The roots of Waltheria indica were sourced from Geji village in Bauchi State, Nigeria in November 2021. The plant was identified and authenticated at the Department of Horticulture and Landscape Technology was given by Mr. Christopher Abok, a taxonomist with Federal College of Forestry, Jos, Nigeria with assigned voucher numbers of FHJ 273 and deposited in the Federal College of Forestry herbarium. The sourced roots of Waltheria indica were cleaned of debris and dried under shade in the department of Pharmacology and Toxicology University of Jos. The dried roots were pulverized using wooden pestle and mortar into coarse powder. Five hundred (500) gram of the pulverized plant material was poured into a 6-L round bottom flask and 3 L of 70% methanol was added to the plant powder to be extracted by maceration. The content of the flask was allowed to stand for 72 h with shaking at intervals of 12 h. After 72 h and was successively passed through sieves of varying pore sizes (800, 500 and 150 μm); followed by filtration using cotton wool plug and finally Whatman No. 1 filter paper to obtain a clear filtrate.

The filtrate was dried by passing a steady stream of air using an exhaust fan to reduce the volume to about one third of the original volume. The remainder was transferred into an oven to dry at a temperature of 40°C after which the dried extract was scrapped and stored in airtight containers at 25 ± 2°C till use.

Healthy sexually experienced male white albino rats weighing between 200-300 g with age ranging from 20-20.5 weeks were obtained from the Animal House Unit of the Department of Pharmacology; University of Jos, Nigeria and used for the studies. Ethical Clearance Certificate was obtained from Ethical Committee Animal Experimental Unit of Department of Pharmacology, FCULTY OF Pharmaceutical Sciences, University of Jos, Nigeria vide reference UJ/FPS/F17-00379 dated 10.01.2022. The male rats were kept in separate cages (Temperature: 22-28⁰C; 12 h natural light and 12 h dark; humidity: 50-55 %) with free access to rat pellets. Ethical clearance was obtained from the animal care and use committee of the Department of Pharmacology and toxicology, Faculty of Pharmaceutical Sciences, University of Jos vide Ethical clearance Certificate No. UJ/FPS/F17-00379 dated 10^th January, 2022.

The method describe by Yakubuet al., 2012 was employed with slight modification. 21 Male rats in group 1 served as control and orally received10ml/kg of normal saline using oro-pharyngeal cannula while male rats in groups 2, 3 and 4 received 50, 100 and 200mg/kg body weight of the extracts respectively, orally daily for 40 days. Male rats were anesthetized, sacrificed on the 41^st day and blood from the rats was collected into clean; dry corked centrifuge tubes. Thereafter, the tubes were centrifuged (Hawksley, England) at 3000 rpm for 15 minutes. The rats were also dissected; testes and epidydimis were collected for testicular studies and sperm analysis

The data were subjected to analysis of variance (ANOVA) using Dunnett’s test to determine the level of statistical significance. Significance level was set at p<0.05 and confidence level at 95%. Statistical analysis was carried out using Graph pad Prism 7.0. Data obtained from statistical analysis were shown as Mean ± Standard Error of Mean.

Table 1 shows that the extract produced an increase in concentrations of serum Testosterone, Follicular stimulating hormone (FSH) and Luteinizing hormone (LH) when compared to the control at (P<0.05).

Table 1

Treatment	Mean ± SEM
	Testosterone nmol/L	FSH (mUI/mL)	LH (mUI/mL)
Normal saline	0.43±0.180	0.07±0.002	0.42±0.002
W. indica 50 mg/kg	5.43±0.440*	0.08±0.005	0.43±0.008
W. indica 100 mg/kg	7.90±2.437*	0.09±0.001*	0.44±0.003*
W. indica 200 mg/kg	3.04±0.534	0.08±0.005	0.43±0.001

n=6, *=P<0.05,when compared with control, nmol/L=nanomoles per liter, mUI/mL = milli-international units per milliliter

Methanolic root extract of Waltheria indica as showed in Figure 1 produced a significant increase in number sperm cell counts when compared to control (P<0.05),

Figure 1

The results in Table 2 show that the extract produced a significant (P<0.05) increase in sperm cells viability male in rats.

Table 2

Treatment	Mean±SEM
	% Live	% Dead
Normal saline	82.10±3.789	17.9±3.789
W. indica 50 mg/kg	82.85±2.260	17.15±2.325
W. indica 100 mg/kg	85.80±1.349	14.2±1.533
W. indica 200 mg/kg	93.63±1.105*	6.37±1.105*

n=6, *=P<0.05, when compared with control

Results in Table 3 reveals that the extract increased the number of active sperms cell in relation to sperm mobile progression.

Table 3

Treatments (mg/kg)	Active (%)	Sluggish (%)	Inactive (%)
Normal saline	58.00 ± 8.00	19.00 ± 4.00	23.00±4.36
50	46.00±4.85	29.00 ±1.00	25.00 ± 4.18
100	69.00±7.81	25.00 ± 4.18	15.00 ± 5.48
200	50.00 ± 6.33	21.00 ± 2.45	29.00 ±7 .48

n=5,* = P< 0.05 when compared with control

Figure 2 shows an increase in Gonado-somatic index.

Figure 2

Results in Table 4 reveals that the extract did not increase the body weight of animals and weight of sexual organ weights.

Table 4

Treatment (mg/kg)	Final Body weight (g)	Testis (g)	Frontal Epidydimis (g)	Caudal Epidydimis (g)
Normal saline	295.40 ± 6.68	1.39 ± 0.11	0.29 ± 0.03	0.23 ± 0.01
50	257.00 ± 24.85	0.89 ± 0.27	0.18 ± 0.05	0.17 ± 0.05
100	258.60 ± 11.26	1.13 ± 0.11	0.25 ± 0.03	0.19 ± 0.03
200	330.70 ± 11.97	1.37 ± 0.07	0.28 ± 0.01	0.22 ± 0.02

n=6, *=P< 0.05 when compared with control

Table 5 shows that the methanolic root extract of Waltheria indica produced an increase in concentration of Gamma glutamyltransferase (GGT), Alkaline phosphatase (ALP), and Acid phosphatase (ACP) which is significant when compared with the control (P<0.05).

Table 5

Treatment (mg/kg)		GGT(U/L )	ALP (U/L )	ACP ( U/L)
Normal saline		10.55±0.76	294.20±15.75	1.68±0.32
50		17.10±0.50	327.60 ±9.96	2.93±0.09*
100		24.27±4.90*	328.90±11.62	1.95±0.05
200		18.30 ±0.35	379.20±31.27*	2.32±0.05*

n=6, *=P< 0.05 when compared with control, U/L= unit per liter

Results in Table 6 show that the methanolic root extract of Waltheria indica produced an increase in testicular cholesterol which was significant.

Table 6

Treatment (mg/kg)	Total Protein (g/dl)	Total Cholesterol (mg/dl)
Normal saline	0.50±0.03	0.12±0.01
50	0.50±0.04	0.20±0.01*
100	0.53±0.02	0.20±0.01*
200	0.38±0.02	0.19±0.02*

n=6, *=P< 0.05 when compared withcontrol

Histology of the testis from rats that received 50, 100 and 200 mg/kg of the methanolic root extract of Waltheria indica reveals that the extract did not cause any morphological changes in the cellular integrity of the testis and spermatozoa cell (Figure 3, Figure 4, Figure 5, Figure 6).

Plate 2

Plate 3

Black stars=seminiferous tubules, White arrow heads = Spermatogonia, Black arrows = spermatids, white stars=Interstitial cells. H&E A: x 100

Waltheria indica has demonstrated significant increase in androgens and spermatogenic indices and therefore, the claimed ethno-medicinal use for managing male sexual dysfunction has been scientifically substantiated.