Elsevier

Food Chemistry

Volume 93, Issue 2, November 2005, Pages 311-317
Food Chemistry

The effects of different extraction solvents of varying polarities on polyphenols of Orthosiphon stamineus and evaluation of the free radical-scavenging activity

https://doi.org/10.1016/j.foodchem.2004.09.028Get rights and content

Abstract

Leaf powder of Orthosiphon stamineus was extracted with the following solvents; distilled water, 50% aqueous methanol, methanol, 70% aqueous acetone and chloroform, at 2, 4 and 8 h, respectively, on a water bath at 40 °C. The extracts were subjected to qualitative and quantitative HPLC analyses of the polyphenols, the most dominant chemical constituents in the leaf. Chloroform extraction from 4 to 8 h at 40 °C gave the highest amount of sinensetin and eupatorin. The extraction with 70% aqueous acetone extracts at 4 and 8 h gave a high yield of 3′-hydroxy-5,6,7,4′-tetramethoxyflavone. The yield of rosmarinic acid was high in 50% methanol extracts at 2, 4 and 8 h of extraction. The extracts were screened for free radical-scavenging potential, using a 1,1-diphenyl-2-picrylhydrazyl in vitro model system. The extracts exhibited significant radical-scavenging activity and the acetone extracts showed the highest 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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