The plant was collected in the commune of Taïba Ndiaye, a town located in the Thiès region of western Senegal, at geographic coordinates 15˚3'0''N and 16˚52'60''W. Identification was carried out at the Fundamental Institute of Black Africa (IFAN) using the LEBRUN & STORK database.

The plant material consists of the leaves and stems of Melothria maderaspatana. After harvesting, these parts were dried in the dark at room temperature (approximately 25˚C) for three weeks at the Bioactive Substances Research Group (GRSB) laboratory of Cheikh Anta Diop University in Dakar. They were then ground into a fine powder using an electric grinder (Silver Crest Powder Grinder SC-200) and carefully stored in jars to prevent contamination.

Adult Wistar rats weighing between 120 g and 150 g were used for this study. These rats were kept in the animal facility of the Pharmacology and Pharmacodynamics Laboratory with free access to food and water. They were maintained at a constant temperature of 22˚C - 28˚C and illuminated according to a 12-hour light-dark cycle. They were acclimated one week before the start of the experiments.

The extracts were prepared by cold maceration with magnetic stirring in ethanol. Specifically, 100 g of finely powdered leaves or stems of the plant were placed in an Erlenmeyer flask with 500 mL of ethanol. The mixture was covered with aluminum foil after 48 hours of magnetic stirring in the dark at room temperature. Filtration was performed using a funnel and Whatman^® brand filter paper.

The operation is repeated twice, in the presence of 300 mL of solvent, with the pomace obtained from the filtration. The resulting filtrates are combined, and the solvent is evaporated using a Buchi R-80 rotary evaporator at 60˚C, yielding the ethanolic extract [7].

Phytochemical screening is a qualitative analysis based on precipitation or color reactions. These reactions allow for the detection of secondary metabolites that may be present in a plant sample. In this work, the screening focuses on the detection of alkaloids, polyphenols, tannins, flavonoids, saponins, sterols and polyterpenes, leucoanthocyanins, catechols, and mucilage. The presence of these different chemical groups is demonstrated by referring to the techniques described in the work of Bedie et al. [8].

Polyphenols and tannins are identified by the FeCl₃ test and Stiasny’s reagent; flavonoids, leucoanthocyanins, and catechols by the cyanidin reaction; saponins by the foam test; sterols and polyterpenes by the Liebermann-Burchard test; mucilage by the absolute ethanol test; and alkaloids by the Mayer test [9].

Anti-inflammatory activity is evaluated using the carrageenan-induced rat paw edema method [10]. Rats, divided into six groups of four, are weighed and then fasted for 12 hours before the experiment. For each rat, the initial thickness (E0) of the left hind leg is measured using stainless-hardened calipers before treatment administration. The different treatments are administered by gavage.

- Batch 1: Physiological saline at a concentration of 10 mL/kg (control);

- Batch 2: Acetylsalicylic acid solution at a concentration of 200 mg/kg;

- Batch 3: Extract of Melothria maderaspatana leaves with ethanol at a concentration of 30 mg/kg;

- Batch 4: Extract of Melothria maderaspatana leaves with ethanol at a concentration of 100 mg/kg;

- Batch 5: Extract of Melothria maderaspatana stems with ethanol at a concentration of 30 mg/kg;

- Batch 6: Extract of Melothria maderaspatana stems with ethanol at a concentration of 100 mg/kg.

One hour after oral administration of the different solutions, each rat received an injection of 100 µl (0.1 mL) of 1% carrageenan solution under the footpad of its left hind paw. The thickness of the injected paws was measured at 1 h, 3 h, and 5 h (T1 h, T3 h, and T5 h) after carrageenan injection, again using stainless-hardened calipers. The degree of edema was assessed by determining the mean percentage increase (% Aug) in paw thickness using the following formula:

% Increase in leg thickness = E f − E i E i × 100

with:

Ef: final thickness of the leg; Ei: initial thickness of the leg.

Results are expressed as means with standard errors (Mean ± SEM). Data were graphically represented using GraphPad Prism 7.0 software (Microsoft USA). Statistical analysis was performed using analysis of variance (one-way ANOVA). Differences between means were determined using Dunnett’s test. A statistically significant difference was defined as p < 0.05.

Table 1. Phytochemical study of the ethanol extract of Melothria maderaspatana leaves and stems.

− = Missing; + = Present.

The phytochemical study of the ethanolic extract of Melothria maderaspatana leaves and stems yielded the results presented in Table 1. This table shows that the ethanolic extract of Melothria maderaspatana leaves and stems contains a significant quantity of polyphenols, catecholic tannins, sterols and polyterpenes, leucoanthocyanins and catechols, and coumarins, which are indicated by intense coloration. Alkaloids are present with moderate coloration in both extracts, as determined by the Dragendorff test. The Shibata reaction revealed an absence of flavonoids in the ethanolic extract of the leaves.

Injection of the 1% carrageenan solution into the left hind leg of control rats causes edema with percentage increases of 40.30% ± 8.34%, 72.70% ± 6.03% and 97.78% ± 4.29% after 1 h, 3 h and 5 h, respectively.

Oral administration of the ethanolic extract of Melothria maderaspatana leaves is associated with a blockage of the increase in edema induced by carrageenan. In the presence of the ethanolic extract of Melothria maderaspatana leaves, the percentage increases in inflammatory edema of the leg are 23.69% ± 6.45%; 34.65% ± 13.93%; 40.87% ± 7.36% for the 30 mg/kg dose and 34.95% ± 5.32%; 48.85% ± 7.28%; 54.62% ± 4.37% for the 100 mg/kg dose. Compared to the controls, these results are highly significant for the ethanolic extract of the leaves at a dose of 30 mg/kg at 3 h (p < 0.001) and 5 h (p < 0.0001). These results are superior to those obtained with the dose of 100 mg/kg (p < 0.001 at 5 h), but identical to those observed with acetylsalicylic acid administered at a dose of 200 mg/kg as illustrated in Figure 1.

From the outset, these results showed that the leaf extract has a more pronounced anti-inflammatory effect at 30 mg/kg than at 100 mg/kg, suggesting an atypical dose-response relationship. This non-linear profile could reflect a biphasic effect, a phenomenon described for several plant extracts, some of whose constituents exhibit optimal activity at low doses, while at higher doses, saturation of the target molecules or the expression of compounds with an antagonistic effect may occur. The chemical composition of the extract may also contribute to this reversal: active but minor molecules could become sufficiently concentrated at high doses to diminish the overall effect. Thus, the maximum effect observed at low doses is an important observation, suggesting that the optimal dose may lie within a submaximal range. Further investigations will be necessary to clarify the mechanisms involved and confirm the biphasic nature of this response.

The results of the anti-inflammatory activity of the ethanolic extract of Melothria maderaspatana stems are shown in Figure 2, which presents the changes in the kinetics of left hind paw edema (LHP) increase in rats, induced by the injection of a 1% carrageenan solution into the aponeurosis of the paw (foot pad). These results are compared to those of acetylsalicylic acid, a non-steroidal anti-inflammatory drug, and to those of the oral administration of the ethanolic extract of Melothria maderaspatana stems, which is associated with a highly significant blockade of the increase in edema induced by carrageenan. The anti-inflammatory activity is assessed by the percentage increase (% Aug) in edema. Indeed, oral administration of the Melothria maderaspatana stem extract resulted in the following percentage increases: 16.08% ± 5.27%; 23.43% ± 8.21%, 22.15% ± 7.00% at a dose of 30 mg/kg (batch 5) and 19.64% ± 7.61%; 33.2% ± 8.40%, 19.61% ± 7.57% at a dose of 100 mg/kg (batch 6). The results obtained, presented in Figure 2, show that the anti-inflammatory profile appears to be better than that of the ethanolic extract of the leaves, with also better activity at the dose of 30 mg/kg, identical to that of aspirin at the dose of 200 mg/kg.

Figure 1. Mean percentage increase in edema after administration of crude ethanolic extract of Melothria maderaspatana leaves at doses of 30 and 100 mg/kg and aspirin at a dose of 200 mg/kg to Wistar rats.

Figure 2.Mean percentage increase in edema after administration of crude ethanolic extract of Melothria maderaspatana stems at doses of 30 and 100 mg/kg and aspirin at a dose of 200 mg/kg to Wistar rats.