Extraction and dual-phase optimization of anticancer doses of selective novel flavonoids from yangmei (Myrica rubra) in CRISPR-KO TRα cell lines of PTC: insights into Traditional Chinese Medicine for targeted therapeutic manufacturing.

Thyroid hormone receptor alpha (TRα) was assessed as a drug target in papillary thyroid carcinoma (PTC) using flavonoids from Myrica rubra. Variant screening identified 20 mutations (14 missense and 6 nonsense) associated with the development of PTC in human tissues regulated by TRα. In both TCGA and GEO datasets, PTC had elevated TRα expression and hypermethylated promoter sequence in association with poor prognostic factors (acute disease, nodal metastasis) and poor survival. In TRα knock-out PTC cell lines (CAL62, FTC133, 8505C, MB1, T3M5) using SW579 as a control (non-cancer cell line), the MTT assay demonstrated that quercetin had the most cytotoxic effect (IC50: 27.13 μM; IC80: 56.77 μM), followed by myricetin and then apigenin. The T3M5 cell line was the most sensitive to the compounds, which was corroborated by advanced ddPCR, gene sequencing, and western blot data. The quantum mechanical calculations identified quercetin as the most electronically stable flavonoid (HOMO-LUMO gap: 0.027 a.u.), followed by myricetin (0.41 a.u.) and then apigenin (0.167 a.u.). The molecular docking and the MMGBSA analysis showed that quercetin had the highest TRα binding affinity (-9.8 kcal/mol, 5 H-bonds, -101.23 kcal/mol). The molecular dynamics simulations also demonstrated that quercetin could stabilize TRα, which was measured by RMSD (the lowest value was 0.24 nm), RMSF (0.14 nm), Rg (1.66 nm), SASA (68.3 nm2), H-bonds (5.12), and PCA (1.11 nm), followed by myricetin in all respective parameters. To recapitulate, these integrated experimental and computational findings highlight quercetin as a strong cytotoxic inhibitor of PTC cell arrest in selective CRISPR-KO TRα models, ranking above myricetin and apigenin despite their having unique cytotoxicity parameters. Highlights Mutation screening of TRα as a potential therapeutic target for thyroid carcinoma.Extraction and characterization of selective novel flavonoids from Myrica rubra.MTT analysis of quercetin, myricetin, and apigenin on CRISPR-KO TRα cell lines.Quercetin resulted most significantly in all in vitro and in silico parameters, despite myricetin and apigenin also showing unique patterns of cell growth arrest properties.GES (±) cell lines were verified using ddPCR, Sanger sequencing, and Western blotting as Post-CRISPR-Cas9 KO molecular screening. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-025-04543-8.