Tropical Journal of Pharmaceutical Research

Original Research Article | OPEN ACCESS

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

Mohammad Jamshed Ahmad Siddiqui , Zhari Ismail

Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia;

For correspondence:- Mohammad Ahmad Siddiqui Email: siddiquijamshed@hotmail.com Tel:006046563443

Received: 10 April 2010 Accepted: 14 November 2010 Published: 14 February 2011

Citation: Ahmad Siddiqui MJ, Ismail Z. Simultaneous Analysis of Bioactive Markers from Orthosiphon Stamineus Benth Leaves Extracts by Reverse Phase High Performance Liquid Chromatography. Trop J Pharm Res 2011; 10(1):97-103 doi: 10.4314/tjpr.v10i1.15

© 2011 The authors.
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Abstract

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