Cocoa tea (Camellia ptilophylla Chang), a natural decaffeinated species of tea – Recommendations on the proper way of preparation for consumption

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Cocoa tea (Camellia ptilophylla Chang), a natural decaffeinated species of tea – Recommendations on the proper way of preparation for consumption Xiao-rong Yanga, Yuan-yuan Wanga, Kai-kai Lia, Jing Lia, Cheng-ren Lia, Xiang-gang Shia, Chun-Hay Kob,c, Ping-chung Leungb,c, Chuang-xing Yea,*, Xiao-hong Songa,* a

Department of Biology, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong, China c State Key Laboratory of Photochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Hong Kong, China b

A R T I C L E I N F O

A B S T R A C T

Article history:

Cocoa tea is a new natural decaffeinated tea. Three types of cocoa tea (green, oolong and

Received 19 February 2011

black) were produced and their sensory evaluation scores accessed. Main components

Received in revised form

and their dissolution rates in ten infusions of the three teas were analyzed by HPLC and

3 June 2011

spectrophotometry. The comprehensive score of sensory evaluations was 85.1, 86.8 and

Accepted 14 June 2011

94.4, respectively. Upon fermentation the contents of water extract (46.67%, 41.8% and

Available online 23 July 2011

40%), tea polyphenols (38.58%, 30.41% and 23.6%) and total catechins (23.51%, 17.68% and 4.02%) in green, oolong and black teas decreased gradually, and theaflavins (0.11%, 0.11%

Keywords:

and 0.17%), thearubigins (4.29%, 5.00% and 9.71%), theabrownins (2.75%, 4.90% and

Three types of cocoa tea (green,

13.52%), water-soluble carbohydrates, flavonoid glycoside and gallic acid in the three teas

oolong and black)

increased gradually. Theobromine (3.52%, 3.43% and 3.71%) was the alkaloid present in

Score of sensory evaluations Main components Content Dissolution rate

cocoa tea and its content did not change upon fermentation. In the three teas, 9–25% of the main components dissolved in the first 10 s-infusion, more than 80% of most components dissolved in five infusions. A recommendation for consuming cocoa tea is to infuse tea leaves (g) with 50 times boiling water (ml) for 3 min, the first infusion should not be discarded and five infusions were sufficient.  2011 Elsevier Ltd. All rights reserved.

1.

Introduction

Tea is probably the most popular beverage besides water in Asia (Wu & Wei, 2002) and is found to be beneficial to human health. Tea polyphenols (PO) have antioxidant effects (Benzie & Szeto, 1999; Ho, Lin, & Shahidi, 2008), as well as anticarcino-

genic and anti-hypercholesterolemia activities (Fujiki et al., 1998; Ikeda et al., 2003). Taking low to moderate doses of caffeine the consumers’ mental alertness, physical energy can be enhanced (Benzie, 1999; Fredholm, Battig, Holmen, Nehlig, & Zvartau, 1999). On the other hand, many people may not wish to consume caffeine, even at low quantities, because

* Corresponding authors: Address: Department of Biology, School of Life Sciences, Sun Yat-Sen University, 135, Xingang West Road, Guangzhou 510275, China. Tel.: +86 20 84112874; fax: +86 20 84036215 (C.-x. Ye and X.-h. Song). E-mail addresses: [email protected] (C.-x. Ye), [email protected] (X.-h. Song). Abbreviations: WE, water extract; PO, tea polyphenols; TH, theanine; GA, gallic acid; TB, theobromine; TP, theophylline; GC, ()gallocatechin; CAF, caffeine; EGC, ()-epigallocatechin; C, (+)-catechin; EC, ()-epicatechin; EGCG, ()-epigallocatechin gallate; GCG, ()gallocatechin gallate; ECG, ()-epicatechin gallate; CG, ()-catechin gallate; Tf, theaflavins; Tr, thearubigins; Tb, theabrownins 1756-4646/$ - see front matter  2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jff.2011.06.001

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of the likelihood of sleep disturbances and may occasionally lead to anxiety (Smith, 2002). Hence decaffeinated tea could be offered to those who are likely to experience such unwanted effects. Methods to produce decaffeinated tea are already available. They include water decaffeination, solvent extraction, adsorption separation, supercritical carbon dioxide extraction, microbial degradation, enzymatic degradation and breeding low caffeine tea plants by transgenic technology, among others. These decaffeination methods have disadvantages and limitations because of high-costs, residues of chemical reagents, and loss of some useful compounds. A new natural decaffeinated tea breed, the cocoa tea, has been domesticated from a wild decaffeinated individual plant (Camellia ptilophylla) by Chuang-xing Ye, Professor of Sun Yat-Sen University, Guangzhou, China. Xiao-rong Yang and others compared alkaloids and catechins of cocoa tea with those of regular tea (Camellia sinensis and Camellia assamica) manufactured in the same way. The major differences between cocoa tea and regular tea are the alkaloids and the major catechins. In cocoa tea the alkaloid and the major catechin are 3–5% theobromine and no caffeine, 9–14% ()-gallocatechin gallate (GCG) while in regular tea there are 2–5% caffeine and a little of theobromine (0.05– 0.5%), and 4–14% ()-epigallocatechin gallate (EGCG) (Gao et al., 2004; Peng, Song, Shi, Li, & Ye, 2008; Yang, Ye, Xu, & Jiang, 2007). Tea pigments include theaflavins (Tf), thearubigins (Tr) and theabrownins (Tb), which are derived from the oxidation of catechins during the fermentation stage of black tea processing (Wan, 2003). Tf is orange or orange-red in color and possesses a benzotropolone skeleton (Roberts, 1958). More than 28 kinds of Tf have been identified. Tf has strong antioxidant activity (Naczk & Shahidi, 2006; Shiraki et al., 1994; Yoshida et al., 1999), anti-inflammatory and cytotoxic activities (Sang et al., 2004). Tr embraces a number of indeterminate structures, from the ‘monomeric’ to the ‘polymeric’, is derived from enzymatic oxidation of the flavan-3-ols (Haslam, 2003). It contributes the reddish color and richness in taste. Tr has antiproliferative effect on growth of human epidermal cell line A432 cells (Liang et al., 1999), antimutagenic effect in vitro (Halder, Pramanick, Mukhopadhyay, & Giri, 2005) and antileukemic effect (Das et al., 2002). Tb is brown and very soluble in water. Tb may be taken as a term embracing amylose, protein, nucleic acid and polyphenols, is derived from the oxidation of Tf and Tr as a result of the excessive withering and anoxic fermentation during black tea processing (Wan, 2003). There is no report yet about water extract (WE) and bioactive components of the three types of cocoa tea. While exploring the chemistry of cocoa tea, one should take into consideration the popular ways of tea processing, so that convincing recommendation could be made on the effective way of consumption of this unique novel species of tea. This study accessed the sensory quality, analyzed the contents of the main components and the dissolution rates of three types of cocoa tea, prepared as unfermented (green tea), semi-fermented (oolong tea) and fully fermented (black tea) products.

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2.

Materials and methods

2.1.

Plant materials – cocoa tea leaves

Cocoa tea leaves were obtained in the tea garden of the Tea Institute of Guangdong Province, China. The fresh leaves were made into green, oolong and black teas with the methods established by the Institute.

2.2.

Apparatus and chemicals

The equipment used included the following: TU-1901 doublebeam UV–Vis spectrophotometer and 1 cm quartz absorption cell (Beijing Puxi General Instrument Co. Ltd., China). The HPLC system: HT-230A column heater (Heng’ao, Tianjin, China), Discovery RP-Amide C16 guard column (4 mm · 20 mm, 5 lm) and Discovery RP-Amide C16 column (4.6 mm · 250 mm, 5 lm) (Supelco, Bellefonte, PA, USA), Waters 515 LC pumps and a Waters 2487 UV detector (Mildford, MA, USA), Yihai data analysis software (Yihai, Guangzhou, China). (+)-Catechin (C), ()-epicatechin (EC), ()-gallocatechin (GC), ()-epigallocatechin (EGC), ()-catechin gallate (CG), ()-epicatechin gallate (ECG), GCG and EGCG were purchased from Sigma–Aldrich Corp. (St. Louis, MO, USA). Caffeine (CAF) was purchased from Wako Pure Chemical Industries Company Ltd. (Osaka, Japan). theanine (TH), theophylline (TP), theobromine (TB) and gallic acid (GA) were purchased from Fluka Chemical Corp. (Milwaukee, WI, USA). HPLC-grade acetonitrile was purchased from Merck Company (Darmstadt, Germany). HPLC-grade water purified using a Millipore MilliQ purification system (Bedford, MA, USA) was used for mobile phase and the preparation of all solutions. Other reagents used were analytical grade.

2.3.

Methods

2.3.1.

Manufacture of green, oolong and black cocoa teas

For green tea preparation, fresh leaves were spread in 4 cm thickness layers on wire netting for 6 h, then blasted with air at room temperature for 1.5 h. The withered leaves were parched in a rotary kiln at about 220 C for 5 min. The leaves were rolled in a rolling machine lightly for 10 min, strongly for 20 min and then lightly for 5 min. Then, the leaves were spread a thin layer and dried in an electric blast oven at 100 C for 30 min, and dried at 80 C for 3 h to make the water content less than 5%. For oolong tea preparation, firstly, fresh leaves were sunwithered for 2 h. Secondly, indoor withering for 10 h, during which, 2 min hand rolling was carried out every 2 h at room temperature and about 80% relative humidity (RH). Then, leaves were parched in a rotary kiln at about 250 C for 2 min. The final steps were 3 min rolling and 5 h roasting at 80–100 C for the first time, and then 5 min kneading and 5 h roasting at 60–80 C for the second time. For black tea preparation, fresh leaves were spread on a wire netting indoors at 30 C, blasted and stirred alternately for 15 h. Then, leaves were rolled in a rolling machine lightly for 10 min, strongly for 20 min and lightly for 10 min again.

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Three grams of manufactured cocoa tea leaves were infused with 150 ml boiling water for 5 min. The sensory evaluation of the three teas was assessed and scored by three professional tea tasters from Tea Institute of Guangdong Province. The full score of 100 was based on a comprehensive assessment, of which 10% for leaves appearance, 10% for infusion color, 30% for infusion aroma, 30% for infusion taste, and 20% for infused leaves (tea leaves were infused once in grading system). This grading system is used to evaluate manufactured tea quality in China.

and CG were dissolved in distilled water, mixed thoroughly and the volume made up to 100 ml, which was then further diluted to five different concentrations with distilled water. Preparation of tea sample solution: One hundred milligrams of each of green, oolong and black cocoa teas, were infused with 100 ml water at 95 C for 30 min and shaken once every 10 min. The volume of filtrate was made up to 100 ml. Standard solutions and tea filtrates were prepared by passing through a 0.45 lm membrane for HPLC analysis. HPLC conditions: A two-gradient elution system was used, mobile phase A was acetonitrile; mobile phase B contained ortho-phosphoric acid (85%) and distilled water (0.05:99.95, v/v), the gradient was as follows: 0–60 min, 2–30% B, postrun time was 10 min. The injection volume was 20 ll, the flow rate was 1.0 ml/min, the column temperature was 35 C and the detection wavelength was 210 nm.

2.3.3. Determination of WE, PO and tea pigments by drying method and spectrophotometry

2.4. Determination of dissolution rate of the components in each infusion

The rolled leaves were pile-fermented for 10 h at room temperature and 70% RH, and wet canvas was laid over the pile to prevent water vaporization. The fermented leaves were first dried in a drying oven at 120 C for 30 min, and then dried at 90 C for 2 h till the leaves could be crushed by kneading.

2.3.2.

Sensory evaluation

The content of WE material was determined in accordance with Chinese National standard GB8305-2002. Ground tea samples 1.5 g were infused with 225 ml boiling water at 100 C for 45 min and shaken twice every 15 min. The volume of infusion was made up to 250 ml. Fifty milliliters infusion was transferred into an evaporating dish and placed in a drying oven until the water evaporated thoroughly, and then cooled and weighted. It was calculated by the equation: WE (%) = [(M1  M2)/V1 · V0/M0] · 100%. In the equation, M1 is the weight of evaporating dish in g; M2 is the weights of evaporating dish add WE in g; V1 is the infusion volume of the transferred into the dish in ml; V0 is the total volume of infusion; M0 is the weight of tea samples in g. The content of PO was determined in accordance with Chinese National standard GB8313-2002. One milliliter of tea infusion from the above 250 ml, 5 ml ferrous tartrate solution, 4 ml distilled water and 15 ml buffer of potassium phosphate (0.067 M, pH 7.5) were transferred into a 25 ml volumetric flask to react for a few minutes. Absorbance readings were made at 540 nm by a PGENRAL TU-1901 spectrophotometer, using distilled water to be a blank solution instead of tea infusion. It was calculated by the equation: PO (%) = 3.914A · (V0/ V2 · M0/1000) · 100%. In the equation, V0 is the total volume of infusion in ml; V2 is the infusion volume used for analysis in ml; M0 is the weight of tea samples in g; A is the absorbance of diluted and dyed infusion at 540 nm; 3.914 means the absorbance at 540 nm will be 1 when the concentration of PO in tea infusion is 3.914 mg/ml. The contents of water-soluble carbohydrates, flavonoid glycosides and tea pigments (Tf, Tr and Tb) were determined in accordance with the methods described by Wu (1998).

2.3.4. HPLC

Determination of alkaloids, catechins and others by

Preparation of standard solution: Twenty milligrams of each of theanine, GA, theobromine, theophylline, caffeine, catechin, EC, ECG, GCG and EGCG as well as 4 mg of each of GC, EGC

Dissolution rate is the percentage of a tea component dissolved in an infusion or infusions compared to the total amount of the component contained in dry tea leaves. Cocoa tea (3 g) was infused 10 times, with 150 ml boiling water each time, 10 s for the first time, 3 min for the rest. The components of each infusion were analyzed one by one with the methods described in Section 2.3.

2.5.

Statistical analysis

All data were analyzed by using SPSS 17.0 made by IBM Company (Chicago, IL, USA). Data were analyzed using ANOVA followed by Duncan’s multiple range tests.

3.

Results

3.1.

Results of sensory evaluation

Green cocoa tea leaves were olive and not vivid green, and its infusion taste had some bitterness and astringency, but infusion color and aroma were good. Oolong cocoa tea leaves were frizzy stripe and brown green, and flower fragrance was not evident, but infusion was bright golden yellow and had some mellow taste with a little astringency. Black cocoa tea had good effects on appearance, color, aroma and taste. The comprehensive scores of sensory evaluation of green, oolong and black cocoa teas were, respectively, 85.1, 86.8 and 94.4 (Table 1).

3.2.

Content of main chemical components

Content of WE in green, oolong and black cocoa teas was 46.67%, 41.83% and 40.0%, that of PO was 38.58%, 30.41% and 23.6%, that of total catechins was 23.47%, 17.68% and 4.02%, respectively. These substances decreased significantly in oolong and black teas compared with those in green tea. Content of water-soluble carbohydrates was 3.06%, 3.66% and 4.1%, that of flavonoid glycoside was 0.45%, 1.17% and

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Table 1 – Results of sensory evaluation of three manufactured cocoa teas (n = 3, mean ± S.D.). Projects Leaves appearance (10%)

Infusion color (10%) Infusion aroma (30%) Infusion taste (30%)

Infused leaf (20%)

Comprehensive score (100)

Green

Oolong

Black

Tight stripe and evident Baekho; olive 83 ± 0.79 Yellow green 95 ± 1.05 Some chestnut fragrance 89.0 ± 0.94 Some bitterness and astringency, a lingering sweetness 76.3 ± 1.31 Olive green 88.5 ± 1.85

Frizzy stripe; dark brown green 80 ± 0.96 Golden yellow 94 ± 1.52 A little flower fragrance 80.7 ± 0.67 Slight mellow taste with a little astringency 91.3 ± 1.02 Slight red 89.0 ± 0.55

Tight and strong stripe; reddish brown 94 ± 0.75 Reddish brown 90 ± 0.82 Evident fruit fragrance 95.3 ± 0.93 Soft and mellow taste 97.0 ± 0.36

85.1 ± 1.73

86.8 ± 1.30

94.4 ± 0.88*

Bright brown 91.5 ± 0.72

* P < 0.05 vs green, mean ± S.D. (n = 3).

1.91%, that of GA was 0.13%, 0.26% and 0.6%, and that of the sum of three tea pigments was 7.11%, 10.53% and 24.23%, respectively. These substances increased obviously with increased fermentation. Content of theobromine (3.52%, 3.43% and 3.71%) did not change upon fermentation (Table 2). Three types of tea pigments, namely Tf, Tr and Tb have complicated structures, and cannot be simultaneously analyzed by HPLC. The HPLC chromatograms of simultaneous analysis of alkaloids and catechins showed good chromatographic peak shape. Reproducibility and precision were good.

Correlation coefficients were between 0.990 and 0.999. Limit of detection and limit of quantification of the 13 compounds varied from 0.0001 to 0.072 ng/ll and 0.0004 to 0.24 ng/ll, respectively. GCG was the major catechin in the three teas (Fig. 1 – standards, green, oolong and black).

3.3.

Dissolution rate of bioactive components in infusions

Dissolution rate of PO in the first infusion for green, oolong and black teas was 6.73%, 4.81% and 15.03%, that of theobro-

Table 2 – Content of main components in green, oolong and black cocoa teas (%: w/w), (n = 3, mean ± S.D.). Components

Contents (%) Green

Oolong

Black

Water extract Tea polyphenols Water-solubility carbohydrate Flavonoid glycoside Theobromine Gallic acid

46.67 ± 0.47 38.58 ± 0.47 3.06 ± 0.68 0.45 ± 0.32 3.78 ± 0.22 0.13 ± 0.44

41.8 ± 0.58*** 30.41 ± 0.49*** 3.66 ± 0.39*** 1.17 ± 0.37*** 3.43 ± 0.07 0.26 ± 0.52*

40.00 ± 0.45*** 23.60 ± 0.44*** 4.10 ± 0.48** 1.91 ± 0.35*** 3.71 ± 0.08 0.60 ± 0.55***

Catechins GC EGC C EC EGCG GCG ECG CG Total

1.74 ± 0.51 0.37 ± 0.56 4.36 ± 0.45 0.22 ± 0.42 3.33 ± 0.49 12.88 ± 0.31 0.57 ± 0.32 0.04 ± 0.45 23.51 ± 0.49

2.32 ± 0.50** 0.12 ± 0.49*** 4.24 ± 0.47* 0.15 ± 0.38** 2.03 ± 0.43*** 8.56 ± 0.34*** 0.26 ± 0.30*** 0** 17.68 ± 0.46***

0.13 ± 0.52*** 0.02 ± 0.41*** 1.32 ± 0.43*** 0.06 ± 0.43*** 0.34 ± 0.42*** 1.96 ± 0.37*** 0.19 ± 0.46** 0** 4.02 ± 0.43***

Tea pigments Theaflavins Thearubigins Theabrown Total

0.11 ± 0.32 4.29 ± 0.40 2.75 ± 0.46 7.11 ± 0.42

0.11 ± 0.39 5.00 ± 0.47** 4.90 ± 0.50*** 10.53 ± 0.47***

0.17 ± 0.30* 9.71 ± 0.45*** 13.52 ± 0.48*** 24.23 ± 0.51***

Abbreviations: GC, ()-gallocatechin; EGC, ()-epicatechin gallate; C, (+)-catechin; EC, ()-epicatechin; EGCG, ()-epigallocatechin gallate; GCG, ()-gallocatechin gallate; ECG, ()-epicatechin gallate; CG, ()-catechin gallate. * P < 0.05 vs green, mean ± S.D. ** P < 0.01 vs green, mean ± S.D. *** P < 0.001 vs green, mean ± S.D.

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Fig. 1 – HPLC chromatograms of standards and three types of cocoa tea. Abbreviations: TH, theanine; GA, gallic acid; TB, theobromine; TP, theophylline; GC, ()-gallocatechin; CAF, caffeine; EGC, ()-epigallocatechin; C, (+)-catechin; EC, ()epicatechin; EGCG, ()-epigallocatechin gallate; GCG, ()-gallocatechin gallate; ECG, ()-epicatechin gallate; CG, ()-catechin gallate.

mine was 19.47%, 8.79% and 25.17%, that of total catechins was 17.31%, 6.61% and 16.33%, that of GCG was 9.52%, 4.53% and 5.00%, respectively (Fig. 2). In the tea samples examined, 95% of polyphenols present dissolved in 10 infusions and 80% of them dissolved in five infusions (Fig. 2 – PO). About 95% of theobromine dissolved in 10 infusions, and more than 90% of it dissolved in five infusions (Fig. 2 – TB). Less than 75% of GA dissolved in 10 infusions, and in oolong only 48.66% dissolved; 43–72% of GA dissolved in five infusions (Fig. 2 – GA); Catechin dissolved 100% in ten infusions and more than 85% of it dissolved in five infusions (Fig. 2 – C). Dissolution rate of EGCG in 10 infusions was 94.35%, 51.58% and 8.93% (Fig. 2 – EGCG). More than 90% of GCG in green and oolong

teas while only 63.30% of it in black tea dissolved in 10 infusions. 57–84% of GCG dissolved in five infusions (Fig. 2 – GCG). About 10% of Tf, Tr and Tb each dissolved in the first infusion of the three teas, and more than 90% of them dissolved in five infusions (Fig. 3 –Tf, Tr and Tb).

4.

Discussion

Results indicated that the main components in the three teas were different because of their different manufacturing processes. Green cocoa tea contained more catechins (23.51%), black cocoa tea contained more tea pigments (the sum of Tf, Tr and Tb was 24.23%), and the contents of total catechins

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Fig. 2 – Dissolution rate of six components in green, oolong and black cocoa teas (%). Each data represents the mean of three experiments. SEM was not shown.

and tea pigments in oolong were between the two types. Some of PO were oxidized into tea pigments or other substances during fermentation. GA increased because of the hydrolysis of ester–catechins such as GCG and EGCG (Dix, Fairley, Millin, & Swaine, 1981). Theobromine did not change upon fermentation. These results are same as those reported previously for manufactured regular tea (C. sinensis or C. assamica) (Hou, Hui, Liu, Tang, & Zhu, 2006; Kilmartin & Hsu, 2003). Total catechins in green cocoa tea are not less than that in regular tea (C. sinensis or C. assamica) (Gao et al., 2004; Peng et al., 2008). Tea pigments are the major polyphenols in fermented tea. The contents of Tf, Tr and Tb are 0.9%, 10.59% and 7.79% in black tea, respectively (Mai, 1985). Tf accounts for 2–6% of the dry weight of solids in brewed black tea (Balentine, Wiseman, & Bouwens, 1997). Tr is at approximately 12–18% of solid extracts of black tea liquors (Graham, 1992). The content of Tf, Tr and Tb was 0.17%, 9.71% and 13.52% in

black cocoa tea, respectively. Regarding fermentation time, it differs greatly from region to region in China. It was used to take 1 h at most and now it can take as long as 10 h or more for fermentation. So, the content of tea pigments differs greatly. The appearance of green cocoa tea leaves was olive green and not vivid green and there was some bitterness and astringency in the taste because of high content of catechins, and these led to its lowest score among the three teas. Obviously, the fermented tea products, oolong and black cocoa teas showed good color, aroma and taste, especially the evident fruity fragrance aroma and mellow taste in black tea. So, the comprehensive score of sensory evaluations of black tea was the highest among the three teas (Table 1). Tb endows tea liquor and leaves with a dark brown color, which has a negative effect on tea quality (Anhui-Agricultural-University, 1999). The order of hydroxyl and 2,2-diphenyl-1-picrylhydrazyl-scavenging ability of Tf, Tr and Tb was

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Fig. 3 – Dissolution rate of teas pigments in green, oolong and black cocoa teas (%). Each data represents the mean of three experiments. SEM was not shown. Abbreviations: Tf, theaflavins; Tr, thearubigins; Tb, theabrown.

Tf > Tb > Tr. These tea pigments showed protection against H2O2-induced damage in HPF-1 cells and suppressed the accumulation of intracellular reactive oxygen species in H2O2-induced damage of HPF-1 cells. Interestingly, Tb, as a further oxidation product from Tf or Tr, showed potent activity, followed by Tf in the above systems (Yang et al., 2007). Tb was 13.52% in black cocoa tea, and its bioactivity should be researched further. Theobromine is found in Theobroma cacao, Theobroma Bicolor, Theobroma angustifolium and other plants. The highest content of it was 3.57–3.67% in the seeds of Th. cacao L. (Nair, 2010). Theobromine is 0.05–0.5% in regular tea (C. sinensis) and 3–5% in cocoa tea (C. ptilophylla). It is a good source of theobromine. Caffeine and theobromine are natural members of methylxanthine family. Caffeine in coffee and regular tea has stimulating effect on the central nervous system (CNS) and causes insomnia in some individuals. Theobromine is used as a bronchodilator or a vasodilator. It has practically no stimulant effect on CNS (Martindale & Reynolds, 1993). Cocoa tea has no stimulant effect on sleep and hence is a good substitute for artificially decaffeinated tea. The dissolution rate of all components in tea leaves decreased by repeated infusions. Re-infusing tea leaves is very common and unfortunately, the first infusion is often discarded for cleaning dust and removing bitterness (Obanda, Okinda Owuor, & Mang’Oka, 2001). Our study showed that 9–25% of the main components in cocoa tea would be lost if the first infusion was discarded. More than 80% of bioactive components in the three teas dissolved in five infusions, but the dissolution rate of GA, EGCG and GCG in oolong or

black was lower. Tf, Tr and Tb dissolved fast and thoroughly, and more than 90% of them dissolved in five infusions. Therefore infusing cocoa tea for five times is enough when using 50 times boiling water each time by the above described method.

Acknowledgements The project was supported by Bureau of Science and Technology of Guangzhou, China (2001-T-021-01) and the National Talent Fund of China (J0730638). Appreciations are expressed to them.

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