Chemical analyses of dark tea: C-13 NMR and IIPLC analysesand isolation of a new constituent.

Wenfei Guol, Di Wul, Kanzo Sakata2

, Shaojun Lu03 and Xiufang Yang3

1: Dept. of Chemistry, Zhejiang University, Hangzhou, P. R. China 3100272: Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611-0011, Japan3: Hangzhou Tea Research Institute of All China Federation of Supplying and Marketing

,Cooperations, 83 Caihe Rd, Hangzhou, P. R. China 310016

SummaryDark tea, e.g., Pu'er tea and Tuocha, etc., is one of main types of tea in China. It is an

indispensable beverage for daily life of the people in the far western part of China. This may bedue to the unique healthy functions of its specific chemical constituents. The processing of dark teaincludes piling, a specific treatnlent includin"g microbial fermentation which produces some specificconstituent(s).

Some representative samples of dark tea produced in several provinces of China were extractedwith boiling water and subjected to C-13 NMR analysis, HPLC and two-dimension paperchromatography, which showed, different from other types of tea, high concentrations of gallate andcaffeine, and, in most cases, very low concentrations of catechins. In addition, a polar spot was

observed in the paper chromatogram, which was not found in other teas. This polar constituent ofdark tea with high molecular weight was separated by chromatography using Toyopearl HW-40Cand Amberlite XAD-2 and showed different properties from catechins.

KeywordsDark tea, NMR, HPLC, Chemical analysis

IntroductionDark tea is a special traditional kind of tea in China. . The processing of dark tea is: fixing,

rolling, piling, "and drying. Piling is the specific processing of dark tea, and nearly all pilingoperations of dark teas are accompanied with natural microbial actions. Different ~rom green teaand black tea, most dark teas are processed from mature leaves and stenlS. Most products of darktea are pressed into masses in special shapes and are also called brick tea. The production of darktea is about 40 thousand tons per year, most of which are provided to the west regions of Chinawhere less vegetables are available. Since centuries ago dark tea has become an indispensablegood in the daily life of the people there. It is believed that drinking dark tea can maintain thehuman health under the hard conditions such as high elevation, strong cosmic radiation, fatty

foodstuffs, etc.The healthy functions of dark tea are attracting the people's interest. In the 1970's Pu'er tea, a

dark tea, was reported to have special function to prevent blood vessel diseases. I ) Since then theconsumption scope of dark tea enlarged to other regions.

The healthy functions of tea have been ascribed mainly to its catechins. However the rawmaterials of dark tea from mature leaves contain lowe~ catechins. This suggests there are otherconstituents responsible to the healthy functions. Here we report the chemical analyses of dark teasand separation of a new constituent.

Session II -288-

Materials and methods

Samples: Representative. dark tea samples processed with the standard methods were collectedfrom the main producing provinces. They were Fangbao (Sichuan), Chitsu Pingcha (Yunnan),

Fuzhuan (f-Iunan), Heizhuan (Hunan), Kangzhuan (Sichuan), Jinjian (Sichuan), Mizhuan (Hubei),Tuocha (Yunnan), Qingzhuan (Hubei), and Pu'er tea (Yunnan).

HPLC analyses of dark tea constituents: A ground sample (0.2 g) was extracted with 70%methanol (5 ml) at 70°C for 10 min. The solution was filtratered through a 0.45 microm~ter

merrlbrane and diluted to 10 ml. For each sample 10 microliters of the filtrate was injected into aShimatzu LC-6A HPLC equipped with an ODS column (4.6 mm x 15 cm) eluted with 0.05 M citric

acid-DMF (78:22) at a flow rate of 1 ml/min.. Quantifications of catechins, caffeine and gallic acidwere performed by external standard method. 2,3)

C-13 NMR analyses of aqueous constituents of dark tea: A dark tea sample was extracted with

water at boiling temperature for 15 min. The filtrate was freeze dried and about 90 mg of it wasdissolved in deuterated water and kept at 50°C. C-13 NMR measurement was carr"ied out on aBruker Avance DMX-500 NMR instrument for 48 hours. TSP was used as the internal standard.

IR: The measurement was carried out on a NEXES 470 FT-IR spectrophotometer.

2D-PC: The compositions of catechins were also analysed by 2D-PC on filter paper (Xinhua)developed at the first direction with acetone-acetic acid-water (10:2:88) and at the second direction

with n-butanol-acetic acid-water (4: 1:5). The catechins were detected by spraying 0.5%ammonium ferrous sulfate in 50% ethanol.4

,5)

Column chromatographies: Two gels, Toyopearl HW-40C (Tosoh Co., Tokyo) and AmberliteXAD-2 (Organo Co., Tokyo) were used to separate the constituents.

Results and discussion

Concentrations of aqueous soluble constituents in dark tea: Analysed by both HPLC and 2D-PC,

the concentrations of gallic acid, caffeine and catechins in the dark tea samples were obtained. The

results are shown in Table 1. The concentrations of (-)-epigallocatechin and (+)-catechin were very

low and are not shown in the table. Among the samples only Tuocha contained relatively high

concentrations of catechins (9.48 %).

Table 1. Concentrations of gallic acid, caffeine and catechins in dark tea (%)

Sample Gallic Caffeine EC EGCG ECG Totalacid catechins

1 Fangbao 0.06 0.84 Trace 0.05 0.02 0.08

2 Chitsu Pingcha 1.12 2.82 0.40 0.14 0.19 0.78

3 Fuzhuan 0.14 1.38 0.11 0.74 0.20 1.064 Heizhuan 0.17 1.79 0.15 1.59 0.36 2.115 Kangzhuan 0.21 1.79 0.20 1.44 0.33 1.99

6 Jinjian 0.18 1.33 0.05 0.23 0.05 0.34

7 Mizhuan 0.49 3.32 Trace 0.10 0.07 0.17

8 Tuocha 0.34 3.82 Trace 4.65 4.37 9.48

9 Qingzhuan 0.i5 1.16 0.05 0.15 0.15 0.36

10 Pu'er tea 1.72 3.93 0.21 0.03 0.01 0.27

Note: EC, (-)-epicatechin; EGCG, (-)-epigallocatechin gallate; ECG, (-)-epicatechin gallate.

Catechins are a group of most important constituents in green tea. The processing and rawmaterial of Tuocha are quite similar to those of green tea. The degree of fermentation of Tuocha is

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light and is reflected in its catechins. Other samples had very low concentrations of catechins,which are much lower than those in green tea from the raw materials of similar maturities. Thissuggests there are reactions of catechins during the processing of dark tea. Mizhuan is a black teatype of dark tea with the sensory characteristics of black tea in which the concentrations of catechinswere very low.

The concentration of caffeine is higher in younger leaves and nearly does not change during teaprocessing. Therefore it can indicate the degree of youth of the raw material.

During the piling (microbial fermentation) of dark tea the concentration of gallic acid increases.As a result, most samples of dark tea had higher concentrations of gallic acid than other teas.

Separation of a new constituent: The HPLC analyses of dark tea showed very low concentrationsof catechins. On the other hand, the C-13 NMR showed there were several main signals similar tothose of catechins (Fig. 1)6). This indicates there must be certain nlain constituent(s) other thancatechins in dark tea, which may be responsible to the healthy functions.

PPMf;'lilfllllIlIlJi"llilJllllil'illillilllJllllllllililIJill/liiliiiii/"l i ii!iir l 'iliii!ijiiliiiillliliiiiilil!i!iiili!!!iii"Ilil'!!!!'li'I!'i!'I"'ill'il'!"'"I!I'!lilll!ili!liilil'

JO 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0

Fig. 1. A C-13 NMR spectrum of aqueous extract of dark tea (100 MHz, 0 20, TSP)The arrows indicate the signals like those ofEGCG.

On the 2D-PC, most samples of dark tea showed a polar spot which was not detected in green tea,black tea or oolong tea. To separate this constituent, a sample (1.5 g) was extracted with boilingwater and the filtrate was subjected to Toyopearl HW-40C column chromatography (eluted with 5%methanol). A fraction with brown color was collected and subjected to Amberlite XAO-2 columnchromatography (eluted with 5%, 20% and 50% methanol). The amount of this constituent was as

high as 7% of the dark tea sample.The constituent from the column showed a single spot, however, in HPLC (ODS,

acetonitrile-water = 80:20) it appeared as a peak with two minor tailing peaks. The LC-ESIMSshowed a base peak at m/z 525.9 and some fragment peaks up to m/z 1008, suggesting the molecularweight is quite high. The IR spectrum showed peaks at 3423, 2931, 2361, 1617, 1417, 1247, 1072

and 1022 cm- l (Fig. 2).

Session n -290-

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Different from catechins, the constituent could not form color complex with ferrous ion. Thedetail elucidation of the structure is in progress.

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Fig.2. IR spectrum of the polar constituent from dark tea (KCI)

References(I) Proceedings of Forum of Yunnan Tea Development, edited by Yunnan Tea Society, Kunming,

April 2001.(2) J. J. Dalluge and 8. C. Nelson, J Chromatography A, 881, 411-424 (2000).(3) H. Wang and K. Helliwell, Food Chemistry, 70, 337-344 (2000).(4) H. P. Singh, S~ D. Ravindranath, and C. Singh, J Agric. Food Chern., 47(3), 1041-1045 (1999).(5) S. Shojaku, K. Ikegaya, H. Takayanagi, and T. Anan, Chagyo Kenkyu Hokoku, 59, 41-44

(1984).

(6) T. Nakabayashi, K. Ina, and K. Sakata, Ryokucha, Kocha, Uroncha no Kagaku to Kino, KogakuPublishing House, Kawasaki, 1991 .

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