2.6. Extraction and Thin Layer Chromatography (TLC) of Kaempferol and Quercetin

Kaempferol and quercetin were extracted using 100 mL liquid culture grown for 7 days. The crude extracts were isolated using different extraction chemicals; their ratios and extraction time are given in Supplementary Table S1. Crude extracts were then separated into different fractions using a 100-cm-long silica gel cylinder. The fractions were dried in a rotary evaporator and dissolved in methanol and ethanol for further analysis on TLC. TLC was performed according to the standard method described by [30], with minor modifications. The fresh solvent was used for each run as they are volatile and also kept at room temperature because increasing the temperature increases evaporation of the solvent. Approximately 5 µL of 1 mg/mL extracts and the same amount of standard were loaded on the TLC plate (20 × 20 cm thickly coated with 0.4–0.5 nm silica gel) and dried with a blow dryer; the extracts were then allowed to run in their respective mobile phases in the TLC chamber for 25 min. The developed plates were fully dried for 20 min at room temperature and then directly visualized in the TLC viewer under 366 nm UV light. The same process was repeated in triplicate. The detected bands were matched with the reference compounds of kaempferol and quercetin. The matching bands were crumb and collected individually and eluted with methanol for further assistance.