Dried samples (50 g) of all selected plant parts were extracted in 100% methanol (300 mL) by cold maceration method. All of the extracts were concentrated using a vacuum pump rotatory evaporator from Buchi, New Castle, USA. The concentrated extracts were dried using a freeze dryer from IIShin Lab Co (South Korea) to obtain the lyophilized extract. Phytochemical screening was performed according to the method reported by Bhatnagar et al.38 Qualitative phytochemical screening of major groups of secondary metabolites, such as alkaloids, anthraquinones, flavonoids, glycosides, phenolics, reducing sugars, saponins, tannins, and terpenoids, was performed.
The Folin–Ciocalteu method was used to estimate the TPC in crude extract samples.39 (link) In brief, 1 mL of 2 N Folin–Ciocalteu phenol reagent was mixed with 1 mL of 1 mg/mL plant extract (prepared by dissolving it in methanol), and then the mixture was diluted by the addition of 5 mL distilled water. After incubating for 5 minutes, 1 mL of 10% Na2CO3 solution was added, and then the mixture was incubated for 1 hour in the dark at room temperature. The absorbance of the final mixture was measured at 725 nm using a UV-visible spectrophotometer (Shimadzu, Japan). TPC was expressed as micrograms gallic acid equivalent per milligram of extract (µg GAE/mg), obtained by calibration curves of gallic acid at 500, 400, 300, 200, 100, and 50 µg/mL concentrations.
The aluminum chloride chelation method was used to approximate the amount of flavonoids in all plant extracts.40 (link) First, a stock solution (1 mg/mL) of each plant extract in methanol was diluted with water in a 1:5 ratio and mixed with 0.3 mL of 5% sodium nitrite solution. Then, the mixture was incubated for 5 minutes and 0.3 mL of 10% of AlCl3 was added to it. This was followed by the addition of 2 mL of 1 M sodium hydroxide. The absorbance of the final mixture was taken at 510 nm using a UV-visible spectrophotometer. Total flavonoid was expressed as micrograms of quercetin equivalent per milligram (µg QE/mg) of the plant extract, obtained by calibration curves of quercetin at 500, 400, 300, 200, 100, and 50 µg/mL concentrations.
The Folin–Ciocalteu method was used to estimate the TPC in crude extract samples.39 (link) In brief, 1 mL of 2 N Folin–Ciocalteu phenol reagent was mixed with 1 mL of 1 mg/mL plant extract (prepared by dissolving it in methanol), and then the mixture was diluted by the addition of 5 mL distilled water. After incubating for 5 minutes, 1 mL of 10% Na2CO3 solution was added, and then the mixture was incubated for 1 hour in the dark at room temperature. The absorbance of the final mixture was measured at 725 nm using a UV-visible spectrophotometer (Shimadzu, Japan). TPC was expressed as micrograms gallic acid equivalent per milligram of extract (µg GAE/mg), obtained by calibration curves of gallic acid at 500, 400, 300, 200, 100, and 50 µg/mL concentrations.
The aluminum chloride chelation method was used to approximate the amount of flavonoids in all plant extracts.40 (link) First, a stock solution (1 mg/mL) of each plant extract in methanol was diluted with water in a 1:5 ratio and mixed with 0.3 mL of 5% sodium nitrite solution. Then, the mixture was incubated for 5 minutes and 0.3 mL of 10% of AlCl3 was added to it. This was followed by the addition of 2 mL of 1 M sodium hydroxide. The absorbance of the final mixture was taken at 510 nm using a UV-visible spectrophotometer. Total flavonoid was expressed as micrograms of quercetin equivalent per milligram (µg QE/mg) of the plant extract, obtained by calibration curves of quercetin at 500, 400, 300, 200, 100, and 50 µg/mL concentrations.
