Abstract
Objective
Aqueous extract of unripe Musa paradisiaca fruit is commonly used for the treatment of ulcers in eastern Nigeria. This study aimed to assess the acute and subacute effects of an aqueous extract of unripe fruit on male and female fertility in rats.
Methods
Aqueous extracts obtained by maceration were analyzed for acute and subacute toxicity and for the presence of phytochemical constituents using standard procedures. The extract (100, 500, and 1000 mg/kg) was administered daily to rats of both sexes for 28 d. Blood samples collected on days 0 and 28 were assessed for follicle-stimulating hormone (FSH), luteinizing hormone (LH), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA). Testes and ovaries were harvested for histopathological analysis. Sperm were also collected to determine the sperm count and motility.
Results
Phytochemical screening revealed the presence of saponins, tannins, alkaloids, and resins. After an oral dose of up to 5000 mg/kg, there were no deaths in the acute toxicity test. The extract (500 mg/kg) significantly (P < .05) enhanced sperm count and motility relative to the untreated control; significantly (P < .05) reduced SOD, CAT, and glutathione levels, while significantly (P < .05) elevated LH, FSH, and MDA levels in male and female rats. Histological examination revealed significant structural damage to the ovaries.
Conclusion
Unripe Musa paradisiaca fruit exhibited an adverse toxicological profile following prolonged administration and caused oxidative stress in rodents.
Introduction
Herbal phytotherapeutic agents are currently used worldwide. When consumed for a long period, there may be a risk of toxic effects on body organs. Toxicity may manifest in substructures such as cells (cytotoxicity) or organs such as the liver (hepatotoxicity). 1 The major evaluation points for toxicological assessments are the basic structural, functional, and biochemical parameters of injury; the dose-response relationships of the agent of toxicity and toxicological parameters; the mechanisms of toxicity (the fundamental biochemical alterations responsible for the induction and maintenance of the toxic response); reversibility of the toxic effect; and possible factors influencing response modification, such as route of exposure, species, and sex. 2 Reports have indicated severe toxicity resulting from the use of herbal medicines or products. 3 These toxicities arise from inherent poisonous phytochemicals, adulteration of medicines, contamination with various chemicals and heavy metals, herb-drug interactions, and poor-quality control of herbal products. The use of animals to predict the risks of various treatments, botanicals, and chemical products in humans is prevalent. However, animal costs can be exorbitant, and tiny changes across species can influence the effects observed. 4 The continuous use of phytomedicines has led to increasing emphasis on the research and development of medicinal plants. The safety, efficacy, and quality of medicinal plants and herbal products are the key issues. Medicinal plants are often assumed to be safe because of their long periods of use and because they have not been subjected to exhaustive toxicological studies. However, phytochemical research has shown that a significant proportion of purported safe plants is toxic. Therefore, it is imperative to conduct extensive toxicity testing to ensure the safety, efficacy, and quality of herbal medicines.
Musa paradisiaca (Musaceae) is traditionally used to treat various ailments like diabetes, cancer, hypertension, atherosclerosis, ulcers, urolithiasis and Alzheimer's infection, and its leaves are widely used in many localities in Nigeria. Alabi et al reported that a low dose of M. paradisiaca improved the quality of male Wistar rat sperm. 5 There is a report on the use of M. paradisiaca fruit stalk in ethno-dentistry. 6 The florets of M. paradisiaca possess antioxidant, antibacterial, and cytotoxic activities.7,8 The use of the aqueous extract of the unripe M. paradisiaca (plantain) fruit as a traditional recipe for ulcer therapy is prevalent in Nigeria. Apart from LD50 and short-term toxicity studies, M. paradisiaca, a popular traditional antiulcer recipe, has not been subjected to extensive toxicological studies. This highlights the need for the present study to investigate the acute and subacute effects of M. paradisiaca aqueous extract on male and female fertility.
Materials and Methods
Plant Material Collection and Identification
Freshly harvested unripe M. paradisiaca fruits were purchased and voucher specimen stored.
Preparation of Extract
Fresh unripe M. paradisiaca fruits (5 kg) were thoroughly washed, peeled, cut into small pieces, and steeped in 25 L of distilled water at ambient temperature for three days, with periodic agitation. The resultant liquid was filtered through Whatman No. 1 filter paper to yield an aqueous extract (MPE) at a concentration of 200 mg/ml. The concentrated extract was stored in a sterile, sealed amber container and refrigerated at 4 °C until further use.
Qualitative Phytochemical Screening
Standard qualitative techniques have been used to screen aqueous extracts of phytoconstituents.9,10
Experimental Animals
Adult Swiss albino rats (120-150 g) and mice (17-25 g) of both sexes were obtained from the Laboratory Animal Facility. The animals were maintained in separate steel cages with unrestricted access to safe water and animal feed pellets. They were kept in a well-aerated facility with a 12/12 h light/dark cycle at room temperature. The minimum number of animals were used for the experiments. The experiments were conducted in accordance with the National Institute of Health's Guide for the Care and Use of Laboratory Animals (Pub. No. 85-23, amended 1985) and the Faculty of Pharmaceutical Sciences, University of Nigeria's ethics committee Ethics Committee on the Use of Laboratory Animals.
Acute Toxicity Test (LD50)
Acute toxicity (LD50) of the aqueous extract of Musa paradisiaca was determined using Lorke's method. 11 This method was performed in two phases using 13 animals (albino mice). In the first phase, nine mice were divided into three groups, each with three animals, and orally administered 10, 100, or 1000 mg/kg. Animals were monitored continuously for 24 h. In the second phase, four mice were divided into four groups, each mice received 1600, 2900, 5000 mg/kg and distilled water (2 mL/kg). Animals were observed for 24 h.
Subacute Male Reproductive Toxicity Test
The model proposed by El-Kashoury et al 12 was employed, with slight modifications. Twenty male albino rats (120-150 g) were randomly selected and divided into four groups (A-D) of five animals each. Groups A, B, and C received 100, 500, and 1000 mg/kg of the extract, respectively. Control group D received 5 mL/kg of distilled water. The animals were weighed weekly. The extract was administered orally once daily for 28 days. Blood was withdrawn via an ocular puncture on days 0th and 28th days. Basal levels of testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), superoxide dismutase (SOD), catalase (CATand malondialdehyde (MDA). Total glutathione (GSH) were measured in the stomach supernatant. In addition, sperms were harvested to assess sperm count and motility. Histopathological examination of the testes was performed.
Subacute Female Reproductive Toxicity
Twenty female albino rats (120-150 g) were randomly selected and allocated to four (A-D) groups (n = 5). 12 Treatment groups A-C received 100, 500, and 1000 mg/kg of extracts, respectively. Control group D received 5 mL/kg of distilled water. The extract was orally administered once daily for 28 days. On days 0 and 28th, blood (2-3 mL) was withdrawn by ocular puncture. Estrogen, progesterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) levels were measured in blood samples. Total glutathione (GSH) were measured in the stomach supernatant. The ovaries were harvested for histopathological assay
Hormonal Assay
Testosterone, Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) Assay
Testosterone, FSH, and LH assays were performed according to the manufacturer's instructions.
Sperm Analysis
An incision was made from the thoracic region to the lesser abdominal area in order to gain access to the peritoneal cavity. The testes were carefully removed and rinsed in 0.9% normal saline solution, and the cauda epididymis was preserved at 37 °C prior to the sperm count and motility assays.
Determination of the Epididymis Sperm Count
Epididymal sperm count was determined according to the protocol described by Freud and Carol. 13 Epididymal spermatozoa were collected by cutting the epididymal caudal area into small pieces and soaking them in 5 mL Ringer's solution at 37 °C. The caudal epididymis from each rat was immersed in 2 mL normal saline prewarmed to 37 °C, where the spermatozoa were collected and maintained in a saline solution. Using a Pasteur pipette, 200 µL of the suspension was introduced into both chambers of a Neubauer hemocytometer by contacting the edge with a coverslip, enabling capillary suction to fill both chambers.
Determination of Sperm Motility
The semen was thoroughly mixed, and a drop was placed on a slide (a method known as wet-mount preparation). The slides were examined under a microscope to visualize the sperm activity. 14
Antioxidant Activity
Assay of Superoxide Dismutase (SOD) Activity
SOD activity was evaluated using spectrophotometric technique. 15 SOD was discovered because of its capacity to prevent superoxide-mediated reduction. One unit was defined as the quantity of enzyme that impeded pyrogallol oxidation by 50%. Activity was measured in units of U/mg protein.
Assay of Catalase (CAT) Activity
The CAT activity was determined using the Aebi method. 16
Glutathione Activity Assay
Glutathione (GSH) activity was estimated using the Flohe and Gunzler technique. 17 A reaction mixture (1 mL) containing 0.3 mL of phosphate buffer (0.1 M, pH 7.4), 0.2 mL of glutathione (GSH) (2 mM), 0.1 mL of sodium azide (10 mM), 0.1 mL of H2O2 (1 mM), and 0.3 mL of stomach supernatant was prepared. The reaction was stopped after 15 min of incubation at 37 °C, with the addition of 5% TCA (0.5 mL). The supernatant was decanted after the tubes were centrifuged at 1500 g for 5 min. The reaction supernatant (0.1 mL) was added to a mixture containing 0.2 mL of phosphate buffer (0.1 M, pH 7.4) and 0.7 mL of 5, 5'-dithio-bis (2-nitrobenzoic acid) (DTNB, 0.4 mg/ml). After mixing, the absorbance at 420 nm was determined, and the activity of the enzyme was estimated as U/mg protein.
Assay of the Tissue Malondialdehyde (MDA) Concentration
MDA concentration in the tissues was measured based on its reactivity to TBA. 18 In tubes, 0.2 mL supernatant from tissues, 0.8 mL phosphate buffer (pH 7.4), 0.025 mL BHT, and 0.5 mL 30% TCA were mixed. The mixture was centrifuged (4000 × g) for 15 min after 2 h of incubation at 20 °C. The supernatant (1 mL) was added to each tube, followed by 0.075 mL of 0.1 M EDTA and 0.25 mL of 1% TBA. The Teflon-lined screw-cap tubes were incubated for 15 min in a water bath at 90 °C prior to cooling to ambient temperature. The absorbance of tissue MDA was measured at 532 nm.
Histological Analysis
According to standard procedure, the testis and ovaries were fixed in formalin solution, dehydrated in ethanol, embedded in paraffin, sectioned at 5 micrometer thickness, and stained with hematoxylin and eosin. A light microscope was used to examine the tissue. 19
Statistical Analysis
The data obtained are presented as mean ± SEM. GraphPad Prism software (version 6.0) was used to analyse the data using one-way analysis of variance and Bonferroni post-hoc tests. Statistical significance was set at P < 0.05.
Results
Extraction Yield
The extraction procedure yielded 1 kg (20% w/w) of aqueous extract.
Qualitative Phytochemical Analysis
Tannins, saponins, alkaloids, and resins are present in the aqueous extracts of M. paradisiaca. No reducing sugars, proteins, oils, flavonoids, cardiac glycosides, or acidic compounds were detected.
Acute Toxicity Test
After 48 h of oral treatment, mice showed no evidence of acute toxicity or death, even at a dose of 5000 mg/kg.
The Effect of the Extract on Testosterone
The extract caused a non-significant (P > .05) increase in testosterone levels at 100 and 500 mg/kg, and a non-significant reduction at 1000 mg/kg relative to the negative control group (Table 1).
The Extract's Effect on Sperm Count, Sperm Motility and Testosterone of Male Rats.
Data are presented as the mean ± standard error of the mean (n = 5). Significance represented as *(P < .05).
Effect of the Extract on Sperm Count
There was a significant increase in sperm count (P < .05) at 500 mg/kg and a non-significant increase (P > .05) at 1000 mg/kg relative to the untreated control group (Table 1).
Effect of the Extract on Sperm Motility
The extract caused a significant (P < .05) increase in sperm motility at 100 and 500 mg/kg and a non-significant (P > .05) increase at 1000 mg/kg compared to the untreated control group (Table 1).
Effect of the Extract on Luteinizing Hormone of the Male Rats
The extract caused a significant (P < .05) increase in luteinizing hormone levels at 100 and 500 mg/kg and a non-significant (P > .05) increase at 1000 mg/kg relative to the untreated control group (Table 2).
Effect of the Extract on LH, FSH and MDA of the Male Rats.
Data are presented as the mean ± standard error of the mean (n = 5). Significance represented as *(P < .05).
Abbreviations: LH, Luteinizing hormone; FSH, Follicle Stimulating Hormone; MDA, Malondialdehyde
Effect of the Extract on Follicle Stimulating Hormone of the Male Rats
Compared to the untreated control group, the extract produced a non-significant (P > .05) increase at 100, 500, and 1000 mg/kg, respectively (Table 2).
Effect of the Extract on Superoxide Dismutase of the Male Rats
The extract caused a significant (P < .05) decrease at 100 and 500 mg/kg and a significant (P < .05) increase at 1000 mg/kg, relative to the untreated control group (Table 3).
Effect of the Extract on SOD, CAT and GSH of the Male Rats.
Data are presented as the mean ± standard error of the mean (n = 5). Significance represented as *(P < .05).
Abbreviations: SOD, Superoxide dismutase; CAT, Catalase; GSH, Glutathione
Effect of the Extract on Catalase of the Male Rats
The extract caused a significant decrease at 100 and 500 mg/kg (P < .05) and a non-significant (P > .05) decrease at 1000 mg/kg relative to the untreated control group (Table 3).
Effect of the Extract on Glutathione (GSH) of the Male Rats
Compared with the untreated control group, the extract induced a significant decrease at 100 and 500 mg/kg (P < .05) as well as a significant increase (P < .05) at 1000 mg/kg. (Table 3).
Effect of the Extract on Malondialdehyde Levels of the Male Rats
Compared to the untreated control, the extract caused a non-significant increase at 100 mg/kg and a significant (P < .05) increase at 500 mg/kg. However, at 1000 mg/kg, it caused a non-significant (P > .05) decrease (Table 2).
Effect of the Extract on the Histology of the Testes
Compared to the control (group A), histopathological findings of the testes of the treated animals indicated no structural damage, as the cellular architecture was normal (Figure 1).
Photomicrograph of the testes showing the normal testicular histomorphology (A, B, C & D). Normal seminiferous tubules lined by a stratified epithelium of spermatogenic cells and Sertoli cells (Sc) were observed. The spermatogenic cells undergo a series of division to give rise to spermatozoa (Sp). Spermatogonia (S), primary spermatocytes (Ps). Early spermatids (Es) and Late spermatids (Ls). Interstitium (I). H&E ×400.
Effect of Extract on Body Weight
The extract did not significantly affect the body weight of the animals (Table 4).
Effects of the Extract on the Body Weight.
The results are expressed as mean ± standard error of mean (S.E.M), n = 5, *P < .05 compared to control (one-way ANOVA, Dunnet post hoc).
Effect of the Extract on Estrogen Level of the Female Rats
The extracts at 100 and 500 mg/kg exhibited a slight and non-significant increase in estrogen levels, but there was a significant (P < .05) increase at 1000 mg/kg relative to the untreated control group (Table 5).
Effect of the Extract on Estradiol, Progesterone, LH and FSH of the Female Rats.
Data are presented as the mean ± standard error of the mean (n = 5). Significance represented as *(P < .05).
Effect of the Extract on Progesterone Level of the Female Rats
The extract at 100 mg/kg produced a slight and non-significant increase in progesterone levels; however, a significant increase was observed at 500 and 1000 mg/kg (P < .05) relative to the untreated control group (Table 5).
Effect of the Extract on Luteinizing Hormone Level of the Female Rats
The extract at 100 mg/kg caused a slight and non-significant increase in luteinizing hormone levels, but there was a significant increase at 500 and 1000 mg/kg (P < .05) relative to the untreated control group (Table 5).
Effect of the Extract on Follicle Stimulating Hormone Level of the Female Rats
Compared to the untreated control group, the extract caused a significant dose-dependent increase in follicle-stimulating hormone levels at 100 mg/kg, 500 mg/kg and 1000 mg/kg (P < .05) (Table 5).
Effect of the Extract on Superoxide Dismutase Level of the Female Rats
The extract induced a significant (P < .05) reduction in SOD levels at 100 and 500 mg/kg, respectively; however, it caused a significant (P < .05) increase at 1000 mg/kg relative to their respective untreated control group (Table 6).
Effect of the Extract on SOD, CAT and GSH of the Female Rats.
Data are presented as the mean ± standard error of the mean (n = 5). Significance represented as *(P < 0.05).
Effect of the Extract on Catalase Level of the Female Rats
The extract induced a significant (P < .05) dose-dependent decrease in CAT at 100 and 500 mg/kg, respectively, and caused a slight non-significant decrease at 1000 mg/kg relative to the untreated control group (Table 6).
Effect of the Extract on Glutathione Level of the Female Rats
The extract produced a significant (P < .05) dose-dependent decrease in GSH levels at 100 and 500 mg/kg; however, it caused a significant (P < .05) increase at 1000 mg/kg relative to their respective untreated control group (Table 6).
Effect of the Extract on Malondialdehyde Level of the Female Rats
The extract at 100 mg/kg produced a non-significant increase in MDA levels; however, it exhibited a significant (P < .05) increase at 500 mg/kg and a non-significant decrease at 1000 mg/kg relative to their respective untreated control group (Table 6).
Effect of the Extract on the Histology of Ovary
Histopathological findings revealed a photomicrograph of the ovary with normal histology (A). The ovarian samples from Groups B, C and D showed several corpora lutea and a few large follicles (Figure 2).
Photomicrograph of the ovary showing the normal ovarian histomorphology (A). The ovarian samples from Groups B, C and D showed several corpora luteal and a few large follicles. Corpora luteal (CL); large follicles (LF); Medulla (M). H&E ×160.
Discussions
Plants have long served as the foundation for medical treatment, and traditional medicine remains widely used. 20 This is because of the affordability, safety, minimal side effects, and ease of sourcing these herbal plants. Unripe M. paradisiaca preparations are commonly used in Nigeria to treat ulcers. The goal of this study was to determine the effects of unripe Musa paradisiaca on male and female reproductive systems.
Although no fatality was documented up to 5000 mg/kg in the acute toxicity study, visible symptoms of acute intoxication, such as reduced activity, depression, and motor incoordination, were observed at all dosages administered, suggesting that the extract may not be completely safe for all practical uses.
Follicle stimulating hormone (FSH) and luteinizing hormone (LH) induce the secretion of estrogen and progesterone, which regulate sexual development, excitement, and conception. 21 Anything that hinders LH or FSH secretion or lowers LH or FSH level will eventually lead to infertility, and this may include hypothalamic suppression, hypopituitarism, hyperprolactinemia, gonadotropin deficiency, gonadal suppression therapies, etc. 21 The increase in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) following treatment with the extract suggests improved reproductive function in treated male and female rats. An increase in progesterone levels hinders ovulation by inhibiting LH production and enhancing the inhibitory effects of estrogen. 22 The progesterone levels increased dramatically in the current study, implying that long-term use of a high dose of the extract can inhibit ovulation. A significant increase in the level of estrogen at high doses may predispose an individual to a higher risk of blood clots, stroke, and thyroid dysfunction when consumed over a prolonged period. Sperm count and motility are the major determinants of sperm quality. 23 At 500 mg/kg, the extract significantly increased sperm count and motility. These large increases in sperm count and motility lead to increased sperm quality over the long term. This outcome is consistent with that reported by Alabi. 5
SOD, CAT, and glutathione peroxidase are endogenous antioxidant enzymes normally found in every living cell, 24 whereas MDA is a biomarker of oxidative stress and lipid peroxidation. Both CAT and SOD are enzymes that aid in the breakdown of potentially toxic oxygen molecules in cells, thereby preventing tissue damage, while GSH is a powerful antioxidant that protects vital cellular components from the harmful effects of reactive oxygen species. 25 At a high dose of 500 mg/kg, the extract significantly elevated oxidative stress in male and female rats owing to an increase in MDA. Oxidative stress mediates male infertility by inducing sperm malfunction. MDA induces oxidative stress, which has a negative impact on sperm function by affecting membrane fluidity and permeability, as well as reducing sperm function efficiency. 26
In both male and female rats, the extracts significantly lowered SOD, CAT, and GSH levels at 500 mg/kg, but significantly increased SOD and GSH levels at 1000 mg/kg. These results suggested that the extract possessed significant antioxidant activity at high doses. This may be attributed to the absence of flavonoids in the extract, which are polyphenolic compounds with high antioxidant activity. 27 However, the level of oxidative stress may not be very high because of the abundance of tannins, which are polyphenolic compounds with mild antioxidant activity. However, care must be taken in cases of prolonged consumption, which may result in serious oxidative stress. The extract mainly yielded tannins, saponins, alkaloids, and resins.
Histopathological analysis of the testes of the treated animals showed no structural damage, as the cellular architecture was normal compared to that of the control group. This suggests that the extract has no significant toxicological effects on the development of the male reproductive system. However, histopathological examination of the ovaries of treated rats showed several corpora lutea and a few large follicles, relative to the untreated control group. This confirms that the long-term use of the extract at high doses can cause estrogen dominance and accompanying side effects. The major limitation of the study was the use of few numbers of animals.
Conclusions
Based on these findings, the aqueous extract of M. paradisiaca has the potential to cause reproductive toxicity because of its dose-dependent ability to cause mild oxidative stress. Therefore, large doses of M. paradisiaca may have deleterious effects on male and female fertility. However, smaller doses (500 mg/kg) may be beneficial because of their ability to improve the sperm quality without causing adverse effects. However, further toxicological studies on unripe M. paradisiaca are ongoing to ascertain its toxicological profile following prolonged administration.
Footnotes
Abbreviations
Availability of Data and Materials
The datasets generated during this study are available from the corresponding author upon request.
Author Contributions
Design, Experimental Procedure, Data collection: [CAO]; writing and editing: [AMO, SCN, PAA]; statistical analysis: [CAO]; experimental parameter analysis: [CAO]; similarity checks: [AMO, IEP]. All authors have read and agreed to the final version of the manuscript.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
