*2.4. Phytochemical Analysis of the Ethanolic Extract*

The phytochemicals present in the ethanolic extract of *S. perfoliata* were determined using the methods described by Mushtaq et al. [15] with minor changes. The stock solutions (1 mg/mL in dH2O) of the ethanolic extract of *S. perfoliata* were used to determine the presence of tannins, saponins and terpenes. The formation of a yellow brown precipitate after the addition of 2 mL of 5% FeCl3 in dH2O, indicated the presence of tannins. The presence of saponins was determined by vigorously shaking the stock solution and observing a stable persistent froth. The development of a reddish-brown interface after the addition of 5 mL CHCl3, 2 mL acetic anhydride, and a few drops of concentrated H2SO4 to 2 mL of the stock solution, indicated the presence of terpenes.

Stock solutions of the ethanolic extract of *S. perfoliata* (1 mg/mL in methanol) were prepared to determine the presence of alkaloids, cardiac glycosides, flavonoids and phenolics. The presence of alkaloids was determined by first adding 1.5 mL of 1% HCl to 2 mL of the stock solution and heating the mixture in a water bath set at 90 ◦C. Following the incubation, 6 drops of Dragendroff's reagent were added and a formation of an orange precipitate was indicative of the presence of alkaloids. The presence of cardiac glycosides was determined by adding 1 mL glacial acetic acid and 1–2 drops of FeCl3, followed by the addition of 1 mL of concentrated H2SO4 to 2 mL of the ethanolic stock solution. The formation of a brown ring at the interface indicated the presence of a deoxysugar, characteristic of cardenolides. The presence of flavonoids was determined by observing the presence of a pink color after adding a few drops of concentrated HCl followed by small pieces of magnesium turnings. The presence of phenolics, on the other hand was indicated by the presence of a blue or green color, after the addition of 1 mL of 1% ferric chloride solution to the stock solution.
