UHPLC ‐ QTOF ‐ MS ‐Based Metabolic Profiling, Estimation of Antioxidants and Anticancer Potential Assessment of Dicliptera bupleuroides Nees by Caspase Activation, and ROS and MMP Evaluation by Using Fluorescence Microscopy

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Dicliptera bupleuroides has been used traditionally for treating various ailments in many countries. Its detailed phytochemical profiling and pharmacological evaluation in treating cancer based on reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) analysis along with caspase activation studies has not been done previously. This study was conducted for detailed phytochemical profiling of D. bupleuroides by using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) and to evaluate its antioxidant and anticancer potential against hepatocellular carcinoma (HepG2) cells. The extract was subjected to UHPLC-QTOF-MS analysis for metabolite profiling, and GNPS Molnet enhancer determined its major phytochemical classes. Phytochemical tests and multimode antioxidant assays were also executed. The extract was pharmacologically evaluated for its anticancer potential against HepG2 cells using CCK-8 assay. Intracellular ROS generation and loss in MMP were studied by fluorescence microscopy. Network pharmacology and molecular docking of selected compounds were executed. The extract contains total phenolic content as 58.37 ± 0.32 μg GAE/mg extract, and remarkable antioxidant potential was revealed in total antioxidant contents (TAC) and total reducing power (TRP) assays, i.e., 70.4 ± 0.4, 83.2 ± 1.9 μg ascorbic acid equilant (AAE) Borges/mg extract. The UHPLC-QTOF-MS analysis showed that the extract contains flavonoid glycosides, phenolic acids, flavans, O-methylated flavonoids, linoleic acids, terpene glycosides, triterpenoid saponins, and certain other phytoconstituents. Maximum inhibition of 77.13% ± 1.60% against HepG2 cells was observed at 24 h. Loss in MMP by JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi- dazolylcarbocyanine iodide) staining and increased ROS generation, and caspase-3, caspase-8 activation by extract significantly illustrated the cancer cell death by apoptosis. D. bupleuroides could be a potential source of safe and therapeutically active compounds that can be purified to develop safer, selective, and efficacious anticancer agents with fewer side effects.

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