3.4. Preliminary Phytochemical Analysis

Crude extract and their fractions were subjected to preliminary phytochemical tests to confirm the presence of flavonoids, phenols, alkaloids, saponins, glycosides and carbohydrates using reported methods [66,67,68]. The crude extract along with different fractions (1 g) were dissolved in the 5 mL DMSO (99.99%, Fischer Scientific, Loughborough, UK) to make a stock solution, and DMSO was further used for re-solubilization (Figure 4).

A few drops from the stock solution of crude extract and different fractions of S. edelbergii were mixed with diluted in 5% NaOH solution, followed by the addition of a few drops of hydrochloric acid (HCl). The presence of flavonoids was detected through color changes from yellow to colorless.

A few drops from the stock solution of crude extract and different fractions were added to the FeCl₃ solution, followed by vigorous shaking. The appearance of a bluish-green color confirms the presence of phenols.

Qualitative detection of alkaloids was performed by mixing 0.5 mL of stock solution of crude extract and fractions with 2% H₂SO₄ solution, followed by heating for 3 min. Then, some drops of Dragendorff’s reagent were added to the mixture and allowed to cool. The appearance of an orange-red color precipitate revealed the presence of alkaloids.

From the stock solution of S. edelbergii extracts and fractions, a few drops were mixed with 0.4 mL of distilled water, followed by shaking. After 5 min, the mixture was boiled and resulted in the formation of small bubbles (froth), which confirmed the presence of saponins.

The presence of carbohydrates was determined by mixing 3 mL of extract solution with 2 mL of research-grade Benedict’s reagent. The mixture was then placed in a hot water bath for 3 min. The appearance of a reddish-brown precipitate revealed the presence of carbohydrates.

A few drops of HCl were mixed with small quantities of extract solutions that caused hydrolysis of glycosides, which were then neutralized through the addition of (how much amount) NaOH solution. After neutralization, about 0.5 mL from Fehling’s A and B solutions were added to each extract mixture. Glycoside’s confirmation was revealed by the appearance of a red color precipitate.

The selected plant was investigated for quantitative estimation of the total phenolic and flavonoids contents in crude extract and subfraction using standard procedure [69].

The phenolic content in S. edelbergii extracts and subfractions was determined using the Folin–Ciocalteu reagent. The total phenolic content was measured as milligrams of gallic acid equivalent (mg GAE/g) per gram of dry sample mass. Different extracts in distilled methanol (5 mg in 5 mL) were mixed with 10 mL distilled water and placed undisturbed for 5 min. About 1 mL from each mixture was taken in a test tube, and its volume was raised to 10 mL by adding distilled water. Then, 1 mL of Folin-Ciocalteu reagent was added to each test tube and incubated for 6 min. After incubation, 10 mL of 7% sodium carbonate solution were added. The final volume of the reaction mixture was raised to 26 mL through the addition of distilled water and incubated for 90 min at room temperature. The absorbance was noted at 760 nm using a UV-visible spectrophotometer.

The total flavonoid content was represented as a quercetin equivalent (mg QE/g) of the dry sample mass of the extract. About 9 mL of the distal water was mixed with 1 mL of each extract stock solution, along with 1 mL of 5% NaNO₂ in a test tube, which was then incubated for 6 min. Then, 2 mL of 10% AlCl₃ was added to each test tube, and the reaction mixture was then placed undisturbed for 5 min. Finally, 2 mL of 1 M sodium hydroxide was added to each test tube, and the absorbance of the resultant mixture was noted at 510 nm via a UV-visible spectrophotometer.