Spectrum–Effect Relationships Between HPLC Fingerprints and In Vitro Antioxidant Activity of the Red Ginseng–Fuzi Herb Pair: A Multivariate Statistical Analysis

To establish an HPLC fingerprint of the red ginseng–Fuzi herb pair, evaluate its in vitro antioxidant activity, and investigate the spectrum–effect relationships between its chemical constituents and antioxidant effects, red ginseng and Fuzi were combined at a mass ratio of 1:2, and nine extracts were prepared by ultrasound-assisted extraction according to an L9(34) orthogonal design. HPLC–UV fingerprints were established, while antioxidant activities were assessed using DPPH radical scavenging and ferric reducing antioxidant power (FRAP) assays. Spearman correlation analysis, partial least squares (PLS) regression, and grey relational analysis were integrated to identify chromatographic peaks potentially associated with antioxidant activity. A total of 51 common peaks were matched across the nine extracts, with fingerprint similarities ranging from 0.960 to 0.999. Among the samples, S7 exhibited the highest DPPH radical scavenging activity and FRAP value, reaching 88.29% and 0.832 mmol/L Fe2+ equivalents, respectively. After false discovery rate (FDR) correction, 17 common peaks remained significantly associated with DPPH activity, with P18 and P39 showing the strongest correlations (ρ = 0.950). P46 and P44 showed relatively high correlations with FRAP, although neither remained significant after FDR correction. Integration of the three statistical approaches provided broadly consistent support for P35 (benzoylmesaconine, BMA), P44 (ginsenoside Rf), P45 (hypaconitine, HA), and P46 (ginsenoside Rb1) as candidate antioxidant-associated peaks, with P44 and P46 showing comparatively consistent evidence across methods. These findings indicate that the in vitro antioxidant activity of the red ginseng–Fuzi herb pair may involve contributions from multiple constituents and demonstrate that the integrated spectrum–effect approach may provide a useful basis for comprehensive quality evaluation and the identification of potential quality markers.