Simultaneous Analysis of Bioactive Markers from Orthosiphon Stamineus Benth Leaves Extracts by Reverse Phase High Performance Liquid Chromatography

Purpose: To develop a reverse phase high performance liquid chromatography (RP-HPLC) method for the analysis of the crude extracts of Orthosiphon stamineus. Methods: A simple and facile analytical method was developed using RP-HPLC with UV detection for the identification and quantitation of bioactive markers present in O. stamineus extracts. Four different bioactive markers were used for the analysis, namely, rosmarinic acid, orthosiphol-A, 3’-hydroxy-5, 6, 7, 4’-tetramethoxyflavone (TMF) and 5, 6, 7, 3’, 4’-pentamethoxyflavone (sinensetin), using an isocratic mobile phase methanol: tetrahydrofuran: water (0.1% H3PO4) (55:5:40) on Nucleosil C-18 column (250 mm x 4.6 mm i.d., 5 µm particle size) at a flow rate of 0.7 ml/min and detection at 330 nm with 30 min separation time. Results: The bioactive marker orthosiphol A was identified and isolated from the water extract of O. stamineus leaves. The standard calibration curves for the marker were linear in the range 0.01 -500 µg/ml with a regression coefficient (r2) > 0.9996. The recoveries of the four markers were in the range 83.2 to 106.4 % at relative standard deviation (RSD) values 5 %. The limit of detection (LOD) and of quantification (LOQ) were 2 and 20 ng/ml, respectively. Conclusion: The developed method is simple, sensitive and specific for simultaneous determination of the indicated marker compounds either qualitatively or quantitatively, and may be used as a fingerprint profile for the standardization of extractives or herbal medicines from O. stamineus.

Keywords
Orthosiphon stamineus, Orthosiphol A, Rosmarinic acid, Sinensetin, Isocratic, Quantification, HPLC