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All content following this page was uploaded by Amra Perva Uzunalic on 26 February 2015.
Received 18 October 2004; received in revised form 11 March 2005; accepted 11 March 2005
Abstract
The eect of dierent extraction set-ups that inuence the extraction eciency of catechins and caeine from green tea leaves
(variety Fanning Belas, China) were studied using dierent aqueous and pure solvents (acetone, ethanol, methanol, acetonitrile,
water), dierent temperatures (60, 80, 95 and 100 C) and times (5240 min). Raw extracts were analysed for contents of major cat-
echins (EC, EGC, ECG, EGCG), caeine, proanthocyanidins and avonols (myricetin, caempherol, quercetin). Starting material
was found to contain 191 g major catechins/kg material, 36 g caeine/kg material and 5.2 g avonols/kg material on a dry mass
basis. The content of major catechins in green tea extracts varied from approximately 280580 g/kg dry extract, with extraction e-
ciencies of major catechins varying from 61% to almost 100%. Content of caeine in extract was in the range of 75 g/kg, where its
extraction eciency varied from 62% to 76%. Average extraction yield was 30% with exceptions when using pure acetone and ace-
tonitrile, where extraction yield was about 3%. Contents of avonols and proanthocyanidins were in the ranges 620 and 1219 g/kg,
respectively. Dierent extraction procedures with water were also investigated and optimal conditions determined: maximum
achieved extraction eciency of catechins with water was obtained at 80 C after 20 min (97%) and at 95 C after 10 min of extrac-
tion (90%). Degradation of catechins was observed at higher extraction temperatures and with prolonged extraction times. Using a
lower ratio of solvent to material, extraction eciencies were increased by applying a multi-step extraction procedure. Optimal
extraction procedure was then performed using decaeinated green tea leaves, which were obtained by high-pressure extraction with
CO2, when 98% of caeine was selectively isolated without signicant impact on valuable catechins.
2005 Elsevier Ltd. All rights reserved.
Keywords: Green tea (Camellia sinensis); Solvent extraction; Raw and decaeinated green tea extract; Catechins; Caeine
1. Introduction mate, season, tea variety, and age of the leaf. Tea also
contains large amounts of tannins or phenolic sub-
Tea (Camellia sinensis), originated in China, dates stances (527%) consisting of catechin (avanol) and
back several thousand years. There are two major kinds gallic acid units, with those in green tea being higher
of tea, black tea and green tea, and they both contain than those in black tea (Leung & Foster, 1996). In gen-
caeine (15%) with small amounts of other xanthine eral, fresh green tea leaves contain 36% polyphenols,
alkaloids also present. Tea composition varies with cli- among which catechins prevail. Pharmacological prop-
erties of tea are due primarily to its alkaloids (caeine)
*
Corresponding author. Tel.: +386 2 2294 461; fax: +386 2 2516 750.
and catechins, which are divided into four primary com-
E-mail addresses: zeljko.knez@uni-mb.si (Z . Knez), forschung@ pounds (Fig. 1), epicatechin (EC), epicatechin gallate
raps-co.de (B. Weinreich). (ECG), epigallocatechin (EGC), epigallocatechin gallate
0308-8146/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2005.03.015
598 A. Perva-Uzunalic et al. / Food Chemistry 96 (2006) 597605
R1 R2
H H (-) epicatechin (EC)
OH OH
OH
H OC OH (-) epicatechin gallate (ECG)
HO O
OH R1
OH H (-) epigallo catechin (EGC)
OH OR2
OC OH
OH OH (-) epigallocatechin gallate (EGCG)
OH
(EGCG), and four secondary compounds, catechin (C), aqueous solvent mixtures (25, 50, 80 vol%), extraction
catechin gallate (CG), gallocatechin (GC), and gallo- temperatures (70100 C) and times were varied in order
catechin gallate (GCG). EGCG is the predominant cat- to investigate the extraction eciency of major catechins
echin present in green tea leaves (4855% of total (EC, ECG, EGC, EGCG) and caeine. Green tea ex-
polyphenols) (Ho, Chen, Wanasundara, & Shahidi, tracts were additionally analyzed for contents of three
1997). avonols (myricetin, caempherol, quercetin), and pro-
Other components present in tea include fats (4 anthocyanidins. Dierent extraction procedures were
16%), amino acids, sterols, vitamins, minerals, avour also conducted with water and optimal extraction tem-
and aroma chemicals, proteins, triterpenoids and others perature and time determined. A multi-step extraction
(Leung & Foster, 1996; Lunder, 1992, chap. 9). Several procedure was performed in order to investigate the
reports have attributed, to green tea, chemo-preventive change of extraction eciency by changing the ratio of
and therapeutic properties (McKay & Blumberg, 2002; solvent to material. A decaeination process with super-
Sueoka et al., 2001). It has also been shown that tea pos- critical CO2 was done at company NATEX Prozesstech-
sesses antimutagenic, anticarcinogenic and anticlasto- nologie GesmbH (Ternitz, Austria) and furthermore,
genic eects (Gupta, Saha, & Giri, 2002; Kuroda & residue material extracted with water in order to obtain
Hara, 1999). For these reasons, green tea has become a water-soluble caeine-free green tea extract enriched in
an attractive research material in the past 15 years. major catechins.
Green tea active ingredients, mainly catechins and
caeine, are usually isolated by extraction with organic
solvents, where extraction conditions (tea and solvent 2. Materials and methods
type, temperature, time, pH, ratio of solvent to mate-
rial.) variously inuence the extraction eciency and 2.1. Materials
quality of obtained extracts. Additionally, during
extraction the epimerization of major catechins can Dry and ground green tea leaves (Fanning Belas,
occur (Komatsu et al., 1993; Wang & Helliwell, 2000; China) were supplied by RAPS GmbH & Co. KG. Ref-
Yoshida, Kiso, & Goto, 1999). In order to obtain caf- erence substances, caeine and major catechins, were
feine-free products of green tea, decaeination processes purchased from Sigma Chemical Co.: caeine
are nowadays used and these include extraction with (CO750), EGCG 80% (E4268), EC98% (E4018), ECG
methylene chloride (Hartmann & Baumberger, 2000), 98% (E3893), EGC 98% (E3768). Reference avonols
ethyl acetate or supercritical carbon dioxide (Chang, were myricetin 95% (Fluka, 70050), caempherol 96%
Chiu, Chen, & Chang, 2000; Lack & Seidlitz, 1992, (Fluka, 60010) and quercetin 99% (Acros, 174070250).
chap. 5). The quality of decaeinated material, obtained All solvents used for analytical and extraction purposes
with these solvents, varies in the amount of isolated caf- were purchased from Merck.
feine, remaining catechins and solvent residue. An Sieve analysis of ground material was performed in
advantage of high-pressure extraction of green tea with order to determine the average particle size, and mois-
CO2 is that a decaeinated material with almost the ture content of samples was measured by a Mettler To-
same amount of catechins as the starting material can ledo DL31 Karl Fischer Titrator.
be obtained.
The authors in this study investigated solvent extrac- 2.2. Analytics
tions of a lower grade green tea (variety Fanning Belas,
China), which is used in teabags. Dierent solvents A method for simultaneous quantication of catechins
(water, acetone, methanol, ethanol, acetonitrile) and and caeine in green tea samples was developed and
A. Perva-Uzunalic et al. / Food Chemistry 96 (2006) 597605 599
supplied by RAPS GmbH & Co. KG (Otto, 2001). The 80%, 100%), methanol (25%, 50%, 80%, 100%), ethanol,
analytical method involved HPLC, wherein, quantica- (25%, 50%, 80%, 90%, 96%, 100%), acetonitrile (50%,
tions of caeine, EC, ECG, EGC and EGCG were per- 100%) and water. Temperature of extraction for aque-
formed via calibration curve. The HPLC system ous solvents was kept at boiling point temperature
consisted of a Varian 9012 HPLC pump and a Varian and, for water, 70, 85 and 100 C. The extraction mix-
9065 diode array detector. ture was constantly stirred with a magnetic stirrer. After
Quantication of proanthocyanidins was obtained 2 h of extraction, the extraction mixture was cooled,
after a hydrolysis reaction of proanthocyanidins to vacuum ltered (0.45 lm) and the solvent evaporated
anthocyanidins in a reaction mixture with n-buthanol under vacuum at 40 C.
and concentrated hydrochloric acid (Bae et al., 1993;
McMurrough & McDowell, 1978; Porter, Hrstich, & 2.3.1.2. Water, dierent extraction times. In order to
Chan, 1986). Repetition of the method was achieved investigate higher ratios of solvent to material
with addition of Fe(III) salt to the reaction mixture. (40 ml:1 g), 2 g of raw material were extracted with
Twenty grams of extract were weighed in a 10 ml ask 80 ml of water in the same manner as described above
and diluted with distilled water to the mark. Two milli- by maintaining constant temperature at 80 and 95 C.
liters of solution were transferred to a 25 ml ask and Dierent extraction times, using water as solvent, were
20 ml of a mixture of n-buthanol:conc. HCl, containing investigated.
FeSO4 H2O (77 mg/500 ml) added. The reaction mix-
ture was incubated for 15 min at 95 C, after which
2.3.2. Extraction with water at dierent starting
the sample was cooled to ambient temperature and ana-
temperature
lysed on UV Varian Cary 1E spectrophotometer at
2.3.2.1. Single extraction step. Hundred milliliters of dis-
540 nm against a reference sample. The absorbance of
tilled water (60, 80 and 95 C) were poured over 1 g of
each sample solution was measured three times in two
green tea leaves in a at-bottom ask and stirred for dif-
parallel trials. The standard deviation value was about
ferent time intervals from 1 to 120 min, without main-
3%.
taining constant temperature. Extract solution was
Quantication of avonols (myricetin, quarcetin,
consequently cooled during this time and its tempera-
caempherol) in green tea samples was obtained by
ture was monitored. The extraction mixture was then l-
an HPLC method (Wang & Helliwell, 2001) the
tered (0.45 lm) and directly analyzed by HPLC. Two
HPLC system consisted of a solvent delivery system,
milliliters of extract solution were evaporated to dryness
constaMetric 3000, a spectromonitor 3100 variable
and concentration of dry matter determined as an aver-
length detector (LDC Analytical), and an HP 3396
age from triple trials.
Series II integrator. Sample preparation for green tea
leaves consisted of weighing 1 g of green tea leaves
in a round-bottom ask and adding 40 ml of 2.3.2.2. Multiple extraction steps. A multi-step extrac-
60 vol% ethanol, and 5 ml of 6 M HCl. The reaction tion procedure, at optimal determined starting tempera-
mixture was thermostatted for 2 h at 95 C and con- ture and time, was performed using 2 g of green tea
stant stirring was applied. The hydrolysed mixture leaves and 80 ml of water (ratio 40 ml:1 g), thus decreas-
was cooled and ltered (0.45 lm) into a 50 ml ask, ing the ratio of solvent to material compared to the pro-
diluted with 60 vol% ethanol to the mark, and twice cedure from Section 2.3.2.1. Four subsequent extracts
analysed. The standard deviation value was about were obtained in dry form and analyzed for major cate-
2%. Fifty milligrams of green tea extract were weighed chins and caeine.
into a 20 ml ask and diluted with distilled water to
the mark. Sixteen milliliters of solution were trans- 2.3.3. Decaeination and further processing of green tea
ferred to a round-bottom ask and 24 ml of absolute Thousand grams of green tea leaves were extracted
ethanol added with 5 ml of 6 M HCl. Hydrolysis and with CO2 by the company NATEX Prozesstechnologie
further dilution were performed as described for green GesmbH (Ternitz, Austria) in a high pressure extrac-
tea leaves. tion pilot plant (Lack & Seidlitz, 1992, Lack, Seidlitz,
& Glanz, 5; Lack et al., 2001). The tea leaves were
2.3. Extraction procedures moistened up to 1550 wt% with water, and extracted
with wet CO2 at 225350 bar and 5080 C. Decaein-
2.3.1. Extraction with dierent solvents at constant ated green tea was dried in an air oven at 40 C to a
temperature humidity content of 4%, analysed for major catechins,
2.3.1.1. Dierent solvents, 2 h extraction. Twenty grams and stored in a cool and dry place until further pro-
of green tea leaves were extracted with 400 ml of solvent cessing. For obtaining a decaeinated green tea extract
(ratio 20 ml:1 g) in a round-bottom ask equipped with enriched in catechins, 150 g of decaeinated green tea
a condenser. Solvents used were acetone (25%, 50%, leaves were extracted with 1400 ml of water in a
600 A. Perva-Uzunalic et al. / Food Chemistry 96 (2006) 597605
Table 2
Extraction of green tea with dierent solvents (T = const., t = 2 h, ratio 20 ml:1 g): extraction yield, extraction eciency of caeine and major
catechins, content of catechins, avonols and proanthocyanidins in dry extracts
Solvent Vol (%) Temperature (C) Extraction yield (%) Extraction eciency Content (g/kg dry extract)
(%)
Caeine Catechins Catechins Flavonolsa Proanthocyanidins
Acetone 25 Boiling point 35.2 75.6 98.5 534 8.7 17.7
50 33.5 72.1 99.3 565 15.8 19.0
80 31.9 67.5 95.2 569 18.8 18.7
100 3.2 6.9 4.6 272 5.8 13.5
Methanol 25 Boiling point 30.9 66.3 79.7 492 13.9 16.3
50 32.5 69.0 81.1 476 16.4 15.7
80 30.3 62.0 77.9 491 16.5 15.9
100 33.4 72.8 87.7 501 16.9 14.7
Ethanol 25 Boiling point 30.3 63.5 62.5 394 16.2 14.9
50 32.8 68.9 76.4 445 15.9 15.7
80 34.5 73.8 89.1 493 17.8 15.6
90 30.0 65.7 74.5 474 15.8 14.3
100 28.2 68.5 77.2 522 10.7 13.4
Acetonitrile 50 Boiling point 36.0 72.8 99.8 530 15.5 18.5
100 2.9 9.2 8.1 534 6.8 13.4
Water 70 29.2 56.0 65.9 430 17.4 16.2
80b 36.5 81.5 84.4 448 9.1 16.9
85 30.4 63.8 61.3 385 17.0 16.8
95b 43.0 89.1 57.2 258 13.4 13.7
100 30.1 57.0 37.3 237 11.4 12.0
a
Sum of myricetin, quercetin and kaempherol contents.
b
Ratio solvent:material = 40 ml:1 g.
A. Perva-Uzunalic et al. / Food Chemistry 96 (2006) 597605 601
500
Major catechins in extract
80
70
400
60
300 50
40
200
30
100 20
80 C 80 C
10
95 C 95 C
0 0
0 40 80 120 160 200 240 0 40 80 120 160 200 240
Time (min) Time (min)
Fig. 3. Extraction of green tea with water (T = const., ratio 40 ml:1 g): (a) content of major catechins in dry extract and (b) extraction eciency of
major catechins.
602 A. Perva-Uzunalic et al. / Food Chemistry 96 (2006) 597605
Fig. 5. Single-step extraction of green tea with water at dierent starting temperatures (Ts) and ratio 100 ml:1 g: contents of major catechins and
caeine in green tea extract solution (mg/100 ml) as a function of extraction time.
A. Perva-Uzunalic et al. / Food Chemistry 96 (2006) 597605 603
700
Ts = 60 C Ts = 80 C Ts = 95 C
600
400
300
200
100
0
0 30 60 90 120 0 30 60 90 120 0 30 60 90 120
Time (min)
CAF EC ECG EGC EGCG total catechins
Fig. 6. Single-step extraction of green tea with water at dierent starting temperatures (Ts) and ratio 100 ml:1 g: contents of major catechins and
caeine in dry green tea extract (g/kg) as a function of extraction time.
which were obtained on the basis of dry matter determi- above 80 C, as well as oxidation and degradation. Con-
nation of extract solutions. tent of caeine in dry extract reached a constant value of
The content of caeine in extract solution was approximately 100 g caeine/kg extract after 30 min of
approximately constant after 2.5 min (30 mg/100 ml) at extraction at all three investigated starting temperatures.
starting 80 and 95 C whereas at 60 C, an extraction By decreasing the ratio of solvent to material (ml:g), a
of 15 min was required to reach this constant value. decrease in extraction eciency of catechins and caeine
Content of major catechins increased up to approxi- was observed. This was conrmed by extracting green
mately 1020 min at all three investigated temperatures, tea leaves for 10 min at starting temperature of 95 C
and it increased with increasing temperature, the highest (optimal parameters) at the applied ratio 40 ml:1 g in-
value being obtained at 95 C and 1015 min (183 mg/ stead of 100 ml:1 g, as described in results in previous
100 ml). Maximum content of major catechins in dry ex- paragraphs. The contents in dry extract were 414 g of
tract, on the other hand, decreased with increasing tem- major catechins/kg and 71.4 g caeine/kg, which were
perature. Content of major catechins in dry extract by 17% for catechins and 24% for caeine lower than
decreased from the maximum value of 682 to approxi- extract obtained at the ratio 100 ml:1 g. Extraction e-
mately 400 g/kg at 60 C after 30 min, and from 654 to ciency for catechins and caeine were therefore 6164%,
500 g/kg at 80 C after 50 min, and further remained compared to 80% obtained at higher ratio.
constant up to 120 min. At 95 C the content of major
catechins decreased from a maximum value of 525 3.3.2. Multiple extraction step
500 g/kg after 20 min and, after the prolonged time of In order to increase the extraction eciency, a mul-
60 min started decreasing to 420 g/kg in 120 min. During ti-step extraction procedure, under the same condi-
this study, it was conrmed that contents of major cat- tions, was investigated, where the residue material
echins decreased after a certain time at elevated starting from the rst extraction step was once again extracted
temperature due to their degradation. These results can with water for 10 min at a starting temperature of
be related to ndings from published data of other 95 C. This was repeated, altogether, four times, and
authors (Komatsu et al., 1993; Wang & Helliwell, results of analyses of extracts obtained in each step
2001), which indicate an epimerization of catechins are shown in Table 3. Extraction yields drastically
Table 3
Multi-step extraction of green tea with water at ratio 40 ml:1 g (Ts = 95 C, t = 10 min)
Extr. step Content (g/kg dry extract) Yield (%) Cumulative amount Cumulative
(g/kg raw material) extraction eciencya
(%)
EGC ECG EC EGCG CAF Catechins CAF Catechins CAF
1 126 47.8 1.2 239 71.4 30.0 124 21.4 65.1 59.4
2 108 59.0 1.1 243 75.5 11.4 156 27.2 81.9 75.5
3 68.5 91.6 1.1 420 54.5 4.0 170 28.5 88.9 79.2
4 42.3 127.6 0.1 484 34.0 1.3 174 28.7 91.3 79.7
a
Regarding the starting material, which contained 191 g catechins and 36 g caeine per kg of raw material.
604 A. Perva-Uzunalic et al. / Food Chemistry 96 (2006) 597605