Discovery and Characterization of a Distinctive Theaflavin-3-Gallate Isomer from Camellia ptilophylla with Potent Anticancer Properties Against Human Colorectal Carcinoma Cells

Theaflavins, as key bioactive compounds of black tea, are garnering increasing attention. However, research predominantly focuses on theaflavin monomers derived from the enzymatic oxidation of <i>cis</i>-type catechins. In this study, we identify a unique stereoisomer of theaflavin-3-gallate (TF-3-G), named isoneoTF-3-G, in black tea from <i>Camellia ptilophylla</i> (<i>C. ptilophylla</i>), which is rich in <i>trans</i>-catechins. IsoneoTF-3-G, a characteristic theaflavin of <i>C. ptilophylla</i> black tea, is formed by the oxidation of gallocatechin gallate and catechin. It exhibits a bright orange-red color and shows an [M+H]⁺ ion at <i>m</i>/<i>z</i> 717.1449 in positive electron spray ionization-mass spectrometry. Furthermore, isoneoTF-3-G demonstrates potent inhibitory effects on the proliferation of human colorectal carcinoma HCT116 cells, with a half-inhibitory concentration of 56.32 ± 0.34 μM. This study reveals that the mitochondrial pathway is involved in the apoptosis induction of HCT116 cells by isoneoTF-3-G. Specifically, isoneoTF-3-G leads to increased reactive oxygen species in HCT116 cells, decreased mitochondrial membrane potential, and the consequent release of cytochrome <i>c</i> from the mitochondria to the cytosol, activating caspase-9 and caspase-3, which further promotes the cleavage of poly(ADPribose) polymerase. The results of this study enhance our understanding of the composition and synthesis mechanisms of theaflavins and provide foundational evidence for the further development of isoneoTF-3-G and <i>C. ptilophylla</i>.