of Chromolaena odorata on body weight.
Citation: Reproduction and Fertility 6, 1; 10.1530/RAF-24-0102
Effect of Chromolaena odorata on testicular and epididymis weight.
Citation: Reproduction and Fertility 6, 1; 10.1530/RAF-24-0102
Effect of Chromolaena odorata on the level of reproductive hormones
Figure 3 shows the effect of C. odorata on the level of reproductive hormones, this result showed a statistically significant reduction (P < 0.05) in levels of LH (P = 0.0425, P = 0.0254), FSH (P = 0.0153, P =0.0106) and Testo (P = 0.0492, P = 0.0171) in groups D and E, respectively, when compared with the control.
Effect of Chromolaena odorata on the levels of reproductive hormones.
Citation: Reproduction and Fertility 6, 1; 10.1530/RAF-24-0102
Effect of Chromolaena odorata on oxidative stress markers and total protein
Figure 4 shows the effects of C. odorata on oxidative stress markers and total protein. The result showed a statistically significant increase in MDA level (P = 0.0422, P = 0.0102) and catalase activity (P = 0.0457, P = 0.0100) and significant decrease in total protein (P = 0.0407, P = 0.0149) in groups D and E, respectively.
Effects of Chromolaena odorata on oxidative stress markers and total protein.
Citation: Reproduction and Fertility 6, 1; 10.1530/RAF-24-0102
Effect of Chromolaena odorata on the histology of the testes
The effects of C. odorata on the histomorphology of the testes are shown in Fig. 5. The result shows an outlined array of interstitial cells, without any observable pathology in the control group A (Fig. 5A), group B (Fig. 5B) and group C (Fig. 5C). The seminiferous tubules from these groups appear intact, with well-defined Sertoli cells seen. In addition, there were spermatogonia cells seen differentiating from primary, secondary and tertiary spermatocytes in a progressive order. No maturation arrest was observed in these groups. The group (D) treated with 500 mg/kg (Fig. 5D) showed mild-to-severe micromorphological alterations that are characterized by observable interstitial space distortion (widened space), pyknotic spermatogonia and interstitial cells, which is an indication for loss of Leydig cells. The group (E) treated with 700 mg/kg of C. odorata (Fig. 5E) showed severe micromorphological alterations that are characterized by observable interstitial space distortion (widened space), pyknotic spermatogonia and interstitial cells, which is an indication for loss of Leydig cells. There is also severe maturation arrest seen in this group.
(A, B, C, D, E) Effect of Chromolaena odorata on the histology of the testes.
Citation: Reproduction and Fertility 6, 1; 10.1530/RAF-24-0102
The interstitial cells (green arrow), lumen (purple arrow) and spermatocytes (found at the core-purple arrow) are all visible across groups. The seminiferous tubules (blue arrow), spermatogonium (containing primary, secondary and tertiary spermatocytes-yellow arrow), basement membrane (black arrow) and interstitial cells (green arrow), are also indicated as shown.
Discussion
This study investigated the effects of C. odorata extract on male fertility in adult male Wistar rats, focusing on testicular and epididymal health, hormonal profiles and oxidative stress markers. The results indicated that administration of C. odorata at high doses had a statistically significant (P < 0.05) negative impact on reproductive health, including a reduction in testicular and epididymis weight, LH, FSH, testosterone and total protein levels. Furthermore, there was a statistically significant increase in catalase activity and malondialdehyde (MDA) levels, suggesting an elevation in oxidative stress.
The significant reduction in testicular and epididymal weights observed in the high-dose group suggests that C. odorata may have a toxic effect on these reproductive organs. The weight of these tissues is closely linked to their functional state, including spermatogenesis and sperm maturation. A reduction in testicular weight is often associated with decreased spermatogenic activity, which may result from testicular atrophy, degeneration of seminiferous tubules or loss of germ cells (Moges 2022). Similarly, the reduction in epididymal weight points to impaired sperm maturation and storage, both of which are vital for male fertility. These findings align with previous studies showing that high doses of C. odorata can induce reproductive toxicity by disrupting cellular functions in reproductive organs (Usunomena & Ewere 2016).
The observed reduction in the levels of LH, FSH and testosterone is consistent with the notion that C. odorata may disrupt the hypothalamic–pituitary–gonadal (HPG) axis. LH and FSH are key regulators of spermatogenesis and testosterone production. LH stimulates the Leydig cells in the testes to produce testosterone, while FSH supports the function of Sertoli cells, which are essential for sperm development (Oduwole et al. 2018). A decline in these hormones suggests that C. odorata may interfere with hormone signaling pathways, potentially by inhibiting the secretion of gonadotropins from the pituitary or through direct toxic effects on the testes (Yakubu 2012).
Testosterone, in particular, plays a critical role in maintaining male reproductive health, including spermatogenesis, libido and secondary sexual characteristics. The reduction in testosterone levels observed in this study corroborates findings from earlier research, which demonstrated that some plant extracts with high concentrations of flavonoids or alkaloids can reduce testosterone levels by disrupting steroidogenesis (Usunomena & Ewere 2016, Luo et al. 2023). The decrease in testosterone likely contributes to the impaired reproductive function seen in the rats, as lower testosterone levels would result in reduced sperm production and testicular degeneration.
The significant reduction in total protein levels in the high-dose group could reflect an overall impairment of metabolic and physiological functions in the testes. Proteins are essential for the maintenance of testicular structure, function and spermatogenesis (Cheah & Yang 2011, Liang et al. 2020). Reduced protein levels might indicate decreased synthesis of vital proteins involved in hormone receptor activity, sperm development and testicular maintenance. Such changes may contribute to the testicular atrophy observed in this study, as protein synthesis is crucial for the proliferation and survival of germ cells.
The statistically significant increase in catalase activity and malondialdehyde (MDA) levels in the high-dose group indicates elevated oxidative stress in the reproductive tissues. MDA is a byproduct of lipid peroxidation, a process that occurs when reactive oxygen species (ROS) attack the lipid membranes of cells. Increased MDA levels are a hallmark of oxidative damage, which can impair sperm membrane integrity, reduce sperm motility and lead to DNA damage, ultimately affecting fertility (Alahmar 2019).
The rise in catalase activity may be a compensatory response to counteract the increased oxidative stress. Catalase is an antioxidant enzyme that detoxifies hydrogen peroxide, a ROS, into water and oxygen. Although catalase helps to mitigate oxidative damage, the excessive production of ROS, as evidenced by elevated MDA levels, may overwhelm the antioxidant defense system, leading to cellular damage. These findings are consistent with previous research showing that high doses of certain medicinal plant extracts can induce oxidative stress in reproductive tissues, resulting in impaired sperm function and reduced fertility (Hosen et al. 2015, Walke et al. 2023).
The histological examination of the testes in male Wistar rats treated with high doses of C. odorata revealed significant distortions in their cytoarchitecture. These structural changes indicate considerable damage to the reproductive tissues, likely contributing to the observed declines in reproductive function and male fertility. The key distortions included the presence of inflammatory cells, collapsed lumen with reduced luminal content, degenerating epithelium and poorly arranged connective tissue fibers in the histological sections.
The presence of inflammatory cells within the testicular and epididymal tissue is an indication of an inflammatory response, which may have been triggered by oxidative stress or tissue injury. Inflammation is a well-documented consequence of cellular stress and damage, and the infiltration of immune cells into these reproductive tissues is likely a defense mechanism against the damage caused by high doses of C. odorata. Inflammatory cells, particularly macrophages and lymphocytes, can impair the function of the testes and epididymis by releasing pro-inflammatory cytokines and ROS, exacerbating tissue damage. This inflammatory response can disrupt the delicate balance required for normal spermatogenesis and sperm maturation. Chronic inflammation may also lead to fibrosis, further damaging the structure and function of these tissues (Dutta et al. 2021).
The observation of a collapsed lumen with reduced luminal content in the seminiferous tubules of the testes suggests a disruption of normal spermatogenesis and sperm transport. Normally, the lumen of the seminiferous tubules and epididymal ducts is filled with developing spermatozoa, which are essential for proper sperm maturation and storage. The collapse of the lumen indicates a structural breakdown of the surrounding tissue, possibly caused by degeneration of the epithelial cells or loss of connective tissue integrity. Reduced luminal content reflects a decline in the number of viable sperm within the reproductive system, likely due to impaired spermatogenesis or the premature destruction of spermatozoa. The absence of mature sperm in the lumen could severely impair fertility, as these cells are crucial for successful fertilization (Dostalova et al. 2017).
The poorly arranged connective tissue fibers, as indicated by the black thick arrow, suggest a breakdown in the structural support provided by the extracellular matrix (ECM) of the testes and epididymis. The ECM is essential for maintaining the organization and stability of reproductive tissues, and disruptions in its arrangement can lead to functional impairments. Connective tissue fibers are crucial for the transport of nutrients, oxygen and other essential molecules to the cells within the testes and epididymis. When these fibers are poorly arranged, the delivery of these essential elements is impaired, further contributing to cellular degeneration. Disorganized connective tissue also compromises the structural integrity of the reproductive organs, which can lead to tissue collapse, as observed in the collapsed lumen (Siu & Cheng 2008, Murdock et al. 2019).
The histomorphological distortions seen in this study may be linked to the bioactive components of C. odorata, such as alkaloids, flavonoids and saponins, which have known cytotoxic and prooxidant effects at high concentrations. These compounds are capable of inducing oxidative stress, a condition in which the balance between ROS and antioxidants is disrupted, leading to cellular damage. The oxidative stress caused by C. odorata likely plays a significant role in the observed degeneration of reproductive tissues, as high levels of ROS can damage proteins, lipids and DNA in cells, impairing their function and leading to cell death (Pizzino et al. 2017, Juan et al. 2021).
In addition, the plant’s bioactive compounds may interfere with hormone signaling pathways, further contributing to the degeneration of reproductive tissues. Disruption of the HPG axis could lead to reduced levels of reproductive hormones, such as testosterone, LH and FSH, which are essential for maintaining the structure and function of the testes and epididymis.
These findings are consistent with previous studies that have demonstrated the toxic effects of C. odorata on male reproductive organs at high doses (Yakubu 2012, Paulose et al. 2016, Olawale et al. 2022). While C. odorata may have therapeutic benefits at lower doses, its administration at high concentrations clearly poses a risk to reproductive health.
Conclusion
This study suggests that high-dose administration of C. odorata extract adversely affects male fertility in Wistar rats by inducing oxidative stress, disrupting hormone regulation and causing significant histological damage to the testes. However, lower doses (120–200 mg/kg) appeared to have minimal adverse effects, suggesting a potential safe range for short-term use. These results highlight the need for caution in the use of C. odorata for medicinal purposes, particularly at high doses. These findings provide a basis for further studies to confirm safety thresholds and long-term implications of C. odorata administration.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the work reported.
Funding
This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Author contribution statement
All authors have contributed significantly to the conception and design of the work, acquisition, analysis and interpretation of data, drafted and substantively revised the work.
Data availability
All data used for this study are available on request from the corresponding author.
Ethics approval
This study was done as an extension of the work on phthalate approved by Research Ethics Committee of the University of Medical Sciences, Ondo, Nigeria.
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