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A reversed-phase high-performance liquid chromatographic method is described for the simultaneous determination of rhinacanthin-C, rhinacanthin-D, and rhinacanthin-N in  Rhinacanthus nasutus  leaves. The method involved the use of a TSK-gel ODS-80Ts column (5 µm, 4.6 × 150 mm i.d.) with the mixture of methanol and 5% aqueous acetic acid (80:20, v/v) as the mobile phase. The parameters of linearity, repeatability, accuracy, and specificity of the method were evaluated. The recovery of the method was 94.3–100.9%, and good linearity ( r  2  ≥ 0.9999) was obtained for all rhinacanthins. A high degree of specificity as well as repeatability and reproducibility (relative standard deviation values less than 5%) were also achieved. The limit of detection and quantification of all rhinacanthins were 0.75 and 3.0 µg/mL, respectively. The solvents for extraction of rhinacanthins from  R. nasutus  leaves were examined in order to obtain the leaf extract with high rhinacanthin content. It was found that ethyl acetate was an appropriate solvent for rhinacanthin extraction. Fractionation of the ethyl acetate extract using a basic anion exchange resin (Amberlite IRA-67) eluted with 10% acetic acid in methanol afforded a rhinacanthin-rich extract (HRn). The total content of rhinacanthins was increased from 37.4% w/w to 77.5% w/w. The antifungal activities of HRn against  Trichophyton rubrum, T. mentagrophytes,  and  Microsporum gypseum  were also improved.

RP-HPLC Analysis of Rhinacanthins in Rhinacanthus nasutus : Validation and Application for the Preparation of Rhinacanthin High-Yielding Extract