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J Sci Food Agric 1990,53, 541-548

A Study on Chemical Composition of Two Special Green


Teas (Camellia sinensis)
Y R Liang, Z S Liu, Y R Xu and Y L Hu
Department of Tea Science, Zhejiang Agricultural University, Hangzhou 310029, China
(Received 30 November 1989; revised version received 16 March 1990;
accepted 26 May 1990)

ABSTRACT
An HPLC method has been used to separate and determine quality-related
chemical components in Zhenong-xiangya and Zhenong-cuiliu, two special
green teas fiom Zhejiang, China. Seventeen or eighteen amino acids and five
catechins were detected. Theamine was the major amino acid by far, its
content reaching 37.7% and 54% of the total amino acids in these teas,
respectively. The catechin content was 154.4 mg g - ' in Zhenong-xiangya
and 170.7 mg g-' in Zhenong-cuiliu. Caffeine contents in the teas studied
were all above 75 mg g-' which was much higher than those previously
reported in green teas. The vitamin C contents were all about 2 mg g - Seven
peaks were resolved in the HPLC projiles of the flavonoid extracts of the two
teas and three of them were identijied as rutin, myricetin and quercetin using
reference compounds.
Key words: Camellia sinensis, special green tea, HPLC, amino acid,
catechin, vitamin C, flavonoid, theamine.

INTRODUCTION
Zhenong-xiangya and Zhenongcuiliu are two special green teas in China which
were first manufactured by the Department of Tea Science at Zhejiang Agricultural
University in 1986 and were awarded prizes for 'Excellent Famous Tea' by the
Zhejiang Tea Science Association in 1987 and 1989. The present research sets out to
investigate the chemical composition of these teas.
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J Sci Food Agric 0022-5142/90/$03.50 0 1990 SCI. Printed in Great Britain

Y R Liany, 2 S Liu, Y R Xu, Y L Hu

542

METHODS AND MATERIALS


Samples
The two samples of tea (Camellia sinensis (L) 0 Kuntze) were provided by the
Research Group of New Tea Breedings at Zhejiang Agricultural University, China.

HPLC analysis of amino acids


Ground tea sample (0.5 g) was placed in a flask with 75 ml boiling distilled water
and extracted for 45 min on a boiling water bath and then allowed to cool to room
temperature. The resultant liquor was filtered through Xinhua filter paper (Xinhua
Papermaking Plant, Hangzhou, China) and the residue was discarded. The filtrate
was dried in a vacuum evaporator to produce a dry powder. The powder was
dissolved in 6 ml 0.1 M HCl containing 300ml methanol litre-'. The dissolved
solution was purified through a C18 column (4.6 x 250 mm) and HA 0.45-pm
Millipore filter. The eluate (20 pl) was blended with reagent (100 pl) and then used
for HPLC analysis. The reagent consists of 80 mg 1,2-dialdehydrobenzene
(1,2-diformylbenzene) (previously dissolved in 1 ml methanol) and 60 pg
8-mercaptoethanol dissolved in 0.4 M sodium borate buffer (pH 9.5). The
chromatographic conditions were as follows:
Injection volume:
Column:
Column temperature:
Mobile phase:
Gradient:
Flow rate:
Detector:
Sensitivity:

10 p1
Amino acid analysis column
62C
Solvent A: 0.2 M sodium citrate buffer, pH 3.0; solvent B:
0.2 M sodium borate buffer, pH 9-6
Pure solvent A to solvent A/B 52:48 v by a linear gradient
during 90 min
0-4 ml min Beckman fluorimetric detector model 157, excitation
338 nm, emission 425 nm filters
0.10 aufs

'

Analysis of catechins and caffeine by HPLC


The tea sample (1.Og) was extracted in a conical flask containing 20ml 950ml
litre- ethanol on boiling water bath for 30 min. The resultant liquor was filtered
through Xinhua filter paper into a 25-ml volumetric flask and was diluted to 25 ml
with 950ml litre-' ethanol when cooled to room temperature. The liquor was
filtered through a 0.45pm millipore filter. The final filtrate was used for HPLC
analysis. The chromatographic conditions were as follows:
Injection volume:
Column:
Column temperature:
Mobile phase:

10 pl
p-Bondapak C18, 3.9 x 300 mm
30C
Solvent A: acetic acid/methanol/water (1:1:98 v); solvent
B:
acetic
acid/methanol/dimethylamine/water
(1:1:50:48 v)

Chemical composition of green teas

Gradient:
Flow rate:
Detector:
Sensitivity:

543

Solvent A/B 2090 v to pure solvent B by a linear gradient


during 25 min
1 ml min-'
Ultraviolet detector, 280 nm
0.05 aufs

Analysis of vitamin C by HPLC


The ground sample (05 g) was macerated in a glass blender containing 5 ml 30%
metaphosphate. The macerate was diluted to 25ml in a graduated flask with
distilled water. The diluted solution was then filtered through a 0.45-pmmillipore
filter and the filtrate was injected directly into the HPLC. The HPLC conditions
were as follows:
Injection volume:
10 p1
Column :
p-Bondapak CN, 3.9 x 150 mm
Column temperature: 30C
Mobile phase:
pH 2.8 200g litre-' (NH4)H,P04
1 ml min-'
Flow rate:
Detector:
Ultraviolet detector, 254 nm
0.1 aufs
Sensitivity:
Analysis of flavonoids by HPLC
The ground sample (10 g) was soaked in a flask containing 50 ml petroleum ether
for over 12 h to remove the pigments. The solvent was filtered and discarded. The
residue was air dried and was then heated and extracted in a flask containing
80 ml ethanol (850 ml litre-') under a reflux condenser for 1 h. The ethanol extract
was filtered through Xinhua filter paper and the residue was re-extracted twice more
as above. The three filtrates were combined and concentrated under vacuum to
approximately 100 ml. An aliquot (5 ml) of the concentrate was filtered through
Sep-Pak column to remove pigments, and the filtrate was injected directly into the
HPLC. The HPLC conditions were as follows:
Injection volume:
Column :
Mobile phase:
Flow rate:
Detector:
Sensitivity:

2 p1
p-Bondapak fatty acid column, 4 x 300 mm
550 ml litre-' methanol adjusted to pH 3.0 with H 3 P 0 4
1.2 ml min-'
Ultraviolet detector, 254 nm
0.1 aufs

Examination and evaluation of tea quality


The two tea samples were examined and evaluated by four senior tea tasters (Prof
Zhang Tangheng and Vice-Prof Hu Yueling from Zhejiang Agricultural University,
Prof Gu Zheng from the Tea Research Institute of the Chinese Academy of
Agricultural Science, and Prof Qian Liang from Shanghai Tea Import and Export
Corporation). The grading system was based on a maximum total score of 100, of

544

Y R Liang, Z S Liu. Y R

XU.

YL

HU

which 30% was awarded for appearance, 25 % for flavour, 10% for liquor colour,
25% for taste and 10% for infused leaf.

RESULTS AND DISCUSSION


Amino acid content
The number of amino acids detected in Zhenong-xiangya was 18 and in Zhenongcuiliu 17. There was a trace of proline in both teas. A trace of methionine was
detected in Zhenong-xiangya but none was found in Zhenongcuiliu. Among other
amino acids the content of theamine was highest, namely 18.97 mg g-' in Zhenongxiangya and 11-79mg g- in Zhenongcuiliu which reached 54 % and 38 % of the
total amino acids, respectively. The second most predominant amino acid was
glutamic acid, 5.29 mg g-' in Zhenong-miangya and 5.09 mg g-' in Zhenongcuiliu. Arginine and histidine came next. These four amino acids amounted to
84.3 % and 79.9 % of the total amino acids in Zhenong-xiangya and Zhenongcuiliu,
respectively (see Table 1 and Fig 1).
Catechin content
Figure 2 and Table 2 show that five catechins were detected in the two teas. The
total content of the five catechins was 154.4 mg g-' in Zhenong-xiangya and
170.7 mg g- in Zhenongcuiliu. The content of epigallocatechin gallate (EGCG)
was highest and that of epicatechin gallate (ECG) ranked second among the five
catechins; that of gallocatechin (GC) was lowest.
TABLE 1
Amino acid composition of two special green teas (mg g-')
Sample

Zhenong-xiangya

Zhenong-cuiliu

Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Theamine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Histidine
Tryptophan
Lysine
Arginine
Total (%)

1.55

0.984
1.13
5.28
Trace
0.467
0.489
18.97
0.023
Trace
0.106
0.24
0.192
0.271
3.1
0.034
0.026
2.29
3.52

1.59
1.18
1.23
5.09
Trace
0.486
0.684
11.79
0.037
0
0.152
0.244
0.188
0.216
3.65
Trace
0.027
4.48
3.13

Chemical composition of green teas

545

14

45

(b)

'i

A
I

90

Retention t i m e (rnin)

Fig 1. HPLC analysis of amino acids in two special green teas A, Zhenong-xiangya;B, Zhenongcuiliu. 1 ,
Asp; 2, Thr; 3, Ser; 4, Theamine; 5, Glu; 6, Gly; 7, Ala; 8, Val; 9, Met; 10, Ile; 1 1 , Leu; 12, Tyr; 13, Phe;
14, His; 15, Trp; 16, Lys; 17, Arg.

Yuan Yucheng et a1 (1962) reported that catechin quality index


([(EGCG +ECG)/EGC] x 100) was directly related to the sensory evaluation of
green teas. The index was 680 in Zhenong-xiangya and 819 in Zhenongcuiliu,
higher than that of second and first grades respectively of Long-jing tea (dragonwell tea).

Y R Liang, Z S Liu, Y R Xu, Y L Hu

546

25
0
Retention time (min)

25

Fig 2. HPLC analysis of caffeine and catechins in two special green teas. A. Zhenong-xiangya; B,
Zhenongcuiliu. 1, Caffeine; 2, L-EGC; 3, DL-GC;4, L-EC;5, L-EGCG;6, L-ECG.

TABLE 2
Catechin composition of two special green teas (mg g-')
Sample

Zhenong-xiangya
Zhenongcuiliu

L-EGC

DL-GC

L-EC

L-EGCG

L-ECG

Total

18.42
16.43

069
2.58

10.11
17.13

76.20
75.93

48.99
58-66

154.4
170.7

L-EGC= L-Epigallocatechin; DL-GC= DL-gallocatechin; L-EC= epicatechin; L-EGCG=


epigallocatechin gallate; L-ECG= L-epicatechin gallate.

TABLE 3
Contents of caffeine and vitamin C in two special green teas (mg g-')
Sample

Caffeine

Vitamin C

Zhenong-xiangya
Zhenong-cuiliu

77.2
75-6

2.17
2.08

Caffeine and vitamin C contents


Table 3 and Fig 2 show that caffeine content was 77.2 mg g - in Zhenong-xiangya
and 75.6 mg g- in Zhenong-cuiliu, much higher levels than in first grade Longjing tea and Huiming tea, two famous special green teas in China (Yuan Yucheng
1982). The vitamin C content was 2.17 mg g-' in Zhenong-xiangya and
2.08 mg g-' in Zhenongciuliu.

(=)L

Chemical composition of green teas


5

547

23.5

10

Fig 3. HPLC analysis of flavonoids in two special

lo green teas. A, Zhenong-xiangya; B, Zhenongcuiliu.

Retention time (min)

4, Rutin; 6, myricetin; 7, quercetin.

TABLE 4
Contents of three identified flavonoids in two special green teas
(mgg-')
Sample

Rutin

Myricetin

Quercetin

Zhenong-xiangya
Zhenongcuiliu

2.53
0.181

1-67
0.404

0
0.075

Results of HPLC analysis of flavonoids


Flavonoids have been shown to make important contributions to the liquor colour
ofgreen tea (Sakamoto 1967,1969,1970).Figure 3 illustrates the HPLC separation
of the flavonoids in the two special green teas. Seven peaks were resolved in the
profile shown in Fig 3; peaks 4, 6 and 7 were identified as rutin, myricetin and
quercetin using reference compounds. Peaks 2,3 and 5 may be major tea flavonoids
but in the absence of suitable reference compounds they remain unconfirmed. The
content of the three identified compounds differed markedly between Zhenongxiangya and Zhenongcuiliu. The rutin content of Zhenong-xiangya, 2-53mg g-',
was 14 times that in Zhenongcuiliu. The myricetin content of Zhenong-xiangya
was 1-67mgg-', three times greater than that of Zhenongcuiliu. A trace of
quercetin was detected in Zhenongcuiliu but none was found in Zhenong-xiangya
(see Table 4).
Xiao Weiqiang (1963)and Nakagawa (1973)reported that the sensory evaluation
ofgreen tea was highly related to thecontents ofcaffeine, amino acids and catechins.
Yuan Yucheng et a1 (1962)showed that the catechin quality index could be used as a
grading standard for green teas: the higher the index, the better the green tea. It is
generally considered that the effects on taste of caffeine, amino acids and catechins
are more important than those of other factors in green tea. Tables 1-3 and 5 show

Y R Liang, Z S Liu, Y R Xu, Y L Hu

548

TABLE 5

Mean scores of the two samples evaluated by four senior tea tasters
Sample

Appearance

Flavour

Liquor
colour

Taste

Infused
leaf

Total

Zhenong-xiangya
Zhenongcuiliu

29.3
28.7

24.3
24.5

9.7
9.8

24.5
24.3

9.8
9-8

97.6
96.9

that Zhenong-xiangya, in which the contents of amino acids, theamine in particular,


and caffeine were higher, had a total catechin content lower than that in Zhenongcuiliu. The former tea was given a slightly higher taste score than the latter. The
catechin quality indices of the two samples were also higher than those (331-436) of
first-grade roasted green teas in Zhejiang (Yuan Yucheng et al 1962). But,
comparing the two samples tested here, we can see that Zhenong-cuiliu, with
the higher catechin quality index, was given a lower score both for taste and overall.
This may be attributed to the high catechin content which made the taste too
astringent. The high quality green teas must be light green and bright in liquor
colour. Flavonoids are generally yellow in colour, and if too much flavonoid were to
be dissolved during tea infusion, the liquor colour would be jeopardised. Tables 4
and 5 provide confirmation of this.
To sum up, we conclude that higher amino acid content, theamine in particular
and caffeine, and lower catechin content are favourable to good taste in green tea.
The flavonoids such as myricetin and rutin are harmful to liquor colour.

REFERENCES
Nakagawa M 1973 Relationship between chemical composition and taste of green tea. Study
of Tea 40 1-9.
Sakamoto Y 1967 Flavones in green tea, Part I. Isolation and structure of flavones occurring
in green tea infusion. Agric Biol Chem 31 1029-1034.
Sakamoto Y 1969 Flavones in green tea, Part 11. Identification of isovitexin and saponin.
Agric Biol Chem 33 959-961.

Sakamoto Y 1970 Flavones in green tea, Part 111. Structure of pigments IIIa and IIIb. Agric
Biol Chem 34 919-925.

Xiao Weiqiang 1963 About chlorophyll in green tea. Tea Communication (4-5) 71-74.
Yuan Yucheng 1982 Chemical composition of Zhejiang tea. Tea in Zhejiang (4) 27-32.
Yuan Yucheng et a1 1962 Chemical and biochemical studies on tea catechins. Ann Rep Tea
Res Inst Chinese Acad Agric Sci pp 217-222.