Determination of red wine flavonoids by HPLC and effect of aging

Abstract

A new method for simultaneous determination of 10 flavonols and 2 flavones by high performance liquid chromatography was developed in this paper. The identified compounds contained quercetin, kaempferol, myricetin, rhamnetin, isorhamnetin, quercetrin, rutin, morin, galangin, fisetin, apigenin and luteolin. The chromatographic separation of these flavonoids was performed in a single run by using the mobile phase gradient elution of acetonitrile–methanol–water mixture (1% tetrahydrofuran, THF) at 20 °C, with the flow rate at 1.0 ml/min and the detection wavelength at 360 nm. With direct injection of wine samples, seven red wine samples, differing in their origin of producing places and time, were analyzed for flavonoids content by this method. The results showed the presence of myricetin, luteolin, quercetin, kaempferol, isorhamnetin and galanin. Additionally, the changes of flavonoids in red wines stored in the three types of oak barrels with aging time were investigated, which indicated that the component of flavonoids in red wine is related to wine aging greatly. These provide a substantial basis for the further research on control of flavonoids during winemaking.

Introduction

In the last decade, polyphenolic compounds have aroused great interest because of their role in assessing the quality of wine (color and taste, etc.) and their importance from a medical point of view (antioxidant, antitumoral and against coronary heart disease (CHD), etc.) (Gronbaek et al., 1995, Hertog et al., 1993a, Knekt et al., 1997, Renaud and de Lorgeril, 1992). These compounds can be classified into two kinds: flavonoids and nonflavonoids (Frankel, 1996, Hertog et al., 1993b). Flavonoids are a large family of over 4000 ubiquitous secondary plant metabolites, which can be further divided into five subclasses including flavonols, flavones, anthocyanins, catechins and flavonones (Merken & Beecher, 2000). Flavonols such as quercetin, myricetin, isorhamnetin, kaempferol and the corresponding flavones, apigenin and luteolin have been well established as potent antioxidants that prevent oxidant of low-density lipoprotein and inhibit lipid peroxidation (Formica and Regelson, 1995, Hertog and Hollman, 1996, Shahidi and Wanasundara, 1992). Evidently, different flavonoids of wines are of biological interest to a different extent. Considering the importance of biologically active flavonoids of wines, it is thus necessary to develop an accurate and rapid method for the analysis of flavonoids.

Determination of flavonols and flavones in food and beverages has been reported by means of various methods including thin layer chromatography, high-performance liquid chromatography (HPLC), gas chromatography and capillary electrophoresis (CE). HPLC analysis revealed that quercetin, myricetin and kaempferol are the major flavonol aglycones in wines (McDonald et al., 1998, Tsanova-Savova and Riharova, 2002) and apigenin and luteolin (Hertog et al., 1993b, Wang and Huang, 2004)and isorhamnetin (Garcia-Viguera & Bridle, 1995) are also present in wines. Recently, a new HPLC method was established for simultaneously determining six flavonoids containing quercetin, myricetin, kaempferol, apigenin, luteolin and galagin (Wang & Huang, 2004). Here, we developed a HPLC method by which 10 flavonols and 2 flavones can be well identified in a single experiment. These compounds contain quercetin, kaempferol, myricetin, rhamnetin, isorhamnetin, quercetrin, rutin, morin, galangin, fisetin, apigenin and luteolin. On this basis, we investigated the changes of flavonols and flavones contents in red wine stored in three different types of oak barrels with the aging time since aging in oak barrels is a very important step in red wine manufacture and great physical and chemical changes had taken place during this period (Fulcrand et al., 1996, Somers, 1971, Zafrilla et al., 2003). These might provide a substantial basis for the further research on control of flavonoids during winemaking.