Simultaneous Determination of Caffeine and Some Selected

Article pubs.acs.org/JAFC

Simultaneous Determination of Caffeine and Some Selected Polyphenols in Wuyi Rock Tea by High-Performance Liquid Chromatography Feng Zhao,†,§ He-Tong Lin,*,† Shen Zhang,† Yi-Fen Lin,† Jiang-Fan Yang,# and Nai-Xing Ye⊥ †

Institute of Postharvest Technology of Agricultural Products, College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China § Fujian Inspection and Research Institute for Product Quality, Fuzhou, Fujian 350002, China # College of Tea and Food Science, Wuyi University, Wuyishan, Fujian 354300, China ⊥ College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China ABSTRACT: The primary taste and healthy benefits of tea are mainly attributed to tea polyphenols and caffeine. Due to very many kinds of flavonoid glycosides in tea and the lack of commercial standards of flavonoid glycosides, it is critical to develop a rapid and cheap method for determining flavonoid glycosides of tea. Contents of myricetin glycosides and quercetin glycosides in Wuyi Rock tea were determined by detecting contents of corresponding myricetin and quercetin. Optimizing hydrolysis conditions for hydrolyzing flavonoid glycosides to their corresponding flavonols including quercetin and myricetin in Wuyi Rock tea was a key technology for detecting contents of corresponding myricetin and quercetin. The results showed that hydrolysis at 2 mol/L HCl solution and at 90 °C for 1 h was an optimizing condition for hydrolyzing flavonoid glycosides to myricetin and quercetin in Wuyi Rock tea. Caffeine and seven kinds of polyphenols (GA, EGC, C, EGCG, EC, ECG, and CGA) in 20 samples of Wuyi Rock tea were simultaneously determined using a simple and fast reverse-phase high-performance liquid chromatography procedure coupled with photodiode array detector (RP-HPLC-PDAD). The results indicated that there were significant (P < 0.05) differences of ECG, CGA, ECG, and myricetin glycosides in ‘Wuyi Rougui’ and ‘Wuyi Shuixian’, which were credited with causing the difference in taste between these two cultivar of Wuyi Rock tea. The study may be useful for clarifying the cause of “cultivated varieties flavor” of Wuyi Rock tea. KEYWORDS: Wuyi Rock tea, tea polyphenols, caffeine, high-performance liquid chromatography, simultaneous determination

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in different tea varieties and grades.13−15 The main classes of tea polyphenols are catechins, flavonols and flavonol glycosides, phenolic acids, and depsides. Generally speaking, catechins are the primary polyphenols of tea. Moreover, the principal naturally occurring catechins in tea leaves are epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), gpicatechin (EC), gallocatechin (GC), and catechin (C).16 They can be determined by high-performance liquid chromatography (HPLC).4,17−25 There is also an ISO method available to give a standardized processing to determine the content of catechins and caffeine with a reverse phase chromatography column and a corresponding isocratic elution system. In addition, the contents of flavonols and their glycosides of tea are next to those of catechins of tea polyphenols. Major flavonols of tea are quercetin, kaempferol, and myricetin, which affect not only the astringent taste but also the color of tea infusion.15,26 Notably, flavonols of tea are usually bound to sugars as O-glycosides, such as quercetin-3-Oglucoside, quercetin-3-O-galactoside, quercetin-3-O-rutinoside, and kaempferol-3-O-rutinoside,27 which can be measured directly by thin-layer chromatography or HPLC system.20,28−31

INTRODUCTION Tea is one of the most popular beverages all over the world. The tea plant (Camellia sinensis L.) is commercially grown in many countries, including China, India, Sri Lanka, and Kenya. These countries account for most of the world’s tea production. According to the manufacturing process, tea can be classified into green tea (nonfermented), white tea (slightly fermented), oolong tea (semi fermented), black tea (fully fermented), and pu-erh tea (postfermented). Wuyi Rock tea, a kind of oolong tea, is one of the 10 top teas in China. ‘Wuyi Rougui’ and ‘Wuyi Shuixian’ are the two primary cultivars of Wuyi Rock tea production. Teas made from these two cultivated varieties, which are manufactured by the same processing technology, are characterized by distinct flavors named “cultivated varieties flavor”. Specifically, ‘Wuyi Rougui’ stands out from other teas for its excellent sharp but pleasingly bitter taste, with a distinctive cinnamon-like aroma and a sweet, milk-like aftertaste. ‘Wuyi Shuixian’ has a woody aroma with a slightly flower-like fragrance, and it tastes smooth with quite long and deep wood and nut flavors. The primary taste and healthy benefits of tea are mainly attributed to tea polyphenols and caffeine. Many studies have proved that tea polyphenols have stimulative effects on antioxidant and anti-inflammatory activities as well as preventive effects against degenerative diseases.1−12 Meanwhile, the specific compositions of tea polyphenols are quite different © 2014 American Chemical Society

Received: Revised: Accepted: Published: 2772

December 15, 2013 February 26, 2014 March 13, 2014 March 13, 2014 dx.doi.org/10.1021/jf4056314 | J. Agric. Food Chem. 2014, 62, 2772−2781

Journal of Agricultural and Food Chemistry

Article

There are also some research studies concerning hydrolyzing flavonol glycosides into their corresponding flavonols and determining them in an indirect way.32 Gallic acid (GA) and chlorogenic acid (CGA), the main phenolic acids and depsides of tea, although quite low in contents relative to other phenolic acids and depsides such as isochlorogenic acid, p-coumaric acid, p-coumaric quinovic acid, and caffeic acid, were detected by Yin et al.13 The pretreatment and evolution system of ISO14502-2 (“determination of content of catechins in green tea − using the method of HPLC) is a promising method for routine analysis of catechins in tea. In addition, the determination of caffeine content of tea by HPLC was reported by Lin et al.,17 Blahova and Lehotay,21 and Prayong et al.24 Previous studies reported that caffeine and polyphenols such as (−)-gallocatechin, (−)-epigallocatechin, (−)-epigallocatechin gallate, (−)-epicatechin, gallocatechin gallate, and (−)-epicatechin gallate in oolong tea, which was obtained from three main production sites in China, that is, Fujian, Taiwan, and Guangdong, were simultaneously determined using HPLC.20 In addition, five catechins in green tea,25 catechins and caffeine in green tea leaves,22 or caffeine and catechins in tea extracts23 were simultaneously determined using HPLC. However, little information is available on the simultaneous determination of caffeine and polyphenols in Wuyi Rock tea. In this paper, the method of high-performance liquid chromatography coupled with photodiode array detector (HPLC-PDAD) at three different wavelengths [detection wavelength at 278 nm (Det.278 nm), reference wavelength at 360 nm (ref.360 nm); detection wavelength at 330 nm (Det.330 nm), reference wavelength at 380 nm (ref.300 nm); and detection wavelength at 370 nm (Det.370 nm), reference wavelength at 290 nm (ref.290 nm)] for the simultaneous determination of caffeine (CAF), catechins (EGC, C, EGCG, EC, GCG, ECG), and polyphenols (GA, CGA, myricetin, myricetin glycoside, quercetin, quercetin glycoside) in ‘Wuyi Rougui’ and ‘Wuyi Shuixian’, the two major cultivars of Wuyi Rock tea, was developed. The objective of this study was to evaluate the tea quality of ‘Wuyi Rougui’ and ‘Wuyi Shuixian’ and provide the method for clarifying the cause of “cultivated varieties flavor” of Wuyi Rock tea.

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Table 2. Linear Range, Linear Equation, Correlation Coefficient (r), and Limit of Quantification (LOQ) of Standard Compounds

factors hydrolysis time (h)

1 2 3

1 2 3

70 80 90

0.5 1.0 1.5

0−100 0−200 0−200 0−100 0−150 0−200 0−200 0−200 0−200 0−100 0−100

Y Y Y Y Y Y Y Y Y Y Y

= = = = = = = = = = =

39.80X + 27.03 1.82X − 3.69 1.45X + 0.86 24.13X − 6.45 42.86X − 235.03 11.33X − 29.49 7.23X − 1.48 13.05X − 14.51 16.27X + 21.84 4.47X − 5.81 11.94X + 17.01

r

LOQb (mg/L)

0.999 0.999 0.999 0.999 0.962 0.996 0.999 0.999 0.999 0.999 0.995

0.012 0.317 0.345 0.021 0.015 0.043 0.064 0.038 0.030 0.431 0.121

quercetin, CGA, and GA were obtained from Chengdu Must Biotechnology Co., Ltd. (China). Caffeine was purchased from Sigma Chemical Co. Ltd. (USA). HCl, acetic acid, vitamin C, and ethylenediaminetetraacetic acid (EDTA) were manufactured by Sinopharm Chemical Reagent Co., Ltd. (China). The above reagents were of analytical grade. Acetonitrile and methanol, of HPLC grade, were the products of Merck Co. Ltd. (Germany). The water used was redistilled and ion free. Selection of Detection Wavelength. Stock solutions (1000 μg/ mL) of each standard were prepared individually by dissolving the required weight of each compound in corresponding solutions. Further diluted solutions (20 μg/mL) of each standard were also prepared individually and then injected into HPLC separately to get their retention time and spectrogram from 200 to 400 nm. After the comparison of each spectrum, the detection wavelength and its reference wavelength were selected. Elution Program. The analyses of polyphenols and caffeine in tea were carried out by the following HPLC method. An Agilent 1200 series liquid chromatography system (Agilent Corp., USA), comprising a vacuum degasser (part G1322A), a quaternary pump (part G1311A), an autosampler (part G1313A), a column oven compartment (part G1316A), and a photodiode array detector (PDAD) (part G1314A), was used. The column was C18 reverse-phase Zorbax Ecllpse XDB-18 (5 μm, 4.6 mm × 250 mm, P/N 990967-902). The elution program was according to method ISO 14502-2 with some modifications. Two mobile phases were employed in gradient HPLC elution. Mobile phase A was prepared with 90 mL of acetonitrile, 20 mL of acetic acid, and 2.0 mL of EDTA water solution (10 mg/mL) and subsequently made up to a volume of 1000 mL with water. Mobile phase B was mixed with 800 mL of acetonitrile, 20 mL of acetic acid, and 2.0 mL of EDTA water solution (10 mg/mL) and subsequently made up to a volume of 1000 mL with water. Both mobile phases (A and B) should be filtered through a 0.45 μm filter before use. Column oven temperature was set at 35 °C. The flow rate of the mobile phase was 1.0 mL/min. The injection volume was 20 μL. All of the above paremeters were concordant with the ISO 14502-2 method. To get a better separation, there were some changes in gradient elution program as follows: 100% (v/v) mobile phase A for 10 min, then (over another 10 min) a linear gradient to 80% (v/v) mobile phase A and 20% (v/v) mobile phase B, then (over 5 min) a linear gradient again to 50% (v/v) mobile phase A and 50% (v/v) mobile phase B, held at this composition for 8 min. Finally, it was reset to 100% (v/v) mobile phase A and allowed to equilibrate before the next injection. Standard Preparation. GA (10 mg), EGC (20 mg), C (20 mg), CGA (10 mg), CAF (15 mg), EGCG (20 mg), E (20 mg), GCG (20 mg), ECG (20 mg), myricetin (10 mg), and quercetin (10 mg) were weighed and mixed together. Then the mixture was dissolved in a 20

Table 1. Factors and Levels of Orthogonal Experiment hydrolysis temp (°C)

GA EGC C CGA CAF EGCG EC GCG ECG myricetin quercetin

linear eqa

Y stands for the area of the peak. X stands for the amount of corresponding compound, mg/L. bSignal to noise ratio (S/N) is 10.

MATERIALS AND METHODS

level

linear range (mg/L)

a

Samples. Wuyi Rock teas (C. sinensis L.), including 10 ‘Wuyi Rougui’ samples and 10 ‘Wuyi Shuixian’ samples, were obtained from

concn of HCl solution (mol/L)

compd

Wuyishan Jiejieqing Tea Co., Ltd., in Fujian province, China; Wuyishan Xiangjiang Tea Co., Ltd., in Fujian province, China; Wuyishan Qinpin Tea Co., Ltd., in Fujian province, China; Wuyishan Yanshang Tea Co., Ltd., in Fujian province, China; Wuyishan Huahui Tea Co., Ltd., in Fujian province, China; Wuyishan Mangtingfeng Tea Co., Ltd., in Fujian province, China; Wuyishan Kaimingxuan Tea Co., Ltd., in Fujian province, China; and Wuyishan Qingpao Tea Co., Ltd., in Fujian province, China. Chemicals. EGC, C, EGCG, E, GCG, and ECG were purchased from Shanghai Ronghe Pharmacy Co., Ltd. (China). Myricetin, 2773

dx.doi.org/10.1021/jf4056314 | J. Agric. Food Chem. 2014, 62, 2772−2781


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according to the above extraction procedure. The collected supernatants from the above extraction were mixed, then diluted to 10 mL with 70% (v/v) aqueous methanol, and filtered through a 0.22 μm filter. The filtrate was collected for the following analysis. Method of Hydrolyzing Flavonol Glucosides to Flavonols in Wuyi Rock Tea. As flavonol glucosides present as O-glycosides with sugars bound at the C3 position and they can be hydrolyzed into their corresponding flavonols,32 the quantity of flavonols in the hydrolyzed solution was used to assess the flavonol glucosides in tea sample according to the method of Wang and Helliwell.32 There were two steps for preparing the sample of hydrolyzed solution from flavonol glucosides in Wuyi Rock tea. The first step was 5 mL of filtrate of tea sample extraction, which was prepared by using the procedure of Extraction Preparation of Sample of Wuyi Rock Tea above, transferred into a tube, and then mixed with 5 mL of HCl solution at different concentrations on the basis of the following test design. The second step was the mixture bathed with different combinations of temperature and time span according to the following test design and then cooled rapidly to 20 °C, after which 70% (v/v) aqueous methanol was added to 25 mL. The hydrolysis conditions for flavonoid glycosides including concentration of HCl solution, hydrolysis temperature, and hydrolysis time are shown in the following Single-Factor Test and Orthogonal Experiment. All hydrolyzed solutions should be filtered through 0.22 μm filters before injection into the HPLC. Single-Factor Test. Effects of different concentrations of HCl solution, hydrolysis temperatures, and hydrolysis times on the content of flavonols and the yield of hydrolysate from flavonol glucosides were conducted, respectively. Specific designs were as follows: the experiment at different concentrations of HCl solution (0, 1.5, 3.0, 4.5, 6.0, 7.5, and 9.0 mol/L) on the flavonol content from the hydrolysis of flavonol glucosides was conducted by hydrolyzing at 80 °C for 1 h, respectively, whereas the experiment of different hydrolysis temperatures (60, 70, 80, 90, and 100 °C) was conducted using 1.5 mol/L HCl solution and hydrolyzing for 1 h, respectively. In addition, the experiment of different hydrolysis times (0, 0.5, 1, 1.5, 2, and 2.5 h) was also conducted using 1.5 mol/L HCl solution and hydrolyzing at 80 °C, respectively. Orthogonal Experiment. On the basis of the result of the above single-factor test, selection of three factors and three levels of orthogonal experiment is shown in Table 1. The test plan of an orthogonal experiment [L9(3)4] design for optimizing parameters of hydrolyzing flavonol glucosides to corresponding flavonols in Wuyi Rock tea is shown in Table 4. Reliability and Quantification of Caffeine and Some Selected Polyphenols in Samples of Wuyi Rock Tea. The UV spectrum of each standard compound after subtraction of the corresponding UV base spectrum was normalized automatically using a computer, and the plots were superimposed. Peaks were considered to be chromatographically pure when there was exact coincidence to their corresponding UV spectra. Chromatographic peaks in the samples were indentified by comparing their retention times and UV spectra with those of the reference standards. The linear relationship, limit of quantification (LOQ), recovery, and relative standard deviation (RSD) are shown in Tables 2 and 3 to evaluate the quality of this method. EGC, C, EGCG, EC, GCG, ECG, GA, CGA, myricetin, quercetin, and caffeine were detected and calculated from the sample extraction directly without hydrolysis. The myricetin glycoside and quercetin glycoside were detected in an indirect way by quantitating myricetin and quercetin in sample extraction after hydrolysis. Statistical Analyses. All statistical analyses, including the design of orthogonal test and independent-sample t test, were carried out with PASW (IBM SPSS Statistics) statistical software (version 19.0).

Table 3. Spiked Recoveries and Relative Standard Deviations (RSDs) of Standard Compounds (n = 3) spiked (g/kg)

recovery (%)

RSD (%)

GA

compd

5 25 50

98.73 97.51 98.90

3.86 2.19 1.93

EGC

5 25 50

97.40 98.92 98.20

3.48 2.27 2.37

C

5 25 50

98.67 99.57 98.13

3.57 2.72 1.26

CGA

5 25 50

98.20 97.75 97.60

3.73 2.09 2.34

CAF

5 25 50

98.93 98.41 98.20

1.11 0.71 1.27

EGCG

5 25 50

99.60 99.43 100.30

2.41 1.79 1.44

EC

5 25 50

99.33 98.30 98.37

2.33 1.50 0.65

GCG

5 25 50

100.10 99.44 99.07

3.21 2.56 1.71

ECG

5 25 50

99.80 99.43 98.70

2.63 1.89 1.41

myricetin

5 25 50

81.13 82.41 83.11

4.52 3.79 3.62

quercetin

5 25 50

88.73 89.89 92.10

4.17 3.14 3.00

mL one-mark volumetric flask with methanol to form the stock mixture solution. Five milliliters of the stock solution was dispensed to 25, 50, and 100 mL one-mark volumetric flasks, respectively. All of these flasks were diluted with mobile phase to form working standard solutions A, B, and C and then injected into the HPLC. The peak area responses were obtained. Each peak was identified by comparing their retention time and UV spectra with those of the respective standard. A standard graph for each component was prepared by plotting concentration versus peak area. Extraction Preparation of Sample of Wuyi Rock Tea. The method of extraction from Wuyi Rock tea was used according to the method of ISO 14502-2 with some modifications. Tea leaves (0.2 g) were mixed with 5 mL of 70% (v/v) aqueous methanol at 70 °C by a vortex mixer. After bathing at 70 °C for 10 min, the tube was removed, allowed to cool, and then centrifuged for 10 min at 3500g and 4 °C, and the supernatant was collected. The precipitation was extracted

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RESULTS AND DISCUSSION Chromatographic Performance of Working Standard Solution. HPLC of caffeine and some selected tea polyphenols in their working standard solution at various concentrations was 2774



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Figure 1. HPLC chromatogram of the working standard solution C mixture containing 25 μg/mL GA (1), 50 μg/mL EGC (2), 50 μg/mL C (3), 25 μg/mL CGA (4), 37.5 μg/mL CAF (5), 50 μg/mL EGCG (6), 50 μg/mL EC (7), 50 μg/mL GCG (8), 50 μg/mL ECG (9), 25 μg/mL myricetin (10), and 25 μg/mL quercetin (11) at different wavelengths under the experimental conditions. (A) GA (1), EGC (2), C (3), CAF (5), EGCG (6), EC (7), GCG (8), and ECG (9) were detected at acquisition wavelength of 278 nm and at reference wavelength of 360 nm. (B) CGA (4) was detected at acquisition wavelength of 330 nm and at reference wavelength of 380 nm. (C) Myricetin (10) and quercetin (11) were detected at acquisition wavelength of 370 nm and at reference wavelength of 290 nm.

C, CAF, EGCG, EC, GCG, and ECG were detected at 278 nm with the reference wavelength at 360 nm. CGA was detected at 330 nm with the reference wavelength at 380 nm. Myricetin and quercetin were detected at 370 nm with the reference wavelength at 290 nm. Reliability of the Method. The linear range, linear equation of the working curve, coefficient of correlation (r), and LOQ for each standard are shown in Table 2. The results

recorded. There were negative peaks in a single wavelength. Therefore, it was important to select the proper wavelength for detection. Figure 1 shows the detection wavelength, reference wavelength, and retention time for each compound of standard solution C. These were slightly overlapping near C (3) and CGA (4), EGCG (6), and EC (7), whereas separation of other compounds performed well at corresponding wavelengths. Considering the sensitivity for quantitative determination, GA, 2775



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lysates from flavonol glucosides, could be influenced by hydrolysis. The mean recoveries and RSDs of the method for determination of myricetin and quercetin in Wuyi Rock tea were 82.1−92.1% and within the range of ±4.5% (n = 3), respectively. By multiplying their corresponding recoveries as the correction coefficient with quantificaiton, more reliable results could be obtained. The above results confirmed that the development of the method in this study was an accurate and precise method for the determination of caffeine and selected polyphenols in Wuyi Rock tea. Factors Affecting Hydrolysis of Flavonol Glucosides to Flavonols in Wuyi Rock Tea. Results of Single-Factor Test. 1. Concentrations of HCl Solution. Figure 2A shows that both myricetin content and quercetin content increased sharply with the concentration of HCl solution from 0 to 1.5 mol/L HCl and reached peak values at 1.5 mol/L HCl, whereas the contents of myricetin and quercetin both decreased gradually with increasing concentration of HCl solution from 1.5 to 9.0 mol/L. These results indicated that the concentration of 1.5 mol/L HCl solution was an appropriate concentration of HCl for hydrolyzing flavonol glucosides to flavonols in Wuyi Rock tea at 80 °C for 1 h. 2. Hydrolysis Temperature. As shown in Figure 2B, contents of myricetin and quercetin in Wuyi Rock tea exhibited a slight change when the hydrolyzing temperature was raised from 60 to 70 °C, whereas both myricetin content and quercetin content increased rapidly with increasing hydrolysis temperature from 70 to 80 °C, reaching peak values at 80 °C and then decreasing sharply with increasing hydrolysis temperature from 80 to 100 °C. These results suggested that 80 °C was a proper hydrolysis temperature for hydrolyzing flavonol glucosides to flavonols in Wuyi Rock tea at a concentration of 1.5 mol/L HCl solution for 1 h. 3. Hydrolysis Time. Quercetin content in Wuyi Rock tea exhibited a sharp increase from 0 to 0.5 h of hydrolysis, a slight decrease from 0.5 to 1 h, a gradual decrease from 1 to 2 h, and a rapid decrease from 2 to 2.5 h (Figure 2C). These results showed that 0.5−1 h of hydrolysis might be a proper hydrolysis time for hydrolyzing flavonol glucosides to quercetin in Wuyi Rock tea at 80 °C and a concentration of 1.5 mol/L HCl solution. As shown in Figure 2C, myricetin content in Wuyi Rock tea exhibited a rapid increase from 0 to 0.5 h of hydrolysis, but almost no change of myricetin content from 0.5 to 1 h, and then showed a gradual decrease from 1 to 2.5 h. From the above results it could be concluded that 0.5−1 h of hydrolysis might be a suitable hydrolysis time for hydrolyzing flavonol glucosides to myricetin in Wuyi Rock tea at 80 °C and a concentration of 1.5 mol/L HCl solution. Results of Design of Orthogonal Experiment. The hydrolysis conditions, including concentration of HCl solution, hydrolysis temperature, and hydrolysis time, for flavonoid glycosides to myricetin and quercetin, which were based on an orthogonal experiment L9(3)4 design, were determined. As shown in Table 4, the order of important factors of the hydrolysis conditions for hydrolyzing flavonoid glycosides to myricetin in Wuyi Rock tea was hydrolysis temperature (factor B), concentration of HCl (factor A), and hydrolysis time (factor C). In addition, Table 4 also shows that hydrolysis at a concentration of 2 mol/L HCl solution and at 70 °C for 1.5 h was the most beneficial hydrolysis condition for hydrolyzing flavonoid glycosides to myricetin in Wuyi Rock tea.

Figure 2. Effect of three factors on the hydrolysis of the flavonols: (A) HCl concentrations for hydrolysis; (B) hydrolysis temperature; (C) hydrolysis time.

indicated that there were low limits of quantification (LOQs) for GA, CGA, CAF, EGCG, EC, GCG, ECG, and quercetin with the range of these being 0.01−0.15 mg/L. Relatively speaking, higher LOQs for EGC, C, and myricetin in the range of 0.30−0.45 mg/L were observed. The correlation coefficient (r) was >0.96 in the analysis of each compound. As shown in Table 3, recoveries were determined in triplicate in Wuyi Rock tea by spiking pure standards to the extraction solution prior to the sample analysis. The mean recoveries and RSDs of the method for determination of GA, EGC, C, CGA, EGCG, EC, and GCG were 97.5−100.3% and within the range ±3.9% (n = 3), respectively. The method of indirect determination for the quantification of myricetin and quercetin, which are flavonols, the hydro2776



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Table 4. Test Plans and Results of Range Analysis Based on an Orthogonal Experiment L9(3)4 Design for Hydrolyzing Flavonol Glucosides to Corresponding Flavonols in Wuyi Rock Tea factors expt 1 2 3 4 5 6 7 8 9 myricetin (μg/mL) k1 k2 k3 R superior level important order of factor quercetin (μg/mL) k1 k2 k3 R superior level important order of factor

results

factor A: concn of HCl solution (mol/L)

factor B: hydrolysis temperature (°C)

factor C: hydrolysis time (h)

error analysis

myricetin (μg/mL)

quercetin (μg/mL)

1 1 1 2 2 2 3 3 3

70 80 90 70 80 90 70 80 90

0.5 1.0 1.5 1.0 1.5 0.5 1.5 0.5 1.0

1 2 3 3 1 2 2 3 1

36.7 29.0 48.8 64.3 41.6 49.5 56.5 42.5 47.7

28.3 38.0 46.4 70.8 73.8 78.6 66.5 67.3 87.0

41.2 55.9 52.7 14.7

56.7 40.7 52.5 15.9

46.3 50.7 52.9 6.6

A2 B>A>C

B1

C3

28.7 56.8 56.1 28.1

42.1 45.5 53.9 11.8

44.3 49.8 47.5 5.5

A2 A>B>C

B3

C2

Table 5. ANOVA of Myricetin and Quercetin Output after Hydrolysis factor

variance

df

F value

myricetin

concn of HCl hydrolysis temp hydrolysis time error

360.3 409.2 68.1 626.4

2 2 2 2

0.575 0.653 0.109

quercetin

concn of HCl hydrolysis temp hydrolysis time error

1546.3 221.4 46.2 3.8

2 2 2 2

Table 4 shows that the order of important factors of the hydrolysis conditions for hydrolyzing flavonoid glycosides to quercetin in Wuyi Rock tea was concentration of HCl (factor A), hydrolysis temperature (factor B), and hydrolysis time (factor C). In addition, Table 4 also shows that hydrolyzing at a concentration of 2 mol/L HCl solution and at 90 °C for 1 h might be the best hydrolysis condition for hydrolyzing flavonoid glycosides to quercetin in Wuyi Rock tea. Further analysis of variance (ANOVA) indicated that there were no significant (P > 0.05) differences among different concentrations of HCl solution for hydrolyzing flavonoid glycosides to myricetin in Wuyi Rock tea; the same results were also found among different hydrolysis temperatures or among different hydrolysis times (Table 5). However, there were significant (P < 0.05) differences among different

402.0 57.5 12.0

F critical value

signif (P < 0.05)

19.0 19.0 19.0

19.0 19.0 19.0

* *

concentrations of HCl solution or among different hydrolysis temperatures for hydrolyzing flavonoid glycosides to quercetin in Wuyi Rock tea, whereas there were no significant (P > 0.05) differences among different hydrolysis times (Table 5). From the above results it could be concluded that the best hydrolysis conditions (hydrolyzing at a concentration of 2 mol/ L HCl solution and at 90 °C for 1 h) for hydrolyzing flavonoid glycosides to quercetin in Wuyi Rock tea could be considered as the best hydrolysis conditions for hydrolyzing flavonoid glycosides to their corresponding flavonols including quercetin and myricetin in Wuyi Rock tea. Typical Chromatographic Performance of Wuyi Rock Tea Sample. As shown in Figure 3A,B, a typical chromatogram of tea polyphenols and caffeine in nonhydrolyzed extraction of Wuyi Rock tea sample was observed at different 2777



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Figure 3. Typical chromatogram of tea sample extraction at different wavelengths under the experimental conditions. The sample contains GA (1), EGC (2), C (3), CGA (4), CAF (5), EGCG (6), EC (7), GCG (8), ECG (9), myricetin (10), and quercetin (11). (A) Tea sample extraction without hydrolysis was detected at acquisition wavelength of 278 nm and at reference wavelength of 360 nm. (B) Tea sample extraction without hydrolysis was detected at acquisition wavelength of 330 nm and at reference wavelength of 380 nm. (C) Tea sample extraction without hydrolysis was detected at acquisition wavelength of 370 nm and at reference wavelength of 290 nm. (D) Tea sample extraction after hydrolysis was detected at acquisition wavelength of 370 nm and at reference wavelength of 290 nm. 2778



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Table 6. Contents of Tea Polypheols and Caffeine in ‘Wuyi Rougui’ and ‘Wuyi Shuixian’ Samples ‘Wuyi Rougui’ (g/kg) compd GA EGC C CGA CAF EGCG EC GCG ECG myricetin glycosides myricetin quercetin glycosides quercetin a

range

av

1.17−2.02 1.50 41.20−61.61 48.34 0−11.69 5.98 0.54−0.85 0.65 22.82−27.26 24.58 59.32−81.04 69.46 3.58−6.55 5.58 0.58−1.13 0.80 8.80−10.78 9.47 2.82−4.91 3.88 none detected 1.19−1.99 1.64 none detected

‘Wuyi Shuixian’ (g/kg) SD 0.24 6.66 4.54 0.10 1.48 6.65 0.97 0.14 0.72 0.61 0.25

range

av

0.91−1.91 1.40 21.08−30.56 26.12 0−12.72 4.58 0−1.68 0.28 19.34−30.22 23.16 57.83−88.49 72.14 3.73−6.98 5.41 0−1.75 0.83 6.44−9.82 8.09 1.00−2.61 1.76 none detected 1.46−2.73 1.86 none detected

asymp signif (2-tailed)a SD

Mann− Whitney U

Kolmogorov−Smirnov Z

0.37 3.03 4.46 0.60 3.18 9.68 1.00 0.68 0.98 0.40

0.353 0.000* 0.613 0.023* 0.165 0.631 0.529 0.971 0.004* 0.000*

0.400 0.000* 0.759 0.003* 0.164 0.988 0.400 0.400 0.003* 0.000*

0.41

0.529

0.759

*, P < 0.05.

sets of Wuyi Rock tea. The results indicated that both the Mann−Whitney U test and Kolmogorov−Smirnov test showed significant (P < 0.05) differences of ECG, CGA, ECG, and myricetin glycosides in ‘Wuyi Rougui’ and ‘Wuyi Shuixian’, which might be credited with causing the different tastes between these two cultivar sets of Wuyi Rock tea. However, there were no significant (P > 0.05) differences of GA, C, CAF, EGCG, EC, GCG, and quercetin glycosides in ‘Wuyi Rougui’ and ‘Wuyi Shuixian’ (Table 6). It also could be found from this effort that contents of C, GCG, and CGA in ‘Wuyi Rougui’ and ‘Wuyi Shuixian’ were at low levels (Table 6). In addition, some peaks of other unknown substances overlapped slightly with the peak of CGA (Figure 3B). The above observation might result in the accuracy of the quantification of C, GCG, and CGA in Wuyi Rock tea. In conclusion, due to the very many kinds of flavonoid glycosides in tea and the lack of commercial standards of flavonoid glycosides, it is critical to develop a rapid and cheap method for determining the flavonoid glycosides of tea. The contents of myricetin glycosides and quercetin glycosides in Wuyi Rock tea could be determined by detecting the content of corresponding myricetin and quercetin. Developing the optimized hydrolysis conditions for hydrolyzing flavonoid glycosides to their corresponding flavonols including quercetin and myricetin in Wuyi Rock tea was regarded as a key technology for determining the content of corresponding myricetin and quercetin. In our work, hydrolysis at a concentration of 2 mol/L HCl solution and at 90 °C for 1 h was found to be optimal for hydrolyzing flavonoid glycosides to myricetin and quercetin in Wuyi Rock tea. Caffeine and seven kinds of polyphenols (GA, EGC, C, EGCG, EC, ECG, and CGA) in 20 samples of Wuyi Rock Tea were simultaneously determined using a simple and fast reverse-phase highperformance liquid chromatography procedure coupled with a photodiode array detector (RP-HPLC-PDAD), which was developed in this effort. This work also showed that there were significant (P < 0.05) differences of ECG, CGA, ECG, and myricetin glycosides in ‘Wuyi Rougui’ and ‘Wuyi Shuixian’, which were credited with causing the different tastes between these two cultivar sets of Wuyi Rock Tea. Therefore, it was suggested that the application of RP-HPLC-DAD, the method developed in this study, might be a promising method for simultaneously determing caffeine and some selected polyphenols in Wuyi Rock tea, evaluating the tea quality of ‘Wuyi

acquisition wavelengths and at different reference wavelengths. Contents of GA, EGC, C, CAF, EGCG, EC, and ECG were detected at an acquisition wavelength of 278 nm and at a reference wavelength of 360 nm (Figure 3A), whereas CGA content was detected at an acquisition wavelength of 330 nm and at a reference wavelength of 380 nm (Figure 3B). No target substances (myricetin and quercetin) in nonhydrolyzed extraction of Wuyi Rock tea sample were detected at an acquisition wavelength of 370 nm and a reference wavelength at 290 nm (Figure 3C). In contrast, contents of myricetin and quercetin in hydrolyzed extraction of Wuyi Rock tea sample were detected at an acquisition wavelength of 370 nm and a reference wavelength of 290 nm (Figure 3D). These results indicated that myricetin and quercetin could be detected in hydrolyzed extraction of Wuyi Rock tea sample, which implied myricetin and quercetin might be hydrolyzed from their corresponding glycosides. Thus, the contents of myricetin glycosides and quercetin glycosides could be determined by detecting the content of corresponding myricetin and quercetin. From the above results it could be concluded that caffeine and some selected polyphenols such as GA, EGC, C, EGCG, EC, ECG, and CGA in nonhydrolyzed extraction of Wuyi Rock tea sample could be simultaneously detected, whereas other polyphenols such as myricetin and quercetin also could be simultaneously detected in hydrolyzed extraction of Wuyi Rock tea sample. Quantification of Caffeine and Selected Polyphenols in Wuyi Rock Tea Samples. In this effort, the developed HPLC method for simultaneous determination of caffeine and some selected polyphenols was successfully applied in the determination of 20 samples of Wuyi Rock tea, including 10 samples of ‘Wuyi Rougui’ and 10 samples of ‘Wuyi Shuixian’. The results showed that, except for myricetin and quercentin, the other nine kinds of polyphenols (GA, EGC, C, EGCG, EC, ECG, CGA, myricetin glycosides, and quercetin glycosides) and caffeine were all detected in the above 20 samples of Wuyi Rock tea (Table 6). Until now, the distribution of caffeine and selected polyphenols in two cultivar sets of Wuyi Rock tea was still unclear. The Mann−Whitney U test and Kolmogorov− Smirnov test, methods of nonparametric statistics, were used for evaluating the differences of caffeine and selected polyphenols in ‘Wuyi Rougui’ and ‘Wuyi Shuixian’, two cultivar 2779



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Rougui’ and ‘Wuyi Shuixian’, and clarifying the cause of “cultivated varieties flavor” of Wuyi Rock tea. In addition, a simple and fast method for determining the flavonoid glycosides in Wuyi Rock tea, except myricetin glycosides and quercetin glycosides, should be further developed.

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AUTHOR INFORMATION

Corresponding Author

*(H.-T.L.) E-mail: [email protected]. Funding

This work was supported by the National Natural Science Foundation of China (Grant 31270735), the Key Science and Technology Program of Fujian Province of China (Grant 2013N5009), the Science and Technology Program of General Administration of Quality Supervision, Inspection and Quarantine of China (Grant 2013QK318), and the Program for New Century Excellent Talents in Fujian Province University of China (Grant NCETFJ-200720). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED RP-HPLC-PDAD, reverse-phase high-performance liquid chromatography procedure coupled with photodiode array detector; GA, gallic acid; EGC, epigallocatechin; C, catechin; CGA, chlorogenic acid; EGCG, epigallocatechin gallate; EC, epicatechin; GCG, gallocatechin gallate; ECG, epicatechin gallate; CAF, caffeine; UV, ultraviolet; PDAD, photodiode array detector; HPLC, high-performance liquid chromatography; ISO, International Organization for Standardization; Det.278 nm, detection wavelength at 278 nm; ref.360 nm, reference wavelength at 360 nm; Det.330 nm, detection wavelength at 330 nm; ref.380 nm, reference wavelength at 380 nm; Det.370 nm, detect wavelength at 370 nm; ref.290 nm, reference wavelength at 290 nm; EDTA, ethylenediaminetetraacetic acid; LOQ, limit of quantification; RSD, relative standard deviation; ANOVA, analysis of variance; asymp signif (2-tailed), asymptotic significance (two-tailed)

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Simultaneous Determination of Caffeine and Some Selected

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