Plant material collection, molecular identification, and sample extraction process

Carissa spinarum L. leaf and root samples were collected from Eor-Ewuaso village, Narok County, Kenya (coordinates −0.9690953596611817, 35.6458956346891). The samples were packed in airtight containers and transported to the University of Nairobi within 12 h of collection. Identification was made by a plant taxonomist, and samples were deposited in the herbarium at the Department of Biology, University of Nairobi (code of voucher specimen MOUNBI 2023/001). The leaf samples were stored at −80 °C for molecular authentication of the plant’s identity. The leaves and root bark of (woody roots) were peeled off and dried for two weeks, then ground into fine powder and stored at 4 °C.

Molecular identification of the plant sample was performed using DNA barcoding, as described by Pere et al. [14]. Genomic DNA extraction was performed using the Qiagen DNeasy® Plant Maxi Kit (Catalogue No. 68163, Qiagen, Germany), which was used to extract genomic DNA from C. spinarum L. leaves according to the manufacturer’s instructions. This was followed by PCR amplification targeting trnH-PsbA and rbcl regions, with the resulting sequences subjected to sequencing using Sanger sequencing chemistry (Macrogen, Netherlands)

The resulting forward and reverse sequences were used to construct contigs in BioEdit software version 5.0.9 [15], followed by manual trimming of low-quality end sequences and correction of miscalled bases based on chromatogram peaks. The cleaned sequences were BLASTed on GenBank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 4^th May 2024), suitable sequences were selected based on the following features: at least 80% identity, ≥ 95% query coverage, and zero expected (E) value. The sequences were then used for phylogenetic analysis using the Bayesian phylogenetic approach of MrBayes, version 3.1.2 [16]. A total of 12,000,000 phylogenetic trees were created, and from every 1000 generations, a tree was sampled. The ‘sum p’ and ‘sum t’ in MrBayer were used in constructing the consensus tree, with the branching confidence being calculated as the posterior probabilities and later visualized in Figtree, version 1.4.3 (http://tree.bio.ed.ac.uk/).

Chemicals

Solvents used in the experiment, including methanol, ethanol, acetone, and hexane, were HPLC grade and purchased from Sigma-Aldrich, St. Louis, MO, USA. Gallic acid, ascorbic acid, tannic acid, rutin, atropine, thiobarbituric acid (TBA), Folin-Ciocalteau reagent (Folin C), trichloroacetic acid (TCA), and poly(vinylpolypyrrolidone) (PVPP) were analytical grade and purchased from Merck Life Science UK Limited, Gillingham, United Kingdom. Distilled water was prepared in the lab and used throughout the study.

Drugs

Drugs used in this study, including vitamin E (EvitTM400), Sustanon 250 mg, and Metronidazole (Megyl-400®), were purchased from Almond Pharmacy in Nairobi, Kenya.

Preparation of crude extracts

To assess solvent efficacy, extraction for phytochemical analysis was performed using varying polarity solvent systems, including ethanol, methanol, hydromethanol (70%), hydroethanol (70%), n-hexane, acetone, and cold and hot distilled water. Extraction was done using the cold maceration method as described by Altemimi et al [17]. Additionally, hot maceration with distilled water (for in vivo studies) was incorporated into the extraction, as reported by Njau et al. [18]. Spectrophotometric assays were done using a UV-Vis spectrophotometer (UVmini-1240, Shimadzu, Duisburg, Germany).

Quantitative phytochemical analysis

Phenolic content was determined in all extracts following the Folin-Ciocalteu (FC) method described by Siddiqui et al. [19], Briefly, 200 µL of crude extracts were transferred into clean test tubes and 1.8 mL of distilled water added. Thereafter, 200 µL of 10% Folin–Ciocalteu reagent was added to each tube, which was shaken before the addition of 7.5% sodium carbonate. The mixture was incubated in the dark at room temperature for 60 min. The absorbance was then read at 725 nm against the blank (containing all reagents except the extract). The Gallic acid standard was prepared from a 1 mg/mL stock with concentrations of 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 µg/mL, respectively. The concentration of phenols was expressed as µg Gallic acid equivalent/gram of dry plant material (GAE/g).

Total flavonoid content was determined based on aluminium chloride reaction as described by Aryal et al [20]. Briefly, 0.25 mL of crude extracts was transferred into clean test tubes, followed by the addition of 1.5 mL of absolute Methanol. A volume of 0.1 mL of 10% Aluminium chloride solution was added to the mixture and shaken, followed by the addition of 0.1 mL of 1M sodium Acetate and 2.5 mL of distilled water, which were also shaken, and then incubated at room temperature for 30 min. The absorbance was measured at 415 nm against a blank (all other reagents except the extract). The standard rutin was prepared from a 10 mg/mL stock at concentrations of 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3, 3.5, and 4.0 mg/mL. Concentrations of flavonoids were expressed in mg Rutin equivalent/gram of dry plant material (RE/g).

The total tannin content of each extract was measured using the Folin-Ciocalteu method as described by Makkar [21]. The concentrations of tannins were determined by first measuring the Total phenolic content using the Folin-Ciocalteu method [19], and expressed as Tannic Acid equivalents. Tannins were then removed from the tannin-containing extracts by mixing 1 mL of it with 1 mL of 1% (w/v) insoluble polyvinyl polypyrrolidone. The mixture was then incubated t at 4°C for 15 min, vortexed, and centrifuged at 3000 × g for 10 min. The supernatant containing simple phenolics was transferred into a clean Eppendorf tube and used to assay TPC, expressed as Tannic Acid equivalents. The pellets containing PVPP-bound tannins were discarded and the difference in concentration due to reduction in absorbance at 725 nm for the two assays was considered as TTC. The tannic acid standard was prepared from a 1 mg/mL stock and used to determine concentrations.

Alkaloid concentration was determined as described by Ajanal et al. [22]. Briefly, previously extracted samples were obtained, and 1 mL of each extract was mixed with 1 mL of 2 N HCl to dissolve the alkaloids. The mixture was shaken vigorously and centrifuged at 1000 xg for 5 min to sediment the unwanted debris. The supernatant (1 mL) from each was transferred to the separating funnel and washed 3 times with 10 mL of chloroform before adding 0.2 N sodium hydroxide dropwise until a neutral pH was attained. Volumes of 5 mL of each 0.1 M phosphate buffer (pH 4.7) and BCG were then added to the mixture, which was shaken to allow the formation of the yellow complex. The mixture was then extracted with 1, 2, 3, and 4 mL of chloroform by vigorous shaking, and the extracts were collected in a 10 mL volumetric flask. The standard was prepared in the same manner, except that the extract was replaced with different concentrations of Atropine from a 1 mg/mL stock.

Evaluation of in vitro antioxidant activity

Ferric Reducing Antioxidant Power (FRAP) was assessed as described by Wang et al. [23], with ascorbic acid (200 mg) used as the standard, where 1mL of each extract was added to clean test tubes, mixed with 2 mL of 0.1M Phosphate Buffered Saline (PBS) (pH 6.6) and 2 mL of potassium ferricyanide (K3[Fe (CN)6]) (1% w/v). The mixture was placed in a water bath maintained at 50 °C for 30 min. The samples were removed and allowed to cool, then 2 mL of 10% Tetrachloroacetic acid (TCA) was added, and the mixture was centrifuged at 1000 xg at room temperature for 10 min. The supernatant (2 mL) was transferred and mixed with equal volumes of ultrapure water and 0.5 mL of 0.1% (w/v) Ferric chloride solution (FeCl3). The mixture was allowed to stand for 10 min, after which the absorbance was read at 700 nm.

Hydrogen peroxide radical scavenging activity was performed according to the method described by Ruch et al. [24], with ascorbic acid (200 mg) used as the standard. Briefly, 0.1 mL of crude extracts (leaves and roots) was separately added into clean test-tubes. This was followed by the addition of 0.3 mL of 1X PBS, pH 7.4, and shaking to mix. A volume of 0.6 mL of 40 mM H2O2 was added to the mixture, and the mixture was shaken to mix. After 10 min of reaction time, the absorbance of the samples, control, and Standard (Vitamin C) was read at 230 nm.

Hydrogen peroxide radical scavenging was calculated according to equation (1):

(1)

Where: Ao is the absorbance of control (containing Buffer, water, and Hydrogen peroxide), A1 is the absorbance of samples/standard antioxidant (Vitamin C).

Compound identification by GC-MS analysis

Root and leaf powdered samples, macerated with ethanol and acetone (the most effective solvents of extraction according to principal component analysis), were analyzed for volatile organic compounds using a GC-MS system (GC-MS-QP2010 SE, Shimadzu, Kyoto, Japan). The program parameters were set as follows before running: the column oven temperature was set to 70°C, the injection temperature to 250°C, the injection mode to split, and the sampling time to 1 min. The carrier gas was helium (99.999%) at 8.0 psi, with a total flow rate of 13.2 mL/min, a column flow rate of 0.92 mL/min, and a split ratio of 10.1. The extracts were separated at programmed GC oven temperatures as follows: 70°C for 5 sec, 10°C/min up to 250°C for 8 min, and 5°C/min up to 270°C for 5 min. The MS adjustments included an ion source temperature of 200°C, an interface temperature of 250°C, and a solvent cut time of 3 min. The total program time dedicated to the separation was 40 min. The separated components were translated into mass spectral peaks and identified by comparison with the built-in National Institute of Standards and Technology (NIST) GC-MS mass spectral library.

Mineral content analysis

Root and leaf samples were first prepared following the standard operating procedure at the Kenya Bureau of Standards (KEBS, TES/ING/TM/05). Briefly, the preparation was initiated by ashing 1 g of each overnight in a muffle furnace. The products were separately mixed with 10 mL of concentrated nitric acid and heated on a hot plate until a clear solution was obtained. The solution was diluted to a total volume of 50 mL using deionized water; this was performed in triplicate. Analysis of each element was performed based on its characteristic maximum absorption wavelength for macro-minerals (Ca, Na, K, Mg, P, S), trace elements (Se, Fe, Zn, Mn, Mo, Cu, Cr, Si), and non-essential elements (Ba and B) were performed using the ICP-OES instrument (5100S, Agilent) following the protocol described by Ngigi and Muraguri [25], and the concentration recorded in parts per million (ppm).

In vivo animal model studies

Animal handling and experimental design

All animals used in this study were handled in accordance with ethical regulations for the use of laboratory animals. Adult male Wistar rats (aged 70–90 days, weighing 130–180 g) were obtained from the Department of Biology, University of Nairobi. They were housed in a clean environment with a 12-hour light/dark cycle, relative humidity of 56–65%, and at room temperature (23 ± 1°C). The animals had free access to water and were fed standard rat pellets. They were acclimatized to the animal house for one week before the start of the study, during which they underwent 5 min of daily handling to familiarize them with human contact. A qualified veterinarian regularly monitored their health, and only healthy animals were used for the research. The study followed the Animal Research: Reporting of in vivo Experiments (ARRIVE) along with European Community guidelines (EEC Directive of 1986; 86/609/EEC). Ethical approval was obtained from the Biosafety, Animal Use, and Ethics Committee of the Faculty of Veterinary Medicine (Reference number FVM BAUEC/2023/528).

Thirty (30) rats were randomly assigned to six experimental groups, with 5 animals per group [27]. They received treatments with different aqueous extract doses, confirmed to be safe based on the dose limit test, conducted following OECD 423 guidelines, with no animals dying or showing signs of toxicity after a single 2000 mg/kg body weight dose, as previously reported by Dossou-Yovo et al. [28] Group 1 (Normal control) received 1 mL/200 g body weight of distilled water, Group 2 (Negative control) received 400 mg/kg metronidazole alone, Group 3 (Low extract dose group) received co-administration of 400 mg/kg body weight of metronidazole and 250 mg/kg body weight of C. spinarum aqueous root extract, Group 4 (Medium extract dose group) received co-administration of 400 mg/kg body weight of metronidazole and 500 mg/kg body weight of C. spinarum aqueous root extract, Group 5 (High extract dose group) received co-administration of 400 mg/kg body weight of metronidazole and 1000 mg/kg body weight of C. spinarum aqueous root extract, while Group 6 (Positive control) received 400 mg/kg body weight of metronidazole, 400 mg/kg body weight of vitamin E, and 0.36 mg/kg body weight of Sustanon. Sustanon (testosterone agonist) for the positive control treatment was administered once a week via intramuscular injection; all other treatments were given orally daily for 56 days.

Sample collection and processing

At the end of the treatment period, the animals were deeply anaesthetized through intraperitoneal injection of 45 mg/kg sodium pentobarbital. The testis was dissected, fat trimmed off, washed in 1 × phosphate-buffered saline (PBS) (pH 7.4), separated from the epididymis, and then weighed. The left testis was immediately fixed in 10% phosphate-buffered formaldehyde (pH 7.4) for histological examination, while the right testis was stored at −20 °C for biochemical assays [11]. The left and right cauda epididymis were separated from the rest of the epididymis, weighed, and dissected for sperm retrieval and analysis. Sperm were collected according to the procedure described by Al-Alami et al. [11]. Briefly, the left and right cauda epididymis were placed in Petri dishes containing 10 mL of PBS buffer (pH 7.4) maintained at 37 °C and minced using a scalpel blade. The spermatozoa were allowed to diffuse into the buffer for 2 min and swirled to obtain a homogeneous suspension for sperm characteristic analysis. The sperm cell suspension was maintained in a pre-warmed water bath at 37 °C throughout the analysis.

Evaluation of sperm characteristics

Sperm count was done according to the method described by Al-Alami et al. [11] and WHO Laboratory Manual for the Examination and Processing of Human Semen [29]. Briefly, an aliquot of 10 µL of the homogenous sperm suspension was loaded on each of the chambers of the haemocytometer covered with a coverslip and allowed to settle for 1 min before counting the sperm cells in the five large squares (four at the corners and the middle one) at ×100 total magnification under a light microscope (Olympus Microscope BH-2, USA). Sperm cells with their heads completely in the 5 medium squares or those lying on the top or left side of the square, but on the line, were counted (excluding those lying on the right or bottom sides of the middle large square). The counts were performed for the two counting chambers and averaged. The concentration of the spermatozoa in 1 mL of the suspension was calculated according to equation (2).

(2)

Sperm viability was assessed based on staining of the sperm cells with eosin-nigrosine stain, according to the WHO Laboratory Manual for the Examination and Processing of Human Semen [29], whereby 50 µL of properly mixed sperm suspension was added to equal volumes of eosin Y-nigrosin stain (prepared according to the WHO manual), mixed, and allowed to stand for 30 seconds on a microscope slide. A cover slip was immediately placed, and 200 spermatozoa were evaluated for each sample at 1000X magnification under oil immersion. Deep pink, red, and dark-stained heads (dead) and unstained/bright heads (live) were counted and recorded.

Sperm morphology was assessed according to the protocol described by Kondracki et al. [30], where thin smears of the spermatozoa were made on a microscope slide, stained with 1% eosin-Y, and counterstained with 5% nigrosin. Spermatozoa (200 for each sample) were examined at 400X light microscope magnification. Head and tail defects were recorded and expressed as a percentage of the total count.

Testicular tissue processing for histology

Testicular tissues were prepared for histological examination according to the protocol described by Slaoui et al. [31]. The formaldehyde-fixed testes samples were washed overnight in running water, followed by dehydration in increasing ethanol concentrations (50%, 70%, 80%, 90%, 95%, and 100%) at 1-h intervals. The tissues were then cleared with methyl benzoate for 1 h, followed by infiltration with molten paraffin wax at 65 °C for 30 min, and finally embedded in paraffin wax and solidified overnight. The wax-containing samples were then placed on a wooden block for blocking, after which 5 µM-thick tissue sections were cut using a Leica Weitzlar® rotary microtome (Leica Microsystems, Germany). The sections were mounted on a glass slide, deparaffinized in xylene, and rehydrated by treatment with decreasing ethanol concentrations (100%, 90%, 70%, 50%) and, finally, water. The rehydrated sections were then stained using Haematoxylin and counterstained with eosin, dried, and examined at 1000 × total magnification using Leica® DM 500 (Leica Microsystems, Germany) light microscope.

Malondialdehyde levels as an oxidative stress marker in the testis

Lipid peroxidation was assessed by measuring malondialdehyde levels in the testis according to the protocol described by Chen & Gallie [32]. Briefly, 0.05 g of testis was weighed and homogenized in 1 mL of ice-cold 0.1% (w/v) Trichloroacetic acid (TCA) using a mortar and pestle. The mixture was transferred to an Eppendorf tube, 0.25 mL of 0.1% (w/v) TCA was added, and the mixture was centrifuged at 000 xg for 5 min at 4°C. Next, 0.25 mL of the supernatant was transferred into clean Eppendorf tubes and mixed with 0.5 mL of 20% (w/v) TCA containing 0.5% (w/v) Thiobarbituric Acid (TBA). The mixture was incubated in a water bath at 95 °C for 30 min, cooled on ice, and then centrifuged again at 4,000 x g for 10 min at 4 °C. Absorbance was measured at 532 nm and 600 nm using a UV-visible spectrophotometer (UVmini-1240, Shimadzu, Duisburg, Germany). Non-specific turbidity was corrected by subtracting the absorbance at 600 nm from that at 532 nm. Malondialdehyde concentration was calculated using its molar extinction coefficient of 155 mM^-1 cm^-1. The results were expressed in nmol of Malondialdehyde per gram of fresh tissue, using the appropriate equation (3)

(3)

Where 600 nm is the wavelength for correcting non-specific turbidity, while 532 nm is the maximum absorbance wavelength of the TBA-MDA complex.

All measurements from the testis tissue homogenate were performed in triplicate.

Measurement of enzymatic antioxidants in the testis

Each testis sample (0.2 g) was homogenized on ice in 1.2 mL of 100 mM potassium phosphate buffer (pH 6.8), 0.2 mM EDTA, and polyvinylpyrrolidone (PVP) using a mortar and pestle. The mixture was then centrifuged at 5,000 xg for 15 min at 4°C. The supernatant was used for assaying catalase and peroxidase enzyme activities.

Catalase (CAT) activity was measured following the protocol by Lall et al. [33]. In a reaction tube, 1 mL of 50 mM PBS (pH 7.2) and 1 mL of 20 mM hydrogen peroxide (H2O2) were combined. The reaction was initiated by adding 50 µL of crude enzyme extract, and the reduction in absorbance due to H2O2 hydrolysis was monitored for 2 min at 240 nm. CAT activity was calculated using equation (4).

(4)

Where: Δt is the time of incubation (min), ΔAbs is the change in absorbance, and ε is the

Molar extinction coefficient of substrates in units of M^-1 cm^-1, and l is the cuvette diameter (1 cm).

Enzyme activity (Unit) was defined as the amount of enzyme that oxidized 1μmol of substrate/min.

Peroxidase (POD) activity was conducted following the protocol described by Lall et al [33]. The assay was started by mixing 50 µL of tissue homogenate with 2 mL of a solution containing 15 mM guaiacol, 20 mM hydrogen peroxide, and 50 mM sodium acetate buffer (pH 7.0). The activity was measured by monitoring the increase in absorbance at 470 nm due to the formation of tetra guaiacol. The molar extinction coefficient of tetra guaiacol (26.6 mM–1 cm–1) was used to calculate the activity. Peroxidase enzyme activity was computed using equation (iv).

Enzyme activity (Unit) was defined as the amount of enzyme that oxidized 1 μmol of substrate/min.

Statistical analysis

Data analysis was performed using R version 4.3.1 [34] and SigmaPlot version 15 [35]. Numeric data obtained from physiological and biochemical assays were subjected to one-way ANOVA, and multiple mean comparisons were done using Tukey’s Honest Significant Difference (HSD) post hoc test. Data is presented as Mean ± Standard Error of Mean (SEM). The results on mean difference were considered significant at P < 0.05.

Molecular identification of the plant sample

The obtained PCR amplicon sizes following PCR amplification were approximately 600 base pairs (bp) with rbcL and 500 bp with trnH-psbA barcode (S1 Fig). A BLASTn search with the rbcL sequences in the database identified our query sequence with Carissa haematocarpa (JQ014161.1), Carissa bispinosa (AJ419738.1), and Carissa bispinosa (JQ014160.1), with percent identities of 99.7%, 99.7%, and 99.56%, respectively. While trnH-psbA identified a query sequence from the Carissa spinarum species, with percent identities ranging from 95.15% to 99.23% (S1 Fig).

A Bayesian phylogenetic inference approach was employed to further understand genetic identity, considering clustering based on sequence similarities. For the rbcl sequences (indicated by blue colour), the query sequence clustered with Carissa bispinosa (AJ419738.1), supported by a low posterior probability value of 69%. Based on the trnH-psbA barcode (indicated by red colour), the query sequence clustered with the three reference C. spinarum species (KR735499.1, KR735438.1, KR735402.1) with a posterior probability value of 99%, thus being identified as Carissa spinarum (S2 Fig).

Phytochemical analysis of C. spinarum leaf and root extracts

Total Phenolic Content (TPC) in C. spinarum root extracts was significantly highest in acetone (130.39 ± 4.45 µg GAE/g), with other polar solvents (ethanol, methanol, Hydromethanol (H.M), Hydroethanol (H.E), distilled water, and hot distilled water) also extracting high phenolics. Hexane extracted a significantly lower concentration of phenolics (36.95 ± 0.16 µg GAE/g) (p < 0.05). TPC in the leaves showed a similar solvent pattern as for the root, with acetone yielding the highest (174.86 ± 1.73 µg GAE/g) and hexane the lowest (19.03 ± 0.31 µg GAE/g). Acetone also produced the highest amounts of Total Flavonoid Content (TFC) in root samples (0.98 ± 0.01 mg RE/g), while Hydromethanol (70%) showed the lowest TFC (0.02 ± 0.00 mg RE/g) (p < 0.05). In leaf samples, TFC was significantly higher in all polar solvents (Table 1).

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Table 1. Summary of TPC, TFC, TTC, and TAC in C. spinarum roots and leaves in different extraction solvents.

https://doi.org/10.1371/journal.pone.0355380.t001

The aqueous (hot maceration) extracts showed the highest tannin concentration in C. spinarum roots (0.205 ± 0.01 mg TAE/g), with hexane recording significantly lower tannin level in roots (0.012 ± 0.00 mg TAE/g) (p < 0.05). In the leaves, methanolic extracts had the highest tannin content (0.502 ± 0.01 mg TAE/g), while hexane had the lowest Total Tannin Content (TTC) (0.027 ± 0.00 mg TAE/g) (p < 0.05). The alkaloid concentration in root samples was highest in distilled water (hot) (0.172 ± 0.00 mg AE/g), while roots macerated with cold distilled water had the lowest alkaloid levels (0.009 ± 0.00 mg AE/g). On the leaves, ethanol extracted significantly higher alkaloids (0.235 ± 0.00 mg AE/g) (p < 0.05), with hexane showing the lowest alkaloid content in the leaves (0.014 ± 0.00 mg AE/g) (Table 1, S5 and S6 Fig).

Comparison of phytochemical concentrations in C. spinarum leaf and root extracts

The results showed that total phenolic content, flavonoids, tannins, and alkaloids were significantly higher in the leaves compared to the roots in the following solvents: acetone, cold distilled water, Hydroethanol, Hydromethanol, ethanol, and methanol (p < 0.05) (S3 Fig).

In vitro antioxidant activity of C. spinarum root and leaf extracts

In the root samples, ferric reducing power was significantly higher in Hydroethanol (6.66 ± 0.02), with hexane showing the lowest ferric reducing power (3.15 ± 0.02), Table 2. For the leaf extracts, Hydromethanol had the highest ferric-reducing capacity (10.88 ± 0.07), while hexane showed the lowest ferric-reducing capacity (p < 0.05), Table 2. The results for hydrogen peroxide (H₂O₂) scavenging activity in the root samples indicated that the acetone extract had significantly higher scavenging activity (34.18 ± 0.26), and hexane had the lowest H₂O₂ scavenging activity (9.70 ± 0.35) (p < 0.05). On the leaf extracts, ethanol demonstrated the highest H₂O₂ scavenging activity (43.53 ± 0.50), whereas hexane exhibited the lowest (18.54 ± 0.28), (p < 0.05) (Table 2, S5 Fig). Pairwise comparison of ferric reducing power between the leaf and root extracts showed that ethanol, methanol, Hydromethanol, Hydroethanol, acetone, cold distilled water, and hot distilled water had significantly higher ferric antioxidant reducing capacity in the leaf samples compared to root samples (p < 0.05). However, there was no significant difference in ferric reducing power in hexane extracts (S4 Fig). H₂O₂ scavenging activity was significantly higher in the leaf samples among the following solvents of extraction: ethanol, methanol, Hydroethanol, Hydromethanol, and acetone. The roots in hot distilled water showed significantly higher H2O2 scavenging activity compared to the leaves (p < 0.05) (S4 Fig).

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Table 2. Effects of C. spinarum extracts on FRAP and HPRS activities.

https://doi.org/10.1371/journal.pone.0355380.t002

Correlation analysis and principle component analysis to determine effective solvents

In the root samples, TPC, TFC, TAC, and TTC had a strong positive correlation with HPRS (p < 0.05), while only TPC and TTC showed a strong positive correlation with FRAP. Alkaloid concentration showed a weak positive correlation with FRAP (P > 0.05). Leaf extracts showed that all metabolite concentrations were positively correlated with both FRAP and HPRS (S3 Fig). For the root and leaf extracts, a similar pattern was observed, with hot distilled water, ethanol, methanol, and acetone clustering together with at least one of the variable. H.E and H.M showed a positive correlation; however, they were not closely associated with any of the variables, whereas hexane was an outlier (S7 Fig).

GC-MS results of C. spinarum leaf and roots, extracted with acetone and ethanol

A total of 24 and 18 volatile compounds were detected in ethanolic and acetonic leaf extracts, respectively (Fig 1A and B). In the root extracts, the ethanolic and acetonic extracts contained 21 and 19 compounds, respectively (Fig 1C and D). Compounds with previous reports of antioxidant activity and male fertility modulation included, squalene [36], lupeol [37], phytol [38], Urs-12-en-3-ol, acetate, (3.beta.)- [39], Olean-12-en-3-ol [40], stigmasterol [41], α-Amyrin/ β-Amyrin [42] and Vitamin E [43,44] (S1-S3 Table and Fig 2).

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Fig 1. GC – MS Spectrograms, showing peaks of A (acetone leaf extract), B (ethanol leaf extract), C (acetone root extract), and D (ethanol root extract).

The peaks are annotated with their respective numbers.

https://doi.org/10.1371/journal.pone.0355380.g001

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Fig 2. Sketches of pharmacologically relevant GC-MS-detected compounds.

These compounds have been reported to reduce oxidative stress and improve male reproductive function. The sketches were made using PubChem Sketcher V2.4.

https://doi.org/10.1371/journal.pone.0355380.g002

Mineral content analysis in C. spinarum leaf and roots

Among the analyzed macrominerals, calcium had the highest abundance in only the roots (57.08 ± 0.146), while potassium reported significantly high concentrations (ppm) in both leaf and root samples (50.01 ± 0.003 and 217.25 ± 0.536, respectively) (p < 0.05). Sodium had the lowest concentrations (ppm) in both root and leaf samples (2.02 ± 0.019 and 1.89 ± 0.049, respectively) (p < 0.05), as shown in Table 3. For trace elements, silicon recorded the highest concentrations in both leaf and root samples (57.45 ± 0.322 and 23.95 ± 3.481, respectively). Conversely, chromium (0.02 ± 0.003) had the lowest concentrations in root and leaf samples (p < 0.05), Table 3. Boron, a non-essential mineral, predominantly exhibited high concentrations in both leaf and root samples, while barium showed the lowest in both leaf and root samples (p < 0.05). A pairwise comparison of each analyzed mineral revealed that the leaves contained significantly higher concentrations of K, Mg, Cu, Zn, P, S, Si, Mn, Fe, B, and Ba, as shown in Table 3.

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Table 3. Mineral content of C. spinarum leaf and root samples.

https://doi.org/10.1371/journal.pone.0355380.t003

Effects of C. spinarum aqueous root extract on reproductive organ and body weights

The testicular weight index assessment showed a significant decrease in the negative control group receiving metronidazole alone (p < 0.05). In contrast, the extract-treated groups, especially those given low and medium doses, showed a significant increase in relative testis weight index (0.95% and 0.98%, respectively) compared to the negative control (0.28%; p < 0.05). The relative testicular weights in the low and medium extract-treated groups were not significantly different from those in the normal control group (1.015%; p > 0.05). Similarly, cauda epididymis weights were significantly reduced among the negative control group (0.28g; p < 0.05). Conversely, the low and high-dose extract-treated groups exhibited increased epididymal weights (0.45g and 0.45 g, respectively), which were not significantly different from the positive and normal control groups (0.52g and 0.55 g, respectively) (p > 0.05) (Table 4, S9 Fig).

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Table 4. Summary table indicating the effects of C. spinarum aqueous root extract on total sperm count, viability, morphology, testis weight index, and epididymis weight.

https://doi.org/10.1371/journal.pone.0355380.t004

Effects of C. spinarum aqueous root extract on sperm qualities

The administration of metronidazole alone in the negative control (group 2) led to a significant decrease in sperm concentration 3.49 × 10⁶/mL) and viability 68.6%, and consequently lowered the levels of morphologically normal sperm cells to only 4.3%. However, treatment with escalating doses of the root extracts significantly (p < 0.05) improved these parameters especially on the low and medium doses of the extracts where sperm concentration was 35.03 × 10⁶/mL and 37.2 × 10⁶/mL respectively, viability 88.5% and 86.7% respectively, while the amounts of sperms with normal morphology were high in these groups (41.90% and 60.5%, respectively). The medium extract dose showed sperm parameters that were not significantly (p > 0.05) different from those of the normal and positive controls. At a high dose of the root extracts, there was a significant reduction (p < 0.05) in sperm parameters compared to all other treatment groups, including the positive and normal controls, but not significantly different from the negative control (Table 4, S10 Fig, and S11 Fig).

Testicular histology

In the normal control group, the testicular parenchyma consisted of regularly shaped seminiferous tubules, which appeared bounded by an intact basement membrane supporting spermatogonia and Sertoli cells. The Sertoli cells are tall and seem to span the entire diameter of the seminiferous epithelium, with apical villi extending into the lumen, while lateral recesses support and contain dividing and differentiating germ cells. The tubular lumen contained spermatozoa with tails pointing toward the center of the lumen and heads attached to the apical villi of the Sertoli cells. The spermatogenic cells appeared to develop in groups at the same stage toward the lumen, indicating a normal spermatogenic cycle and wave. The interstitial tissue of the normal control displayed interstitial cells, blood vessels, and lymphatic vessels. Among these interstitial cells are Leydig cells, which appear polyhedral (Fig 3A).

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Fig 3. Photomicrographs of rat seminiferous tubules.

A) Normal control with normally aligned spermatogenic cells at different stages of development interspersed among Sertoli cells (SC) lying on intact basement membrane (BM) and whose apical villi attach the elongate spermatids (ES) and appear to project into the lumen (L). B) Negative control subjected to metronidazole alone showing significantly disorganized seminiferous epithelial alignment within the seminiferous tubules with accompanying increased luminal diameter (L) that appears empty. Vacuolations can also be seen (thick blue arrow), C and D) represent low and medium doses of C. spinarum root extracts, respectively. The seminiferous tubular integrity in both treatment groups appears intact, with the cellular ultrastructure resembling that of the normal control. E) represents the high dose extract-treated group. Note the loss of seminiferous epithelial integrity characterised by disintegration and general swelling of the seminiferous epithelial diameter. The basement membrane (BM) appears irregular, and the lumen (L) is empty. F) represents the positive control, its seminiferous tubular ultrastructure resembles that of the normal control with intact BM and spermatogenic cells at different stages of development interspersed among the Sertoli cells (SC) with spermatozoa (S) in the lumen. The interstitium of the positive control group also shows a cellular outline comprising Leydig cells (LC) among other interstitial cells and blood vessels (BV). Haematoxylin and eosin stain used and the magnification bar represents 50 µm.

https://doi.org/10.1371/journal.pone.0355380.g003

The negative control, which was treated with metronidazole 400 mg/kg/bw alone, showed significant structural changes in the seminiferous tubules. The seminiferous epithelium generally appeared thinner with prominent vacuolation in spermatogenic cells and a lack of Sertoli cells. There was evidence of complete seminiferous tubular degeneration, with dissociation from the basement membrane, which appeared irregular and showed cell exfoliation in some areas. The interstitial tissue appeared abnormally enlarged, with signs of edema and irregular distribution of interstitial cells (Fig 3B).

Seminiferous tubules in animals treated with low and medium doses of extract exhibited normal structural features, including an intact germinal epithelium and basement membrane. The germinal epithelium within the seminiferous tubules was characterized by well-organized spermatogenic cells at various stages of development, intermingled with Sertoli cells and spermatozoa (Fig 3C and D).

At high extract dose treatment, the basement membrane of seminiferous tubules appeared irregularly shaped with folds in certain areas (Fig 3C and D), and the seminiferous epithelium seemed dissociated from the basement membrane and from each other. There was evidence of cellular degeneration, as indicated by pyknotic nuclei in the affected cells. The seminiferous epithelial cells were also exfoliated from the basement membrane (Fig 3E).

The seminiferous tubules in the positive control group exhibited features of seminiferous epithelium resting on an intact basement membrane, with a lumen filled with spermatozoa, indicative of a normal seminiferous tubule (Fig 3F).

Effects of C. spinarum aqueous root extract on oxidative stress markers and enzymatic antioxidants in the testis

To understand the in vivo antioxidant properties of aqueous root extracts of this plant in relation to male infertility treatment, testicular levels of malondialdehyde (MDA) and the activity of peroxidase and catalase were measured in right testis homogenates. The negative control group, which was treated with metronidazole alone, showed the highest concentration of MDA (5.35 ± 0.69 nanomoles/g of testicular tissue) (p < 0.05). The treatment groups, especially those given medium (500 mg/kg) and high (1000 mg/kg/bw) extract doses, exhibited significantly lower MDA levels of 1.38 ± 0.14 and 2.77 ± 0.23 nanomoles/g of testicular tissue (p < 0.05). The animals treated with medium and high extract doses showed MDA levels that were not significantly different from those in the positive and normal control groups (p > 0.05) (Table 4).

The results for catalase enzyme activity showed a significant reduction among the negative control group (8.45 ± 0.09 nmol g^-1 testis tissue × 10⁻²) treated with metronidazole alone. However, treatment with low and medium doses led to a significant (p < 0.05) increase in the levels of catalase enzyme activity of 13.88 ± 0.21 and 14.56 ± 0.19 nmol g^-1 testis tissue × 10⁻², respectively. The results also indicated that those MDA levels from the low and medium extract-treated animals were not significantly different from the normal and positive control group animals (p > 0.05) (Table 4).

The test for peroxidase enzyme activity reported normal levels of the enzyme activity in the negative control animals (17.26 ± 0.11 nmol g^-1 testis tissue × 10⁻²) with reference to the normal control group (p > 0.05). However, there was an increase in the activity of peroxidase enzyme activity among the low (28.07 ± 0.24 nmol g^-1 testis tissue × 10⁻²), medium (30.95 ± 0.11 nmol g^-1 testis tissue × 10⁻²), and high (33.24 ± 0.14 nmol g^-1 testis tissue × 10⁻²) extract-treated groups. This increase in peroxidase enzyme activity among the extract-treated groups was not significantly different with reference to the positive control animals (p > 0.05) (Table 4).