Quantification on fire-processing intensity effects on selaginellae herba carbonisata quality using integrated colorimetric and chemical analysis

Objective: To investigate the quantitative correlation between color parameters and chemical component variations in Selaginellae Herba (SH) processed products under different firing intensities, integrating chromatographic and microscopic imaging techniques to establish objective quality control indicators for the traditional Chinese medicine processing theory of “preserving the herbal drug's nature after carbonization by stir-heating”. Methods: We employed a Multivariate analytical approach to analyze samples representing distinct processing stages: crude (Selaginellae Herba crude, SH-Cr), optimally carbonized (Selaginellae Herba Carbonisata, SH-Ca), insufficiently carbonized SH-Ca (ISH-Ca) and excessively‑carbonized SH-Ca (ESHCa). These samples underwent macroscopic and microscopic colorimetric analysis using CIE L*a*b* measurements. Their contents of three bioactive biflavonoids—amentoflavone, sotetsuflavone, and isocryptomerin—were quantified via high-performance liquid chromatography (HPLC). Data integration and analysis were performed using partial least squares-discriminant analysis (PLS-DA) alongside correlation studies to identify key discriminative markers. Results: The integrated analytical results demonstrated systematic changes across multiple scales during carbonization. Macroscopic and microscopic colorimetric analysis showed progressive darkening, with L* values decreasing significantly from SH-Cr to ESHCa, accompanied by tissue-specific colorimetric responses. HPLC quantification revealed distinct degradation profiles of the bioactive biflavonoids, indicating that amentoflavone exhibited greater thermal stability than sotetsuflavone, the latter of which degraded completely in ESHCa. Multivariate statistical modeling effectively discriminated processing stages (R2Y = 0.664, Q2 = 0.656), and identified microscopic color parameters, particularly epidermal cell a* values (VIP > 1.5), as more effective quality markers than macroscopic features. Conclusion: This study establishes that microscopic color parameters, particularly the a* value of epidermal cells, serve as sensitive indicators of the chemical transformations occurring during SH carbonization. These findings provide a scientific basis for utilizing microscopic colorimetry in the quality control of carbonized traditional medicines.