*2.4. Detection of Total Flavonoid Content (TFC) of Plants*

Flavonoids are secondary metabolites having multiple physiological activities in plant development, pigmentation, and UV protection, and in defense and signaling pathways between plants and microbes [39,40]. The total flavonoid contents of *A. aspera* (aerial and root), *C. procera*, *C. gigantea* and *C. gigantea ash* parts were analyzed in water, methanol, ethyl acetate, ethanol, and hexane fractions, as shown in Figure 1. The highest TFC was found in the root-based ethyl acetate fraction (103.76 <sup>±</sup> 14.8 mg RE g−<sup>1</sup> ) and the aerial ethanol fraction (97.61 <sup>±</sup> 10.65 mg RE g−<sup>1</sup> ). Similarly, the highest TFC was found in the ethyl acetate fraction of *C. gigantea* ash (184.28 <sup>±</sup> 11.64 mg RE g−<sup>1</sup> ), followed by the ethyl acetate fractions of *C. gigantea* (112.48 <sup>±</sup> 4.28 mg RE g−<sup>1</sup> ) and *C. procera* (118.89 <sup>±</sup> 0.44 mg RE g−<sup>1</sup> ).

**Figure 1.** Total flavonoid content determination (**a**) *A. aspera* (**b**) *C. gigantea* and *C. procera*.
