2.8. Determination of Alkaloids

The total alkaloid contents in the samples were quantified according to the procedure developed by [42]. In the analysis, 40 mL of extract at a 1000-µg/mL concentration was used and acidified to pH 2–2.5 with 1-N HCl and 4 mL of Dragendorff reagent and centrifuged at 2400 rpm for 30 min. The supernatant was discarded, and the residue was treated with 1 mL of solute ethyl alcohol; 2 mL of 1% sodium sulfite was added, and the mixture was centrifuged at 2400 rpm for 30 min. The supernatant was then discarded, and the residue was treated with 2 mL of concentrated nitric acid; the resulting contents were transferred to a 50-mL volumetric flask and brought to volume with distilled water. Then, 1 mL of this solution was taken, and 5 mL of 3% (w/v) thiourea was added; the mixture of nitric acid and thiourea was used as a blank, and the sample’s absorbance read at 435 nm was measured. Berberine (Sigma-Aldrich, St. Louis, MO, USA) was employed as the standard, and linearity was obtained between 40 and 200µg/mL. The alkaloid contents are expressed in milligrams per gram of dry weight of extract.