*2.5. Measurement of Total Flavonoids Content (TFC)*

The total flavonoids content (TFC) was measured using the aluminium chloride colourimetric method reported by with slight modifications [25]. First, to 0.5 mL of the sample solution, 0.15 mL of NaNO2 (5%, *w*/*v*) was added. After the mixture was stirred for 5 min, 0.15 mL of (10%, *w*/*v*) AlCl3 was added. After another 5 min, 1 mL of NaOH (1 M) was added. The solution was then made up to a final volume of 5 mL by the addition of ultrapure water. After complete addition, the reaction mixture was further incubated at ambient temperature for 20 min, and the absorbance was immediately recorded at 510 nm with an UV-vis Spectrophotometer (UV-2600, Shimadzu, Kyoto, Japan). Rutin was used as a reference, and the TFC was calculated according to the regression equation: *y* = 11.2736*x* − 0.0028 (*R*<sup>2</sup> = 0.9997, final rutin concentration 18.12–72.48 μg/mL), where *y* is the absorbance, *x* is the content (μg/mL), and the TFY was calculated by the equation given below:

$$\text{Extraction yield } \left( \% \right) = \frac{\mathbb{C} \times V}{W} \times 100 \tag{2}$$

where *C* represents the TFC (mg/mL); *V* represents the total volume of extractive solution (mL); and *W* represents the dry weight of plant material (mg).
