The plant material consists of Solanum torvum roots. These specimens were collected in their natural habitat in Bouaké, a city located approximately 347 kilometers north of Abidjan. The identification of the plant was carried out by the National Center for Floristics (CNF) of the Félix Houphouët-Boigny University. This identification was carried out by comparing it with the herbarium of S. torvum archived under the number CNF 8377.

In this study, white albino rats of the species Rattus norvegicus, belonging to the subfamily Muridae and more specifically to the Wistar strain, were used as experimental subjects. The average age of the subjects was 12 weeks, with an average weight of 225 ± 25 g. The animals were fed pellets (IVOGRAIN^®) and had free access to water. Their acclimatization took place in a controlled environment, namely a laboratory, where the temperature was maintained at 22˚C and a 12-hour light cycle was established.

The roots of Solanum torvum were washed, cut into small pieces, and dried in the shade at room temperature for two weeks. The powder obtained by grinding this dry material was used for the preparation of the aqueous extract according to the method of [10] . Thus, 100 g of the powder were homogenized in one liter (1 L) of distilled water using a Blender-type mixer. This operation was repeated three times. The obtained homogenates were successively filtered, twice through hydrophilic cotton and once through Whatman 3 mm filter paper. The filtrates were dried in an oven at 50˚C for five days. The brown-colored evaporate obtained constituted the extract of Solanum torvum roots coded EX used for experimentation.

Qualitative phytochemical analysis is based on the one hand on the formation of insoluble complexes using precipitation reactions and on the other hand on the formation of colored complexes using staining reactions [11] .

1) Test for polyphenols

The polyphenols in the extract were detected by the ferric chloride (FeCl₃) reaction. Indeed, 2% ferric chloride oxidizes in the presence of polyphenols and gives a blue-black coloration. For this test, 2 mL of the EX extract were taken in a test tube, and then a drop of alcoholic solution was added. The tube was homogenized and observed for the appearance of a blue-black or green coloration.

2) Test for flavonoids

Flavonoids were detected using the cyanohydrin reaction. In the presence of flavonoids, cyanidin changes from a red color to a pink-orange color. Thus, 2 mL of the EX extract were evaporated on a sand bath, and the residue was dissolved in 5 mL of hydrochloric acid diluted twice. To this solution, 3 magnesium chips were added, followed by three drops of isoamyl alcohol. The tube was observed for the release of heat and the appearance of a rose orange color.

3) Test for tannins

Catechin tannins were detected from the intense black in the presence of the Stiasny reagent. In this method, the tannins are characterized by a blue coloration. To do this, five (5) mL of the extract were evaporated in the oven. The obtained residue was dissolved in 15 mL of the Stiasny reagent. The mixture was maintained in a water bath at 80˚C for 30 minutes and the tube was observed for the appearance of a blue color.

4) Test for alkaloids

The alkaloids were characterized using Buchard’s reagent (iodo-iodide reagent) and Dragendoff’s reagent (potassium iodo-bismuthate reagent). Indeed, in the presence of alkaloids, Buchard and Dragendoff reagents produce a reddish-brown precipitate. For this test, 6 mL of the extract were evaporated to dryness. The residue was dissolved in 6 mL of alcohol at 60˚C, and 2 drops of Dragendorff’s reagent were added to the alcoholic solution. The tubes are observed for the presence of a precipitate or an orange coloration.

Iron content of the extract was determined according [12] . To do this, 1 g of powdered Solanum torvum roots was taken in a crucible and then calcined in a muffle furnace for two hours. The obtained ash was dissolved in a test tube containing 4 mL of concentrated nitric acid, then the solution was homogenized for 1 minute. The homogenate was evaporated to dryness on a hot plate at 100˚C, then the evaporate was calcined at 550˚C in a muffle furnace for 1 hour. After cooling in a desiccator, the ash from the evaporate was taken up in 10 mL of hydrochloric acid, then filtered into a standard 50 mL dilution tube. The filtrate was homogenized for 2 minutes, then the homogenate was preserved for iron dosage. A calibration curve was established using a range of concentrations from 0 to 20 μg/mL, obtained from a stock solution of iron concentrated at 100 μg/mL. The reading of the samples was done using a Flame Atomic Absorption Spectrophotometer (AAS) (Pekin Elmer AAnalyst 400) at 248 nm, and the iron content was calculated using the expression below and then expressed in mg/100 g of extract:

$\text{Iron content}\left(\text{mg}/100g\text{extract}\right)=\frac{C\times 50\times 100}{PE\times 1000}$

C: concentration of the element to be measured in mg/l read at the AAS.

PE: Test run in g.

1) Induction of malaria clinical signs in rats

In this study, three symptoms associated with malaria, namely anemia, pain, and fever, were induced in 25 healthy rats. Their temperature and contortion were recorded, and then the blood levels of anemia markers such as hemoglobin (Hb), red blood cells (RBC), and hematocrit (Hct) were measured. Subsequently, the 25 rats were divided into two distinct groups: a control group of five untreated sick individuals and a test group of 20 specimens.

Anemia was induced in the animals according to the method of [13] , which involves administering 1 mL of phenylhydrazine (20 mg/kg) by gavage for 7 consecutive days to the test group, while the control group received only distilled water. At the end of this period, analgesia was induced in the anemic rodents by intraperitoneal injection of 1 mL of a 1% acetic acid solution [14] .

To cause hyperthermia, the ill animals were starved for a whole day [15] . The rats were given a single subcutaneous injection of 1 mL of a 20% brewer’s yeast (Saccharomyces cerevisiae) slurry as part of the experiment. Eighteen hours after induction, the temperature and the number of writhes were recorded.

2) Treatment of animals

The selected anemic animals were divided into 5 groups of 5 rats each. This number of rats is sufficient to observe significant pharmacological effects of the substances, takes into account ethical considerations and allow difference between the mean of two groups. The treatment lasted 7 days for all the animals. Lot 1 (healthy control) and 2 (sick no treated) receive no treatment, lot 3 (EX100) receives the extract at a dose of 100 mg/Kg.BW, lot 4 (EX500) receives the extract at a dose of 500 mg/Kg.BW, and lot 5 (FTP 10) receives the references molecules at a dose of 10 mg/Kg.bw (Folifer for anemia, Tramadol for pain, and Paracetamol for fever). At the end of the treatment, the animals’ blood was collected for the determination of hematological parameters including red blood cell (RBC) count, hemoglobin (Hb) and hematocrit (HCT). Rectal temperatures are taken every hour for 4 hours after administration of the test substances, using an accurate electronic thermometer. Reference molecules dose is slightly lower than those found by the conversion equations proposed by [16] and adjusted according to the Institutional Animal Care & Use Committee’s guidelines [17] .

The statistical analysis and graphs were performed using GraphPad Prism version 8.0 software. The values expressed are the means of at least three experiments accompanied by the standard error of the mean (Mean ± SEM). The one-way analysis of variance (ANOVA) followed by Dunnett’s non-parametric multiple comparisons test was used for comparing the means with a significance level of 5%.

The results of the qualitative characterization of the secondary metabolites of the extract are recorded in Table 1 . According to the data in this table, the observation of the tubes for polyphenols shows the appearance of a blue-black coloration, indicating a high polyphenol content in the extract. Also, the extract is very rich in flavonoids, identified in the tubes by a release of heat followed by the appearance of an intense pink-orange coloration. Regarding the tannins, the presence of a faint blue coloration indicates a medium content of the extract. Furthermore, the extract shows a high alkaloid content, indicated by the presence of a very dark brown-reddish coloration.

Moreover, the iron assay showed that the extract has an iron content of 44.07 mg/100g of extract.

Table 2 shows the variation of symptomatic markers of malaria between the control group and the test group. Indeed, the serum values of white blood cells

+: presence; +++: High content; ++: Average content.

Value = Mean ± SEM (n ≥ 5); With asterisk (* or ***) in the column indicates a significant difference compared to the control batch value respectively with p < 0.05 and p < 0.001. RBC: Red Blood Cells; WBC: White blood cells; Hb: Hemoglobin; HCT: Hematocrit.

(WBC), red blood cells (RBC), hemoglobin (Hb), hematocrit (HCT), the number of writhings, and the rectal temperature in healthy control rats are respectively (7.25 ± 0.05) × 10⁶/mm³; (9.05 ± 0.26) × 10⁶/mm³; 15.12 ± 0.03 g/dl; 39.25% ± 0.06%; 56 ± 0.02 writhings; and 35.98˚C ± 0.02˚C.

Once anemia is established, a significant variation in the evaluated hematological markers is observed in the animals. Thus, a significant increase in the number of WBCs was observed in the study subjects, rising from (7.25 ± 0.05) to (10.62 ± 0.03) × 10⁶/mm³, which corresponds to a relative increase of 46.48%. In contrast, anemia, unlike white blood cells, induces a significant reduction (p < 0.001) in other hematological markers. Thus, a decrease in hemoglobin, hematocrit, and red blood cell count was observed. Indeed, the initial values, respectively 15.12 ± 0.05 g/dl, 39.25 ± 0.06, and 49.25% ± 0.06%, respectively decreased to 8.53 ± 0.2 g/dl, 22.09% ± 0.55%, and 43.71% ± 0.1%. These results suggest a significant decrease in hemoglobin and hematocrit, with respective variations of 41.76% ± 0.5% and 43.71% ± 0.1%.

Moreover, the pain induced in the animals leads to a significant increase (p < 0.05) in writhing, from 56 ± 2.35 to 108 ± 1.25, which is an increase of 92.85%. As for the fever, it was characterized by an increase in rectal temperature from 35 ± 0.02 to 39.45 ± 0.09˚C, which is an increase of 12.27%.

The effect of the extract on the symptoms of malaria is represented by Figures 1-6 . According to these results, the extract at a dose of 100 mg/kg pc leads to a modest non-significant increase (p > 0.05) in red blood cells from 5.27 ± 0.03 to 5.38 ± 0.01.106/mm³ ( Figure 1 ); hemoglobin from 8.53 ± 0.2 to 8.67 ± 0.01 g/dl ( Figure 2 ); hematocrits from 22.09% ± 0.55% to 22.35% ± 0.37% ( Figure 3 ) compared to the untreated sick batch, with respective percentage increases of 2.08%; 1.64%; 1.17%; 2.89%.

The same applies to white blood cells ( Figure 4 ), contortions ( Figure 5 ), and temperature ( Figure 6 ), changing respectively from (10.62 ± 0.56) to (10.15 ± 0.02) × 10⁶/mm³, from 108 ± 1.55 to 100 ± 0.06, and from 39.45 ± 0.09 to 38 ± 0 when to compared to the untreated sick batch.

However, compared to the healthy control group, a significant variation (p < 0.05) of the different markers is observed at this same dose. Indeed, this dose causes a significant decrease in the number of white blood cells from 9.05 ± 0.26 (control value) to 5.38 ± 0.01 (value at 100 mg/kg pc), hemoglobin from 15.12 ± 0.03 (control value) to 8.67 ± 0.01 (value at 100 mg/kg pc), and hematocrit from 39.25 ± 0.65 (control value) to 22.35 ± 0.37 (value at 100 mg/kg pc), as well as a significant increase in the number of white blood cells from 7.25 ± 0.03 (control value) to 10.15 ± 0.01 (value at 100 mg/kg pc), contortions from 56 ± 2.25 (control value) to 100 ± 0.65 (value at 100 mg/kg pc), and temperature from 35 ± 1.25 (control value) to 38 ± 1.65 (value at 100 mg/kg pc).

Furthermore, the extract at a dose of 500 mg/kg bw significantly normalized (p < 0.05) the levels of the studied markers compared to the healthy control group. In the context of the analysis conducted, the red blood cell (RBC) count was determined to be (09.21 ± 0.03) × 10⁶/mm³, hemoglobin (Hb) at 15.65 ± 0.045 g/dl, hematocrit (HCT) at 40.06% ± 0.01%, white blood cell (WBC) count at (7.85 ± 0.05) × 10⁶/mm³, contortions at 54, and body temperature at 36.05˚C.

The extract at a dose of 500 mg/kg pc significantly normalized (p < 0.05) the RBCs to (09.21 ± 0.03) × 10⁶/mm³, Hb to 15.65 ± 0.045; HCT to 40.06% ± 0.01%; white blood cells to (7.85 ± 0.05) × 10⁶/mm³; contortions to 54, and temperature to 36.05˚C compared to the healthy control lot.

The reference molecules (Folifer, Trémadol, and paracetamol) at doses of 10 mg/kg bw normalized the clinical signs associated with anemia, pain, and fever in the treated rats.

In untreated sick animals (SNT), the RBC, Hb, and HCT significantly decreased (p < 0.05) to (3.57 ± 0.03) × 10⁶/mm³; 16.26 ± 0.1 g/dl, and 16.26% ± 0.06%, respectively, while the white blood cell count, contortions, and temperature significantly (p < 0.05) increased to (15.32 ± 0.06) × 10⁶/mm³; 127 ± 2.47, and 39.86˚C ± 2.35˚C, respectively, compared to the healthy control group.