Two and a Bud 59(2):148-151, 2012 RESEARCH PAPER 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity and total catechins content of fifteen grades of Indonesian black teas Dadan Rohdiana', A. G. Suganda2 and K. R. Wirasutisna2 'Research Institute for Tea and Cinchona, Indonesia 2 School of Pharmacy, Institute Bandung of Technology, Indonesia ABSTRACT The antioxidant activities offifteen grades oflndonesian black teas was determined by the 1,I-diphellyl-2-picrylhydrllzyl (DPPH) free-radical scavenging assay. PF (Pekoe Fanning), one of the first grades oflndonesian black teas showed the higher DPPH free radical scavenging activity, with lowest ICso 218 /lg/ml followed by BOPF, Dust, BOP and BT each 227, 240,252 and 260 /lg/ml respectively. The catechin content was determined by High Performance Liquid Chromatography (HPLC). The total catechins content was found to vary from 6.27 to 10.18 %. The antioxidant activities were well correlated with the total catechin content (r) 0.7947 for the 1,I-dipllenyl-2-picrylllydrazyl free-radical scavenging assay. The high correlation between free radical scavenging activity and total catechin contents indicated that catechin compounds could be one of the main components responsible for antioxidant activities of these Indonesian black teas INTRODUCTION Tea (Camellia sinensis L. Kuntze) is a popular beverage worldwide. Depending on the technology of manufacturing, tea can be classified into three major types: green tea, black tea and Oolong tea. Black tea, which is one of the most widely drunk beverages in the world, is produced by fermentation. During the fermentation process the constituents of the leaves are enzymatically converted to numerous secondary products that contribute to the characteristic color and flavor of black tea i.e theaflavin and thearubugin (Kusano et al., 2008). Black tea infusion is characterized by a high content of flavonoids. Flavonoids are a large group of phenolic products of plant metabolism with a variety of phenolic structures that have unique biological properties and may be responsible for many of the health benefits attributed to tea (Rietveld and Wiseman, 2003). Many in vitro studies show that the flavonoids, especially of catechins present in tea have strong antioxidant and metal-chelating properties and may, therefore, protect cells and tissues against free oxygen radicals (Ell inger et aI., 20 I I; Xu et al., 201 I; Fu et al., 20 II). E-mail address:[email protected]148 Catechins are flavonoid phytochemical compounds that appear predominantly in tea. Four polyphenol catechins in green tea include gallocatechin (GC), epigallocatechin (EGC), epicatechin (EC), and epigallocatechin gallate (EGCG). Due to their potent antioxidant capabilities, catechins, often referred to as "tea flavonoids," are being investigated for their ability to prevent cancer and heart disease. In experimental models, catechins show a wide range of protective effects, including cardioprotective (Velayutham et al., 2008; Archivio et al., 20 I 0), chemoprotective (Marnewick el al., 2009; Shimizu et al., 20 II), antidiabetic (Abeywikrama et al., 20 I I; Zegarac et al., 20 I 0), anticancer (Cross et al., 20 I I), antiobesity (Brown et al., 2011) and antimicrobial properties (Finotti et al., 20 II, Yoo et al., 20 II; Hull el al., 20 II). In this study, the free radical scavenging activities of 15 grades of Indonesian black teas and its total catechin content were evaluated. MATERIALS AND METHODS Materials: 15 grades oflndonesian black teas (BOP, BOPF, PF, Dust, BP, BT, PF2, Dust2, BP2, BT2, PF3, Dust3, BM, BBL, BTL) manufactured by Indonesian Research Institute
Transcript
Two and a Bud 59(2):148-151, 2012
RESEARCH PAPER
1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity and totalcatechins content of fifteen grades of Indonesian black teas
Dadan Rohdiana', A. G. Suganda2 and K. R. Wirasutisna2'Research Institute for Tea and Cinchona, Indonesia
2 School of Pharmacy, Institute Bandung of Technology, Indonesia
ABSTRACT
The antioxidant activities offifteen grades oflndonesian black teas was determined by the 1,I-diphellyl-2-picrylhydrllzyl(DPPH) free-radical scavenging assay. PF (Pekoe Fanning), one of the first grades oflndonesian black teas showed the
higher DPPH free radical scavenging activity, with lowest ICso 218 /lg/ml followed by BOPF, Dust, BOP and BT each 227,240,252 and 260 /lg/ml respectively. The catechin content was determined by High Performance Liquid Chromatography(HPLC). The total catechins content was found to vary from 6.27 to 10.18 %. The antioxidant activities were wellcorrelated with the total catechin content (r) 0.7947 for the 1,I-dipllenyl-2-picrylllydrazyl free-radical scavengingassay. The high correlation between free radical scavenging activity and total catechin contents indicated that catechincompounds could be one of the main components responsible for antioxidant activities of these Indonesian black teas
INTRODUCTION
Tea (Camellia sinensis L. Kuntze) is a popular beverageworldwide. Depending on the technology of manufacturing,tea can be classified into three major types: green tea, blacktea and Oolong tea. Black tea, which is one of the mostwidely drunk beverages in the world, is produced byfermentation. During the fermentation process theconstituents of the leaves are enzymatically converted tonumerous secondary products that contribute to thecharacteristic color and flavor of black tea i.e theaflavin
and thearubugin (Kusano et al., 2008).
Black tea infusion is characterized by a high content offlavonoids. Flavonoids are a large group of phenolicproducts of plant metabolism with a variety of phenolicstructures that have unique biological properties and maybe responsible for many of the health benefits attributed totea (Rietveld and Wiseman, 2003). Many in vitro studiesshow that the flavonoids, especially of catechins presentin tea have strong antioxidant and metal-chelating propertiesand may, therefore, protect cells and tissues against freeoxygen radicals (Ell inger et aI., 20 I I; Xu et al., 201 I; Fu etal., 20 II).
Catechins are flavonoid phytochemical compounds thatappear predominantly in tea. Four polyphenol catechins ingreen tea include gallocatechin (GC), epigallocatechin(EGC), epicatechin (EC), and epigallocatechin gallate(EGCG). Due to their potent antioxidant capabilities,catechins, often referred to as "tea flavonoids," are beinginvestigated for their ability to prevent cancer and heartdisease. In experimental models, catechins show a widerange of protective effects, including cardioprotective(Velayutham et al., 2008; Archivio et al., 20 I 0),chemoprotective (Marnewick el al., 2009; Shimizu et al.,20 II), antidiabetic (Abeywikrama et al., 20 I I; Zegarac etal., 20 I0), anticancer (Cross et al., 20 I I), antiobesity (Brownet al., 2011) and antimicrobial properties (Finotti et al., 20 II,Yoo et al., 20 II; Hull el al., 20 II). In this study, the freeradical scavenging activities of 15 grades of Indonesianblack teas and its total catechin content were evaluated.
MATERIALS AND METHODS
Materials: 15 grades oflndonesian black teas (BOP, BOPF,PF, Dust, BP, BT, PF2, Dust2, BP2, BT2, PF3, Dust3, BM,BBL, BTL) manufactured by Indonesian Research Institute
for Tea and Cinchona, individual catechin standard (Sigma),chloroform, ethyl acetate, DPPH standard, absolute ethanoland methanol.
Determination of the free radical scavenging activity bythe 1,I-diphenyl-2-picrylhydrazyl free-radical scavengingassay: Scavenging activities of 15 grades of Indonesianblack teas on the stable free radical DPPH were assayed byAnesini et al. (2008) in which the bleaching rate of DPPHwas monitored at a characteristic wavelength in presenceof the sample. A volume of 0.1 ml of an aqueous dilution ofthe extracts was mixed with 0.5 ml ofa 500,uM DPPH solutionin absolute ethanol and 0.4 ml ofa 0.1 M Tris-CIH buffer at
pH 7.4. The mixture was kept for 30 min in darkness, andthen the absorbance was read at 517nm. The percentage ofdecrease of DPPH was calculated by measuring theabsorbance of the sample and applying the followingequation:
% of inhibition = [ 1 - (As/Ao)] x 100
where, As is the absorbance of sample and Ao is theabsorbance of the DPPH solution.
Method of catechin content analysis: 25g of Indonesianblack teas was extracted by 250ml hot boiling water andshaken for 10 minutes. Filtrate was extracted with chloroform
1: I (v/v) and then centrifuged to obtain clear filtrate. Lowerlayer was removed and top layer was extracted for threetimes. This liquid extract is named flavonoid fraction.Furthermore, flavonoid fraction was extracted by ethylacetate I: I (v/v). This solution was separated in twofractions, top and bottom layers. Top layer was ethyl acetateextract of catechin and bottom layer was removed. Ethylacetate was evaporated by rotary evaporator and dried byfreeze drier for 48 hours to obtain crude catechin. 15 mg ofcrude catechin was dissolved in 25 ml water. It was shaken
to homogenize and filtered through the Whatman filter paper(0.45 fIm). Furthermore this filtrate was taken in a test tubeto analyze by HPLC (Price and Spitzer, 1993).
RESULTSAND DISCUSSIONS
Antioxidant activity: In the test antioxidant activity (AOA)
was expressed as ICso (AOA is expressed as inhibitionconcentration of absorbance ofDPPH of50%). PF (PekoeFanning) showed the highest antioxidant activity followedby BOPF (Broken Orange Pekoe Fanning), Dust, BOP(Broken Orange Pekoe) and BT (Broken Tea) showing theinhibition concentrations of218 fIg/ml, 227 Ilg/ml, 240 Ilg/ml, 252 fIg/ml and 260 Ilg/ml respectively as shown in Fig. I.
BP (Broken Pekoe) was the one and only the first grade teahaving a low activity (375 fIg/ml). Similar with BP, BP2 (thesecond grade of black tea) had a low activity (373 Ilg/mI)too. Whereas BT2 (second grade of black tea) had a good
I -- - ------;'-;-'~;he~yi--;~i;;;'h;~~;;;r:~-;~~i~~I-ScaVenging :
I Activity of Fifteen Grades of Indonesian BlackTeasl'"
Il~II II II Ii BoP BOPF PF OUST BP III F'F2 OVST2 8P2 811 PFJ 0UST3 8M an
I .¥l9lrlllsl ~! 'Z27 i 2"18._ 2«1 • 375 I 26lJ 269 3}4 32'2 263 309 300 28ll 373 2ell
Fig. I. 1,1-diphenyl-2-picrylhydro7,)l1 free radical scavengingactivity of fifteen grades of Indonesian black teas
activity (263 Ilg/ml) compared to BP (first grade of blacktea) and slightly better than PF2 (269 Ilg/ml). Even, BTL(Bohea Tulang-Indonesian Language, the third grade) hada fairly strong activity (280 Ilg/ml) and slightly better thanBM (Broken Mixed, I2881lg/ml).
Total Catechin Content: The total catechin content was
found to vary from 6.27 to 10. I 8 %. PF had 10.18% of totalcatechin content followed by BOPF, Dust, BOP and BTrespectively having 10. I3%,9.76%,8.6% and 8.07% of totalcatechin (Fig. 2).
----,.Total Catechins Content of Fifteen Grades 'I
Fig. 2. Total catechins content of fifteen grades ofIndonesian black teas
Generally, the total catechin content of first grade tea washigher than second and third grade. Among all the samples,both BP and BBL were the grades which had the lowesttotal catechin contents. BP had 6.27% and BBL had 6.81 %.
Actually, BP is the first grade in the international grading.The result analysis showed that BP was the lowest in bothactivity and total catechin content. BP produced fromyoung stem contained less catechin than the tea made fromyoung tea leaves. So far, grading is ascertained consideringthe color, shape, size and density. BP had a black colorgraded as first grade. But chemically BP is an inferior grade.Therefore, grading should not be based only on existingphysical parameters and chemical parameters should beconsidered to strengthen of quality determination.
Correlation between antioxidant activity and total catechincontent: correlation between antioxidant activity and totalcatechin content is shown on Fig. 3.
149
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.-...-.--......-. ~ -----. --.------ -.-.- .
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400
Total Catechin (%)
Cross, S.E., Jin, Y.S., Lu, Q.Y., Rao, J. and Gimzewski, J.K.(2011). Green tea extract selectively targetsnanomechanics of live metastatic cancer cells.
Nanotechnology. 22(21 ):2151 0 1.
Brown, A.L., Lane, J., Holyoak, c., Nicol, B., Mayes, A.E.,Dadd, T. (2011). Health effects ofgreen tea catechins in overweight and obese men:a randomised controlled cross-over trial. British1. Nutr. 7: 1-10
10.189398
y ·34.273ll:+S57.46R'" 0.1947
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6.1.7 7052
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Fig. 3. Correlation between antioxidant activjty and totalcatechin content
In this work, a high correlation was demonstrated betweenthe total catechin content and antioxidant activity.Pearson's correlation coefficient r2was 0.7947. The total
catechin content and the antioxidant activity of made teaare the biological properties, and both assays should beapplied for the quality control of manufactured and importedteas.
Ellinger, S., MUlier, N., Stehle, P. and Ulrich-Merzenich, G.(201 I). Consumption of green tea orgreen tea products: Is there an evidencefor antioxidant effects from controlled
Finotti, E., Bersani, E. and Friedman, M. (2011). Applicationof a functional mathematical index
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CONCLUSION
The high correlation betwee,n free radical scavengingactivity and total catechin contents indicated that cate:chincompounds could be one of the main componentsresponsible for antioxidant activities of these Indonesianblack teas. Generally, Indonesian black teas hadhigh antioxidant activity and total catechin contents, andcould be an important dietary source of antioxidantcatechins for prevention of diseases caused by oxidative
stress. Ac.cording to the resuHs obtained, tea fromIndonesia is of good quality.
Hull, V.S., Tucci, M. and Benghuzzi, H. (2011). Evaluationof the antimicrobial efficacy of green tea extract(egcg) against Streptococcus pyogenes in vitro biorned. Biomed Sci instrum. 47.:177-82.
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