Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2008
Volume 11
Issue 4
Topic:
Food Science and Technology
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Duda-Chodak A. , Tarko T. , Sroka P. , Satora P. 2008. ANTIOXIDANT ACTIVITY OF DIFFERENT KINDS OF COMMERCIALLY AVAILABLE TEAS – DIVERSITY AND CHANGES DURING STORAGE, EJPAU 11(4), #07.
Available Online: http://www.ejpau.media.pl/volume11/issue4/art-07.html

ANTIOXIDANT ACTIVITY OF DIFFERENT KINDS OF COMMERCIALLY AVAILABLE TEAS – DIVERSITY AND CHANGES DURING STORAGE

Aleksandra Duda-Chodak, Tomasz Tarko, Paweł Sroka, Paweł Satora
Department of Fermentation Technology and Technical Microbiology, Food Technology Institute, Agricultural University in Cracow, Poland

 

ABSTRACT

Tea is a very popular beverage rich in phenolic compounds of pro-heath properties. In the study the abilities to scavenge ABTS and DPPH radicals by tea infusions were evaluated. Sixteen different black, green or red loose-leaf teas, which were commercially available, had been used in the research. Analyses were performed twice: immediately after opening the package and after 1 month of storage. The results revealed that: (1) antioxidant properties of tea infusions depended on manufacturing processes of tea leaves. The highest antioxidant activity was shown for unfermented green tea, the lowest one for semifermented red tea, (2) green tea abilities to scavenge ABTS and DPPH radicals differed significantly from that of black tea, (3) there was no relationship between antioxidant activity of tea and its country of origin, (4) the properties of high antioxidant activity teas significantly decreased (up to 35%) when stored in opened packages at room temperature.

Key words: green tea, black tea, Pu-Erh tea, fermentation process, catechins, antioxidant activity, storage.

INTRODUCTION

The scientific researches prove that many civilization diseases are caused by excessive exposure to reactive oxygen species (ROS). Oxidative damages that occur in different tissues and cell structures lead to functional disturbances and have negative effect on whole organism [3].

Many defense mechanisms, which allow limiting ROS generation or attenuating their damaging action, were created in the course of evolution. However, prolonged stress, tiredness, or diminished immunity is the situations when inner antioxidant system may be insufficient. In such cases the exogenous antioxidants supply with a diet is necessary. Polyphenols are the most numerous group of antioxidant components, and they are present in fruit and vegetables, leguminous plants, grains, teas, herbs, spices and wines [4,6,16].

Tea is second only to water in terms of worldwide popularity as a beverage, and includes large amounts of antioxidant compounds. All teas are derived from the leaves of Camellia sinensis, but different processing methods produce different types of tea. Hence tea infusions differ in taste and aroma as well as in their effect on organism (therapeutic, sedative or stimulating) [15]. The tea plant contains many kinds of polyphenols, catechins being particularly prolific. Catechins present in tea leaves are responsible for astringent taste and are divided into two classes: the free catechins, (+)-catechin, (+)-gallocatechin, (–)-epicatechin, (–)-epigallocatechin; and the esterified or galloyl catechins, (–)-epicatechin gallate, (–)-epigallocatechin gallate, (–)-gallocatechin gallate [10,32]. Those monomers predominate in green tea (up to 20% of the dry matter), which is unfermented. During the rolling process, polyphenol oxidase and catechins, which exist separately in the tea leaf, mix. The polyphenols are located in vacuole in the palisade layer of the tea leaves, whereas the enzyme is located in the epidermal layer [30]. In the process of manufacturing black tea (fermentation), catechins are mostly oxidized to form dimmers (theaflavins) or highly complexed groups of compounds (thearubigins). These oxidized products represent the reddish-brown colors of black tea [18].

Besides the manufacturing processing some other factors affect tea properties. The country of origin should be taken into account, because it is connected with insolation, temperature and climatic condition in the region. The cultivation position, time of harvest, age and quality of tea leaves are equally important, as well as the transport and storage condition of raw material and final product.

Many in vitro and in vivo researches showed that tea catechins play protective role and exert anticancer, antibacterial, antiviral, antitoxin or antifungal activity [7,9,11,21,28,31]. Moreover it was proved that even a huge amount of catechin intake over a lifetime would not harm human beings [10].

The aim of the study was to evaluate antioxidant properties of infusions prepared from 16 different types of commercially available in Poland teas. We try also to assess how the abilities to scavenge ABTS and DPPH radicals during storage change. For this purpose antioxidant activity was evaluated immediately after tea package opening and compared with values obtained after 1 month of storage at room temperature, which simulated the conditions typical of household.

MATERIALS AND METHODS

Chemicals
ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; DPPH (1,1-diphenyl-2-picrylhydrazyl radical); Trolox (±)-6-hydroxy-2,5,7,8-tetramethyl-chromane-2-carboxylic acid; and a phosphate buffer (PBS): 0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride; pH 7.4 at a temperature of 25°C. All the chemicals listed were purchased from the Sigma-Aldrich Company. Potassium persulfate (K2S2O8) was obtained from the POCh Company, Poland, and a 96% ethanol from the ChemPur Company, Poland.

Materials
Sixteen different types of loose-leaf teas sold in 100-g packages were the material used in the research. We decided to test products, which are generally purchased by consumers. Hence we chose teas offered by 4 companies with the most diversified assortment of goods, which were readily available in hypermarkets and local shops. There were: 9 different kinds of black teas, 4 different green teas, and 3 red teas (Pu-Erh). Characteristics of teas chosen (type, name, producer or distributor name and country of origin) are presented in Table 1.

Table 1. Characteristics of teas used in the research

Producer/Distributor

TEA

Green

Red

Black

Name

Tea country of origin

Name

Tea country of origin

Name

Tea country of origin

BIO-Active, Ltd

Gun-Powder
Silver Moon

China
China

Gingko
Pu-Erh lemon

China
China

Darjeeling
Assam
Ceylon
Iguana

China
India
India
ND

Zas-Pol, Ltd

Madras
Yunnan
Assam

Sri Lanka
China
India

Oskar International
Trading, Ltd

Oskar Yunnan Green

China

Oskar Pu-Erh

China

Oskar Yunnan Black

China

Mokate SA

Loyd Tea Green

ND

Loyd Tea Black

ND

ND – shipping country of tea was not determined by the producer/distributor

Sample preparation
An amount of 2.00 g ± 0.01 g of tea leaves was weighed and tea infusions were prepared by pouring hot distilled water (100 ml) over the leaves. After 5 min the solution was seeped and the filtrate was cooled. Then the infusions were diluted with distilled water and immediately assayed for antioxidant activity. The fresh infusions were prepared in the same way after 1 month of storage in opened packages at a room temperature, and antioxidant activity was determined again.

ABTS radical cation decolorization assay
Using this method, it is possible to quantitatively assess the capacity of compounds to scavenge radicals. The antioxidant activity was assayed on the basis of a modified protocol represented by Re et al. [22]. A stable ABTS radical was generated during a chemical reaction between the 7 mM ABTS+ solution and the 2.45 mM potassium persulfate. The solution was kept at a room temperature in darkness throughout the night, in order to complete the reaction and to stabilize the ABTS cation-radical (ABTS+). To investigate antioxidant activity of tea infusion, a concentrated solution of the radical was diluted by a phosphate buffer (PBS, pH 7.4), in such a way that allowed to obtain a final absorbance of the solution, measured at a wavelength of 734 nm, of A = 0.70 ± 0.02 (ABTS0.7).

An amount of 100 µl of the 250-fold diluted tea infusion or of Trolox solutions (their concentration rates ranging from 0 to 10 mg 100 ml-1) was added to a 1 ml of ABTS0.7; next, the absorbance was measured in the 6th minute upon mixing. The antioxidant capacity of tea infusions under study was calculated using a standard curve drawn up for solutions of the synthetic vitamin E (Trolox) and expressed as mg of Trolox 100 ml-1.

DPPH radical-scavenging assay
In the determination procedure with a DPPH method used, antioxidants present in the sample investigated reduce a stable DPPH radical and cause a drop in the absorbance value measured at a wavelength of 515 nm. The scavenging capacity of DPPH radical was assessed on the basis of modified methods by Schlesier et al. [26] and Nicoli et al. [17]. An amount of 200 ľl of the infusion analyzed (properly diluted with a re-distilled water) or Trolox solutions (their concentrations ranging from 0 – 2.5 mg 100 ml-1) was added to 800 µl of a 225 ľM ethanol solution of DPPH and, then, the rate of absorbance disappearance was measured in the 10th minute upon the mixing of reagents in a cuvette. The antioxidant capacity of tea infusions was calculated using a standard curve developed for Trolox, and expressed as mg of Trolox 100 ml-1.

Statistical analysis
The results were shown as an arithmetic mean (± standard deviation) of six independent determinations. A single-factor Analysis of Variance test (ANOVA) with a post hoc Tukey test was applied to perform a statistical analysis. A Kołmogorov-Smirnov test was applied to examine the normality of distribution. Differences were considered to be significant at p < 0.05.

RESULTS AND DISCUSSION

The antioxidant activity of 16 different kinds of teas, that were available in shops throughout Poland, has been evaluated in the study. The results revealed that green teas showed the highest antioxidant activity among all the teas examined (Table 2).

A)
Table 2. Antioxidant activity of tea infusions determined by ABTS (A) or DPPH (B) method. The assays were performed immediately after opening the packages (fresh tea) and after 1 month of storage in room temperature (stored tea), arithmetic mean ± SD, n = 6

Group of products

Tea
(Producer/Distributor
& Name of product)

ABTS [mg of Trolox 100 ml-1 of infusion]

 

fresh tea

stored tea

 

mean ± SD

mean ± SD

Green
teas

Bio-Active Silver Moon

2068.1 ± 71.8

1313.8 ± 92.4 *

Bio-Active Gun Powder

785.6 ± 0.8

625.5 ± 26.3*

Oskar Yunnan

1800.7 ± 81.2

1813.4 ± 38.5

Mokate Loyd Tea Green

1017.6 ± 49.9

790.1 ± 45.7 *

Red
teas

Bio-Active Gingko

505.0 ± 0.6 a

513.9 ± 29.7 A

Bio-Active Pu-Erh

595.9 ± 4.5 a

575.8 ± 1.1 A

Oskar Pu-Erh

535.0 ± 31.2 a

576.4 ± 9.1 A

Black
teas

Bio-Active Assam

865.7 ± 47.4 b

762.5 ± 47.3 B

Bio-Active Ceylon

898.8 ± 32.9 b

650.7 ± 53.1 C *

Zas Madras

1129.9 ± 35.4 c.d

988.9 ± 53.8 D.E *

Zas Yunnan

396.3 ± 6.1 e

439.4 ± 34.4 F

Zas Assam

760.2 ± 17.1

849.5 ± 58.9 B.G *

Oskar Yunnan Black

462.6 ± 6.6 e

424.8 ± 6.6 F

Bio-Active Darjeeling

1160.4 ± 23.7 c

1072.9 ± 25.8 D *

Bio-Active Iguana

1064.5 ± 18.9 d

900.3 ± 27.9 E.G *

Mokate Loyd Tea Black

591.1 ± 6.1

604.6 ± 29.8 C

The same letters in columns mean that differences within a particular group of products are not statistically significant (at p < 0.05).
a. b. c – denotation for fresh teas. A.B.C – denotation for stored teas;
* indicates statistical significance (p < 0.05) compared with control (fresh tea) – in rows.

B)

Group of products

Tea
(Producer/Distributor
& Name of product)

DPPH [mg of Trolox 100 ml-1 of infusion]

fresh tea

stored tea

mean ± SD

mean ± SD

Green
teas

Bio-Active Silver Moon

707.3 ± 6.4

450.9 ± 39.3 A *

Bio-Active Gun Powder

328.4 ± 20.8

235.1 ± 7.5 B *

Oskar Yunnan

622.9 ± 5.8

503.0 ± 44.5 A *

Mokate Loyd Tea Green

257.9 ± 14.3

271.8 ± 6.9 B

Red
teas

Bio-Active Gingko

178.9 ± 5.9 a

234.2 ± 27.4 C *

Bio-Active Pu-Erh

209.1 ± 0.2 a

222.4 ± 38.1 C

Oskar Pu-Erh

192.6 ± 3.7 a

227.2 ± 14.0 C

Black
teas

Bio-Active Assam

309.1 ± 20.1 b

247.9 ± 4.4 D *

Bio-Active Ceylon

353.9 ± 4.0 c.d

246.3 ± 10.9 D *

Zas Madras

382.3 ± 22.5 c.e

328.8 ± 31.8 E *

Zas Yunnan

133.0 ± 12.5 f

171.0 ± 17.5 F

Zas Assam

236.1 ± 16.2

282.6 ± 21.2 D.E *

Oskar Yunnan Black

160.2 ± 16.5 f

162.0 ± 16.4 F

Bio-Active Darjeeling

400.0 ± 17.0 e

330.2 ± 46.8 E *

Bio-Active Iguana

323.3 ± 4.1 b.d

162.6 ± 15.6 F *

Mokate Loyd Tea Black

140.1 ± 9.8 f

139.9 ± 11.4 F

The same letters in columns mean that differences within a particular group of products are not statistically significant (at p < 0.05).
a. b. c – denotation for fresh teas. A.B.C – denotation for stored teas;
* indicates statistical significance (p < 0.05) compared with control (fresh tea) – in rows.

The mean antioxidant activity of green teas determined by ABTS method was 1418 mg of Trolox 100 ml-1 and 479 mg of Trolox 100 ml-1 of infusion when DPPH method was used. The intermediate values were obtained for black tea infusions (814 and 271, respectively), and the lower ones for red tea (545 and 194). The antioxidant activity rates of teas as achieved in the experiments with an ABTS radical involved are always higher than the respective rates obtained with a DPPH radical applied. This is attributed to the dissimilar nature of the two radicals, since they enable the determination of hydrophobic antioxidant substances only (as in the case of DPPH) or of hydrophilic and hydrophobic (as in the case of ABTS).

The achieved results comply with former reports that postulated that antioxidant properties of tea infusion depended on tea leaves treatment [5]. Many researches indicate that unfermented teas (white and green) show higher antioxidant activity than fermented ones [2,14]. Tea leaves destined for green tea are dried only, so almost all bioactive compounds remain unchanged. Thermal inactivation prevents polyphenols' oxidation and transformation and reduces degradation of vitamins, which are present in tea shoots. Therefore the final product is abounding with C, K and B-group vitamins and polyphenols compounds, mainly catechins, epicatechins, epigallocatechins and phenolic acids. It is estimated that in green tea leaves catechins and their derivatives constitute 20-42% of dry weight, while in red and black teas it is only 8-20% and 3-10%, respectively [1,5,8]. According to Rice-Evans et al. [23] catechins and gallic esters are responsible for even 78% of green tea antioxidant activity.

There is no unanimity among researches. Wiseman et al. [35] presented different opinion and demonstrated that black and green teas were similar in antioxidant properties. However, Yen and Chen [36] revealed that fermented tea had higher ability to scavenge free radicals than semi- and unfermented products. It is postulated that theaflavins and thearubigins, which are formed by polymerization of catechins during fermentation of black tea, are responsible for the ability of scavenging reactive oxygen species [12,27,34]. In black tea the theaflavins content is from 0.3 to 2.0% of dry matter of leaves, and thearubigins from 10 to 20%. In unfermented tea those compounds are not present at all, while catechins remain unchanged [13,24,33]. Moreover, gallic acid is found in large amounts in black tea; this strong antioxidant is released from catechin gallate during fermentation process [20]. Therefore, many researches consider the thesis that drinking of any kind of tea may yield significant health benefits [12,25,29]. It should be highlighted, that our results showed a great diversification of antioxidant properties among teas of particular kind provided by different producers or suppliers (Table 2). In the group of green teas examined a significant dispersion of results was found, from 786 to 2068 mg Trolox 100 ml-1 (in ABTS assay) and from 258 to 707 (DPPH assay). Black teas were also characterized by great variability, from 463 to 1160 and from 133 to 400 mg Trolox 100 ml-1, respectively.

According to literature references, chemical constitution of tea is depended on country of origin and technological processes of tea leaves, and it results in different antioxidant properties [2,12,14,19]. Our results indicated no relationship between antioxidant activity of tea infusion and shipping country of the tea. We can not say that teas originated from China have higher antioxidant activity than those from India, for example. In each group of tea – irrespective of region – good and bad radical scavengers can be found. Hence, there is no possibility to predict antioxidant activities on the basis of country of origin. We postulate, that differences of antioxidant activity of tea infusions result mainly from the quality of raw material used. Of course, different climatic and soil condition in particular cultivation region, as well as harvesting time, and transport or storage condition of raw materials from plantation to factory and of final product from factory to distributor, can matter.

In the group of green teas examined, the highest antioxidant ability to scavenge free radicals had "Silver Moon" and "Oskar Yunnan" teas, with values 2068 and 1801 (for ABTS method, respectively) and 707 and 623 mg Torlox 100 ml-1 infusion (DPPH). Other teas under study showed lower antioxidant properties (Table 2). The "Silver Moon" and "Gun Powder" teas are the products of the same company – Bio-Active Ltd. that tea leaves imports from China Co., Ltd exclusively. Regular, constant supplier guarantees relatively repeatable quality of goods. Therefore differences between antioxidant activities of those two teas are connected mainly with differences in early stages of technological processes: the season and method of harvest, quality of leaves, hand-operated or mechanical treatment. The transport, packaging, and storage condition would have less influence in that case, because they are similar in the whole company. In the case of "Loyd Tea Green" we only know that is imported from China. The differences in antioxidant activity between black teas studied were less significant, but again the Bio-Active products were best ones. The teas of Zas-Pol or Oskar showed lower antioxidant activities. There were no differences among red teas studied.

During analysis an assumption was made that products under study conformed requirements, and had been produced only from the best quality raw material. But adulteration by dishonest producer or distributor could not be excluded, and teas offered may contain admixture of leaves of inferior quality or stems ground instead of leaves. Each additive mentioned would cause decreased antioxidant properties and worse aroma and taste of tea infusion.

The determination of antioxidant activity was repeated after 1 month of storage at room temperature. The tea samples were stored in opened packages to simulate the conditions typical of household. Our results confirmed the assumption that antioxidant properties depend on storage condition. Untight packages that enable unlimited oxygen access as well as sunlight and high temperature (eg. exhibition place at shop heated by sun) can accelerate oxidation reactions and cause a decrease of pro-healthy values of tea. The tea infusions prepared after 1 month of storage almost always showed lower antioxidant activity than that prepared immediately after opening package of tea. Significant decrease of scavenging ability (even up to 35%) was noted especially in case of teas of high initial antioxidant activity. Whereas, properties of teas that were characterized by low initial activity had not change considerably. Our results indicated, that tea contained antioxidant components, which are very sensitive to oxidation process occurred during storage. Teas that immediately after opening the package showed low antioxidant properties, probably had been improperly stored, packed or transported. Therefore the degradation of labile components could proceed before final product reach consumer. As a consequence of this the values of antioxidant activity received in the study could be underrated.

In case of red teas, the tendency of increasing antioxidant activity was observed. Those teas undergo semi-fermentation process and their composition differs from that of green and black teas. It seems that red teas better tolerate the storage. The detailed analysis of components should be performed before and after storage, but the results achieved in DPPH method suggest that some hydrophobic compounds were formed.

CONCLUSIONS

The supplementation of diet with antioxidants may contribute to immunity rise, lifetime elongation and disease risk reduction. Tea is considered as one of the richest sources of antioxidants. The results received in the study indicated that different kinds of teas comprised different elements. Generally, green teas were characterized by highest antioxidant properties than black or red teas. In our research we revealed significant diversification of ability to scavenge ABTS and DPPH radicals in green (from 786 to 2068 and from 258 to 707 mg Trolox 100 ml-1 of infusion, respectively) and black teas (from 463 to 1160 and from 133 to 400 mg Trolox 100 ml-1 of infusion). Significant decrease of antioxidant potential (up to 35%) was found during storage of teas with high initial antioxidant activity. It testifies that antioxidant properties of tea infusions depend on the quality of the raw material used to production and condition of storage and transport as well. Therefore, in order to keep the beneficial values of tea the products of reliable companies and shops that provide good storage condition should be chosen. Moreover, small packages of tea should be preferred to shorten the time period between opening the tea and its consumption, because on exposure to light, oxygen and moisture tea loses its valuable properties. Those are also the hints for tea producers how to improve inexpensively the health values of their products.

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Accepted for print: 8.10.2008


Aleksandra Duda-Chodak
Department of Fermentation Technology
and Technical Microbiology,
Food Technology Institute,
Agricultural University in Cracow, Poland
Balicka 122, 30-149 Cracow, Poland
Phone: (+48 12) 662 47 92
email: aduda-chodak@ar.krakow.pl

Tomasz Tarko
Department of Fermentation Technology
and Technical Microbiology,
Food Technology Institute,
Agricultural University in Cracow, Poland
Balicka 122, 30-149 Cracow, Poland
Phone: (+48 12) 662 47 92

Paweł Sroka
Department of Fermentation Technology
and Technical Microbiology,
Food Technology Institute,
Agricultural University in Cracow, Poland
Balicka 122, 30-149 Cracow, Poland
Phone: (+48 12) 662 47 92

Paweł Satora
Department of Fermentation Technology
and Technical Microbiology,
Food Technology Institute,
Agricultural University in Cracow, Poland
Balicka 122, 30-149 Cracow, Poland

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.