World Journal of Agricultural Sciences 9 (3): 266-270, 2013

ISSN 1817-3047
IDOSI Publications, 2013
DOI: 10.5829/idosi.wjas.2013.9.3.1737

Tea Polyphenols and Alkaloids Content Using

Soxhlet and Direct Extraction Methods
1

Fui-Seung Chin, 1Khim-Phin Chong, 1Atong Markus and 2Nyet Kui Wong
School of Sustainable Agriculture, Universiti Malaysia Sabah,
Jalan UMS, 88400 Kota Kinabalu Sabah, Malaysia
2
School of Science and Technology, Universiti Malaysia Sabah,
Jalan UMS, 88400 Kota Kinabalu Sabah, Malaysia
1

Abstract: Extraction efficiency of extraction methods on the content of polyphenols (catechins and gallic acid)
and alkaloids (caffeine, theobromine and theophylline) from tea leaves is studied. Level of polyphenols and
alkaloids in samples (leaf buds, young leaves, old leaves and Sabah black Tea) from Sabah Tea Plantation were
evaluated by HPLC. Leaf buds were found to have a higher polyphenol and alkaloid contents compared to
young and old leaves. Fermentation process during manufacturing of Sabah Tea significantly reduced the
levels of catechins, gallic acid and alkaloids by approximately 2-fold in comparison to fresh tea leaves.
Degradation of catechins was also observed at high extraction temperatures. By increasing the heating
temperature from 40C in direct extraction to 70C in soxhlet extraction, concentrations of total polyphenols and
total alkaloids decreased. However, no significant change was observed in the Sabah Black Tea, with the only
exception for epigallocatechin that was 3-fold lower in soxhlet extraction. It is recommended that for preparation
of oxidation-sensitive plant samples like tea, extraction should be carried out at 40C or lower using direct
extraction method couple with a multi-step extraction procedure, rather than percolating with high boiling point
(> 40C) organic solvents that are routinely used in plant extraction.
Key words: Tea leaves

Soxhlet extraction

Direct extraction

INTRODUCTION

Alkaloids

made. Yield of polyphenols and alkaloids in different

extraction parameters such as heating temperatures,
extraction solvents, pH and filter membranes have been
analyzed [6, 8-12]. However, comparison of difference
extraction methods for the extraction yields of tea leaf
polyphenol and alkaloid contents from different
maturation stages has not been reported.
In the present paper, the changes in polyphenol and
alkaloid components in tea leaves (leaf buds, young
leaves and old leaves) and in the commercial Sabah Black
Tea (SBT) in two different extraction methods are
examined.

In tea (Camellia sinensis L.) plant, polyphenols and

alkaloids have been considered as the main active
components in many pharmacological studies [1-5].
Lost of potential activities in plant extracts after prolong
extraction and heating were usually reported and that
would be barrier to further study the functionality and
potential of these extracts. Hence, a good extraction
method must be able to extract compounds of interest
completely and simultaneously avoid chemical
modification [3]. Hot water (70100C) was frequently
used in tea extraction in many studies [1, 2, 4-6]. However,
the purpose of these studies focused more on the sensory
evaluation of tea quality by colour, flavour and taste [7],
provided little quantitative information about the actual
polyphenols and alkaloids present in the solid samples.
Many attempts have been made to investigate the factors
attributed to extraction yields and great progress has been
Corresponding Author:

Polyphenols

MATERIALS AND METHODS

Chemicals: Authentic (+)-catechin hydrate (C), ()epicatechin (EC), ()-epicatechin gallate (ECG), ()epigallocatechin (EGC), ()-epigallocatechin gallate
(EGCG), gallic acid (GA) and caffeine (Caf) were bought

Nyet Kui Wong, School of Science and Technology, Universiti Malaysia Sabah, Jalan UMS,
88400 Kota Kinabalu Sabah, Malaysia.

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World J. Agric. Sci., 9 (3): 266-270, 2013

from Sigma Chemical Co. (St. Louis, MO). Acetonitril

(ACN) and methanol (MeOH) were HPLC grade.
Dichloromethane (DCM) and n-hexane (Hex) were GC
grade. Ortho-phosphoric acid-85% (H3PO4) was reagent
grade chemical. High-purified water was from Millipore
(Billerica, MA, USA).

gallate (CG)] and minor alkaloids [theobromine (Tb) and

theophylline (Tp)] were not available in this study, the
concentration of CG was quantified as the equivalent of
its epimer ECG, while concentration of Tb and Tp were
quantified as the equivalent of Caf.
Extraction Methods
Soxhlet Extraction (SE): Each plant samples (50 g) were
separately filled in four sets of soxhlet apparatus, each
containing 350 mL of Hex as the first extraction solvent.
The temperature was brought to boiling point (69C) and
percolation was carried out for 24 h. On the next day, Hex
extract was discharged and all plant materials were
allowed to dry before the second solvent DCM (350 mL)
was refilled in and percolation was continued for another
24 h. The same process was repeated for MeOH (350 mL).
At the end of extraction period, DCM and MeOH extracts
of LB, YL, OL and SBT were individually distilled off at
40C under reduced pressure in rotary evaporator.
Once concentrated to a small volume, each extract was
transferred to pre-weighed beaker and was allowed to dry
completely in a bench top oven (30C) overnight. A final
concentration of 1 mg.mL 1 of each DCM and MeOH
extracts of LB, YL, OL and SBT were prepared in 20%
aqueous ACN and filtered prior to HPLC analysis. Final
concentration of each identified compound was expressed
in mg.g 1 DW 1 (milligram per gram of dried weight).

Samples Preparation: Fresh tea leaves of the variety

assamica were harvested from the field of Sabah Tea
Plantation (STP). They were categorized into three main
groups, consisting of tea leaf buds (LB), young leaves
with light green in colour (YL) and old leaves with dark
green in colour (OL). Commercial SBT was from the
factory of STP. They were transferred to Laboratory in the
same day and cleaned before being dried in a ventilated
oven at 40C for 48 hours. Dried plant samples were
pulverized into powders and were used throughout in this
study.
Identification and Quantification of Tea Polyphenols and
Alkaloids
Analytical Condition of High Performance Liquid
Chromatography: An Agilent Technologies 1200 series
high performance liquid chromatography (HPLC)
system was used. The column used was an Eclipse
XDB-C18 reversed phase (5 m, 250 x 4.6 mm) with a C18
(5 m, 30 x 4.6 mm) guard column. Analytical conditions
were optimized based on the methods of Yao et al. [13]
and Owuor and Obanda [14]. The mobile phase
composition started at 92% solvent A (0.1% aqueous
H3PO4) and 8% solvent B (100% ACN) and linearly
increased to 18% solvent B in 25 minutes, following
by a post-run of 3 minutes before the next analysis
cycle. Column temperature was set at 35C and the
effluent was monitored at 280 nm. All standards and
samples were filtered through 0.20 m PVDF membrane
filters and aliquot of 20 L was injected into the HPLC
system.

Direct Extraction (DE): Direct extraction of plant samples

(LB, YL, OL and SBT) were carried out with 20% aqueous
ACN under continuous stirring (120 rpm) for 24 h in the
dark. Ten grams of each plant sample were placed in 200
mL capped bottles, each of which was extracted with 100
mL x 3 of 20% aqueous ACN. In the fourth day, all 20%
aqueous ACN extracts of LB, YL, OL and SBT were
filtered prior to HPLC analysis. The mean concentration
was taken from 3 subsequent extractions of each sample
in 3 replications to indicate the final concentration
(mg.g 1 DW 1) of individual compound.

Establishment of Calibration Curves: Quantification of

catechins (C, EC, ECG, EGC and EGCG), caffeine (Caf) and
gallic acid (GA) in sample extracts were carried out using
HPLC with external standard. Stock standard solutions
of 1 mg.mL 1 of catechins, GA and Caf were prepared. A
10 different concentration of each authentic standard
(0.05-50.0 g.mL 1) was generated by plotting linear
regression of peak area in relation to known concentration
(g.mL 1) injected into the HPLC system. The correlation
coefficient (R2) of linear regression curve for all of the
standards was then calculated using Microsoft Excel.
Authentic standards of minor catechin [(-)-catechin

Data Analysis and Statistics

Statistical Analyses: Statistical Package for Social
Science software (SPSS) 12.0 Version for Windows was
chosen in this study. The means and standard deviation
of all the studied parameters within the samples were
calculated with Microsoft Excel. Statistical significance
differences of the extraction yields between SE and DE
were analyzed with one-way ANOVA (analysis of
variance) and the difference of means between groups
was analyzed with post hoc test Tukey HSD. A p-value of
0.05 or less was considered statistically significant.
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World J. Agric. Sci., 9 (3): 266-270, 2013

Fig. 1: (a) HPLC Chromatogram of seven external standards. (b) A typical chromatogram profile of leaf buds sample.
Polyphenol and alkaloid contents in various
extracts of leaf bud (LB)

Polyphenol and alkaloid contents in various

extracts of Sabah Black Tea (SBT)
39.07

43.29

44.02

50
40

22.01

30
20
10
0

37.92

10.42

8.59
3.48

5.36
0.21 2.36 10.9
0.01 0.27
Tp

Tb

5.56

Caff

GA

EC

2.92

1.38 12.87 10.76 24.84

0.86 2.76
C

SE DCM

8.89

40

Concentration
(mg.g-1 DW-1)

Concentration
(mg.g-1 DW-1)

58.77

60

CG

ECG

SE MeOH

24.54

30
20
10
0

EGC EGCG

3.44

Tp

3.48

0.25 1.04
0.91
Tp

Tb

Caff

SE DCM

2.97

0.75

0.01
GA

2.86

EC

SE MeOH

4.92 7.85

12.77 12.78

1.93
0.02

CG

0.02
ECG

EGC

1.45
C

6.18
4.29

EC

13.11
11.78
14.13
12.05
8.82
5.1

4.14

CG

ECG

SE MeOH

EGC

EGCG

DE 20% ACN

21.62

22.25

Concentration
(mg.g-1 DW-1)

Concentration
(mg.g-1 DW-1)

10

7.9
4.37

GA

2.54

Polyphenol and alkaloid contents in various

extracts of old leaf (OL)

22.31

8.98

Caff

SE DCM

15.01
3.76 3.22

5.75
0.02

35.78

25.38

20

Tb

DE 20% ACN

37.45

30

5.87

3.2

0.22 2.58
5.48
0.01 0.28

Polyphenol and alkaloid contents in various

extracts of young leaf (YL)

40

30.51

EGCG

DE 20% ACN

25

16.3

20

9.21

15

10.9 9.03

10
5
0

Tp

Tb

Caff

SE DCM

6.05
2.73

1.712.62
0.85
0.03

0.75

0.99
GA

2.53

EC

SE MeOH

1.67

CG

2.97
2.59

6.56

6.41

0.52

ECG

EGC

EGCG

DE 20% ACN

Fig. 2: Distribution of individual catechins, gallic acid and alkaloids in leaves of Sabah Tea Plantation obtained by
soxhlet and direct extraction methods.
RESULTS AND DISCUSSION

Yields of Individual Polyphenol from

Soxhlet and Direct Extraction Methods: The SE-DCM,
SE-MeOH and 20% ACN aqueous DE extractions from all
samples (LB, YL, OL and SBT) were subjected to HPLC
to determine their concentration of polyphenol and
alkaloid contents and were shown in Fig. 3. The largest
portion of total polyphenols content was EGC, with
LB (DE, 43.3 mg.g 1; SE-MeOH, 10.8 mg.g 1) and
YL (DE, 35.8 mg.g 1, SE-MeOH, 12.8 mg.g 1) being the
highest. The second largest portion of total
polyphenols is EGCG, with LB and YL contained the
highest (DE, 22.3-37.9 mg.g 1; SE, 12.8-24.8 mg.g 1).
Major catechin ECG made up the third largest portion in
the total polyphenols content; with LB contained the
highest in concentration (DE, 22.0 mg.g 1; SE-MeOH, 12.9
mg.g 1), followed by YL (DE, 15.0 mg.g 1; SE-MeOH, 7.9

Analytical Conditions: Complete baseline separation of

tea polyphenols (C, EC, CG, ECG, EGC, EGCG and GA)
and alkaloids (Caf, Tb and Tp) was achieved in 25 min.
All calibration curves obtained were linear over the
concentration ranges indicated in Section 2.3.2, with R2
ranged from 0.9977 to 1.0000 (results not showed).
The HPLC chromatogram of a standard (C, EC, ECG, EGC,
EGCG, GA and Caf; 3 g per standard) and a typical
chromatogram of LB are shown in Fig. 1a and 1b,
respectively. The major polyphenols and alkaloids present
are labeled in Fig. 1a and 1b. The additional 3 peaks were
unknown but they are most probably the minor alkaloid
theobromine (Tb), theophylline (Tp) and minor catechins
(-)-catechin gallate (CG) [13, 15, 16].
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World J. Agric. Sci., 9 (3): 266-270, 2013

90

Concentration (mg.g-1 DW-1)

127.09

GA
EGC
CG
C

120

93.15
b

80

61.41

59.03

42.58

69.12
51.55

40

EGCG
ECG
EC

19.58
f

0
SE

DE
LB

SE

DE
YL

SE

DE
OL

SE

DE

Concentration (mg.g-1 DW-1)

160

74.55

80
70
60

Tb

36.56
d

40

37.15
d

26.58

20.81

30

Caf

47.64

44.43

50

20
10
0
SE

DE
LB

SBT

Tp

58.12

SE

DE
YL

SE

DE
OL

SE

DE
SBT

(a)
(b)
Fig. 3: Total concentration of (a) polyphenols and (b) alkaloids in tea leaves obtained from soxhlet and direct extraction
methods. Bars with the same letter are not significantly different (P = 0.05).
mg.g 1), SBT (DE, 11.8 mg.g 1; SE-MeOH, 12.1 mg.g 1)
and OL (DE, 9.2 mg.g 1; SE-MeOH, 2.6 mg.g 1). EC was
the fourth highest tea major catechins of total
polyphenols content and follows the same trend with LB
being the highest, followed by YL, SBT and OL.

were varied between 26.6-74.6 mg.g 1 in DE and 20.8-58.1

mg.g 1 in SE. Interestingly, the percentage variation of
alkaloid contents from DE method to SE method in YL and
OL were 64.3% and 64.2%, respectively. Prolong
percolation of LB at high temperature (b.p. MeOH 65C)
in the SE method yielded polyphenols content that is
closed to the content obtained in SBT, 59.0 and 51.5
mg.g 1 respectively.

Yields of Individual Alkaloid from

Soxhlet and Direct Extraction Methods: Yield of total Caf
from both SE and DE methods remains in the range of
54.9-58.8 mg.g 1 in LB, 34.4-37.4 mg.g 1 in YL and
19.9-22.2 mg.g 1 in OL; with highest content obtained
from DE method. In contrast, Caf content obtained from
SBT was higher in the method of SE (44.5 mg.g 1) in
comparison to DE (30.5 mg.g 1). For the minor alkaloids
Tb and Tp, their present was in much lower quantities
than Caf. The Tb contents in leaf samples were found to
be approximately 10-fold lower than major alkaloid Caf,
with a relative ratio of 11.0 in LB, 11.6 in YL, 8.5 in OL and
9.5 in SBT, whereas Tp was found to decrease from LB >
YL > SBT > OL. Overall, total Caf content in all of the
samples were mainly constant irrespective to maturity
stages.

CONCLUSIONS
This study has demonstrated that direct extraction
can extract polyphenol and alkaloid which its content is
as similar to green tea, but when it is using soxhlet
extraction, the polyphenol content is as similar to black
tea. The yields of both polyphenol and alkaloid were
significantly higher in the extract obtained by direct
extraction method using 20% aqueous acetonitril than to
the soxhlet extraction method using dichloromethane and
methanol solvents. This indicated that total polyphenol
and alkaloid contents in tea extracts are affected by the
extraction methods used in sample preparation as well as
maturity stages of the tea leaves used, with their contents
decreased in the order of LB > YL > SBT > OL for
polyphenol, while alkaloid follows the order of LB > SBT
> YL > OL.

Contents of Polyphenol and Alkaloid in Sample Extracts:

Fig. 3 shows the total concentration of all types of
polyphenols and alkaloids obtained. Tea leaves were high
in both polyphenols and alkaloids in general. Method of
DE consistently yields higher and greater polyphenols
and alkaloids in comparison to SE. Polyphenols
content in LB, YL and OL obtained from DE method were
61.4-127.1 mg.g 1, but decreased by 53.6-68.1% when SE
method was used. The impact of extraction methods on
SBT was small but yet significant, with a 25.4% and 22.0%
decrease in polyphenol and alkaloid contents,
respectively. Total alkaloid contents in LB, YL and OL

ACKNOWLEDGEMENTS
The authors would like to thank Universiti Malaysia
Sabah (Grant number: FRG0054-51-1/2006) and the
Ministry of Science, Technology and Innovation
(Scholarship: M/0229/01/2006/BIO) for financial support
and to the Sabah Tea Plantation for providing tea samples
that support this study.
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World J. Agric. Sci., 9 (3): 266-270, 2013

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