A novel zebrafish In vivo phenotype-based high-resolution biochromatography with automated melanin quantification for high-efficient screening natural anti-melanogenic constituents.

High-resolution biochromatography (HRBC) is an efficient technique which couples HPLC separation with bioactivity assays to pinpoint active compounds in herbal extracts. Conventional HRBC mainly relies on in vitro enzyme assays or cellular activity tests and therefore has limited in screening the active compounds with no definite enzyme target or whole-cell models. In this study, a zebrafish in vivo phenotype-based HRBC strategy was developed for the rapid screening anti-melanogenic constituents from the fibrous roots of Bletilla striata. HPLC eluates collected from 30 to 63 min were micro-fractionated into 96-well plates and incubated with zebrafish embryos. Melanin deposition in the zebrafish head region was quantitatively analyzed using an automated image-analysis workflow integrating deep-learning-based foreground segmentation, grayscale conversion, melanin segmentation, and morphological filtering. By correlating chromatographic retention time with melanin deposition, a zebrafish in vivo phenotype-based HRBC was established. Under optimized conditions, including a sample concentration of 5 mg/mL, a collection rate of 14 s/well corresponds to HRBC resolution of 4.29 points/min, 11 potential anti-melanogenic constituents were screened and identified by UHPLC-Q-Orbitrap-MS/MS. Two active compounds were further isolated by high-speed counter-current chromatography and semi-preparative HPLC and identified as coelonin and batatasin III by HRMS and NMR. In zebrafish validation assays, batatasin III and coelonin reduced the melanin area ratio in the head region from 19.60 ± 2.06% (blank group) to 9.03 ± 1.27% and 14.76 ± 1.44%, respectively. These two compounds have been reported to possess anti-melanogenic activity for the first time. This strategy provides a biologically relevant and sensitive approach for the rapid discovery of anti-melanogenic constituents from complex herbal matrices. It retains high-resolution activity localization while introducing a whole-organism evaluation dimension, enabling rapid and intuitive screening of active components without well-defined targets.