Uvaria chamae leaves and Sida linifolia whole plant were collected respectively in Togo respectively in Agoè Apessito (6˚17'42.8''N; 1˚09'49.0''E), on March 13, 2022, and in Dalavé (6˚22'43.7''N; 1˚10'35.2''E), on August 22, 2022. The plants were identified by the Laboratory of Botany and Plant Ecology of the Faculty of Sciences in the University of Lomé (Togo), and a sample was kept in the herbarium of the said Laboratory under numbers TOGO15906 and TOGO15907. The harvested plant materials were dried and ground into powder at the Department of Pharmaceutical Sciences of the Faculty of Health Sciences in the University of Lomé (Togo).

Experiments were conducted following the institutional guidelines and ethics of the Laboratory of Physiology/Pharmacology of the University of Lomé, Togo (ref: 003/2022/CB-FDS-UL).

Wistar rats of both sexes weighing 80 to 150 g were pooled and used for ex vivo tests. The study animals were obtained from the Department of Pharmaceutical Sciences. They were all maintained in hygienic environmental conditions with a light/dark period of 12/12 h in standard cages and fed with food and water ad libitum.

DPPH, TPTZ, quercetin, metformin, glucose, gallic acid, and ascorbic acid were purchased from Sigma Aldrich (USA). Ethanol and methanol were purchased from VWR (France).

The extracts were obtained using cold maceration in alcohol at 10% (m/v) with hydro-ethanolic solutions (80% for Sida linifolia and 50% for Uvaria chamae). The mixtures were subjected to mechanical stirring for 72 hours. The macerate was filtered and evaporated under reduced pressure at 45˚C. The crude extracts obtained were stored in the refrigerator at 4˚C until use.

Two methods were used to fractionate our extracts:

For Uvaria chamae, the extracts were dissolved at 10%, m/v in distilled water and shaked with n-hexane, chloroform, ethyl acetate, butanol, (1:1) for 24 hours and then allowed to stand for 48 hours before separating the phases in a separating funnel. The fractions were evaporated under reduced pressure at 45˚C. The fractions were respectively labelled U5HE, U5CH, U5AE, U5BU, and U5ED for the hexane, chloroform, ethyl acetate, butanol, and aqueous fractions (Figure 1(a)).

For Sida linifolia, the extracts were fractionated in solvents of increasing polarity. The extracts were mixed with the solvents (10%, m/v) under mechanical stirring for 24 hours, then filtered before being redissolved in the solvent of the next polarity. The solvents were used in this order: n-hexane, chloroform, ethyl acetate, butanol, and distilled water. The resulting fractions were evaporated under reduced pressure at 45˚C. The fractions were respectively labelled S8HE, S8CH, S8AE, S8BU, S8ED for the hexane, chloroform, ethyl acetate, butanol, and aqueous fractions (Figure 1(b)).

The yield was calculated using the following formula:

Yield (%) = (mass of fraction obtained)/(mass of extract used) × 100.

The chemical screening consisted of the search for large chemical groups such as alkaloids, tannins, sterols, triterpenes, phenols, flavonoids, saponins, and reducing sugars through the described methods [10].

Figure 1. Fractionation of Uvaria chamae (a) and Sida linifolia (b).

To 0.1 ml of aqueous solution of the fraction was added 2 ml of a sodium carbonate solution (2%). After five minutes, 100 μL of Folin-Ciocalteu reagent (1 N) was mixed with it, kept for 30 minutes at room temperature, and the absorbance was read against a blank using a spectrophotometer at 750 nm. A calibration curve is made simultaneously under similar conditions with gallic acid as a positive control (10 - 100 µg/ml). The results were expressed as milligram gallic acid equivalent per gram of dry fraction (mg GAE/g) [11].

To 2 mL of methanol, AlCl₃ (2%), 2 mL of the fraction was added. The mixture was incubated for 10 minutes away from light and the absorbance was measured at 415 nm against a blank using a UV-Visible spectrophotometer. The standard range of 10 to 100 μg/ml was prepared under the same conditions as the fraction. The calibration curve was plotted using the different concentrations of quercetin, and the results were expressed as milligram quercetin equivalent per gram of dry fraction (mg QE/g) [12].

A volume of 50 μL of the sample was added to 1500 μL of 4% vanillin methanolic solution and mixed vigorously. Then, a volume of 750 μL of concentrated HCl was added. The resulting mixture was left in the dark at room temperature for 20 minutes. The absorbance was measured at 550 nm. Ranging concentrations of catechin (0 - 1000 μg/ml) as standard were used to draw the calibration curve, and the results were expressed in mg Catechin Equivalent per gram of dry fraction (mg CE/g) [13].

3.6.1. Effect of Glucose Uptake by Rat Muscle

The animals were sacrificed after 12 hours fasting, and the psoas muscle (0.5 g) was harvested. The muscles were then rinsed in cold Krebs’ Ringer bicarbonate buffer and placed in tubes containing Krebs’ Ringer bicarbonate buffer with 11.1 mM glucose and incubated for 60 minutes at 37˚C. After incubation, the muscles were taken out. The glucose content of the incubated medium was measured by the GOD-POD method. The uptake of glucose was calculated in mg/g tissue. Glucose uptake per gram of tissue was calculated as the difference between the initial and final glucose content in the incubated medium. The effect of fractions at the doses of 0.1 and 0.2 mg/ml was measured. A 2 mg/ml metformin solution was used as a positive control [9].

3.6.2. Effect on Glucose Uptake by the Jejunum

The reduction of glucose concentration in an incubation solution containing 5 cm of freshly isolated rat jejunum (sacrificed after 12 hours of fasting) and different fractions’ concentrations was measured using Chukwuma et al. (2018) method [14]. Briefly, a 5 cm jejunal segment from the isolated rat gut intestinal gut was first inverted to expose the villi and then incubated in 8 ml of Krebs’ buffer containing 11.1 mM glucose and fractions (0.1 and 0.2 mg/ml). Glucose with Krebs’ buffer was used as a control. Glucose concentrations were measured in all incubation solutions before and after the 120 min incubation period at 37˚C by using a commercial assay kit. The intestinal glucose absorption was calculated as the amount (mg) of glucose absorbed per cm of rat jejunum using the following formula:

Intestinal glucose absorption per cm of jejunum = (G1 − G2)/length of jejunum used in cm, G1 and G2 are glucose concentrations before and after the incubation, respectively.

3.7.1. DPPH Test

The anti-radical activity of the different fractions was evaluated using 2,2’-diphenyl-1-picrylhydrazyl (DPPH) as a relatively stable free radical. One hundred microliters (100 μl) of the fraction were added to 2 mL DPPH (0.004% prepared in methanol). The mixture was homogenized, and the absorbance reading was taken with a spectrophotometer at 517 nm after 30 minutes of incubation at room temperature in the dark. Three tests were carried out for each sample. The standard was quercetin (100 to 1000 mg/ml) [15][16].

The percentage of inhibition of radical activity was calculated according to the formula:

Inhibition (%) = (Absorbance (control) − absorbance (sample))/(absorbance (control)) × 100

The IC₅₀ was generated by GraphPad Prism 8.

3.7.2. Ferric Reducing Antioxidant Power (FRAP) Assay

The iron III reducing capability of the extract was assessed using the Eloh et al. (2024) method with some modifications. The FRAP test involves assessing the extract capacity to release an electron, thereby converting Fe³⁺ into Fe²⁺. This conversion can be quantified by measuring the resulting formation of Fe²⁺ ions. The FRAP solution was prepared by mixing 25 ml of acetate buffer, 2.5 ml of Fe³⁺-TPTZ (10 mM) in HCl (40 mM), and 2.5 ml of FeCl₃·6H₂O (20 mM/l). To 900 μL of the FRAP solution, 30 μL of the fraction (1 mg/ml) and 90 μL of distilled water were incorporated. The optical density was measured at 593 nm when the intense blue color became apparent. Calibration was conducted using an iron sulfate solution (FeSO₄). Each concentration (250 - 1000 µg/ml) was tested three times, and the results were expressed in EC₅₀, which is the median effective concentration. The standard used was the ascorbic acid [17].

3.7.3. Statistical Analysis

Statistical analysis was performed using GraphPad Prism 8 (USA). Results were reported as mean ± standard error of the mean or standard deviation. The replicates (n = 3) were samples from different animals. Data were subjected to one-way analysis of variance (ANOVA). These analyses were followed by the Tukey post-test, which performs multiple comparisons. Differences were considered significant if p < 0.05.

The following yields were obtained after the plants’ extraction (Figure 2).

Figure 2. Yields of the fractions of the extracts; (a): Uvaria chamae; (b): Sida linifolia.

Phytochemical analysis revealed the presence of several groups in the fraction (Table 1), and the determination of total phenols and flavonoids contents showed that the highest levels of phenols and flavonoids were found in S8ED for Sida linifolia and U5BU for Uvaria chamae (Table 2).

Table 1. Phytochemical screening of Sida linifolia and Uvaria chamae fractions.

+: presence; -: absence.

Table 2.Uvaria chamae and Sida linifolia total phenolic and flavonoid contents.

Values are expressed as means ± standard errors; GAE: gallic acid equivalent; QE: quercetin equivalent; CE: catechin equivalent.

The effects of Uvaria chamae and Sida linifolia extract fractions on glucose uptake in isolated rat muscle are presented in Figure 3. Results showed that U5ED (0.2 mg/ml), U5AE (0.1 and 0.2 mg/ml) increased significantly glucose absorption by muscles (p < 0.001, 0.01, and 0.5), respectively, compared to control (Figure 3(a)) for Uvaria chamae. For Sida linifolia, glucose uptake was significantly higher for the groups that received S8ED (p < 0.01) compared to the control (Figure 3(b)).

The results showed that all doses of U5ED, U5BU for Uvaria chamae decreased significantly (P < 0.01, P < 0.05, P < 0.01, P < 0.001, respectively) the absorption of glucose by the rat jejunum (Figure 4) compared to the control group (Figure 4(a)). For Sida linifolia, almost all fractions decreased the glucose absorption by the jejunum compared to the control group (Figure 4(b)).

Figure 3. Effects of fractions on glucose uptake in isolated rat muscle (a): Uvaria chamae; (b): Sida linifolia; M: metformin;(Values are expressed as means ± standard error of the mean (n = 3); *P < 0.05, ** P < 0.01, ***P < 0.001 vs control).

Figure 4. Effects of fractions on intestinal glucose absorption, (a): Uvaria chamae; (b): Sida linifolia; (values are expressed as means ± standard error of the mean (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001 vs control).

The scavenging capacity of the fractions was assessed and presented in the following table (Table 3).

Table 3. Antioxidant activity of Sida linifolia and Uvaria chamae fractions.

Values are expressed as means; GAE: gallic acid equivalent; QE: quercetin equivalent; CE: catechin equivalent.