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.
