The plant material for this work consists of the aerial parts (leaves and seeds) of Chenopodiumambrosioides collected at Tsévié-Dalavé (about 50 Km north of Lomé) on October 31, 2019 at the following coordinates: North latitude 6˚23'0.042'' and longitude East 1˚14'7.692'' during the fruiting period. The harvested plants were authenticated at the Botanical and Plant Ecology Laboratory of University of Lomé (Togo). The leaves and seeds harvested were washed with water and then dried in the dark for a month in order to lower the water content, avoid contamination and keep the active ingredients at room temperature laboratory which is 20˚C. They are then ground into a powder using a grinder and stored away from air and moisture.

The reagents used are: DPPH (1,1-diphenyl-2-picryl-hydrazyl), quercetin, iron III trichloride (FeCl₃) come from the supplier Carlo Erba. Sodium carbonate (Na₂CO₃) and gallic acid are purchased from Sigma Aldrich. Other chemicals such as chloroform, soda, hydrochloric acid, sulfuric acid have been used. Ethanol, methanol and distilled water served as solvents. All the reagents and solvents were purified according to the methods described in the work published in the scientific literature.

Obtaining the ground material

The aerial parts of Chenopodium ambrosioides (leaves and seeds) collected are washed and dried in the open air and protected from light for a month in order to lower the water content and avoid contamination, and to keep the active ingredients at ambient temperature (20˚C). They are then ground into a powder and stored away from air and moisture.

Preparation of extracts

The aqueous and ethanolic extracts are prepared based on two extraction methods.

Maceration

Maceration consists of keeping the drug in contact with a solvent at room temperature for a period of 30 minutes to 48 hours. In the case of herbal teas the solvent is water. This method allows gentle extraction of the active ingredients, especially when they are thermolabile.

The aqueous extraction was carried out by introducing 8 g of vegetable powder in 80 mL of distilled water, the mixture is stirred and left to macerate for 24 hours then filtered.

The same steps were followed for the ethanolic extract (95% ethanol).

Infusion

It is prepared by pouring the boiling solvent over the parts of fresh or dried plants and soaking them well to extract their active ingredients, leaving the mixture to stand a few minutes at laboratory temperature (no more than 15 minutes).

For the aqueous extract, 8 g of vegetable powder is introduced into 80 mL of boiling distilled water and left to infuse for 15 minutes.

The same steps were followed in boiling ethanol (95% ethanol).

The filtrates obtained are used for the phytochemical and anti-radical studies.

Phytochemical screening makes it possible to highlight the presence or absence of groups of chemical families in a plant material. It is based on precipitation and coloring reactions. The method applied is that adopted by Haddouchi et al. [12]; EL-Haoud et al. [13] and Halla et al. [14] modified and adapted to the conditions of the Laboratory of Organic Chemistry and Natural Substances of the University of Lomé.

Detection of flavonoids

To 0.5 × 10⁻³ liter of each extract, 10 drops of concentrated HCl and a few mg of magnesium turnip are added. After 3 minutes of incubation at room temperature, a pink-red or yellow color indicates the presence of flavonoids.

Detection of tannins

To one milliliter of each extract, add 8 drops of a dilute 1% FeCl₃ solution. At room temperature, the mixture is incubated for a few minutes. In the presence of catechic tannins the coloring is greenish while in the presence of Gallic tannins it is blackish blue.

Detection of alkaloids

To one ml of each extract are added 2 ml of 1% HCl. It is heated in a water bath (60˚C) and then the extract is divided into two equal volumes. One volume is treated with Mayer’s reagent—(potassium mercuric + iodide solution) and the other with Wagner’s reagent—(solution of iodine in potassium iodide). A yellowish-white precipitate by Mayer’s reagent is formed if alkaloids are present in the extract. An orange-red to brown staining by Wagner’s reagent leads to the same conclusion.

Detection of saponins

In a test tube, place 2 ml of each extract and make up with distilled water to obtain 5 ml of solution. The tubes are shaken lengthwise for 15 seconds. Leave to stand for 20 minutes. If the height of the foam is more than 1 cm, the test is positive.

Detection of terpenoids

To 1 ml of each extract are added, successively, 0.5 ml of chloroform and 0.7 ml of concentrated H₂SO₄. Steroidal heterosides are characterized by a green-blue color and the presence of terpene heterosides gives a green-purple.

Detection of terpenes

The sample (1 mL) was placed in a test tube with 1 mL of concentrated sulfuric acid and heated for two minutes. The appearance of a grayish color of the supernatant indicates the presence of terpenes.

Reducing compounds

Their detection involves placing 2 mL of the aqueous extract in a test tube, then 2 mL of Fehling’s liquor (A + B) is added. Then, the whole is brought to a boiling water bath for 8 minutes. Reducing compounds are detected by obtaining a brick red precipitate.

Cardiac glycosides

Two (2) mL of chloroform are poured into 1 mL of the extract, the appearance of a reddish-brown color after the addition of H₂SO₄ indicates the presence of cardiac glycosides.

Free anthracene derivatives

To 1 ml of the extract dissolved in chloroform (1 g of powder in 10 ml), dilute NH₄OH (1 ml) is added. Free anthracene derivatives are detected by a more or less red color after shaking.

Detection of quinines

In a test tube containing 1 ml of 1% sodium hydroxide (NaOH), 1 ml of the sample is introduced. Everything is well mixed. The presence of quinines is characterized by a blue, green or red coloration.

A mass of 20 g of powder was macerated for 24 hours and infused in 200 mL of distilled water and 95˚ ethanol. The resulting mixture is filtered through cotton and then through filter paper. The extracts are evaporated at a temperature of 45˚C.

The yields of dry extracts are determined according to the following formula:

Efficiency (%) = [P1 − P2/P3] × 100

P1: weight of the flask after evaporation; P2: weight of the empty flask; P3: product mass.

The method to be adopted is that used by Ali-Rachedi et al. [15] where a volume of 200 μL of each extract was added, with a mixture of 1 mL of Folin-Ciocalteu reagent diluted 10 times, and 800 μL of a solution kept for 30 minutes. The absorbance is read at 765 nm.

DPPH (2,2-diphenyl-1-picrylhydrasyl) is a free radical that has an odd electron on one of these nitrogen atoms and is the basis of most antioxidant tests. The violet-colored DPPH radical, brought into contact with an extract containing antioxidant molecules, changes color and becomes yellow. This color change is due to the reduced form which no longer absorbs at this wavelength 517 nm (Scheme 1).

The method described by Dieng et al. [5] to determine the anti-radical activity allowed us to test our different extracts.

The DPPH solution was prepared in ethanol with a concentration of 100 μmol·L⁻¹. Each extract is diluted according to concentrations of (1.8; 2; 2.2; 2.5; 2.8; 3.3; 4; 5; 6; 7; 10) mg/mL and the reference (quercetin) according to the following concentrations: (1.29; 1.38; 1.5; 1.8; 2; 3) µg/mL. Then, to 150 μL of extract and the reference are added 3 mL of the DPPH solution; then the absorbances were measured at 517 nm after incubation for 30 minutes in the dark. Three tests were carried out for each concentration of product tested.

In order to determine the means and the correlation coefficients, an analysis of the results is carried out using Microsoft Office Excel 2010 software. The graphs and histograms were produced using this same software. The IC₅₀ were calculated from the linear or logarithmic equations of the plotted curves.