Qualitative phytochemical screening for alkaloids, carbohydrates, flavonoids, protein, phenols, saponins, steroids, and tannins was performed according to standard methods [25].

Alkaloids. Dragendrorff’s test determined the presence of alkaloids in the sample. About 1 mL of a sample solution was mixed with few drops of Dragendorff’s reagent. The formation of orange-yellow precipitate indicated the presence of alkaloids.

Carbohydrates. Benedict’s test determined the presence of carbohydrates in the sample. The sample solution was mixed with few drops of Benedict’s reagent (alkaline solution containing cupric citrate complex) and boiled in a water bath, forming a reddish-brown precipitate indicated a positive result for the presence of carbohydrate.

Flavonoids. The Shinoda test did detection of flavonoids in the sample. A small quantity of the sample was dissolved in 5 mL of 95% v/v ethanol. Then few drops of concentrated hydrochloric acid and 0.5 g magnesium turnings were added. The formation of pink or magenta color indicated the presence of flavonoids.

Protein. Firstly, the Biuret test was applied. About 1 mL of sample solution was treated with 10% sodium hydroxide solution, and two drops of 0.1% copper sulphate solution, the formation of violet/pink color indicated the presence of protein. Secondly, a Ninhydrin test was used. 2–3 drops of Ninhydrin reagent were added to the test tube containing 1 ml of the sample solution. A purple color formation indicates the presence of amino acids.

Phenols. The phenols were tested using Ferric Chloride and lead acetate. In the former test, about 1 mL of a sample solution was mixed with two drops of aqueous 0.1% Ferric Chloride. The formation of blue-black coloration indicated the presence of phenols. Whereas the later test involved about 1 mL of a sample solution mixed with three drops of lead acetate solution. The formation of precipitate indicated the presence of phenolic compounds.

Saponins. The foam test was used to determine saponins. According to this test, about 1 mL of sample solution was shaken with 5 ml of distilled water for 5 minutes. The formation of a stable, characteristic froth that lasts for at least 30 minutes indicated saponins’ presence.

Steroids. Salkowski test was used to determine steroids. To a 2 mL of the sample solution, about 5 mL of chloroform was added. Then, 1 mL of 98% concentrated sulphuric acid was added to the above mixture carefully along the test tube walls. The formation of a reddish-brown ring at the junction of two layers indicates the presence of steroids.

Tannins. The gelatin test was used for Tannins. A sample solution was treated with 1% w/v gelatin solution containing 10% sodium chloride. The formation of white precipitate determined the presence of gelatin.

TPC of the extract was determined according to the Folin-Ciocalteu method [26]. About 1 mg/mL of the sample solution was oxidized with 2 mL of 10% v/v Follin-Ciocalteu reagent (FCR) and 2 mL of 7.5% sodium carbonate. The mixture was incubated for 40 minutes at 45°C, and absorbance was measured at 765 nm using a UV/VIS spectrophotometer (JENWAY, UK). The TPC was calculated as a Gallic acid equivalent based on a standard calibration curve.

The extract’s total flavonoid content was determined according to the aluminium chloride colorimetric method [27]. Briefly, 1 mg/mL of crude extract was mixed with 4 mL of distilled water and 0.5 mL of 5% NaNO2, then allowed to stand for 5 minutes. Later, 0.5 mL of 10% AlCl3 was added and allowed to stand for 6 minutes before adding 1 mL of 1 M NaOH and made the mixture 10 mL with distilled water. The mixture was allowed to stand for 15 minutes at room temperature, and absorbance was measured at 510 nm using a UV/VIS spectrophotometer (JENWAY, UK). The total flavonoid content was calculated as quercetin equivalent based on the standard calibration curve.

According to the previously developed and validated HPLC method, the extract’s fingerprint was determined [28]. The chromatographic system consisted of a Shimadzu LC-10AT equipped with an SPD-20A UV/VIS detector (Tokyo, Japan), communicator CBM-20A (Tokyo, Japan), and degassing unit DGU-20A5R (USA) with an isocratic binary system of the mobile phase. Chromatographic separation was performed on a Lunar® C18 column (5 μm; 250 x 4.6 mm; Phenomenex, USA) maintained at a temperature of 40°C in a Shimadzu column oven (CTO-20AC, Tokyo Japan). For chromatographic fingerprint, the mobile phase used was methanol: water: acetonitrile (60:30:10 v/v) containing 0.01% trifluoroacetic acid and a flow rate of 0.6 mL/min with a detection wavelength of 210. For quantification of quercetin, the mobile phase was methanol: 0.1% orthophosphoric acid water (60:40) and a flow rate of 1.0 mL/min with a detection wavelength of 370. In both fingerprint and quantification, the volume injected was 10 μL at a pressure of 2,596.17 psi. The sample was prepared in triplicate using methanol to a concentration of 1 mg/ml. Samples, mobile phase, and standard quercetin solution were filtered through 0.45μm membrane filters (EZ-Pak®, France) before loaded into the system.

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