Quantitative analysis of rhinacanthins
The RRn extract was accurately weighed to 5 mg and dissolved in 10 ml methanol. The solution was filtered through 0.45 μm membrane filter and subjected to determination of rhinacanthin content by HPLC (Panichayupakaranant et al. 2009). HPLC analysis was carried out using Agilent 1100 series equipped with photodiode-array detector and autosampler (Palo Alto, U.S.A). Separation was achieved isocratically at 25 °C on a 150 mm×4.6 mm i.d. TSK-gel ODS-80Ts column (Tosho Bioscience, Japan) (Fig. 2). The mobile phase consisted of methanol and 5% aqueous acetic acid (80:20, v/v) and was pumped at a flow rate of 1 ml/min. The injection volume was 20 μl. The quantification wavelength was set at 254 nm. The calibration curves were established from the standards rinacanthin-C, -D and -N at the concentration between 12.6 – 201.0 μg/ml for rhinacanthin-C and 3.1 – 51.0 μg/ml for rinacanthin-D and -N. The linear equations of Y=40.569X+35.263 (r2=1.0000), Y=79.615X–4.168 (r2=1.0000) and Y=112.810X+18.292 (r2=0.9999) correspond to rinacanthin-C, -D and -N, respectively.