Objectives: Bidens pilosa and Trianthema portulacastrum are noteworthy weeds with a series of bioactive flavonoid constituents, hence, they can be utilized as potential health supplements and readily available sources of natural antioxidants, as well as effective constituents in medicinal applications. The current study aims to assess the anti-proliferative activity of B. pilosa and T. portulacastrum extracts using the HepG2 cell line. Methods The prepared extracts were evaluated for their cytotoxic influence and their potential CC50 in HepG2 cell lines and normal hepatocytes using the MTT assay. Using quantitative real-time polymerase chain reaction (qRT-PCR), the relative gene expression of Raf-1, MEK-1, LC3B, and Atg12 was quantified in treated cells to detect the expression levels of cell proliferation factors and autophagy-related genes. The quantification analysis of the released interleukin-1beta (IL-1β) and interleukin-1alpha (IL-1α) was also done using an ELISA assay. Results: The activities of B. pilosa extract showed an anti-proliferative influence on HepG2 cell lines upon treatment as compared to normal cells. It was assessed for cytotoxicity using molecular studies against both Raf-1 and MEK-1 as proposed anticancer mechanisms and showed promising inhibitory activity against Raf-1 and MEK-1 gene expression. Likewise, the reduction of autophagy-related genes, Atg12 and LC3B, in HepG2 cells pre-treated with B. pilosa extract, further confirmed its influence in the induction of programmed cell death (PCD). The ELISA assay revealed a substantial elevation of the pro-inflammatory cytokines IL-1α and IL-1β upon treatment. Conclusion: This study found that B. pilosa extract, without any detectable cytotoxic effects, had potential inhibitory activities against both Raf-1 and MEK-1 gene expression, and a significant reduction in autophagic machinery upon treatment.
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