Study Setting

This experimental study was conducted at the animal facility of the National Institute of Biomedical Research (INRB) and the Biochemistry and Hematology Laboratory, Faculty of Pharmaceutical Sciences, University of Kinshasa (UNIKIN), Kinshasa, Democratic Republic of Congo.

2.2.1. Experimental Animals

A total of 25 adult Wistar rats (male and female), an outbred albino strain widely used in biomedical research, were used in this study.

Animals were randomly assigned and acclimatized under standard laboratory conditions with ad libitum access to a standard commercial diet and water. The sample consisted of 25 adult male and female Wistar rats divided into groups. These 25 rats were first used to evaluate hypoglycemic activity and were subsequently kept for two weeks or one month for toxicology testing and biochemical analysis. The rats were fed a cake made from standard complete livestock feed. Their average weight was 204 g before extract administration and 195 g after extract administration.

Rats were individually identified using non-toxic body marking applied to different anatomical sites (head, tail, back, and flank). Body weight was measured to determine dosing, assess toxicity, perform oral glucose tolerance tests, and evaluate changes in body mass throughout the experiment.1.2.2 Blood samples.

Blood samples were collected from the tail vein for glycemic measurements and from cardiac puncture for biochemical analyses.

2.2.2. Plant Material

Fresh fruits of Solanumaethiopicum Lyophilized Solanumaethiopicum

Fresh fruits of Solanumaethiopicum were purchased from Rond-Point Ngaba market in Kinshasa, Democratic Republic of Congo. Taxonomic identification was conducted at the Herbarium of the National Institute for Agronomic Study and Research (INERA), Faculty of Sciences, University of Kinshasa. Lyophilized whole-fruit extracts were subsequently prepared and used for all experimental procedures. The Solanumaethiopicum samples were prepared by cleaning the edible part of the fruit, cutting it into pieces, and placing them in an Erlenmeyer flask.

After adding distilled water, the mixture was brought to a boil, filtered, allowed to cool, and then transferred to trays.

Subsequently, the extract was frozen at −18˚C and placed in a freeze-dryer for 48 hours at −55˚C; the freeze-dried product was stored in a dry place.

The freeze-drying yield was 1.89%, based on the weight of the fresh Solanumaethiopicum sample (P1) = 2300 g and the weight of the freeze-dried Solanumaethiopicum sample (P2) = 43.48 g.

3.1.1. Principle

Acute oral toxicity was evaluated by administering graded doses of S.aethiopicum extract via oral gavage. Animals were monitored for mortality and clinical signs of toxicity for 7 days following treatment. Dose selection was based on OECD acute oral toxicity guidelines and focused on the dose range corresponding to moderate toxicity (0.5 - 5 g/kg body weight).

3.1.2. Experimental Procedure

Lyophilized fruit extract was dissolved in distilled water to prepare a series of concentrations of 5000 mg/kg, 3750 mg/kg, 2500 mg/kg, 1250 mg/kg, and 650 mg/kg. Animals were divided into six groups:

Group I: 5000 mg/kg (n = 5)

Group II: 3750 mg/kg (n = 5)

Group III: 2500 mg/kg (n = 4)

Group IV: 1250 mg/kg (n = 4)

Group V: 650 mg/kg (n = 4)

Control group: distilled water (n = 3)

Following a 24 h fasting period, the extracts were administered orally via an orogastric gavage tube at doses normalized to body weight.

Animals were observed continuously at 30-minute intervals the first 8 hours, reassessed at 24 hours, and monitored daily thereafter for 7 days. Mortality and clinical signs of toxicity were systematically recorded throughout the study period.2.1.3 LD₅₀ calculation.

The median lethal dose (LD₅₀) was calculated using the Kärber and Behrens-Tainter method and expressed as mg/Kg body weight according to the following equation:

where:

DL₁₀₀ = lowest dose causing 100% mortality

a = difference between consecutive doses

b = mean number of deaths between two doses

c= number of animals per group

∑ = sum of all intervals

3.2.1. Animal Grouping and Treatment

After acclimatization and a 24-hour fasting period, rats were randomly assigned to five groups (n = 5 per group):

Group 1: S. aethiopicum extract (500 mg/kg)

Group 2: extract (250 mg/kg)

Group 3: extract (125 mg/kg)

Group 4: glibenclamide (5 mg/kg; positive control)

Group 5: distilled water (negative control)

3.2.2. Test Substances

Glucose solution: 4 g/kg (oral load)

Test extract: S. aethiopicum (125 - 500 mg/kg)

Reference drug: glibenclamide (5 mg/kg)

Vehicle: distilled water

A fixed administration volume of 2 mL was used for a 200 g rat.

3.2.3. Oral Glucose Tolerance Test (OGTT)

The hypoglycemic activity was evaluated using an oral glucose-induced hyperglycemia model. Following a fast, baseline blood glucose levels were measured prior to oral glucose administration (4 g/kg body weight). Immediately after glucose loading, the test substances were administered according to the experimental group allocation. Blood glucose concentrations were subsequently determined at 30, 60, 120, and 180 minutes post-administration.

3.2.4. Blood Glucose Measurement

Blood glucose concentration were measured using a Sinocare® glucometer based on the glucose oxidase method coupled with a potassium ferricyanide-mediated redox reaction [16].

Blood samples were obtained from the tail vein, and glucose concentrations were expressed as mg/dL.

3.3.1. Blood Collection

Following a 24-hour fasting period, animals were anesthetized using ketamine hydrochloride at a dose adjusted to body weight. Blood samples were collected via cardiac puncture and centrifuged at 1000 rpm for 10 minutes. The resulting serum was separated and stored until biochemical analysis.

3.3.2. Method for the Analysis of Biochemical Parameters

Serum aspartate aminotransferase (AST/TGO), alanine aminotransferase (ALT/TGP), urea, and creatinine concentrations were determined using standard spectrophotometric assays. AST and ALT: kinetic IFCC method (340 nm)

Urea: enzymatic Berthelot reaction (546 nm)

Creatinine: Jaffé reaction (490 nm) [17].

Statistical analyses were conducted using SPSS version 21 (IBM Corp., Armonk, NY, USA). Results are reported as mean ± SD. Group differences were evaluated using one-way ANOVA with Bonferroni post hoc correction, while paired Student’s t-tests were used to assess changes in body weight. A significance level of p < 0.05 was adopted throughout.

4.1.1. Toxicity Results

A total of 25 Wistar rats were included in this study. The sex distribution (Table1), showed a predominance of females (17/25, 68%) relative to males (8/25, 32%).

Table 1. Distribution of Wistar rats by sex.

A total of 25 Wistar rats were included in this study. The sex distribution (Table 1), showed a predominance of females (17/25, 68%) relative to males (8/25, 32%).

Table 2. Determination of the lethal dose (LD50) of *Solanum aethiopicum* extracts.

As shown in Table 2, no mortality or treatment-related clinical signs of toxicity were observed in rats administered Solanumaethiopicum extract at doses ranging from 650 to 5000 mg/kg. The absence of mortality across all treatment groups (0/25) indicates that the medianLD₅₀ exceeds 5000 mg/kg, suggesting that the extract exhibits very low acute toxicity.

Table 3. Comparison of mean weight before and after dose administration.

No statistically significant differences in body weight were observed before and after treatment among the experimental groups (Table 3).

4.1.2. Glycemia Results

Table 4. Description of blood glucose levels over the observation period.

Blood glucose levels varied significantly over time following glucose administration (F = 41.765, p < 0.05), as detailed in Table 4. Mean glycemia increased sharply from baseline (59.12 ± 12.88 mg/dL) to 30 minutes (127.32 ± 27.05 mg/dL), followed by a progressive decline at 60 minutes (116.24 ± 25.95 mg/dL), 120 minutes (74.88 ± 27.99 mg/dL), and 180 minutes (67.76 ± 21.32 mg/dL).

Table 5. Comparison of the difference in means between the times, pairwise.

1) Bonferroni test; the difference is significant at the 5% level; 2) ***: significant; 3) NS: not significant; 4) Bi: Lower bound; 5) Bs: Upper bound.

Pairwise comparisons using the Bonferroni test (Table 5) revealed significant differences primarily between the early (0 - 60 min) and late (120 - 180 min) time points, indicating a pronounced time-dependent glycemic response to oral glucose loading.

Table 6. Description of blood glucose levels in relation to *Solanum* extracts, with Glibenclamide as a reference sample and distilled water as a control.

Significant differences were also observed among treatment groups (F = 3.754, p = 0.006), as shown in Table 6. Treatment with glibenclamide resulted in a marked reduction in blood glucose levels compared with distilled water and all extract-treated groups.

Table7. Comparison of the difference between means across pairwise samples.

1) Bonferroni test; the difference is significant at the 5% level; 2) ***: significant; 3) NS: not significant; 4) Bi: Lower bound; 5) Bs: Upper bound.

In contrast, administration of Solanumaethiopicum extract at doses of 125, 250, and 500 mg/kg did not significantly reduce blood glucose levels relative to the control group, as confirmed by Bonferroni pairwise comparisons (Table 7).

4.1.3. Determination of Certain Biochemical Parameters

Serum biochemical parameters are summarized in Table 8. Urea, creatinine, AST, and ALT levels in treated animals remained within, or were close to, established physiological reference ranges.

Table 8. Comparison of biochemical parameters against reference ranges.

Serum biochemical parameters are summarized in Table 8. Urea, creatinine, AST, and ALT levels in treated animals remained within, or were close to, established physiological reference ranges.

Table 9. Comparison of biochemical parameters.

Overall comparisons among experimental groups revealed no significant differences in urea, creatinine, AST, or ALT concentrations (p > 0.05), as shown on Table 9, indicating that administration of the extract did not induce detectable hepatic or renal toxicity under the experimental conditions.

Table 10. Comparaison des paramètres biochimiques par rapport au sexe.

Sex-based analysis showed no significant differences in urea, AST, or ALT levels (Table 10). In contrast, creatine concentrations differed significantly between male and female rats (p = 0.039), although all values remained within physiological limits.

In the present study, oral administration of Solanumaethiopicum extract at doses of 650 - 5000 mg/kg produced neither mortality nor observable clinical signs of toxicity in 22 Wistar rats. Accordingly, the median lethal dose (LD₅₀) was estimated to be greater than 5000 mg/kg, indicating that the extract possesses low acute oral toxicity under the conditions of this study.

Following oral glucose administration, blood glucose concentrations in Wistar rats increased markedly, reaching a peak at 30 minutes with a mean value of 127.32 ± 27.05 mg/dL (95% CI: 116.15 - 138.49 mg/dL). Thereafter, glucose levels decreased progressively to 116.24 ± 25.95 mg/dL at 60 minutes and 67.76 ± 21.31 mg/dL at 180 minutes (Table 4).

The overall mean blood glucose concentration during the experimental period was 89.1 ± 36.1 mg/dL (95% CI: 82.68 - 95.45 mg/dL), indicating a clear time-dependent regulation of glucose homeostasis following oral glucose. loading. These findings are broadly consistent with previous reports on plant-derived hypoglycemic agents. For example, Masunda et al. demonstrated that extracts of Raphiagentiliana significantly reduced blood glucose levels approximately two hours after administration. In the present study, however, the decline in blood glucose was evident as early as 60 minutes, suggesting a more glycemic response. This observation is in agreement with physiological evidence showing that postprandial blood glucose typically peaks within 30 - 60 minutes and gradually returns toward baseline within 60 - 120 minutes after carbohydrate ingestion [18].

Among the treatment groups, the lowest mean blood glucose concentration was recorded in the glibenclamide-treated group (65.56 ± 31.03 mg/dL), followed by the group receiving 125 mg/kg of S.aethiopicum extract (92.04 ± 34.28 mg/dL) (Table 6).

Statistical analyses revealed a significant effect of time on blood glucose concentrations (p < 0.05), indicating substantial temporal variation throughout the 180-minute observation period (Table 5).

Furthermore, significant differences were also observed among treatment groups (p < 0.05), as shown in Table 7. Nevertheless, the hypoglycemic effect of S.aethiopicum extract was less pronounced than that of glibenclamide, suggesting that the extract does not exhibit glucose-lowering activity comparable to the reference drug under experimental conditions employed.

This finding contrasts with the study of Masunda et al. (2014), who reported hypoglycemic activity of Raphiagentiliana comparable to glibenclamide. Likewise, while Achyut et al. [19] observed significant reductions in blood glucose in animals treated with Aeglemarmelos extracts relative to controls, no significant differences were detected between the distilled water group and the S.aethiopicum extract-treated groups in the present study. These results suggest that S.aethiopicum extract exhibits limited or no direct hypoglycemic activity at the tested doses. In addition, a significant reduction in body weight was observed during the acute toxicity study (p < 0.05). This decrease is likely attributable to the 24-hour fasting period imposed prior to LD₅₀ determination rather than to a toxic effect of the extract itself.

The analysis of biochemical parameters, including urea, creatinine, AST (TGO), and ALT (TGP), showed no statistically significant differences among the treated groups (p > 0.05), as shown in Tables 8-10. These findings indicate that S.aethiopicum extract did not induce detectable hepatic or renal toxicity in Wistar rats under the experimental conditions employed.

The present results differ from those reported by Srikanth and Muralidharan [20], who observed significant dose-dependent alterations in biochemical markers following administration of Sapindusemarginatus extracts. Similarly, Achyut et al. [19] (2006) reported changes in biochemical parameters varied according to both dose and duration of treatment with Aegle marmelos. These discrepancies may reflect differences in plant species, phytochemical composition, extraction procedures, administered doses, and experimental designs.

Conversely, several studies have highlighted beneficial metabolic effects of medicinal plants. For example, Beddazekri and Ghemam [21] demonstrated that Ajugaiva and Matricariapubescens significantly reduced blood glucose levels and improved body weight following repeated administration. Likewise, Djedioui and Messareh [22] reported that Inulaviscosa extract reduced blood glucose levels, improved hepatic function, and enhanced antioxidant status without causing adverse effects in healthy rats.

More recently, [23] showed that consumption of S.aethiopicum preserved pancreatic histoarchitecture, improved glycemic control, and reduced body weight in diabetic rats. Although the present study similarly observed a reduction in body weight in Wistar rats, no direct hypoglycemic effect was detected. This discrepancy may be attributed to differences in experimental conditions, particularly the form of plant material used and extraction procedures, as previous studies frequently employed fresh or differently processed fruits, whereas the present study utilized lyophilized extracts.