The effects of different extraction solvents of varying polarities on polyphenols of Orthosiphon stamineus and evaluation of the free radical-scavenging activity
Introduction
Orthosiphon stamineus (OS), Benth, (Lamiaceae) is used in Southeast Asia, for the treatment of eruptive fever, epilepsy, gallstone, hepatitis, rheumatism, hypertension, syphilis and renal calculus. In Malaysia, the tea prepared from the leaves is taken as beverage to improve health and for treatment of kidney, bladder inflammation, gout and diabetes (Hegnauer, 1966, Wangner, 1982). Lipophilic flavonoids isolated from OS showed radical-scavenging activity towards the diphenylpicrylhydrazyl radical and inhibition of 15-lipoxygenase from soybeans used as a model for mammalian 15-lipoxygenase (Lyckander & Malterud, 1996). Research indicates that the flavones, sinensetin and 3′-hydroxy-5, 6, 7, 4′-tetramethoxyflavone isolated from OS exhibited a diuretic activity in rats after intravenous administration of 10 mg/kg body weight and, therefore, the diuretic effect of OS extracts could be partially due to its lipophilic flavones content (Schut & Zwaving, 1993).
O. stamineus contains several chemically active constituents, such as terpenoids (diterpenes and triterpenes), polyphenols (lipophilic flavonoids and phenolic acids), and sterols (Tezuka et al., 2000). The therapeutic effects of OS have been ascribed mainly to its polyphenol, the most dominant constituent in the leaf, which has been reported to be effective in reducing oxidative stress by inhibiting the formation of lipid peroxidation products in biological systems (Hollman & Katan, 1999). Caffeic acid derivatives, including rosmarinic and 2,3-dicaffeoyltartaric acids, are the most abundant polyphenols in an aqueous methanol extract of OS leaf, which predominate over polymethoxylated flavones. The polymethoxylated flavones, principal flavonoid aglycones, present in OS leaf are unique, with a methoxy group at C-5, a structural feature rare in flavonoids.
The stability of different extracts from the same material depends on the extraction solvent used for removal of the polyphenolic compounds, and it is apparent that extracts from the same plant material may vary widely with respect to their antioxidant concentrations and activities. Therefore, the aim of this study was to identify the effect of different extraction solvents of varying polarities on three main polymethoxylated flavones in O. stamineus leaves, sinensetin (SEN), eupatorin (EUP), 3′-hydroxy-5,6,7,4′-tetramethoxyflavone (TMF) and the major phenolic acid, rosmarinic acid (RA) and scavenging effects of these extracts on free radical species.
Section snippets
Chemicals and reagents
Standard compounds of sinensetin, eupatorin, 3′-hydroxy-5,6,7,4′-tetramethoxyflavone and rosmarinic acid were purchased from Indofine Chemical Co. (Hillsborough, NJ, USA). Solvents used for chromatography were tetrahydrofuran, methanol (HPLC grade), water (HPLC grade), ethanol (80%) obtained from Merck (Darmstadt, Germany). 1,1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma Chemical Co. (St. Louis, MO). Membrane filters (0.45-μm pore size) from Millipore were used for filtration of
HPLC analysis
In the present study, all the markers (Fig. 1) were determined in a single run of HPLC by using the isocratic condition of methanol:water:tetrahydrofuran (45:50:5 v/v). The markers were separated within a total time of 30 min (Fig. 2). The peaks of RA, EUP, TMS and SEN were confirmed by comparison of their retention times with reference standards. The standards were resolved and eluted at 5.6, 13.3, 17.8 and 25.9 min with respect to RA, TMF, SEN and EUP (Fig. 2). The markers were shown to
Conclusion
Different solvent systems of varying polarities were used for the extraction of major polyphenols in OS leaves. The chloroform extraction at 2, 4 and 8 h gave the highest amount of SEN and EUP. The extraction with 70% aqueous acetone at 4 and 8 h gave a higher yield of TMF and the yield of RA was higher in 50% aqueous methanol extracts at 8 h of extraction on a water bath at 40 °C. The solvent extraction systems and the HPLC method developed in this study could be applied to analyse
Acknowledgements
This study was supported by Intensifying Research Priority Areas (IRPA) Grant from the Ministry of Science, Technology, and Environment, Malaysia.
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