Published:Journal of Chromatographic Science,
ISSN 0021-9665 Volume
47, Number 8, September 2009, pp. 705-708
RP-HPLC Analysis of Rhinacanthins in Rhinacanthus
nasutus: Validation and Application for the Preparation of Rhinacanthin
High-Yielding Extract
P. Panichayupakaranant1, T. Charoonratana1,
and A. Sirikatitham2
1Department of Pharmacognosy and Pharmaceutical Botany, Faculty
of Pharmaceutical Sciences, Prince of Songkla University, Songkhla 90112,
Thailand,
2Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical
Sciences, Prince of Songkla University, Songkhla 90112, Thailand
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 (r2 ≥ 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.
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