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专业: 生物化学

[交流] HPLC前言实验内容纠错

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Tea is generally consumed in one of three forms: green, oolong, or black.
Approximately 3.0 million metric tons of dried tea is produced annually, 20% of which is green tea, 2% is oolong, and the remainder is black tea (International Tea Committee, 2002). Many studies have shown that the black tea confers great beneficial effects to the health of consumers. Theaflavins, which are dimerization products of catechins, have been considered as the main players concerning antioxidant (Frei, B. and Higdon, J. V, 2003; Yoshida et al., 1999; Rechner et al., 2002; Leung et al., 2001) , antibacterial (Friedman et al., 2006), antimutagenic (Shiraki et al., 1994), and hypocholesterolemic functions (Maron et al., 2003). Theanine, as the characteristic amino acid in tea, was shown to down-regulate cerebral function, reduce blood pressure and inhibit the excitatory effects of caffeine (Terashima et al., 1999; Yokogoshi et al., 1995; Kakuda et al., 2000) The main purine alkalois which includes Caffeine, theobromine, theophylline are reported to exhibit beneficial effects to enhance mental activity and to treat asthma(McLellan et al., 2005; Barnes et al., 2005). The latest research showed theacrine, the main purine alkalois in C. assamica var. kucha, has the properties of sedative and hypnotic (Xu et al., 2006).
Interest in investigating the health-promoting activities of black tea has created a need for analytical methods which are reliable for the simultaneous determination of the levels of these beneficial ingredients in black tea. Separation by high performance liquid chromatography (HPLC) is the most widely used method for analysis of tea flavonoids. The majority of recently published analytical HPLC methods have focused on optimization of separation of tea catechins and theaflavins. (Andrew ET AL., 2006; Bee-Lan Lee and Choon-Nam Ong, 2000; Ya et al., 2003) These methods appear to have not taken full advantage of HPLC column since it can separate only very limited active ingredients in black tea. Recently, a method developed by Friedman et al. for simultaneous analysis of seven catechins, three purine alkaloids and theaflavins in black tea is relatively comprehensive(Friedman et al., 2006) while it still couldn’t determine theacnine and GA which are also important constituents in black tea.
No methods have been carried out concerning a simultaneous analysis of all these 18 beneficial ingredents, including theanine, gallic acid, four purine alkaloids, eight catechins and four theaflavins for routine analysis of black tea. Based on our previously work using C16 column to analyze green tea (Peng et al., 2008), we developed a method using reverse-phased HPLC systems for the separation of 18 important compounds including eight catechins: (+)-Catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-catechin gallate (CG), (-)-epicatechin gallate (ECG), (-)-gallocatechin gallate (GCG), (-)-epigallocatechin gallate (EGCG); four purine alkaloids: Caffeine (CAF), Theophylline (TP), Theobromine (TB), Theacrine (TC); four types of theaflavins : theaflavin (TF), theaflavin-3-monogallate (TF-3-MG), theaflavin-3’-monogallate (TF-3’-MG), theaflavin-3,3’-digallate (TFDG) ; gallic acid and theanine in black tea ( Fig.1).
Leaves of C. ptilophylla and C. assamica var. kucha，two new resource of tea plants in China mainly contain theobromine and theacrine respectively were firstly made into black tea and analyzed. Seven kinds of world-famous black teas including Darjeeling, Assam, Nierjeeli, Keemum, Yunnan black tea, Yingde black tea and Uva were also successfully analyzed.

3.1
Various methods of sample preparation were tested to select an efficient extraction of the 18 compounds from black tea leaves. Extractions in ethanol–water at different proportion by refluxing for 30min were determined. 40% ethanol was chosen since it not only shows better extraction efficiency than other ethanol/water mixtures and showed the significant differences in the levels of TFs than water which are the most important active ingredients in black tea, however, the extraction of catechins with 40% ethanol was lower than with boiling water (Table 1). The method, extracted with boiling water at 90℃ for 30 min was also choosed since this condition was similar to an actual brewing condition.
Catechins in green tea could be obtained with UV at 280nm (H.Horie et al., 1997), on the other hand, it was reported that most HPLC methods monitored TFs at 375nm (A.Finger et al., 1992). In the present study it was observed that with the use of acetonitrile, a much higher UV absorbance could be detected at 205-210 nm instead of 280 or 375nm(Fig 2). Theanine shows no absorbance at 280 or 375 nm. As a result , 210nm was used as the detection wavelength at which all 18 compounds showed good absorption.

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