Source and Authentication of Mucuna pruriens
The source of the Mucuna pruriens pods was Gbolasere Farms in Sagamu, Ogun State, Nigeria. The voucher specimen number IFE 18235 was given to the botanical identification after it was verified at the Herbarium, Department of Botany, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria [13].
Experimental Animals
The study employed male Wistar rats that weighed 120 ± 20 g and were 7 ± 1 weeks old. The animals were kept in typical laboratory settings, with a 12-hour light/dark cycle, a temperature of 22 ± 2°C, and a relative humidity of 55 ± 5%. They were given unlimited access to water and regular pelletised feed. The Ethical Committee for Animal Experimentation at the University of Lagos, Nigeria, examined and approved all experimental procedures in compliance with the National Institutes of Health's (NIH) recommendations for the use and care of laboratory animals [14].
Extraction and Phytochemical Characterization of Mucuna pruriens Trichomes
Mucuna pruriens pods were carefully scraped of their trichomes, and the bioactive components were extracted by macerating the trichomes in methanol and distilled water at a ratio of one gramme per 100 mL of solvent. During the 96 hour extraction procedure, the temperature was kept at 27 ± 2°C and the agitation was kept constant at 130 rpm. After filtration, the extract was concentrated at reduced pressure at 40°C using a rotary evaporator, the extracts were kept at 4°C prior to further analysis [15].
Phytochemical Analysis
The presence of secondary metabolites was evaluated through qualitative phytochemical screening using standard procedures, such as Wagner's test for alkaloids, ferric chloride (FeCl₃) tests for tannins and phenolic acids, Trim-Hill test for terpenoids, sodium hydroxide (NaOH) test for flavonoids, and a chloroform and sulphuric acid (H₂SO₄) test for steroids [16].
Animal Grouping and Treatment
Forty-two (42) male Wistar rats were randomly assigned into seven experimental groups (n = 6 per group):
Group 1: Control (no treatment)
Group 2: Methanol control (vehicle)
Group 3: Water control (vehicle)
Group 4: 200 µL aqueous extract
Group 5: 400 µL aqueous extract
Group 6: 200 µL methanolic extract
Group 7: 400 µL methanolic extract
Behavioral Assessments
The open field test was used to evaluate exploratory behaviour, locomotor activity, and anxiety-related responses. In order to measure exploratory and anxiety-like behaviors, rats were kept in a circular arena with a diameter of 40 cm for five minutes. The number of line crossings, rearing, and grooming activities were noted and examined [17 ]. Using the tail suspension test, the extracts' antidepressant potential was assessed. The total amount of time the rats were immobile was measured after they were each suspended by their tails for six minutes. A decrease in immobility time was thought to have an effect similar to that of antidepressants [18].
Toxicity Studies
To determine whether the extracts were safe, both acute and sub-acute dermal toxicity tests were performed. A single application of the extract was used to treat acute dermal toxicity, and the animals were observed for symptoms such as lethargy, diarrhoea, tremors, and discoloured fur [19]. Applying the extracts topically for 14 days in a row allowed for the assessment of sub-acute dermal toxicity. According to Rhiouani et al. [20], major organs were removed for histopathological analysis, and blood samples were obtained for biochemical analysis.
Immunohistochemical Analysis
After being collected and preserved in 10% neutral buffered formalin, skin and brain samples were subjected to tissue processing and staining. The tissues underwent processing before being sectioned at a thickness of 5 µm and embedded in paraffin. After sections were deparaffinized, endogenous peroxidase activity was inactivated by treating them with 0.3% hydrogen peroxide (H2O₂). The brain and skin tissue sections were treated with specific antibodies that target important neurochemical markers. Also, immunoreactivity was observed under a light microscope and staining intensity was measured to evaluate neurochemical changes brought on by extract application [21].
Statistics
The collected datasets were analysed using Microsoft Excel and Graphpad Prism 5.0. Variations within the datasets were illustrated using bar charts with error bars representing the standard error of mean (SEM), followed by the Turkey-Kramer post-hoc test. Differences between groups were assessed through Analysis of Variance (ANOVA). Statistical significance was determined at 95% confidence interval with a p-value threshold of 0.05.