Cytotoxic Activity and Phytochemical Screening of Eco-Friendly Extracted Flavonoids from Pueraria montana var. lobata (Willd.) Sanjappa & Pradeep and Trifolium pratense L. Flowers Using HPLC-DAD-MS/HRMS

Increasing prospective phytochemical investigations and biological activities on Pueraria lobata and Trifolium pratense flowers exhibited their nutritional (food industry) pharmaceutical (anticancer, anti-inflammatory, anti-microbial, etc.) potentials. Previous studies paid great attention to the screening of isoflavones profile from phytochemicals. This study, in contrast, aimed at identifying the flavonoids from Pueraria lobata flowers or kudzu flower (KF) and Trifolium pratense flowers or red clover (RC) flowers and determining their cytotoxic activities on normal; (HEK-293) and cancer cell lines; human glioblastoma (A-172), osteosarcoma (HOS), embryonic rhabdomyosarcoma (Rd), lung carcinoma (A-549) and liver carcinoma (HepG2). The phytochemical screening using a high-performance liquid chromatography-diode array detector coupled with quadrupole time-of-flight mass spectrometry (HPLC-DAD-Q-TOF/MS) showed that 15 flavonoids, including isoflavones, flavones, flavonols, and flavanones, were identified in KF and 8 flavonoids, including isoflavones and flavonols, were found in RC. Nevertheless, the majority of flavonoid chemical constituents in KF or RC were found to be isoflavones (66.6%) and (62.5%), respectively. HPLC-DAD analysis following eco-friendly extraction of phytochemicals showed that KF contains mainly daidzein and genistein, while RC contains primarily formononetin and biochanin A. Cytotoxic activities evaluated according to IC50 values exhibited the most pronounced dose-dependent antiproliferative effect of KF and RC extracts were against HOS and Rd cancer cell lines, respectively. Accordingly, the morphological observation carried out using acridine orange/ethidium bromide and Giemsa stains revealed apoptotic activities and cell death in HOS and Rd cell lines when subjected to KF or RC extracts. Cytotoxic activities and apoptotic changes were pronounced among all cancer cell lines except for the control cells (HEK-293). Additionally, various polyphenols and flavonoids were identified and quantified in KF and RC extracts and exhibited potent radical scavenging activities. Overall, this study suggests that KF and RC could be valuable edible sources of flavonoids. The comprehensive flavonoid profiles for KF and RC may explain their remarkable biological activities and contribution to inducing antioxidant and cytotoxic activities against cancer cell lines.