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COMPARISON OF THE COMPOSITION AND ANTIOXIDANT CAPACITIES OF GREEN TEAS PRODUCED FROM
THE ASSAM AND THE CHINESE VARIETIES CULTIVATED IN THAILAND
Theerapong Theppakorn 1*, Atika Luthfiyyah 1 and Karnjana Ploysri 2
Address(es): Theerapong Theppakorn,
1 School of Agro-Industry, Mae Fah Luang University, Chiang Rai 57100, Thailand.
2 Tea Institute, Mae Fah Luang University, Chiang Rai 57100, Thailand.
*Corresponding author: theerapong@mfu.ac.th
ABSTRACT
Keywords: Antioxidant, caffeine, catechins, green tea, polyphenols
INTRODUCTION
Tea is made from the young tender shoots (flushes) of Camellia sinensis (L.) O.
Kuntze and it is the most widely consumed drink after water, due to its refreshing
and mildly stimulant effects. Freshly harvested tea leaves are processed
differently to produce specific types of tea (green, oolong, and black tea). Green
tea is heated to avoid enzymatic oxidation. Oolong tea is semi-fermented to
permit a moderate level of enzymatic oxidation during processing and then dried.
Black tea is the most thoroughly oxidized enzymatically. Approximately 76-78%
of the tea produced and consumed worldwide is black tea, 20-22% is green tea
and less than 2% is oolong tea (McKay and Blumberg 2002).
The predominant constituents of tea leaves, accounting for up to 30% of the dry
weight, are the polyphenols, which include flavonols, flavones, and flavan-3-ols.
Of these, 60–80% are the flavan-3-ols commonly known as catechins. Some
studies support the idea that among all tea types, green teas contain the highest
amount of catechins (Lin et al., 2003). The catechins include (-)-gallocatechin
(GC), (-)-epigallocatechin (EGC), (+)-catechin (C), (-)-epicatechin (EC), (-)-
epigallocatechin gallate (EGCG), (-)-gallocatechin gallate (GCG), (-)-
epicatechin gallate (ECG) and (-)-catechin gallate (CG). These compounds are
primarily responsible for many of the health benefits, resulting from the
antioxidant properties. Antioxidative tea components are reported to have
beneficial protective effects against cancers (Trudel et al., 2012; Khan et al.,
2009; Shukla 2007), obesity (Li et al., 2013; Ueda and Ashida 2012),
cardiovascular disease (Yamada and Watanabe 2007) and diabetes (van
Woudenbergh et al., 2012; Fiorino et al., 2012). The radical-scavenging and
antioxidant properties of tea polyphenols are frequently cited as important
contributors to these beneficial effects (Higdon and Frei 2003). However,
studies have shown that the compounds having beneficial health effects in tea is
influenced by many factors such as the cultivars, harvest season, age of the plant,
climate, environmental conditions and processing conditions (Owuor et al.,
2008).
In Thailand, tea is cultivated in the north part of the country in the provinces of
Chiang Rai and Chiang Mai (accounting for 93% of tea production in Thailand).
About 30% of the production is commercialized in the domestic market, whereas
the remaining 70% is exported. Commercially grown teas in Thailand are the
Assam cultivar (Camellia sinensis var. assamica) and the Chinese cultivar
(Camellia sinensis var. sinensis). The Assam tea is usually used for green and
black tea production, while the Chinese tea is used for green and oolong tea
production. Green tea processing in Thailand occurs in five steps: harvesting,
roasting, rolling, drying and blending/packing. The amount of tea polyphenols
has been regarded as a quality indicator of tea (Owuor et al., 2006). Research
concerning the Thai tea has been conducted on the caffeine content
(Suteerapataranon et al., 2009) and volatile flavor constituents (Pripdeevech
and Machan 2011). Data based on chemical constituents are complementary
indicators of the quality of tea, regarding its biological activities. The growing
popularity of tea in recent years on the basis of beneficial health effects requires
additional data concerning its chemical constituents and biological activities. As
polyphenols are the most important group of tea leaf components, especially the
catechins, the objectives of this study were to investigate and compare the
chemical content and antioxidant capacities of green teas produced from C.
assamica and C. sinensis cultivated in Thailand. The contents of caffeine, total
polyphenols, total catechins and eight catechins (GC, EGC, C, EC, EGCG, GCG,
ECG and CG) together with the radical scavenging capacity and reducing power
capacity are also investigated.
MATERIAL AND METHODS
Green tea samples
A total of 13 green teas of 2 major cultivars, C. assamica and C. sinensis (cv.
oolong no.12) were collected from factories in Thailand from April to June 2013.
The samples were analyzed within 6 months of their production. For each
factory, three samples were sampled.
Chemicals and reagents
The standards , which include gallic acid (G, ≥ 99%), (-)-gallocatechin (GC, ≥
98%), (-)-epigallocatechin (EGC, ≥ 98%), (+)-catechin (C, ≥ 98%), (-)-
epicatechin (EC, ≥ 90%), (-)-epigallocatechin gallate (EGCG, ≥ 95%), caffeine
(CF, ≥ 99%), (-)-gallocatechin gallate (GCG, ≥ 98%), (-)-epicatechin gallate
Tea (Camellia sinensis L.) has attracted much attention due to the antioxidant capacities of bioactive compounds present in it, which are
considered beneficial to consumer health. In Thailand, commercially cultivated teas are the Assam and the Chinese cultivars which both
are processed as green teas. The aim of this study was to investigate the chemical composition and antioxidant capacities of these teas.
Samples were collected from tea factories and analyzed for caffeine content (CF), total polyphenol content (TPC), total catechin content
(TCC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (+)-catechin (C), (-)-epicatechin (EC), (-)-epigallocatechin gallate (EGCG),
(-)-gallocatechin gallate (GCG), (-)-epicatechin gallate (ECG), (-)-catechin gallate (CG), the 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical-scavenging activity (DPPH-assay) and the ferric reducing antioxidant power (FRAP-assay). The results showed that green tea
produced from the Assam cultivar was significantly higher in CF and TPC than the Chinese cultivar. There was no significant difference
in the TCC. Both cultivars had the same level of radical scavenging capacity (DPPH). As opposed to the radical scavenging capacity,
there was a significant difference in the reducing power capacity (FRAP). The FRAP of the Assam was significantly higher than that of
the Chinese tea. There was a significant difference in the individual catechins; the Assam cultivar mainly contained ECG, EGCG, and
EC, whereas the major catechins in the Chinese cultivar were EGCG and EGC. Results from this study suggest that the profiles of
catechins in green teas manufactured in Thailand might contribute to the cultivars.
ARTICLE INFO
Received 25. 11. 2013
Revised 9. 1. 2014
Accepted 10. 2. 2014
Published 1. 4. 2014
Regular article
J Microbiol Biotech Food Sci / Theppakorn et al. 2014 : 3 (5) 364-370
365
(ECG, ≥ 98%) and (-)-catechin gallate (CG, ≥ 98%), were purchased from
Sigma-Aldrich (St. Louis, Missouri, USA). Trolox ((±)-6-Hydroxy-2,5,7,8-
tetramethylchromane-2-carboxylic acid) and DPPH (2,2-diphenyl-1-
picryhydrazyl) were purchased from Aldrich (Steinheim, Germany). Folin–
Ciocalteu’s phenol reagent, monosodium phosphate monohydrate, disodium
phosphate heptahydrate, acetonitrile, trifluoroacetic acid (TFA), trichloroacetic
acid (TCA) and methanol (HPLC-grade) were purchased from Fluka (Buchs,
Switzerland). Potassium hexacyanoferrate [K3Fe(CN)6] and anhydrous sodium
carbonate were purchased from Merck (Darmstadt, Germany). Ascorbic acid and
ferric chloride (FeCl3) were purchased from Ajax Finechem (Seven Hills,
Australia). Water was purified with a Milli-Q water purification system
(Millipore, Bedford, USA). All other chemicals and solvents in this study were
of analytical grade.
Sample extraction
Collected tea samples were ground and samples (5 g, weighed to the nearest
0.001g) were extracted with 100 mL of distilled water at 80 °C for 30 min
(Vuong et al., 2013). The extraction mixture was filtered through a filter paper
(Whatman No. 4). The residue was washed with distilled water (3x10mL). The
tea solution was cooled to room temperature and adjusted to 250 mL with
distilled water. All samples were extracted in triplicate.
Determination of moisture content
Tea samples, 5 g, weighed to the nearest 0.001g, were placed in a moisture can
and heated in an oven at 103±2C for at least 16 h to constant weight. The
percentage of moisture content (%MC) and dry matter (%DM) in the samples
were then calculated from the weight differences. All tests were performed in
triplicate.
Determination of total polyphenol content (TPC)
The total polyphenol content (TPC) was determined by spectrophotometry, using
gallic acid as a standard, according to the method described by the International
Organization for Standardization (ISO 14502–1, 2005). Sample extracts were
diluted 50-fold with distilled water and 1.0 mL portions of the diluted solution
were transferred in duplicate to separate tubes containing 5.0 mL of 10%v/v
dilution of Folin-Ciocalteu’s reagent in water. Then, 4.0 mL of sodium carbonate
solution (7.5% w/v) was added with mixing. The tubes were then left to stand at
room temperature for 60 min before absorbance at 765 nm was measured. The
concentration of polyphenols in samples was derived from a standard curve of
gallic acid ranging from 10-100 μg/mL. The TPC was expressed as gallic acid
equivalents (GAE), i.e. GAE g/100 g dry weight (DW).
Determination of individual catechins, total catechins and caffeine
Individual catechins, total catechins content (TCC) and caffeine (CF) were
determined by ISO method (ISO 14502–2, 2005) with slight modifications. The
individual standard solutions of GC, EGC, C, EC, EGCG, GCG, ECG, CG and
CF were prepared by dissolving them in methanol to generate a stock
concentration of 1,000 µg/mL. The mixed stock standard solution was prepared
by mixing an equal volume of each stock standard. Working standard solutions
were prepared by dilution of the mixed stock solution and then filtered through a
0.45 µm polytetrafluoroethylene (PTFE) filter (Millipore Ltd., Bedford, USA.
HPLC analysis of standards and samples was conducted on Water 966 high
performance liquid chromatography comprising vacuum degasser, quaternary
pump, auto-sampler, thermostatted column compartment and photo diode array
detector. The column used was a Platinum EPS C18 reversed phase, 3µm (53x7
mm) with the column temperature of 30°C. Mobile phase was water/acetonitrile
(87:13) containing 0.05% (v/v) trifluoroacetic acid (TFA) with the flow rate of 2
mL/min. The injection volume was 20 µL. Absorption wavelength was 210 nm.
Validation was carried out in compliance with the AOAC International guidelines
for single Lab. validation of chemical methods for dietary supplements and
botanicals (AOAC, 2002). Individual catechins and caffeine were identified by
comparing their retention times and UV spectra in the 190-400 nm range with
standards. Calibration graphs were constructed using nine levels of concentration
which covered the concentration ranges expected in the tea samples. The
characteristics of the calibration curves, including the regression equation,
correlation coefficient (r), range of linearity, limit of detection (LOD), limit of
quantitation (LOQ) were determined. The LOD and LOQ were based on a signal-
to-noise (S/N) ratio as 3:1 and 10:1, respectively. The linearity of the HPLC
method was checked for caffeine and individual catechins (0.10-51.9, 0.09-43.1,
0.20-100.0, 0.19-97.0, 0.16-80.0, 0.20-100.0, 0.11-52.9, 0.20-98.0, 0.09-44.1
µg/mL for GC, EGC, C, EC, EGCG, CF, GCG, ECG and CG, respectively. All
the analytes exhibited good linearity (r) over the range tested with correlation
coefficient from 0.9965 for GCG to 0.9997 for CF. Individual catechins and
caffeine were quantified using calibration curves. The results were expressed as
g/100 g DW. The total catechin content (TCC) as g/100 g DW was determined by
the summation of individual catechins (GC, EGC, C, EC, EGCG, GCG, ECG and
CG).
Determination of DPPH radical scavenging activity (DPPH assay)
Tea extract was diluted (50-fold) with distilled water. The diluted tea extract (50
µL) was mixed with an aliquot of 2,000 µL of 60 µM DPPH radical in methanol.
Distilled water was used as a control instead of the extract. The reaction mixture
was vortex-mixed and let to stand at 25C in the dark for 60 min. Absorbance at
517 nm was measured using methanol as a blank. The control and standard were
subjected to the same procedures as the sample except that, for the control, only
distilled water was added, and, for the standard, the extract was replaced with 0-
1,000 µM Trolox standard. The percentage inhibition values were calculated
from the absorbance of the control (Ac) and of the sample (As). The calibration
curve was plotted between Trolox concentration (µM) and % inhibition. The
DPPH radical scavenging activity of tea sample was expressed in terms of
millimole equivalents of Trolox (TE) per 100 grams of dry sample, i.e. mmol
TE/100 g DW) (Roy et al., 2010) .
Determination of ferric reducing antioxidant power activity (FRAP assay)
A 1-mL aliquot of each extract (50-fold dilution) was mixed with 2.5 mL of
phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of a 1% aqueous potassium
hexacyanoferrate [K3Fe(CN)6] solution. After 30 min incubation at 50C, 2.5 mL
of 10% trichloroacetic acid were added, and the mixture was centrifuged for 10
min. A 2.5-mL aliquot of the upper layer was mixed with 2.5 mL of water and
0.5 mL of 0.1% aqueous FeCl3, and the absorbance was recorded at 700 nm. The
control and standard were subjected to the same procedures as the sample except
that, for the control, only distilled water was added, and, for the standard, the
extract was replaced with 0-1,000 µM ascorbic acid standard. The calibration
curve was plotted between ascorbic acid concentration (µM) and absorbance at
700 nm. Iron (III) reducing activity was determined as millimole equivalents of
ascorbic acid (AA) per 100 grams of dry sample, i.e. mmol AA/100 g DW)
(Hajimahmoodi et al., 2008).
Statistical analysis
Data were expressed as means ± standard deviation. The data were also subjected
to analysis of variance (ANOVA) and Duncan’s multiple range tests using SPSS
16.0 for Windows. The significance level of P<0.05 was considered significantly
different.
RESULTS AND DISCUSSION
Elution order of compounds analyzed by HPLC
The modification of the analytical method separated a mixture of standards of
caffeine (CF) and 8 catechins (GC, EGC, C, EC, EGCG, GCG, ECG and CG)
within 20 min. Typical chemical profiles identified by HPLC in green teas are
shown in Fig. 1. The elution order was GC, EGC, C, EC, EGCG, CF, GCG ECG
and CG (Fig. 1A). As can be seen from Fig. 1 (B and C), green teas from C.
assamica (Assam tea) and C. sinensis (Chinese tea) contained CF and 7 catechins
(GC, EGC, C, EC, EGCG, GCG and ECG), but CG was not detected in green
teas produced from either cultivars.The disappearance of CG in both varieties
might be related to the quality of tea grown in Thailand having soil and climatic
conditions different from other regions. Moreover, cultivation practices, post-
harvesting handling and processing techniques of Thai green teas may contribute
in this disappearance.
J Microbiol Biotech Food Sci / Theppakorn et al. 2014 : 3 (5) 364-370
366
GC
A
B
C
EGC C
EC
EGCG
CF
GCG
ECG
CG
Figure 1 Typical chromatogram of standards (A), green teas from C. assamica
(B) and C. sinensis (C). Peak identification and approximate retention times in
minutes in parentheses are as follows: GC (1.25); EGC (1.59); C (1.88); EC
(2.56); EGCG (2.94); CF (3.40); GCG (4.36); ECG (5.81); and CG (8.71).
Chemical compositions and antioxidant capacities of green teas from C.
assamica
Data on the caffeine content, total polyphenol content, total catechin content and
antioxidant capacities (DPPH-assay and FRAP-assay) of the green teas produced
from C. assamica are presented in Table 1. The caffeine content ranged from
3.27 to 3.71 g/100 g DW, with a mean of 3.44±0.51 g/100 g DW. There was no
significant difference in caffeine content of all samples analyzed (P≥0.05). Total
polyphenol content varied slightly, ranging from 16.63 to 20.83 GAE g/100 g
DW with an average of 18.95±1.45 GAE g/100 g DW. Among these teas, A-GT-
004 taken from Ming Dee Tea had the highest concentrations of phenolic
substances, whereas A-GT-006 taken from Mae Ka Tea had the lowest
concentrations. Catechins are the main phenolic compounds and are present in
large amounts in green tea. In this study, total catechins varied slightly, ranging
from 9.02 to 14.08 g/100 g DW with an average of 12.42±2.41 g/100 g DW.
There was no significant difference in total catechins in all samples (P≥0.05).
Among the 6 tested samples, the DPPH values ranged from 132.32 to 247.11
mmol TE/100 g DW, with a mean value of 183.90±46.68 mmol TE/100 g DW.
A-GT-001 collected from Raming Tea had the highest DPPH value (247.11±2.02
mmol TE/100 g DW), followed by A-GT-002 (240.56±4.77 mmol TE/100 g
DW) collected from Choui Fong Tea. A-GT-006 collected from Mae Ka Tea had
the lowest DPPH value (132.32±0.89 mmol TE/100 g DW). The FRAP values
ranged from 1.22 to 2.13 mmol AA/100 g DW, with a mean value of 1.66±0.32
mmol AA/100 g DW. A-GT-004 collected from Ming Dee Tea had the highest
FRAP value (2.13±0.06 mmol TE/100 g DW).
Table 1 Mean values of caffeine, total polyphenol, total catechin content and antioxidant capacities of 6 green teas produced from C. assamica
No.
Code
Company
Caffeine ns
(g/100 g DW)
Total polyphenols
(GAE g/100 g DW)
Total catechins ns
(g/100 g DW)
Antioxidant capacities
DPPH
(mmol TE/100 g DW)
FRAP
(mmol AA/100 g DW)
1
A-GT-001
Raming Tea
3.71±0.21
19.69±0.62 ab
13.08±0.54
247.11±2.02 a
1.44±0.00 d
2
A-GT-002
Choui Fong Tea
3.43±0.05
18.27±0.25 c
12.87±0.32
240.56±4.77 a
1.22±0.01 e
3
A-GT-003
Thai Tea Suwirun
3.44±0.01
19.71±0.87 ab
13.51±0.21
150.69±3.20 c
1.91±0.05 b
4
A-GT-004
Ming Dee Tea
3.35±1.59
20.83±0.80 a
11.97±0.98
178.92±1.58 b
2.13±0.06 a
5
A-GT-005
Choui Fong Tea
3.27±0.04
18.57±0.06 bc
14.08±0.17
153.80±11.40 c
1.74±0.06 c
6
A-GT-006
Mae Ka Tea
3.45±0.23
16.63±0.07 d
9.02±0.29
132.32±0.89 d
1.53±0.08 d
Minimum
3.27
16.63
9.02
132.32
1.22
Maximum
3.71
20.83
14.08
247.11
2.13
Mean
3.44
18.95
12.42
183.90
1.66
Standard deviation
0.51
1.45
2.41
46.68
0.32
Values are expressed as means ± SD (n=3). Different letters in the same column indicate significant difference at p < 0.05.
ns = not significant.
Comparison of the individual catechins of 6 tea manufactures is shown in Table
2. Green teas produced from C. assamica contained GC, EGC, C, EC, EGCG,
GCG and ECG. Surprisingly, CG was not detected in all tested samples. Among
quantified catechins, ECG was predominant, ranging from 1.65 to 3.53 g/100 g
DW with an average of 2.94±0.78 g/100 g DW, followed by EGCG (ranging
from 1.16 to 3.69 with an average of 2.62±0.94 g/100 g DW), EC (ranging from
1.31 to 2.88 with an average of 2.23±0.60 g/100 g DW), EGC (ranging from 0.70
to 2.48 with an average of 1.77±0.61 g/100 g DW), C (ranging from 1.41 to 2.15
with an average of 1.65±0.38 g/100 g DW) and, to lesser extents, GC and GCG.
The tested samples possessed slightly varied quantities of individual catechins
depending on the tea company sources. A little variation in catechins composition
among samples may be the result of cultivation practice, postharvest handling
and processing techniques of each manufactures (Cooper et al., 2005).
However, our results indicate that the major catechins in C. assamica green teas
were ECG, EGCG and EC.
J Microbiol Biotech Food Sci / Theppakorn et al. 2014 : 3 (5) 364-370
367
Table 2 Mean values of individual catechins present in 6 green teas produced from C. assamica
No.
Code
Company
Individual catechins (g/100 g DW)
GC ns
EGC
C ns
EC
EGCG
GCG ns
ECG
1
A-GT-001
Raming Tea
0.77±0.06
1.60±0.36 c
1.70±0.04
2.88±0.16 a
2.20±0.61 cd
0.39±0.08
3.53±0.14 a
2
A-GT-002
Choui Fong Tea
1.01±0.02
1.95±0.01 bc
1.51±0.03
2.29±0.05 ab
2.74±0.05 abc
0.48±0.04
2.90±0.14 ab
3
A-GT-003
Thai Tea Suwirun
0.89±0.11
1.70±0.20 bc
2.15±0.13
2.62±0.04 ab
2.44±0.16 bc
0.25±0.02
3.45±0.00 a
4
A-GT-004
Ming Dee Tea
0.99±0.56
2.16±0.19 ab
1.41±0.95
1.31±0.91 c
3.69±0.92 a
0.76±0.58
1.65±1.41 b
5
A-GT-005
Choui Fong Tea
0.96±0.03
2.48±0.02 a
1.61±0.01
2.39±0.02 ab
3.47±0.06 ab
0.34±0.02
2.84±0.04 ab
6
A-GT-006
Mae Ka Tea
0.39±0.03
0.70±0.01 d
1.53±0.05
1.87±0.01 ab
1.16±0.02 d
0.10±0.14
3.24±0.10 a
Minimum
0.39
0.70
1.41
1.31
1.16
0.10
1.65
Maximum
1.01
2.48
2.15
2.88
3.69
0.76
3.53
Mean
0.84
1.77
1.65
2.23
2.62
0.39
2.94
Standard deviation
0.28
0.61
0.38
0.60
0.94
0.28
0.78
Values are expressed as means ± SD (n=3). Different letters in the same column indicate significant difference at p < 0.05.
CG was not detected in any sample. ns = not significant.
Chemical compositions and antioxidant capacities of green tea from C.
sinensis
Results of the caffeine content, total polyphenol content, total catechin content
and antioxidant capacities of the green teas from C. sinensis are presented in
Table 3. The caffeine ranged between 1.69 to 3.51 g/100 g DW with a mean of
2.50±0.71 g/100 g DW. This mean value was significantly lower than in
C.assamica samples (P<0.05). Among 7 samples analyzed, O12-GT-007 taken
from Doi Chang Tea had the highest concentrations of caffeine, whereas O12-
GT-004 taken from Sing Long Shop had the lowest concentrations than other
samples. Total polyphenols varied from 11.52 to 17.34 GAE g/100 g DW with an
average of 14.58±1.87 GAE g/100 g DW. This value was significantly lower than
in C.assamica samples (P<0.05). Total catechins ranged from 8.32 to 13.20 g/100
g DW with an average of 10.86±1.47 g/100 g DW. This value was not
significantly different from C.assamica samples (P≥0.05). Among 7 samples
analyzed, there were significant differences in total polyphenols and total
catechins (P<0.05), in which the highest total polyphenols (17.34±0.29 GAE
g/100 g DW) and total catechins (13.19±0.32 g/100 g DW) were found in the
OT-GT-003 sample taken from Jing Jing Tea shop. Among the tested samples,
the DPPH values ranged from 80.08 to 241.39 mmol TE/100 g DW, with a mean
value of 146.97±56.40 mmol TE/100 g DW. O12-GT-007 collected from Doi
Chang Tea had the highest DPPH value (241.39±0.71 mmol TE/100 g DW). The
FRAP values ranged from 0.61 to 1.08 mmol AA/100 g DW, with a mean value
of 0.85±0.16 mmol AA/100 g DW. O12-GT-003 collected from Jing Jing Tea
shop had the highest FRAP value (1.08±0.17 mmol AA/100 g DW).
Table 3 Mean values of caffeine, total polyphenol and total catechin content and antioxidant capacities of 7 green teas produced from C. sinensis
No.
Code
Company
Caffeine
(g/100 g DW)
Total polyphenols
(GAE g/100 g DW)
Total catechins
(g/100 g DW)
Antioxidant capacities
DPPH
(mmol TE/100 g DW)
DPPH
(mmol TE/100 g DW)
1
O12-GT-001
Rai Boon Rawd
2.54±0.11 c
14.31±0.23 d
10.97±1.08 ab
119.35±3.52 d
0.79±0.08 cd
2
O12-GT-002
Wong Pud Tan Tea
2.81±0.27 bc
15.09±0.11 c
10.91±0.78 ab
116.18±1.19 de
0.89±0.02 abc
3
O12-GT-003
Jing Jing Tea Shop
3.17±0.08 ab
17.34±0.29 a
13.19±0.32 a
147.34±1.03 c
1.08±0.17 a
4
O12-GT-004
Sing Long Shop
1.69±0.10 d
13.71±0.37 d
10.29±1.26 bc
111.80±0.56 e
0.82±0.00 bcd
5
O12-GT-005
Thai Sanguan Tea
1.93±0.36 d
11.52±0.09 e
8.32±1.62 c
80.08±2.19 f
0.61±0.04 d
6
O12-GT-006
Yao Sing Shop
1.82±0.03 d
13.91±0.23 d
10.52±0.53 bc
212.64±5.73 b
0.78±0.07 cd
7
O12-GT-007
Doi Chang Tea
3.51±0.07 a
16.14±0.44 b
11.66±0.35 ab
241.39±0.71 a
1.00±0.09 ab
Minimum
1.69
11.52
8.32
80.08
0.61
Maximum
3.51
17.34
13.20
241.39
1.08
Mean
2.50
14.58
10.86
146.97
0.85
Standard deviation
0.71
1.87
1.47
56.40
0.16
Values are expressed as means ± SD (n=3). Different letters in the same column indicate significant difference at p < 0.05.
ns = not significant.
Comparison of the individual catechins of 7 tea manufactures is shown in Table
4. Green teas produced from C. sinensis contained 7 catechins as found in C.
assamica. CG was again not detected in any sample. Among quantified catechins,
the most abundant catechin was EGCG. The EGCG ranged from 2.75 to 5.49
with an average of 3.99±0.92 g/100 g DW, Among samples analyzed, O12 -GT-
003 taken from Jing Jing Tea Shop had the highest concentrations of EGCG
(5.49±0.29 g/100 g DW), followed by O12-GT-007 taken from Doi Chang Tea
(4.62±0.30 g/100 g DW). The second most abundant catechin was EGC, ranging
from 2.17 to 3.18 with an average of 2.75±0.48 g/100 g DW. There was no
significant difference in EGC among samples analyzed. The remaining catechins,
GC (0.94±0.14 g/100 g DW), C (0.83±0.17 g/100 g DW), EC (0.92±0.14 g/100 g
DW), GCG (0.65±0.23 g/100 g DW), and ECG (0.77±0.29 g/100 g DW), were
found in small amount when compared to EGCG and EGC. These results
demonstrate that the two major catechins in C.sinensis green teas were EGCG
and EGC.
J Microbiol Biotech Food Sci / Theppakorn et al. 2014 : 3 (5) 364-370
368
Table 4 Mean values of individual catechins present in 7 green teas produced from C. sinensis
No.
Code
Company
Individual catechins (g/100 g DW)
GC
EGC ns
C
EC
EGCG
GCG
ECG
1
O12-GT-001
Rai Boon Rawd
1.13±0.04 a
2.87±0.41
0.90±0.02 ab
0.95±0.07 b
3.74±0.61 bc
0.76±0.18 b
0.63±0.01 c
2
O12-GT-002
Wong Pud Tan Tea
0.91±0.04 bc
2.95±0.18
0.87±0.14 bc
0.91±0.08 bc
4.01±0.27 bc
0.51±0.10 bc
0.78±0.06 b
3
O12-GT-003
Jing Jing Tea Shop
0.91±0.03 bc
2.41±0.13
1.04±0.01 a
1.09±0.01 a
5.49±0.29 a
1.06±0.11 a
1.19±0.04 a
4
O12-GT-004
Sing Long Shop
1.08±0.05 a
3.18±0.56
0.64±0.03 d
0.80±0.01 cd
3.50±0.63 bc
0.60±0.12 bc
0.52±0.03 cd
5
O12-GT-005
Thai Sanguan Tea
0.99±0.05 ab
2.17±0.63
0.64±0.01 d
0.70±0.01 d
2.75±0.85 c
0.66±0.10 b
0.41±0.06 d
6
O12-GT-006
Yao Sing Shop
0.80±0.01 cd
3.16±0.22
0.72±0.04 cd
0.91±0.04 bc
3.83±0.34 bc
0.36±0.06 c
0.76±0.01 b
7
O12-GT-007
Doi Chang Tea
0.74±0.14 d
2.50±0.40
1.03±0.07 a
1.09±0.08 a
4.62±0.30 ab
0.58±0.01 bc
1.12±0.06 a
Minimum
0.74
2.17
0.64
0.70
2.75
0.36
0.41
Maximum
1.13
3.18
1.04
1.09
5.49
1.06
1.19
Mean
0.94
2.75
0.83
0.92
3.99
0.65
0.77
Standard deviation
0.14
0.48
0.17
0.14
0.92
0.23
0.29
Values are expressed as means ± SD (n=3). Different letters in the same column indicate significant difference at p < 0.05.
CG was not detected in any sample. ns = not significant.
Comparison of green teas from C. assamica and C. sinensis
The chemical compositions and antioxidant capacities of green teas produced
from C. assamica and C.sinensis are shown in Table 5. The caffeine of the
Assam cultivar (3.44±0.51 g/100 g DW) was significantly higher than the
Chinese cultivar (2.50 ±0.71 g/100 g DW). Obuchowicz et al (Obuchowicz et
al., 2011) reported that caffeine content in green tea was 1.44-4.03 g/100 g DW
(average 2.60, n=287). Our values are in the range of previous reports. The
comparison between the Assam and the Chinese cultivars indicated that cultivars
affected the level of caffeine in green teas. However, there have been reports that
there was no significant difference in the caffeine contents of tea infusions
brewed from these two varieties (Suteerapataranon et al., 2009). This might be
because this study used only one tea sample and taken from a manufacturer who
cultivated the variety assamica in the area near to the variety sinensis, indicating
that cultivation areas may also affect the caffeine content. It is suggested that the
determinations of the chemical profile of teas should follow sampling protocols
that include tea samples from various sources and manufacturers.
Phenolic compounds are a class of chemical constituents containing one or more
hydroxyl residues attached to an aromatic (phenyl) ring. They are one of the most
effective antioxidative constituents that contribute to the antioxidant capacities of
teas. Hence, it is important to quantify phenolic content and to assess its
contribution to antioxidant activity. Green tea from the Assam cultivar
(18.95±1.45 GAE g/100 g DW) had levels of total polyphenols significantly
(p<0.05) higher than the Chinese cultivar (14.58±1.87 GAE g/100 g DW). This
result is in agreement with Jin et al. (Jin et al., 2005) who reported that the
Assam cultivar had significantly higher polyphenols content than the Chinese
cultivar. Previous reports have stated that the total polyphenols in green tea was
9.95-25.79 GAE g/100 g DW (average 16.35, n=347) (Obuchowicz et al., 2011),
10.10-22.20 GAE g/100 g DW (average 17.00, n=51) (Engelhardt 2005), 8.70-
10.60 GAE g/100 g DW (average 8.63, n=3) (Khokhar and Magnusdottir
2002). Our results are in reasonable agreement with these previous reports.
The levels of total catechins in the Assam and the Chinese cultivars were
12.42±2.41 g/100 g DW and 10.86±1.47 g/100 g DW, respectively. This result is
in accordance with mean values obtained in previous studies, 6.57-21.38 g/100 g
DW (average 11.70, n=358) (Obuchowicz et al., 2011), 8.50-20.60 g/100 g DW
(average 15.10, n=51) (Engelhardt 2005) and 2.80-22.80 g/100 g DW (average
10.90, n=18) (Unachukwu et al., 2010). There were no significant differences in
total catechins of green teas from these two varieties. Among 8 catechins
analyzed in two varieties, CG was not detected and GC was in a similar level in
both tea types. The levels of the EGC, C, EC, EGCG, GCG and ECG showed
apparently significant differences (p<0.05). The ECG (2.94±0.78 g/100 g DW),
EGCG (2.62±0.94 g/100 g DW), and EC (2.23±0.60 g/100 g DW), were the 3
predominant catechins in the Assam cultivar, whereas the EGCG (3.99±0.92
g/100 g DW), and EGC (2.75±0.48 g/100 g DW), were the 2 predominant
catechins in the Chinese cultivar. The four major catechins present in green tea
were reported as EGCG, EGC, ECG, and EC (Peterson et al., 2005; Ananingsih
et al., 2013; Obuchowicz et al., 2011). According to the study by Obuchowicz
et al., (2011), the greatest amount of individual catechins in green teas was
EGCG followed by EGC, which is similar to our result with the Chinese cultivar.
Most studies of individual catechins in green teas have been carried out on the
Chinese tea. This may be because the Chinese tea is usually used to produce
green teas. There are a few countries that grow both the Assam and the Chinese
tea in order to process as the green tea. Therefore, most data on individual
catechins in green teas is obtained from the Chinese cultivar. Khokhar and
Magnusdottir (2002) had cited variations in the abundance of compounds in
teas as being dependent on sample tea subtypes and the different subtypes result
from different tea processing protocols, horticultural practices, and geographic
settings (Sultana et al., 2008).
The radical scavenging capacity and reducing power capacity of a sample is
regarded as a significant indicator of its potential antioxidant activity. FRAP
measures the ability of compounds to act as an electron donor while DPPH
measures their ability to act as hydrogen donors. In DPPH assays, the antioxidant
capacity of the Assam cultivar was slightly higher than the Chinese cultivar.
However, no significant differences between cultivars were found with the
Duncan test. Although the Assam cultivar had the total polyphenols significantly
higher than the Chinese cultivar, both cultivars had the same level of radical
scavenging capacity (DPPH). This is likely due to the presence of different
antioxidant compounds in green teas. As opposed to the radical scavenging
capacity, there was a significant difference in the reducing power capacity
(FRAP) (P<0.05). The FRAP of the Assam was substantially higher than the
Chinese (P<0.05). According to a study by Stewart et al., (2005), the greatest
antioxidant contribution to green tea comes from catechins. The different result in
the DPPH and the FRAP found in this study can be demonstrated by the
difference in mechanisms between the scavenging and chelating for measuring
the antioxidant capacity. The antioxidant capacity of tea may not be determined
by one or few phytochemical compounds, but is widely distributed among a
range of phenolic compounds including catechins (Horzic et al., 2009) and
additional antioxidant compounds such as glycosylated flavonols,
proanthocyanidins, and phenolic acids and their derivatives (Lin et al., 2008) in
green tea as evidenced by higher TPC levels in the Assam tea.
Table 5 Comparison of green teas produced from C. assamica and C. sinensis
Specifications
C. assamica
C. sinensis
Caffeine content
3.44±0.51 a
2.50±0.71 b
Total polyphenol content
18.95±1.45 a
14.58±1.87 b
Total catechin content
12.42±2.41
10.86±1.47
GC
0.84±0.28
0.94±0.14
EGC
1.77±0.61 b
2.75±0.48 a
C
1.65±0.38 a
0.83±0.17 b
EC
2.23±0.60 a
0.92±0.14 b
EGCG
2.62±0.94 b
3.99±0.92 a
GCG
0.39±0.28 b
0.65±0.23 a
ECG
2.94±0.78 a
0.77±0.29 b
CG
nd
nd
DPPH assay
183.90±46.68
146.97±56.40
FRAP assay
1.66±0.32 a
0.85±0.16 b
Values are expressed as means ± SD.
Different letters in the same row indicate significant difference at p < 0.05.
nd = not detected.
J Microbiol Biotech Food Sci / Theppakorn et al. 2014 : 3 (5) 364-370
369
As found in this study, the difference in chemical constituents and antioxidant
capacities might be related to the cultivars. Thus our hypothesis is that the pattern
displayed by catechins enables and observer who is given a tea extract, but is not
told from which tea species the extract was prepared, to determine this with
reasonable confidence purely from analyzing the pattern displayed by these
compounds. At present, the data do not enable a decision as to whether this is just
true for the region where the work was performed, or if is generally applicable.
The challenge is therefore to tea scientists in other geographical regions where
teas are produced comparable to those studied here, to determine if their regional
products are classifiable by the same criteria, and by their results to verify or
falsify this hypothesis as something processing general applicability.
It also should be noted that these differences could be related to the quality of tea
grown in different regions having their soil and climatic conditions different,
cultivation practices, post-harvesting handling and processing techniques by
different manufacturers. Because health effects may depend on the tea consumed
and because there is a lack of composition data on tea produced in different parts
of the world, there is a need to know the compositions. Our results provide
information that the consumer may be able to select green teas containing high
levels of beneficial compounds and avoid those containing low amounts of these
compounds.
CONCLUSION
This study provides evidence of the dependability of amounts of compounds and
antioxidant capacities in green tea manufactured in Thailand. Present findings
show that green tea from the Assam cultivar had caffeine and total polyphenols
levels significantly higher than the Chinese cultivar. The ECG, EGCG and EC
were the 3 predominant catechins in the Assam cultivar, whereas the EGCG and
EGC were the 2 predominant catechins in the Chinese cultivar. The difference in
catechins found in this study might be related to the cultivars used to produce
green teas. The results reported here were derived from the analysis of samples
collected from representative tea manufacturing establishments in Thailand. It is
well recognized that uncontrolled variables, such as the geography, climates,
season, cultivation practice, postharvest handling and processing techniques may
affect the chemical composition and antioxidant capacities of green teas.
However, this investigation provides basic information on important chemical
compounds present in Thai green tea, which can be added to the national and
regional databases. The data can also be used as a guideline for establishing tea
standards and for further detailed studies.
Acknowledgments: The authors warmly thank Mae Fah Luang University for a
support.
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