Citrulluslanatusseeds were purchased from a local market in Abidjan, Côte d’Ivoire, and identified at the National Floristic Center (voucher specimen UCJ004346). The seeds were air-dried and ground into a fine powder. The aqueous extract (EACL) was prepared by macerating 200 g of the powder in 2 L of distilled water under magnetic stirring at 70˚C for 24 hours. The macerate was then filtered and dried in an oven at 60˚C for 72 hours.

A preliminary phytochemical screening of EACL was performed using standard qualitative tests to identify the presence of secondary metabolites, including sterols, polyphenols, flavonoids, saponins, quinones, alkaloids, and tannins [13][14].

The acute oral toxicity of EACL was evaluated in female Swiss mice (20 - 30 g) according to the OECD Guideline 423 [15]. Mice were administered a single dose of 2000 or 5000 mg/kg body weight (b.w.) and observed for signs of toxicity and mortality for 14 days.

2.4.1. Effects on Normoglycemic Rats

Twenty (20) male Wistar rats (100 - 160 g) were divided into five groups (n = 4) and treated orally for 14 days as follows:

Group 1 (Control): 2 mL distilled water.Groups 2 - 5: EACL at doses of 500, 1000, 1500, and 2000 mg/kg b.w., respectively.

Body weight and fasting blood glucose were measured weekly (Day 0, 7, 14). On Day 14, blood was collected for the determination of total cholesterol.

2.4.2. Effects on Hyperglycemic Rats

The study focused on normoglycaemic animals that had received a glucose load, rather than on a model of chronic diabetes. No mechanistic biomarkers were measured.

Sixteen (16) rats were divided into 4 groups of 4 rats each, with an average weight of between 100 and 150 g. Three distinct models were used to evaluate the anti-hyperglycemic effect of EACL (2000 mg/kg) compared to a standard drug, glibenclamide (10 mg/kg), in overnight-fasted rats.

Model1(Pre-treatment): The test substances were administered 30 minutes before oral glucose (4 g/kg).Model2(Post-treatment): Oral glucose (4 g/kg) was administered 30 minutes before the test substances.Model3(Co-administration): The test substances were co-administered with glucose (4 g/kg).

In all models, blood glucose was measured at 0, 30, 60, 90, 120, and 150 minutes.

2.4.3. Statistical Analysis

All data were expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 5.01. Differences between groups were assessed using one-way ANOVA followed by Dunnett’s and Turkey-Kramer’s multiple comparison tests. A p-value < 0.05 was considered statistically significant.

The phytochemical screening of EACL revealed the presence of sterols and polyterpenes, polyphenols, flavonoids, saponins, quinones, and catechic and gallic tannins. Screening carried out using Dragendorff’s reagent (positive) and Bouchardat’s reagent (negative) does not confirm the presence of alkaloids. Further analyses (CCM or spectrophotometry) are essential to reach a definitive conclusion (Table 1). The acute toxicity study showed no mortality or signs of toxicity at doses up to 5000 mg/kg b.w., indicating that the oral LD50 of EACL is greater than 5000 mg/kg.

Table 1. Phytochemicals in the aqueous extract of Citrulluslanatus (Cucurbitaceae) seeds.

(+): Presence of the compound; (−): Absence of the compound.

Treatment with EACL for 14 days did not lead to a significant change in body weight compared to the control group (p > 0.05) (Figure 1).

Figure 1. Changes in body weight in normal rats during 14 days of treatment with an aqueous extract of Citrulluslanatus seeds. n = 5; p > 0.05.

However, there was a dose-dependent and statistically significant decrease in fasting blood glucose levels, with the greatest reduction of 45.9% observed at the highest dose of 2000 mg/kg by Day 14 (Figure 2).

Figure 2. Evolution of fasting blood glucose in normoglycemic rats treated with EACL for 14 days. Values are mean ± SEM (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001 vs control at same time point. (Data presented as mean ± SEM).

Furthermore, EACL significantly reduced total cholesterol. Following administration of doses of 500, 1000, 1500 and 2000 mg/kg body weight, after 14 days, the total cholesterol levels in the rats fell to 0.648 ± 0.02; 0.636 ± 0.03; 0.608 ± 0.04 and 0.588 ± 0.04 g/L, respectively (Figure 3).

Figure 3. Changes in total cholesterol concentration in normal rats treated for 14 days with an aqueous extract of Citrulluslanatus seeds. n = 5; *: p < 0.05; **: p < 0.01; ***: p < 0.001 Statistically significant compared with the control.

The anti-hyperglycemic activity of EACL (2000 mg/kg) was evaluated in three distinct experimental models of glucose-induced hyperglycemia in rats, and compared to the standard drug glibenclamide (10 mg/kg) (Figures 4-6).

3.3.1. Pre-Treatment Model (EACL Administered before Glucose)

In the pre-treatment model, EACL and glibenclamide significantly reduced blood glucose levels at T30 (0.73 ± 0.04 g/L and 0.68 ± 0.03 g/L, respectively, p < 0.01) compared to the positive control group (0.94 ± 0.02 g/L). Following glucose administration at T30, both treatments significantly attenuated the hyperglycemic peak at T60 compared to the positive control (1.19 ± 0.5 g/L for EACL vs. 1.45 ± 0.5 g/L for positive control, p < 0.01). Blood glucose levels continued to decline progressively, reaching 0.82 ± 0.4 g/L for EACL and 0.62 ± 0.3 g/L for glibenclamide by T150 (Figure 4).

Figure 4. Effect of EACL and glibenclamide on blood glucose levels in hyperglycaemic rats, administered prior to glucose over a period of 150 minutes.

3.3.2. Post-Treatment Model (EACL Administered after Glucose)

Figure 5. Effect of EACL and glibenclamide on blood glucose levels in hyperglycaemic rats following glucose administration over a 150-minute period.

In the post-treatment model, glucose loading significantly increased blood glucose in the positive control group, reaching a peak of 1.51 ± 0.80 g/L at T30 (p < 0.001). Administration of EACL (2000 mg/kg) at T30 induced a significant reduction in blood glucose at T60 (1.28 ± 0.6 g/L, p < 0.01), with levels continuing to decline to 0.76 ± 0.4 g/L by T150. This effect was comparable to glibenclamide, which reduced blood glucose from 1.53 ± 0.5 g/L at T30 to 0.63 ± 0.3 g/L at T150 (Figure 5).

3.3.3. Co-Administration Model (EACL Combined with Glucose)

When EACL was co-administered with glucose (4 g/kg), it significantly attenuated the hyperglycemic peak at T30 compared to the positive control group (0.8 ± 0.3 g/L vs. 1.28 ± 0.4 g/L, p < 0.001), representing a 37.5% reduction. This effect was superior to that of the glibenclamide + glucose mixture, which showed a 22.6% reduction at the same time point. Blood glucose levels then continued to decline progressively, reaching 0.75 ± 0.4 g/L for EACL and 0.54 ± 0.3 g/L for glibenclamide by T150 (Figure 6).

Figure 6. Effect of the combinations (EACL + glucose) and (glibenclamide + glucose) on blood glucose levels in rats over a 150-minute period.

Overall, these results demonstrate that EACL possesses potent anti-hyperglycemic activity in all three models, with effects comparable to the reference drug glibenclamide. Notably, co-administration of EACL with glucose almost completely prevented the establishment of postprandial hyperglycemia.