Phytomedicine Plus 3 (2023) 100465 Contents lists available at ScienceDirect Phytomedicine Plus journal homepage: www.sciencedirect.com/journal/phytomedicine-plus Moderate doses of Mucuna pruriens seed powder is safe and improves sperm count and motility Samuel Adjei a, Perpetua Dagadu b, Brodrick Yeboah Amoah b,*, G.N.A. Hammond c, Ezekiel Nortey d, Richard Obeng-Kyeremeh a, Ifeoma C Orabueze e, George A. Asare b,** a University of Ghana, Noguchi Memorial Institute for Medical Research (NMIMR), Legon, Ghana b University of Ghana School of Biomedical and Allied Health Sciences (SBAHS), Department of medical Laboratory Sciences, Korle Bu, Ghana c University of Ghana, Department of Anatomy, Legon, Ghana d University of Ghana, Department of Statistics, Legon, Ghana e University of Lagos, Department of Pharmacognosy, Faculty of Pharmacy, Lagos, Nigeria A R T I C L E I N F O A B S T R A C T Keywords: Background: Conventional remediation techniques involving male fertility include hormonal therapy, in vitro Mucuna pruriens fertilization and surgery. However, the use of natural products continues to be a popular option. Emerging new Sperm count products that have not been well investigated is the use of Mucuna pruriens seed powder. Motility Aim: This study aimed at determining the efficacy and safety of Mucuna pruriens (MP) seed powder on the male fertility using normal animal models. Methodology: Four groups of seven (7) male Sprague-Dawley rats were used. Groups comprised Control (distilled water), Low dose (500 mg/kg b.wt MP), Medium dose (1000 mg/kg b.wt MP) and high dose (2000 mg/kg b.wt MP). Test groups were administered aqueous crude extract of MP by gavage over 90 days. Upon sacrifice, the following assays were performed: FSH, testosterone, oestrogen, PSA, semen analyses, histology of reproductive organs, and general haematological and biochemical analyses. Results: FSH increased, whilst oestrogen decreased, across groups: however, it was not statistically significant between groups. Although testosterone increases were not statistically significant, increases were dose- dependant. Sperm count increased significantly between the Control and Medium dose groups (p<0.001). Motility significantly increased with the Medium and High dose groups compared to Control group (p = 0.022, p = 0.029, respectively). Additionally, immotility significantly decreased in all treatment groups compared to control group (p = 0.013). No abnormality was observed in biochemical, haematological and histological analyses. Conclusion: This study demonstrates a margin of safety and improved spermogram pattern between the low and medium dose administration of Mucuna pruriens seed powder. Introduction identified (65–80%), followed by a situation of normal spermogram in which causes remain unknown (10–20%). Hormonal and sperm trans- Infertility is conventionally diagnosed following unprotective sexual port may account for another 10% (Winters, and Walsh, 2014). Lifestyle intercourse by a couple over a period of one year or longer, without factors such as obesity which imbalances the testosterone and oestrogen conception. According to WHO, about 72.4 million people are affected ratio is also responsible for male infertility (Bieniek et al., 2016). and further suggests that half of 9% of couples struggling to conceive are Smoking, abuse of drugs and illicit substances also have a role to play, attributable to men (Boivin et al., 2007). though a causal association has not yet been established (Yu et al., al., The causes of male infertility are numerous but can be categorized 2018). into abnormality with spermogram of which the aetiology may not be Other lifestyle factors such as diet, use of caffeine and the level of * Corresponding author at: University of Ghana School of Biomedical and Allied Health Sciences (SBAHS), Department of medical Laboratory Sciences, Korle Bu, Ghana. ** Corresponding author. E-mail addresses: abybrodrick@gmail.com (B.Y. Amoah), george.asare@gmail.com (G.A. Asare). https://doi.org/10.1016/j.phyplu.2023.100465 Available online 25 May 2023 2667-0313/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). S. Adjei et al. P h y t o m e d i c i n e P lus 3 (2023) 100465 activity also contribute to infertility. Lifestyle changes with a departure significant sperm motility and density increase as well as reproductive from junk food, smoking and excessive use of alcohol, departure from organs and accessories (testes and epididymes). excessive red meat, consumption of more vegetable have been reported Similar results in sperm motility and density were observed in the to improve sperm quality (Gameiro et al., 2016), (Cairo Consensus oligospermic cohort. Furthermore, these significant changes in the Workshop Group, 2020). Environmental factors such as pesticides and human study were attributed to serum testosterone increase. cell phones have also been implicated in infertility (Gameiro et al., As the search for newer and safer plant products continues, Mucuna 2016), (Bieniek et al., 2016). The role of stress has been recognized pruriens seed is one of such with insufficient empirical data albeit pur- (Gollenberg et al., 2010) considering that physical or economic pres- ported to improve libido and testosterone levels (Choowong-In et al., sures affect hormonal levels and subsequently, spermatogenesis and 2021). Furthermore, controversies surround the appropriate safe dose of semen quality (Anderson et al., 2010). The role of oxidative stress and M. pruriens due to dopamine levels contained in its seeds. Again, animal mutational damage to sperm DNA seem to suggest that dietary vitamin studies demonstrating its fertility outcomes amongst normal subjects do supplement with antioxidant capacity have the potential of improving not exist. The aim of the present study was therefore to determine the male fertility (Suresh et al., 2013). The scrotum and the temperature of safety, fertility potential and appropriate dose of M. pruriens using ani- the sac have its impact on spermatogenesis through hyperthermia; mal models. It was hypothesized that Mucuna pruriens if indeed aphro- scrotal skin temperature affects sperm output whilst scrotal hyperther- disiac, could improve male fertility hormones and subsequently affect mia results in impairment of spermatogenesis (Jung and Schuppe, semen production, motility and viability, at a dose that would not be 2007). toxic. Treatment modalities employed in infertility treatment include sur- gical techniques aimed at improving sperm production, sperm delivery, Materials and method correcting retrograde ejaculation or retrieving sperms for in vitro fertil- ization (Lopushnyan, 2012). The use of stem cell therapy for male Plant extract preparation infertility treatment is still in the evolutionary stage of inducing pluripotent stem cells and mesenchymal stem cells to counter azoo- M. pruriens seed were donated by Prof. George Awuku Asare. Seeds spermia (Saha et al., 2021). were received from Catholic Missionaries visiting Battor Catholic Hos- Herbal extracts such as Epimedii Herba and Angelicae Gigantis Radix pital (Sogakofe Volta Region), and identified as such, by the National are known to reduce oxidative stress and subsequently increase the Herbarium, University of Ghana. Seeds were sun-dried for 3 days and production of sperm (Park et al., 2017). Other medicinal plants highly roasted lightly to break the hard coat followed by grounding with an used in the Palestine and the West Bank rural areas include Ferula her- electronic high-speed multifunctional grinder (Zhejiang, China) into monis roots (96.08%), Phlomis brachyodon leaves (88.24%) and Phoenix fine powder. The powder was used to prepare the crude extract of dactylifera pollen grains (86.27%) (Jaradat and Zaid, 2019). Ashwa- concentrations 500 mg/kg b.wt, 1000 mg/kg b. wt and 2000 mg/kg b. gandha (Withania somnifera) an Indian herb, is known for its aphrodisiac wt. The selected dose was based on a previous study that determined the properties. Furthermore, studies have shown that the root extract of LD50 to be above 2000 mg/kg b.wt (Jayakumar et al., al.,2016). Withania somnifera is said to treat oligospermia (Ambiye et al., 2013) as well as improve concentrations of lactate, citrate and essential amino Experimental animals acids in semen (Gupta et al., 2013) and thus, consequently improve overall semen quality. Additionally, it has been reported to reduce The Ethics and Protocol Review Committee of the School of oxidative stress and improve testosterone, Luteinizing Hormone and Biomedical and Allied Health Sciences, University of Ghana approved Follicle Stimulating Hormone, thereby reversing male infertility the study and issued ethics number SBAHS/AA/MLAB/10,607,885/ (Ahmad et al., 2010). 2020–2021. A total of 28 male Sprague-Dawley (SD) rats were obtained Aspalathus linearis (rooibos) a well-known tea in South Africa for its from the Noguchi Memorial Institute of Medical Research (NMIMR), antioxidative properties when treated on sperm in vitro using the fer- University of Ghana and housed at the Department of Animal Experi- mented form, attenuated the production of reactive oxygen species, DNA mentation. Rats were housed in stainless steel cages with soft wood fragmentation and improved sperm motility and vitality (Takalani et al., shavings and fed rodent feed pellet (AGRIFEEDS, Kumasi) with access to 2021). drinking water ad libitum. Four (4) groups were established (7 rats per Mono herbal preparations of aphrodisiac plants and plant products group) after being randomized. Animals were allowed to acclimatize for have been explored over the years. Chlorophytum borivilianum- an seven days under 12-hour light and 12-hour darkness. Temperature and aphrodisiac plant is said to have positive effects on semen profiles and humidity were 22±3 ◦C and 4–45%, respectively. Groups comprised the improve plasma biochemical profiles in infertile men. C. borivilianum is control group (distilled water), low dose group (500 mg/kg b.wt referred to as nature’s wonder drug mainly because of its aphrodisiac M. pruriens), medium dose group (1000 mg/kg b.wt M. pruriens) and properties. At doses of 125 and 250 of Chlorophytum borivilianum using high dose group (2000 mg/kg b.wt M. pruriens) administered by gavage Wistar albino rats, the lower dose of 125 demonstrated aphrodisiac over a period of 90 days. Rats were weighed weekly, and the dose properties during the 3-hour observation. Furthermore, sperm count adjusted to the weight. On the 91st day, animals were sacrificed, and continued to increase for the next 3 weeks (Ray et al., 2014). However, blood samples taken by cardiac puncture for various haemato- basic science research on this plant for its aphrodisiac and fertility po- biochemical analyses. Organs such as testes, prostate and seminal tential is very minimal. Similarly, Eulophia campestris though reported to vesicle were harvested alongside systemic organs. Epididymal sperms have similar effects on male infertility has little or no scientific data samples were taken for laboratory analysis. available on its fertility and aphrodisiac potential as a monoherbal plant. The art of combining monoherbal therapeutic plants to produce a Physical weights of rats and organ poly herbal product is an ancient art in Ayurvedic medicine. Such combinations have been seen for disease such as diabetes (Madić et al., Weights of animals were determined initially. Subsequent weights 2021) and hypertension (Tian Shen et al., 2019). were taken on weekly basis throughout the period of study. Chlorophytum borivilianum, Eulophia campestris and Mucuna pru- riens was investigated as a combined product for the improvement of Blood collection semen quality and motility in infertile males involving administration of the poly herbal product to male albino rats for 40 days and oligospermic The rats were sacrificed on the 91st day. Rats were first anaesthetised males for 90 days. Results from the former (albino rats) resulted in using ethyl ether and seven millilitres (7 ml) of the blood samples were 2 S. Adjei et al. P h y t o m e d i c i n e P lus 3 (2023) 100465 drawn by cardiac puncture with 25 G needles and discharged into gel Results separator and EDTA tubes for various assays. From Table 1, all groups gained significant weight after 90 days. The greatest change was observed in the high dose group. Organ harvesting and tissue processing For relative organ weights Post hoc analysis on the liver and spleen showed significant difference (p < 0.05) between the control and or Organs harvested included prostate, testes, seminal vesicle and other other dose groups. For the kidney, differences were between the control systemic organs such as liver, kidney, and heart. Organs were weighed, group and low dose group. For the prostate, differences occurred be- rinsed in normal saline solution, and were preserved in 10% buffered tween the control and low dose, low and medium dose and medium and formalin except for testes which were preserved in Bouin’s solution for high dose (p < 0.05, in all cases). histological analysis. The histological examination of organs was ac- cording to the study of (Owagboriaye et al., 2017). Tissues were pro- Hormonal analyses cessed using Leica TP 1020 tissue processor (Wetzlar, Germany) which employed the routine paraffin embedding procedure. The embedded Testosterone showed a gradual increase across the groups. However, tissues were subjected to serial sectioning of 4 μm thickness using a this change was not significant. Oestrogen declined but not in a dose rotary microtome and were subsequently processed in alcohol-xylene dependant manner with the greatest decline occurring in the medium series which were stained with hematoxylin and eosin (H & E). Slides dose. FSH on the other hand increased only with the high dose group but prepared were examined at x4, x10 and x40 objective lens using was not statistically significant (Table 3). Olympus CX23 light microscope (Tokyo, Japan). Semen analyses Semen collection From Table 4, motility increased significantly for all the experi- mental groups with the highest seen in the medium dose group. Caudal epididymis from each side of the testis of the rats were Conversely, immotility decreased across the experimental groups with removed and placed into 2 mls of 0.85% warmed physiological saline. the lowest in the medium dose groups. Analysis of variance (ANOVA) for Sterile needle was used to puncture the caudal epididymis several times motility and immotility was statistically significant with p-values 0.013 to allow sperms to seep into the saline to produce a saline sperm sus- and 0.013, respectively. A decrease in sperm count was observed in the pension. A volume of 10 μl was dispensed onto a pre-warmed micro- low and high dose groups with the highest observed in the medium dose scope slides and were covered with cover slips. Slides were observed group. Again, the difference was statistically significant (p < 0.01) immediately under Olympus CX23 light microscope (Tokyo, Japan) for (Table 4). However, no correlation existed between sperm count and the motility of the sperms. Sperm count estimation, was carried out by hormonal levels. thoroughly mixing saline semen suspension and semen diluting fluid Post hoc analysis for motility (Table 5), demonstrated that significant (sodium bicarbonate 5 g, formalin 1 ml, and distilled water 99.0 ml). A difference existed between the control and the medium dose group (p = drop of sperm suspension was loaded into a haemocytometer and was 0.022) as well as control and high dose groups (p = 0.029). Post hoc allowed to settle in a humid place for 5 to 10 min afterwards, sperma- analysis of immotility, showed that significant changes occurred be- tozoa were counted under the light microscope [Olympus CX23 (Tokyo, tween the control and the medium dose group (p = 0.03) as well as Japan)]. Sperm count was estimated by using the formula (Sperm count control and the high dose group (p = 0.004). = number of sperm counted x dilution factor / volume x 1000). For sperm count, post hoc analysis revealed significant differences between the control group and the medium dose group as well as the low dose group and medium dose groups (p < 0.05 respectively). Further- Haemato-biochemical analyses more, significant difference was observed between the medium and high dose groups (p = 0.000). Hormonal analyses Hormones were analysed using the MAGLUMI 600 chemistry auto- Liver function test analyser (Shenzhen, China) with Testosterone, FSH, LH and Oestrogen hormonal test kits (Shenzhen, China). The testosterone test was based on Liver function test (TBIL, DBIL, IBIL, ALT, AST, ALP, GGT, AST/ALT, a competitive chemiluminescence immunoassay. Oestrogen estimation TP, ALB, Glo, Alb/Glo) did not show any significant difference for the was a competitive immunoluminometric assay employed. LH and FSH various analytes except for ALT (p = 0.042), (Table 6). assays were based on a sandwich chemiluminescence immunoassay. Renal function test Determination of haematological and biochemical parameters Full blood count (FBC) was performed on whole blood samples using For renal function test (Na, k, Creatinine, Urea), only creatinine was Sysmex haematology autoanalyzer (Kobe, Japan). Liver function tests (Total and conjugated bilirubin, liver enzymes, total protein, and albu- Table 1 min) as well as renal function tests (sodium, potassium, urea and serum Body weight profile of rats within groups after 90 days administration of the creatinine) were determined using SELECTRA JUNIOR Version 04 crude aqueous extract of Mucuna pruriens. autoanalyzer (Vital Scientific, Spankeren, The Netherlands). Groups Initial body Weight after 90 days of P – value weight administration Statistical analysis (g) Repeated measures design was used to control factors that caused Control 147.7 ± 13.9 273.53 ± 18.2 <0.001 variability between subjects. Continuous variables were expressed as Low dose 196.3 ± 7.9 291.7 ± 35.3 <0.001 mean ± SEM and statistical significance determined by ANOVA. Bon- Medium 190.9 ± 14.28 298.0 ± 27.9 <0.001 ferroni post hoc was used to determine specific groups where significant dose differences were found. Pearson correlation was used to determine the High dose 185.0 ± 27.9 317.0 ± 16.3 <0.001 association between hormonal levels and sperm count. The significant Values are expressed as mean±SEM; n = 7 per group; statistically significant at p level was set at p < 0.05. < 0.05. Data were analysed by a paired T-test. 3 S. Adjei et al. P h y t o m e d i c i n e P lus 3 (2023) 100465 significant (p = 0.043) for ANOVA (Table 7). Discussion To the best of our knowledge, this present study is the only experi- Post hoc analysis of biochemical parameters mental investigation involving M. pruriens seed powder that has been conducted over a 90-day period regarding its efficacy and safety in the Post hoc analysis of ALT (showed significant difference (p = 0.033) remedy of male infertility. All previous studies created pathological between the medium dose and high dose group with a reduction in the conditions of some disease, mainly diabetes. This present study however high dose group. However, post hoc analysis did not reveal any signif- demonstrates findings on non-pathological conditions, thereby resolving icant difference in creatinine (Table 8). the incline of what happens when normal people use M. pruriens to boost fertility. Effect of extract on haematological parameters Rats exhibited normal growth weight over the 90-day period (Table 1). M. pruriens on its own does not appear to cause weight in- Red cell indices in Table 9 (RBC, HGB, HCT, MCV, MCH, MCHC, crease. The weight increase is the normal growth pattern as even seen in RDWCV, RDWSD) showed no statistically significant difference with the the control group. Indeed, M. pruriens improves body composition and exception of RDWCV and RDWSD and (p = 0.007; 0.002) respectively. subsequently prevents obesity (Tavares et al., 2021). Brain size From Table 4.12, WBC and platelet parameters [WBC (tov tal), Neu, increased non-significantly in a non-dose dependant manner but not in Lym, Mon, Eos, Bas, Bas%, PLT, PDW, PCT, PLCC, PLCR] did not show the relative organ weight (Table 2). M. pruriens therefore does not have any significant change with the exception of basophils% which showed detrimental effects on the brain. In one study, remarkable protection significant change (p = 0.036). was observed in the brains of experimental rats treated with M. pruriens as opposed to those not treated who suffered from cerebral ischaemia (Nayak et al., 2017). Post hoc analysis for haematological parameters Spleen organ weight demonstrated a reduction in all groups with the lowest weight in the medium dose group. For relative organ weigh the Post hoc analysis in Table 11 revealed differences existed between change was still significant (p < 0.05). The spleen is responsible for WBC the control and the medium dose group as well as conrol and low dose production. This did not reflect in the total WBC; indeed, all parameters group for RDW-SD (p = 0.013; p = 0.012, respectively). Significant of WBC were normal except for basophils which showed a significant differences in RDW-CV existed between low dose and medium dose reduction comparing the high dose group to the control group groups, as well as medium dose and high dose groups (p = 0.039; p = (Table 10). A reduction in basophils is often associated with some al- 0.022) respectively. For BAS% differences existed between the Control lergy or infection. However, all animal were produced pathogen-free. group and the High dose group (0.046). This change may not be pathological. Other haematological parame- ters that showed differences included the red cell distribution width Histological analysis coefficient of variation and Red Cell Distribution Width SD (Table 9). Slight decreases were observed from medium dose and high dose, Histology so far does not confirm any adverse effects of the aqueous compared to the control. These parameters are associated with anaemia. crude seed extract of Mucuna pruriens on systemic organs. Furthermore, A decrease will suggest an improvement in the haemoglobin level. reproductive organs herein presented also showed normal architecture Indeed, haemoglobulin levels improved slightly in all groups receiving (Figs. 1-2). M. pruriens in a dose dependant manner, although not significant Fig. 1. . Photomicrograph of cross sec- tion of the testis (400X). This figure shows the cross section of the testis in S- D rats following treatment with low dose (500 mg/kg), medium dose (1000 mg/kg) and high dose (2000 mg/kg) of M. pruriens for 90 days. In the control group, the testicle appeared covered by a capsule of connective tissue (the tunica albuginea). Testicular paren- chyma consisted of seminiferous tubules which appeared rounded or oval with regular contour. The interstitial spaces in-between the tubules contain a deli- cate loose connective tissue and Leydig cells. H and E-stained testicular samples of the low dose group, medium dose group, and high dose group revealed the same histological features of the testis. (Hematoxylin and Eosin stain, 400X). 4 S. Adjei et al. P h y t o m e d i c i n e P lus 3 (2023) 100465 Fig. 2. Photomicrograph of the seminal vesicles (100X). This figure shows the effect of the crude extract of M. pruriens on the histology of the seminal vesicles (Hematoxylin and eosin). All groups showed normal tissue histology. All groups showed mucosal folds/ridges extending into the lumen. The lining epithelium of seminal vesicle was pseudo-stratified columnar epithelium, composed mainly of a single layer of tall columnar principal cells and triangular shaped basal cells. The lumen of seminal vesicle was filled with eosinophilic secretion. The epithelium lies on a thin layer of connective tissue lamina propria. The lamina propria was lined by inner circular and outer longitudinal smooth muscle fibres. Mucosal alveoli or crypts showed small amount of secretion. with a sharp decrease in the high dose group (Table 6). Post hoc analysis Table 2 revealed that differences were between the high dose and the medium Effect of the crude aqueous extract of Mucuna pruriens on relative organ weight (g) of vital organs of rats after 90 days administration by gavage. dose groups (Table 8). However, histologically there were no liver le- sions. In one study, when liver tissues were damaged by cobra venom in Organs (g) Control Low Medium High p- rats, the pre-treatment of rats with M. pruriens extract protected vessels dose dose dose value in these organs from damage as seen histopathologically (Jung and Heart 0.350 0.388 0.388 0.362 NS Schuppe, 2007), thereby suggesting hepatoprotective properties of Liver 3.400β 3.100§ 2.900§ 3.000§ ˂0.05 M. Kidney 0.315θ 0.290α 0.270 3.000 ˂0.05 M. pruriens. Lungs 0.735 0.750 0.750 0.670 NS Although creatinine was significantly elevated in the medium dose Spleen 0.330ɸ 0.230* 0.200* 0.220* ˂0.05 groups (Table 7 & 8), this did not lead to any concomitant increase in Brain 0.550 0.577 0.925 0.880 NS urea nor were histological lesions observed. On renal function, when M. Testes 0.880 0.935 0.935 0.853 NS rats were fed with high fructose to induce renal damage by oxidative Seminal 0.850 0.750 0.725 0.800 NS vesicle stress mechanisms (increased renal MDA), 200 mg/kg daily M. pruriens Prostate 0.270Ψ 0.375Ω 0.263‡ 0.385χ ˂0.05 modulated the kidney nuclear transcription factors thereby restoring kidney function after 8 weeks of deterioration (Ulu et al., 2018). Addi- NS = Statistically not significant. Statistically significant at p < 0.05. M. Kidney= Mean of kidneys. M. Testes Mean of testes. Post hoc revealed: ɸ tionally, the protective effect of M.pruriens against arsenic-induced liver = compared to * 0.05; θ compared to α 0.05; β compared to 0.05; Ψ and kidney dysfunction has been recorded (Concessao et al., 2020). < < § < compared to Ω <0.05; Ω compared to ‡ <0.05; ‡ compared to χ < 0.05. Contrary findings have however been presented by (Gbotolorun et al., al.,2018) in which the authors observed that medium dose was associ- (Table 9). One study also alludes to the fact that M. pruriens could ated with mesengial expansion, hypercellularity of the glomeruli as well improve anaemia. (Abidemi and Fadeyibi 2011). The only other study as tubular necrosis. These adverse effects differences may be due to the that examined the effect of M. pruriens on haematological parameters in use of M. pruriens methanolic extract, if the methanol was not properly a 28-day study showed that PCV, Hb, RBC, MCV, HCHC, MCHC and total cleaned. Furthermore, the apparent nephrotoxicity was not accompa- WBC were normal as similarly observed in this study. However, nied by increase in creatinine and was observed only in the medium dose regarding red blood cell indices, platelet count decreased at a dose of group (100 mg/kg. b.wt) for 2 weeks. 400 mg/kg b.wt. Furthermore at doses of 50 and 200 mg/kg b.wt neu- In this study, hormonal assays demonstrated a dose-dependant in- trophils decreased (Abidemi and Fadeyibi 2011). Contrarily, neua- crease in testosterone, though not statistically significant. However, trophils increased in this study although not significantly. other hormones such as oestrogen and FSH did not show any dose- Biochemically, the increase in ALT was in the medium dose group, dependant changes (Table 3). Perhaps the positive effects of the testosterone increase can be supported by data from semen analysis. 5 S. Adjei et al. P h y t o m e d i c i n e P lus 3 (2023) 100465 Table 3 Table 5 Effect of Mucuna pruriens crude aqueous seed extract on hormonal parameters Post hoc analysis for motility (%), Immotility (%),sperm count of rats after 90 (Testosterone, Oestrogen and Follicle Stimulating Hormone (FSH). days oral administration with low dose (500 mg/kg), medium dose (1000 mg/ Parameter Control Low dose Medium dose High dose p- kg) and high dose (2000 mg/kg) of crude aqueous seed extract of Mucuna (500 mg/ (1000 mg/kg) (2000 mg/ Value pruriens. kg) kg) Parameter Low dose (500 Medium dose (1000 High dose (2000 Testosterone 5.59 ± 6.58 ± 0.7 6.65 ± 0.4 7.58 ± 0.6 NS mg/kg) mg/kg) mg/kg) 0.4 Motility (%) Oestrogen 210.6 ± 180.8 ± 135.7 ± 16.6 152.6 ± NS Control 0.208 0.022* 0.029 22.4 26.0 28.0 Low dose 1.000 1.000 FSH 2.9 ± 2.9 ± 0.2 2.9 ± 0.2 3.0 ± 0.3 NS Medium dose 1.000 0.2 Immotility Values are expressed as mean (%) ± SEM. n = 7; NS = Statistically not significant. Data were analysed. Control 0.061 0.003* 0.004* by one-way ANOVA. Low dose 1.000 1.000 Similar positivity of semen analysis and improvement in sexual behav- Medium dose 1.000 iour was also reported by (Suresh and Prakash 2012) where the levels of Sperm count Control 1.000 0.003* 1.000 FSH and LH in diabetic rat models administered M. pruriens improved Low dose <0.001** 1.000 significantly. This was coupled with improvement in daily sperm pro- Medium duction. The authors also reported improved spermogram parameters. dose In this present study, sperm count was highest in the medium dose group *statistically significant at p < 0.05; **statistically significant at p < 0.001. (1000 mg/kg b.wt) and statistically significant. Motility was also significantly high in this group (Tables 4 & 5). In mice models, M. pruriens seeds at a dose of 600 mg/kg positively affected degenerative Table 6 seminiferous epithelium. Testosterone levels and sexual behaviour also The effect of the crude aqueous seed extract of Mucuna pruriens on liver function improved (Choowong-In et al., 2021). after 90 days administration by gavage. In humans 5 g of M. Pruriens administered over 3 months in infertile Control Low dose Medium dose High dose P- men increased testosterone levels to the levels of controls (Gupta et al., (500 mg/ (1000 mg/kg) (2000 mg/ Value 2011). At this same dose, sperm count, and motility increased (Shukla kg) kg) et al., 2009). These results are similar to our results where normal rats TBIL 0.746 ± 0.865 ± 0.872 ± 0.08 0.773 ± NS had testosterone increase dose-dependently (but not significantly) over 0.05 0.04 0.11 3 months. It is proposed that levodopa in M. Pruriens antagonizes pro- DBIL 0.104 ± 0.232 ± 0.163 ± 0.04 0.122 ± NS 0.05 0.07 0.01 lactin. Increase prolactin level supresses testosterone and libido (Shukla IBIL 0.638 ± 0.605 ± 0.713 ± 0.09 0.618 ± NS et al., 2009). Similarly in this study, sperm count and motility increased 0.07 0.08 0.11 significantly thereby mimicking the study of (Gupta et al., 2011) in ALT 104.6 ± 106.6 ± 151.7 ± 27.3 74.6 ± 12.8 0.042* humans. 10.2 13.2 Although behavioural studies were not undertaken in this study, the AST 400.9 ± 286.1 ± 420.02 ± 122.9 273.4 ± NS 110.6 38.6 66.8 increasing testosterone and sperm count would suggest improved sexual ALP 314.7 ± 305.5 ± 333.1 ± 49.4 289.7 ± NS behaviour. Improved sexual behaviour was observed as increase in 20.0 40.4 28.4 mounting frequency, intromission frequency, and ejaculatory frequency GGT 5.2 ± 4.03 ± 0.80 5.28± 0.9 5.32 ± 0.6 NS amongst others. Sperm quality and daily sperm production improved. 0.25 AST/ 3.58 ± 2.76± 0.32 2.63 ± 0.36 3.53 ± 0.41 NS M. pruriens at 200 mg/kg of M. pruriens seed extract is said to have ALT 0.69 restored mitochondria dysfunction and DNA damage of sperm in dia- TP 65.8 ± 73.1 ± 2.65 76.03 ± 2.97 70.43 ± NS betic rats over a period of 60 days (Suresh et al., 2013). The authors of 2.24 4.32 that study indicated that activities of sexual behaviour were however ALB 34.16 ± 37.12 ± 36.75 ± 1.14 40.0 ± 1.7 NS maximized and effective at their medium dose of 200 mg /kg. in rats 0.87 1.30 Glo 31.7 ± 36.07 ± 39.28 ± 2.42 34.5 ± 2.7 NS 1.41 1.76 Table 4 Alb/ 1.08 ± 1.033 ± 0.967 ± 0.06 1.05 ± 0.03 NS Effect of Mucuna pruriens on semen analysis (motility, immotility and sperm Glo 0.02 0.05 count) of rats after 90 days oral administration with low dose (500 mg/kg), Values are expressed as mean ± SEM. n = 7. NS = Statistically not significant; medium dose (1000 mg/kg) and high dose (2000 mg/kg) of crude aqueous *statistically significant at p < 0.05. Data were analysed by one-way ANOVA. extract of Mucuna pruriens. TBIL: Total bilirubin (μmol/L); DBIL: Direct bilirubin (μmol/L); IBIL: Indirect Parameter Control Low dose Medium dose High dose p – bilirubin (μmol/L); ALT = Alanine aminotransferase (U/L); GGT = Gamma- (500 mg/ (1000 mg/kg) (2000 mg/ Value glutamyl transferase (U/L); ALP = Alkaline phosphatase (U/L); AST: Aspartate kg) kg) transaminase (U/L); Glo: Globulin (g/L); TP: Total protein (g/L); Alb: Albumin Motility 0.36 0.50 0.57 0.07 0.56 0.013 (g/L). ± ± ± ± (X 0.04 0.01 0.04 100%) (Suresh et al., 2009). Immotility 0.64 ± 0.50 ± 0.43 ± 0.05 0.44 ± 0.013 Erectile dysfunction in aged rats observed by a reduction in the (X 0.03 0.01 0.03 100%) number of myelinated fibres, indentation of the myelin sheath with Sperm 273.0 ± 260.3 ± 339.3 ± 14.5 257.8 ± <0.001 degenerative changes of the dorsal nerve of the penis with its implica- Count 12.1 10.7 6.4 tion on erectile function, were all recovered following M. pruriens sup- (n x plementation (Seppan et al., 2020). 10^6) Histologically, no abnormality was found in the testicles of rats Values are expressed as mean ± SEM (n = 7); statistically significant at p < 0.05. receiving M. pruriens in this study (Fig. 1). A study on testicular damages Data were analysed by one-way ANOVA 6 S. Adjei et al. P h y t o m e d i c i n e P lus 3 (2023) 100465 Table 7 Table 10 Effect of the crude aqueous extract of Mucuna pruriens on renal function test (Na, The Effect of the crude aqueous seed extract of Mucuna pruriens on White blood K, Urea, Crea.) after 90 days administration by gavage. cells and platelets after 90 days of oral administration by gavage. Control Low dose Medium dose High dose P- Control Low dose Medium dose High dose p- (500 mg/kg) (1000 mg/kg) (2000 mg/kg) Value (500 mg/kg) (1000 mg/kg) (2000 mg/ Value kg) Na 142.6 ± 142.8 ± 1.6 141.7 ± 0.56 143.8 ± 1.08 NS 0.9 WBC 3.7 ± 1.0 7.1 ± 1.8 7.0 ± 1.1 7.4 ± 1.9 NS K 3.56 ± 3.62 ± 0.10 3.55 ± 0.04 3.57 ± 0.09 NS Neu 0.6 ± 0.2 1.3 ± 0.3 1.4 ± 0.1 1.3 ± 0.3 NS 0.11 Lym 2.9 ± 0.8 5.6 ± 1.6 5.4 ± 0.9 5.6 ± 1.5 NS Urea 6.64 ± 6.23 ± 0.37 6.96 ± 0.27 6.72 ± 0.38 NS Mon 0.17 ± 0.17 ± 0.09 0.14 ± 0.03 0.50 ± 0.20 NS 0.43 0.06 Crea 102.8 ± 106.5 ± 6.2 120.5 ± 4.7 103.7 ± 2.5 0.043* Eos 0.02 ± 0.01 ± 0.01 0.01 ± 0.0 0.02 ± 0.01 NS 3.8 0.01 Bas 0.01 ± 0.004 ± 0.01 ± 0.004 0.0 ± 0.0 NS Values are expressed as mean ± SEM (n = 7); NS = Statistically not significant; * 0.002 0.002 statistically. Bas 0.3 ± 0.1 0.1 ± 0.1 0.2 ± 0.1 0.02 ± 0.01 0.036 significant at p < 0.05. Data were analysed by one-way ANOVA. Na: Sodium % (mmol/L); K:. PLT 409.0 ± 571.0 ± 401.9 ± 101.5 677.4 ± 35.6 NS Potassium (mmol/L); Crea: Creatinine (mmol/L); Urea (mmol/L). 172.1 73.0 PDW 15.7 ± 15.4 ± 0.1 15.5 ± 0.1 15.4 ± 0.1 NS 0.3 Table 8 PCT 0.28 ± 0.42 ± 0.05 0.29 ± 0.07 0.50 ± 0.04 NS 0.11 Post hoc analysis of ALT and creatinine of rats after 90 days oral administration PLCC 41.7 ± 65.6 ± 9.02 48.6 ± 11.7 85.2 ± 11.4 NS with low dose (500 mg/kg), medium dose (1000 mg/kg) and high dose (2000 12.9 mg/kg) of crude aqueous extract of Mucuna pruriens. Values are expressed as mean ± SEM (n = 7). NS = statistically not significant; Parameter Low dose Medium dose High dose *statistically significant at p < 0.05. Data were analysed by one-way ANOVA. (500 mg/kg) (1000 mg/kg) (2000 mg/kg) WBC: white blood cell count (x103); Eos: Eosinophils (U/L) count; Mon: ALT Monocyte (U/L); Bas: Basophils count (U/L); Neu: Neutrophils count (U/L); Control 1.000 0.508 1.000 Lym: lymphocytes (%); PLT: platelet count (%); LYM: lymphocyte (%); MPV: Low dose 0.500 1.000 mean platelet volume (fL); PDW: platelet distribution width (fL); PCT: Platelet Medium 0.033* (x103); PLCR: Platelet large cell ratio; PLCC: Platelet Large cell count (fL). dose Creatinine Control 1.000 0.092 1.000 Low dose 0.240 1.000 Table 11 Medium 0.095 Post hoc analysis for haematological parameters (RDW-SD, RDW-CV, basophils dose % and PLT) of rats after 90 days oral administration with low dose (500 mg/kg), *Statistically significant at p < 0.05. medium dose (1000 mg/kg) and high dose (2000 mg/kg) of crude aqueous extract of Mucuna pruriens. Low dose Medium dose High dose Table 9 (500 mg/kg) (1000 mg/kg) (2000 mg/kg) . Effect of the crude aqueous extract of Mucuna pruriens on red cell indices after RDW- 90 days of oral administration by gavage. SD Control 1.000 0.013* 0.070 Control Low dose Medium dose High dose P- Low dose 0.012* 0.074 (500 mg/ (1000 mg/kg) (2000 mg/ Value Medium 1.000 kg) kg) dose RDW- RBC 7.6 ± 8.2 ± 0.4 8.47± 0.1 8.3 ± 0.3 NS CV Control 1.000 0.249 0.132 0.3 Low dose 0.039* 0.022* HGB 14.6 ± 15.3 ± 0.4 15.4 ± 0.3 15.4 ± 0.5 NS Medium 1.000 0.5 dose HCT 42.5 ± 44.7 ± 0.8 44.2 ± 0.8 44.6 ± 1.3 NS BAS% 1.5 Control 0.413 1.000 0.046* MCV 56.1 ± 54.6 ± 1.4 52.2 ± 0.8 54.0 ± 1.1 NS Low dose 1.000 1.000 0.6 Medium 0.168 MCH 19.6 ± 18.7 ± 0.4 18.2 ± 0.3 18.6 ± 0.3 NS dose 0.2 MCHC 34.5 ± 34.3 ± 0.2 34.8 ± 0.2 34.5 ± 0.2 NS *Statistically significant at p < 0.05. 0.2 RDWCV 15.2 ± 15.5 ± 0.4 14.2 ± 0.3 14.0 ± 0.2 0.007 induced by ethanol ingestion through MDA accumulation in rats was 0.2 RDWSD 32.5 ± 32.3 ± 1.0 28.3 ± 0.8 29.0 ± 0.8 0.002 subsequently reversed by M. pruriens seed extract administration. This is 0.6 in agreement with our testicular histology. Indeed, testicular damage will not support the several findings that suggested that M. pruriens Values are expressed as mean ± SEM, n = 7; NS = Statistically not significant; could improve spermatogenesis. Histologically, the seminal vesicles did Statistically significant at p <0.05. Data were analysed by one-way ANOVA. RBC: red blood cell count (x10⁶); HGB: haemoglobin concentration (g/dL); HCT: not show any abnormality. hematocrits (%); MCV: mean corpuscular volume (fL); MCH: mean corpuscular The concept of polyherbalism has been in existence since the era of haemoglobin (pg); MCHC: mean corpuscular haemoglobin concentration (g/dL); Ayurvedic medicine as exemplified by triglize usage (Parasuraman et al., RDW-CV= red cell distribution width coefficient of variation (%); RDW-SD (fL): 2010). In Ayurvedic medicine it is believed that combining plants boosts red cell distribution width-standard deviation. the levels of certain phytocompounds which otherwise would exist in minimum quantities. The phenomenon of synergy suggestively led to combinations such as black pepper, long pepper and ginger (mucous-- reducing as well as “heating” effect) or ginger and neem to take care of 7 S. Adjei et al. P h y t o m e d i c i n e P lus 3 (2023) 100465 “warmth” and “cold” extreme conditions (Pole et al., 2013). Scientifi- Concessao, P., Bairy, L.K., Raghavendra, A.P., 2020. Protective effect of Mucuna pruriens cally, the concept of pharmacodynamics and pharmacokinetics synergy against arsenic-induced liver and kidney dysfunction and neurobehavioral alterations in rats. Vet. World 13 (8), 1555–1566. in most cases support polyherbalism (Spinella et al., 2002). The efficacy Gameiro, S., Boivin, J., Dancet, E., Emery, M., Thorn, P., Van den Broeck, U., Venetis, C., of Mucuna pruriens (Linn), Chlorophytum borivillianum (Sant and Fer- Verhaak, C.M., Wischmann, T., Vermeulen, N., 2016. Qualitative research in the nand), and Eulophia campestris (Wall) as a polyherbal drug for the ESHRE Guideline ’Routine psychosocial care in infertility and medically assisted reproduction - a guide for staff’. Human Reproduction 31 (8), 1928–1929. management of male infertility has been demonstrated and fully dis- Gbotolorun, S.A., Isah, P.K., Adebajo, O.A., 2018. Toxicity of Mucuna pruriens seed cussed elsewhere (Mahajan et al., 2012). extract on the kidney of adult Sprague-Dawley rats. Afr. J. Pharm. and Therap. 7 (1), 27–33. Gollenberg, A.L., Liu, F., Brazil, C., Drobnis, E.Z., Guzick, D., Overstreet, J.W., Conclusion Redmon, J.B., Sparks, A., Wang, C., Swan, S.H., 2010. Semen quality in fertile men in relation to psychosocial stress. Fertil. 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