1. Introduction
Infertility is defined as the inability to achieve pregnancy after 12 months of regular, unprotected sexual contact [
1]. It is now considered a global health issue due to its increasing prevalence in couples in their reproductive years, especially those in developing nations [
2]. According to a recent prolonged systematic meta-analysis study over 30 years (2022), the infertility prevalence estimates reported by male respondents were generally lower than those reported by female respondents, suggesting potential differences in the experience and reporting of infertility between sexes [
3]. Male infertility is influenced by diverse factors including diet [
4], stress [
5], smoking [
6], excessive exercise [
7,
8], genetic aberration [
9], chemical exposure that disrupts the hormonal system [
10,
11], heat [
12], genital inflammation [
13], food additives such as monosodium glutamate (MSG) [
14], and several other factors [
15]. MSG, derived from the sodium salt of glutamic acid, is commonly used as a seasoning and flavor enhancer in food products to enhance taste, quality, and shelf-life. Although it is generally regarded as safe, in terms of safety, it remains controversial for use in feed additives [
16]. Due to the efficient metabolism of glutamate by enterocytes, only a minute portion of ingested MSG is absorbed into the bloodstream, increasing plasma glutamate levels transiently. However, when consumed excessively, it may pose a danger to health. Studies have highlighted that the excessive consumption of MSG is associated with adverse effects such as headaches, numbness, flushing generalized weakness, dizziness, and muscle pain [
16]. In recent years, the detrimental impact of MSG on the male reproductive system and its functions has been extensively detailed by Kayode et al. [
17]. Some of the identified mechanisms and consequent effects of MSG include spermatogenic alteration resulting in oligospermia, teratozoospermia, and necrospermia. Oxidative damage, histological alteration (hemorrhage, distorted germ and Sertoli cells), as well as gonadotropin imbalance (reduced testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) concentrations) have likewise been reported [
18]. Although these adverse effects can be managed with conventional therapy such as hormonal treatment or surgical interventions, the use of natural compounds with stimulatory effects on male reproductive health may also serve as an alternative treatment strategy.
Spirulina platensis (Spirulina), a type of photosynthetic cyanobacteria, commonly known as blue-green algae, belongs to the Oscillatoriaceae family. It is multicellular, filamentous, and has a distinctive spiral shape. It is classified under the Cyanophyceae class.
Spirulina platensis is cultivated globally and serves as a primary dietary supplement for numerous individuals [
19]. Likewise, it is utilized in aquaculture and poultry farming as a feed supplement [
20]. It has a wide range of prophylactic and curative nutrients, including vitamins, minerals, proteins, linolenic acid, carotene, and undiscovered bioactive compounds [
21]. Along with its nutritional benefits,
S. platensis also possesses antibacterial, antifungal, antiviral, anticancer, anti-inflammatory, and antioxidant properties [
22].
Studies have reported its protective and ameliorative potential on spermatogenesis in animals exposed to cadmium [
23] and silver particles [
24]. Similarly, supplementation of
Spirulina to semen extender has been shown to influence the quality and antioxidant parameters of chilled or cryopreserved Arabian stallion spermatozoa [
25].
Lepidium meyenii (Maca), belongs to the family Brassicaceae, growing widespread in various South American nations. It is rich in many chemical constituents with bioactivity including glucosinolates, macamides, macaenes, thiohydantoins, and alkaloids. For centuries, the Andes have used Maca as an adaptogenic plant to treat anaemia, and for balancing of female hormones. More recently, Maca has been introduced to Japan, Europe, and the US, and it is now being used more frequently throughout the world [
26].
Tribulus arabica is an annual herbaceous plant in the caltrop family (Zygophyllaceae), that grows all over the globe. The major active ingredients of
T. arabica are furostanol saponins known as protodioscins. Both Indian and Chinese traditional medicine have used it to treat a variety of diseases [
27].
Fenugreek, scientifically known as Trigonella foenum-graecum, is an annual plant belonging to the Fabaceae family. A single leaf consists of three small obovate or oblong leaflets. This plant is cultivated globally as a semiarid crop. The seeds and leaves of this plant have been used as a culinary ingredient in the Indian subcontinent for a long time and are a popular addition to dishes in that region. The seeds are composed of protein, starch, sugar, mucilage, minerals, risky oil, constant oil, nutrients, and enzymes.
While Fenugreek is commonly offered as a nutritional supplement and utilized in conventional medicine [
28], several studies have indicated its potential significant adverse effects, including allergic reactions [
29]. Therefore, there is a lack of medical evidence supporting the healing properties of Fenugreek.
While the medicinal potential of Maca, Fenugreek (in low quantity), Spirulina and Tribulus have been reported in diverse pathophysiological conditions, studies investigating their effect on MSG-induced reproductive function impairment are limited. Additionally, data on the phytochemical composition of these plant extracts are lacking. Thus, the present study aimed to examine the phytochemical constituents of Lepidium meyenii, Trigonella foenum-graecum, Spirulina platensis and Tribulus arabica, extracts and to assess their potential effect on monosodium glutamate-induced male reproductive dysfunction in adult Wistar rats
4. Discussion
Male infertility is becoming a global health problem, and it is attributable to more than 50% of couples’ infertility. Various factors have been linked to subfertility, encompassing inadequate dietary intake, adverse effects of medications, pathological changes or disease states, and intoxication, which may involve excessive consumption of MSG. Although various conventional treatment strategies are available for treating subfertility, these methods are sometimes too expensive, invasive, and cumbersome. In the search for alternative treatment methods with minimal invasion and side effects, the use of natural herbs and plants has gained more traction [
38]. Thus, the present study evaluated the potential effect of natural herbs, including
Lepidium meyenii,
Trigonella foenum-graecum,
Tribulus arabica, and
Spirulina platensis in treating male reproductive parameters impairment in MSG-induced reproductive dysfunctions in male rats in.
This current study revealed that
Lepidium meyenii,
Trigonella foenum-graecum, and
Tribulus arabica, respectively, have potential benefits for male fertility with the most significant results seen with
Lepidium meyenii. These plants offer various constituents that can contribute to male reproductive health and fertility.
Lepidium meyenii exhibited the highest antioxidant activity, with a free radical inhibition of 63% which may reduce oxidative stress (OS) and improve sperm quality.
Trigonella foenum-graecum, which has been shown to have phyto-estrogenic effects, provides nutrients and helps regulate blood sugar levels [
39], exhibited a free radical inhibition of 50%. Although
Tribulus arabica exhibited a free radical inhibition of 30%, and
Spirulina platensis showed a 25% free radical inhibition.
Following MSG administration, animals displayed reduced sperm motility (total and progressive), which is consistent with findings from other studies [
40]. On the other hand, treatment with
Lepidium meyenii ameliorated the adverse effects of MSG by restoring sperm total motility and progressive motility to levels comparable to the control group. This is in line with several studies that reported the positive effects of
Lepidium meyenii on sperm quality, including sperm count, motility, and morphology [
41,
42]. This ameliorative effect may be attributed to its antioxidant activity, which can help reduce OS. Although reactive oxygen species (ROS) levels were not assessed in this study, it is widely documented that oxidative stress (OS) detrimentally affects sperm motility through elevated ROS generation. The sperm plasma membrane has a high percentage of polyunsaturated fatty acids [
43] which are essential for sperm motility. However, these fatty acids are susceptible to ROS, leading to lipid peroxidation and subsequent membrane invasion [
44]. Lipid peroxidation takes place when reactive oxygen species (ROS) interact with fatty acid chains, resulting in the formation of lipid peroxyl radicals. These peroxyl radicals then further react with fatty acids, generating additional ROS. This cascade of free radical reactions results in lipid breakdown. However, studies have shown that protecting against OS-induced damage requires addressing the underlying cause [
45] and subsequently suppressing pro-oxidants with antioxidants [
46]. Thus, it is suggested that the improved sperm motility observed following treatment with
Lepidium meyenii may be partly attributed to its antioxidant activity, which could mitigate the stress induced by excessive MSG consumption. Invariably, the antioxidant properties of
Lepidium meyenii may help protect spermatozoa from oxidative damage and improve overall fertility. Maca’s secondary metabolites, such as macamides and macaenes, along with other lipid-soluble components, may influence the reproductive system by altering the antioxidant-oxidant balance, potentially improving semen quality possibly through the stimulation of glucosinolates. However, the relationship between macamides, macaenes, glucosinolates, and the medicinal effects of
Lepidium meyenii on male gonads remains unclear due to insufficient data. Further research is needed to explore the specific macamides and glucosinolates unique to
Lepidium meyenii particularly their role in activating androgen signaling based on their distinct structures. Additionally, a study on mice revealed that Maca could counteract the effects of ketoconazole, which inhibits the synthesis of adrenal steroids and testosterone by blocking the P-450 enzyme system. By restoring cytochrome P450 functionality,
Lepidium meyenii appears to enhance sperm motility, suggesting its potential to reverse chemical subfertility related to cytochrome P450 and androgen production. By restoring cytochrome P450 functionality, Maca appears to enhance sperm motility, suggesting its potential to reverse chemical subfertility related to cytochrome P450 and androgen production [
47].
Notably,
Trigonella foenum-graecum has been utilized to promote reproductive well-being and address male infertility concerns [
39]. Several factors contribute to the potential benefits of
Trigonella foenum-graecum for fertility, including its phytoestrogenic properties [
48].
Trigonella foenum-graecum encompasses constituents such as trigonellin, which have undergone scrutiny for their phytoestrogenic attributes. Phytoestrogens are plant-derived compounds capable of binding to estrogen receptors in the body and eliciting estrogen-like effects. These phytoestrogens are believed to aid in hormonal regulation and support reproductive health [
49]. Furthermore,
Trigonella foenum-graecum boasts a significant nutritional profile, serving as a source of various nutrients, vitamins, minerals, and antioxidants [
50]. The present study demonstrated substantial antioxidant activity, evidenced by a 50% inhibition of free radicals. These nutrients and antioxidants play a vital role in overall health and contribute to the optimal functioning of the reproductive system [
39]. Additionally,
Trigonella foenum-graecum has been examined for its potential antidiabetic effects, primarily attributed to the presence of 4-hydroxyisoleucine. Maintaining stable blood sugar levels is crucial for reproductive health, as imbalances in blood sugar can adversely impact fertility [
51].
Of interest,
Tribulus arabica has potential benefits for male fertility. It may regulate hormone levels, including testosterone, which is important for reproductive function [
52]. Moreover, it may also improve sperm health, motility, and morphology, contributing to successful conception [
51]. Its antioxidant properties can protect spermatozoa from oxidative stress [
53]. The present study showed that Tribulus exhibited an antioxidant activity with a free radical inhibition of 30%. Additionally, Tribulus may promote nitric oxide production, improving blood flow to reproductive organs [
54]. It is also known as a natural aphrodisiac, potentially enhancing libido and sexual function, indirectly supporting fertility [
55].
It is noteworthy to mention that myristic acid has been identified in three of these plants, including
Lepidium meyenii, Tribulus arabica, and
Trigonella foenum-graecum, which have demonstrated potential anti-infertility effects. Additionally, a study indicated that the administration of myristic acid offers a promising therapeutic strategy for protecting the testes against oxidative stress and inflammation induced by hyperglycemia. This compound exhibits efficacy in preserving reproductive function and enhancing fertility in males with diabetes [
56].
The presence of myristic acid, a fatty substance, has been found to be crucial in enhancing sperm motility and acrosome reaction (AR). Notably, a study observed a minor decrease in the levels of saturated fatty acids, along with a significant decrease in monounsaturated and polyunsaturated fatty acids within the phospholipid fraction, which is a primary constituent of sperm membranes. This alteration significantly influences the quality of semen, wherein these fatty acids play a pivotal role. Analyzing the quantity and quality of fatty acids present in sperm lipid composition can potentially provide an explanatory framework for unexplained infertility. Polyunsaturated fatty acids play a significant role in sperm motility and the ability to penetrate the egg [
57,
58,
59].
Of note,
Tribulus arabica,
Trigonella foenum-graecum, and
Lepidium meyenii in the current study demonstrated the presence of total flavonoid in a percentage of 0.12%, 0.08%, and 0.01%, respectively (
Table 2). Additionally, HPLC analysis of these three plants detected rutin, apigenin, quercetin, syringic acid, and ferulic acid (
Figure 2 and
Figure 3). The combination of these flavonoids and phenolic acids exhibits synergistic activity, as they all possess antioxidant and anti-inflammatory properties. These compounds are believed to combat oxidative stress, which can damage sperm DNA and impair sperm function. Therefore, this combination may potentially improve sperm quality and reduce inflammation in the testes [
60].
Furthermore, flavonoids have been reported to improve male reproductive system dysfunction, such as testicular structural disruption and spermatogenesis disturbance, owing to their antioxidant, anti-inflammatory, immunity-boosting, anti-apoptotic, anticarcinogenic, anti-allergic, and antiviral properties. In a study where spermatogenesis was inhibited due to testicular weight loss and structural disruption, which led to a decline in sperm quantity and quality, a flavonoid supplement of 20 mg/kg significantly enhanced testicular histology and reduced germ cell apoptosis by reversing the vacuolation of the germinal epithelium, separation of germ cells from the basal lamina, and shedding of immature germ cells [
61].
In the present study, the histological features of the testis reflected normal morphology and were not affected by the administration of phytochemical constituents to MSG-induced Wistar rats. Although exposure to MSG causes histological alternations to the interstitium of the testis and the germinal epithelium and lumina of seminiferous tubules, this is in contrast to our observations [
62,
63] (rat male reproductive system (NF-kB) levels in response to monosodium glutamate; environmental factor). However, our findings are in agreeance with Abd-Elkareem et al. [
64] and Baradaran et al. [
65]. Histopathological and biochemical effect of quercetin on monosodium glutamate supplementation-induced testicular toxicity [
66], both of whom reported that the addition of phytochemical agents
Nigella sativa L. seeds and Quercetin, respectively, induced visible transition of the seminiferous tubule epithelium to normal in the initial MSG-treated groups. It may be postulated that a similar reparative process occurred with the phytochemical agents in the present study. It is noteworthy that our study is the first to describe the architecture of the seminiferous tubule epithelium in relation to the combined administration of multiple phytochemicals in MSG-treated Wistar rats.
Although previous studies highlighted the individual alleviative role played by phytochemical agents such as
Spirulina, Quercetin flavonoid and
Nigella sativa L. seeds on reproductive function, none have incorporated a combination of phytochemicals into their investigations [
64,
66,
67]. Moreover, very few have shed light on the reparative effect of these phytochemicals on histomorphometric parameters in MSG-induced animals. This applies specifically to the TL and NCST of the present study as no comparative mean values exist. On the contrary, our mean STD and GEH values were higher than studies exploring the effects of
Tribulus and
Nigella sativa L. seeds [
64,
68]. Statistically significant differences in mean STD were yielded for the control vs. MSG + H20 and MSG + H20 vs.
Tribulus comparisons which may be indicative of the markedly higher mean STD noted in the MSG + H20 group of our study. Although the STD is expected to increase from a control group to one that was treated with MSG, the aforementioned statistical significance does not explain the lower STD value obtained for the
Tribulus group as it is known to exert a protective effect against testicular damage [
69]. The mean GEH values were recorded to be greater in the Maca and Fenugreek groups of our study, but lower than that documented by Kianifard et al. [
67] who concluded that MSG promotes gonadotoxic effects of the antimalarial drug, Quinine, on testicular tissue. This may be attributed to the cellular characteristics of testicular tissue which renders it vulnerable to factors that are unique to an environment [
70]. Interestingly, a comparison of the mean GEH between control and Fenugreek animal groups resulted in a statistically significant difference which may be due to the increases seen when GEH magnitude was traced from the Control and MSG + H20 groups to the Fenugreek group. This improvement in histomorphometrics of the testis is suggestive of the protective effects afforded by Fenugreek. These results are also similar to Arafa et al. [
71] who showed that the antioxidant, anti-inflammatory and antifibrotic effects of Fenugreek on cadmium-induced testicular injury and hepatic dysfunction make it a promising medicinal herb. With the exception of the STD parameter, treatment of the phytochemical agents diminished histomorphometric alternations of the TL, GEH, and NCST, which were caused by the initial administration of MSG.
Additionally, exposure to MSG reduced serum testosterone levels, while LH and FSH levels remained unchanged. Several studies have similarly reported decreased serum testosterone levels following MSG exposure [
14,
40]. However, findings on LH and FSH expression after MSG exposure remain controversial. For instance, Koohpeyma et al. reported a decrease in both serum LH and FSH following MSG administration [
40], while Abd-Elkareem et al. reported an elevation in serum LH [
64]. The discrepancies may be due to differences in the concentration of MSG administered. Nonetheless, studies have consistently reported a decrease in testosterone concentration, whether in serum or testicular tissue, after MSG administration.
However, treatment with Lepidium meyenii, Spirulina platensis, Tribulus arabica, and Trigonella foenum-graecum improved serum testosterone levels, although not significantly.