bk-2000-0754.ch013

Chapter 13

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Antioxidative Phenolic Compounds in Green-Black Tea and Other Methylxanthine-Containing Beverages Ulrich H . Engelhardt, Christiane Lakenbrink, and Svenja Lapezynski Institut fuer Lebensmittelchemie, Technical University of Braunschweig, D-38106 Braunschweig, Germany

More than 90 tea samples (green and black, from different origins) have been analyzed for the total polyphenols and for individual catechins, flavonol glycosides, theaflavins, and flavone C glycosides. The contents of total polyphenols in green and black tea samples were about the same while catechins in green tea samples were in average roughly 3 times as in the black tea samples (15 vs 4 %), however, in some Darjeeling samples the catechin amount (ca 10%) was in the same order of magnitude as in green teas with low catechin contents. The contents of flavonol glycosides (0.58 - 2.11 % ) and flavone C glycosides (0.013 - 0.26 %) were only little higher in green teas. - The amounts of theaflavins in black teas were between (0.3 - 2.4 %) for the 4 main theaflavins and 100 - 200 mg/100 g for the 5 minor theaflavins determined. Extraction experiments with tea bags showed that ca 70 % of the total flavonol glycosides were extracted in a tea brewed for 2 minutes ("total" extract was a 2 stage extraction using 70 % methanol).

Health benefits of flavonoids from tea and other sources have been published in a great extent in the past few years. Catechins, especially epigallocatechin gallate (EGCG), theaflavins and flavonol glycosides are thought to be responsible for antioxidative properties in tea (e.g. 1-3). Flavonol glycosides (FOG) have activities against myocardial infarct and stroke. The properties of the compounds, including their absorption characteristics, have been reviewed recently (1-3).

© 2000 American Chemical Society

In Caffeinated Beverages; Parliment, Thomas H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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Newly identified polyphenols


Proanthocyanidim In tea many proanthocyanidins have been described by Japanese groups, e.g. [46]. Several publications deal with the isolation and identification of proanthocyanidins from tea but only one paper uses H P L C which has been used in other foodstuffs, e.g. [7, 8]. It is likely that the proanthocyanidins contribute to the health benefits but data on the occurrence and the amounts in tea are scarce. In a L C - M S study we found at least 12 proanthocyanidins in a green tea sample which could be tentatively identified (9). More recently the structural elucidation of the compounds in figure 1 was possible using various N M R techniques (10,11). Three of them (bold printed in figure 1) have not been described previously, EAG-4->8-EGCG was identified i n tea for the first time. Figure 1 gives a H P L C separation of a green tea sample (RP-18; detection at 280 nm, eluent A was acetic acid (2 %, aq.), eluent Β acetonitrile, gradient: 5 - 26 % Β in 75 min). The sample pretreatment consists of an extraction from ground tea (75 % acetone) and (after removing acetone, sample dissolved in water) solid phase extraction using polyamide cartridges. The interfering compounds (catechins and flavonol glycosides) are removed from the column by washing with methanol and acidified methanol and finally the proanthocyanidins are eluted using 75 % acetone. A method for the quantification of the compounds is in progress.

s

1 2 3 S T 4

Bisflavanol Β EGC-4-8-EGCG GC-4-8-EGCG Strictinin EC-Trimere C-4-8-EGCG GC-4-8-ECG 5 EC-4-8-EGCG 6 EGC-4-8-ECG 7 Bisflavanol A 8 EGCG-4-8-EGCG 9 EC-4-8-ECG 10 EGCG-4-8-ECG 11 ECG-4-8-EGCG 12 ECG-4-8-ECG 13 EAG-4-8-EGCG 14 EAG-4-8-ECG 15 ECG-4-6-EGCG 16 EGCG-4-6-ECG 17 EAG-4-6-EGCG 18 ECG-4-6-ECG 19 EAG-4-6-ECG

8

10

13 12| 2

20

30

40

50

60

70

80

retention time [min]

Figure 1. HPLC -separation of proanthocyanidins (details see text). Abbreviations: EGCG: epigallocatechin gallate; EC: epicatechin; GC: gallocatechin; ECG: epicatechin gallate; EAG: epiafzelechin gallate; EGC: epigallocatechin


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Flavonol glycosides (FOG) Recently novel flavonol glycosides have been identified by oui" group. These are kaempferol-3-0-[a-L-rhamnopyranosy l-( 1 -»3 )-a-L-rhamnopyranosyl-( 1 - > 6 ) - β - ϋ glucopyranoside], kaempferol-3-0-[a-L-rhamnopyranosyl-( 1 ->3)-(4' ' '-0-acetyl)-aL-rhamnopyranosyl-(l-^6)-p-D-glucopyranoside] and K-rrgal (kaempferol-3-0-[a-Lrhamnopyranosyl-( 1 ->3)-a-L-rhamnopyranosyl-(l—>6)-p-D-galactopyranoside]). The structural elucidation was accomplished by N M R after isolation of the compounds by polyamide clean up, gel chromatography and preparative H P L C (12). Figure 2 shows a chromatogram (RP-18; eluent A was 2 % acetic acid (aq.), eluent Β was acetonitrile; 6 - 2 0 % Β in 50 min, detection at 354 nm). K-rrgal (peak 12 in figure 2) was present in almost all China samples and only one from Japan. L C - M S data show the presence in Darjeeling and Assam samples of another flavonol trisaccharide (K-rrg) which coelutes with K-rdg (Peak 13 in fig.2): it contains two rhamnose and one glucose or galactose moiety (possibly branched). A similar observation was made for quercetin trisaccharides (peaks 6 and 9) tentatively identified to contain 2 rhamnose and a glucose or galactose moiety. The compounds from the Assam and the China tea seem to contain the same sugar moieties but their retention behavior is different. The structural elucidation is in progress. Obviously the triglycoside patterns differ characteristically for samples of different origins. It has to be stressed that we did not succeed in resolving all F O G from tea with a single H P L C system. A system for the resolution of quercetin rutinoside and kaempferol glucorhamnogalactoside has been published (13). 1 Myr-rut 2 Myr-gal 3 Myr-glu 4 Q-grg 5 Q-rdg 6 Q-rrg 7 Q-rgal 8 Q-rut + K-grg 9 Q-rrg 10 Q-gal 11 Q-glu 12 K-rrgal 13 K-rdg+K-rrg 14 K-gal + K-rrglu 15 K-rut 16 K-g!u

13

20

25

30

35

45 40 retention time [min]

50

55

60

Figure 2. FOG determinations by HPLC. Q: quercetin, Myr: myricetin, K: kaempferol, glu: glucose, gal: galactose, r: rhamnose; rut: rutinose; rdg: rhamnodiglucoside


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Data on polyphenols in green and black tea samples In a survey study we analyzed green and black tea samples (blends and unblended samples) representing samples typical for the German tea market for total polyphenols and several groups of individual polyphenols (14). The majority of the black tea samples was from India (Darjeeling: 16, Assam: 11, South India: 1, Sri Lanka: 4, Indonesia: 4, Afrika: 3, China: 3), while the green tea samples were primarily from China (China: 38, Japan: 7, Taiwan: 2 along with 1 green Darjeeling, 1 Vietnam and 2 green Assam samples). The methodology for the determinations has in principle been published. Thermospray L C - M S was used to confirm findings i f necessary and for method development (15, 16). The final analytical method in case of catechins, theaflavins, flavonol glycosides and flavone C glycosides was R P - H P L C (13, 17 -19). In case of the catechins only an extraction (2-stage, 70 % methanol, 70 °C) but no clean-up was carried out. The results in detail will be published in the near future (14). Going to details means that catechin data consist of at least 4 individual compounds, theaflavins are up to 11, flavonol glycosides 13, flavone C glycosides 7. A n overview of the data generated can be found in table I.

Table I. Polyphenols in green and black tea samples

total polyphenols Catechins Caffeine Theogallin Gallic acid Theaflavins Flavonol glycosides * Flavone C glycosides*

Green tea 10.1- -22.2 8 . 5 - 20.6 1.5- -5.2 0.1- -1.4 0.01 - 0.19 n.i d. 0.28- -0.95 0.005 -0.14

Average 17.0 15.1 3.4 0.6 0.09 n.d. 0.64(1.38) 0.086(0.16)

Black tea 8 . 3 - 24,8 0 . 7 4 - 10.00 2.0- -5.4 0.1- •1.0 0.16--0.60 0.30- -2.41 0.24--0.87 0.02- -0.12

average 16.5 4.2 3.5 0.6 0.26 0.94 0.47 (0.89) 0.051 (0.09)

Note: Units are g/100 g; n. d. = not determined, * = calculated as aglycones; in parentheses calculated as glycosides Total polyphenols (TPP) The total polyphenols data were determined by the Folin-Ciocalteu method (20) currently under validation for tea by ISO. The data are expressed as gallic acid equivalents as gallic acid was the calibrant. A s can be seen, the average levels found were only slightly lower in black than in green tea. More extensive ISO data on green and black tea confirm this result (average levels in green and black teas were 17.5 and 14.4 % respectively).


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Catechins The data of the catechins given are the sum of E G C G , E G C , E C and E C G . It appears for black teas that these figures are high in Darjeeling samples while those are much lower in Assams (average: 6.6 vs 1.9 %; data not shown). Some of the Darjeeling black teas are higher in catechins compared with some green teas lower in catechins. Table II gives a comparison of black teas high in catechins and green teas low in catechins. In earlier studies we found black teas with even higher catechin levels (up to 12.5 %) and green teas with even lower catechin levels (down to 5%) than those reported in Table II. Even in those black teas high in catechins the comparison of the sum of catechins and the total polyphenols showed that in nearly all cases the proportion of catechins was less than 55 %. In green tea this proportion was more than 55 %, (1 exception). As regards black teas Assams are quite low in catechins (average 1.9% with a percentage of total polyphenols ca 20 %).

Table II. Catechins and caffeine in selected green and black teas

Note: Data are given in %. Abbreviations: EGC egigallocatechin; EC epicatechin; EGCG epigallocatechin gallate; ECG epicatechin gallate

Flavonol glycosides (FOG) We determined up to 14 F O G (the novel F O G are not included) using two different H P L C systems after polyamide clean-up (13). The F O G concentration (sum, calculated as glycosides) in the tea samples was between 0.6 and 2.1 %. The determination of the individual glycosides is important as the sugar moiety seems to affect the absorption of these flavonoids (21). There was no difference in principle between green and black teas. The average content was higher in the green teas compared with black teas, but this seems at least in part to be due to the origins. The tea with the highest concentration was a China green Pekoe with 2.12 % (calculated as glycosides). Darjeelings had amounts between 0.5 and 1.21 % (average was 0.94 %) while Assam samples were between 0.43 and 0.7 (average was 0.57). Taking a look at the pattern of these flavonoids (calculated as aglycones) there is no significant difference between the average data of green and black tea (kaempferol 30/29 %; myricetin 21/17 %; quercetin 54/50 %) compared with the sum of the aglycones. Theaflavins (TF) Up to 11 theaflavins were determined in extracts of black teas prepared by a


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newly developed continuous extraction procedure employing a rotary perforator (22). These are the 4 main theaflavins (theaflavin, the two monogallates and the digallate), theaflavic (only detected in Assams as yet) and epitheaflavic acids, epitheaflavic acid3-gallate, neotheaflavin, epitheaflagalline, epitheaflagalline-3-gallate. The method is also capable of detecting isotheaflavin but this compound was not detectable in the samples analyzed as yet. The sum of theaflavins was between 0.30 and 2.40 %. It was obvious that the Darjeeling samples had much lower contents of theaflavins (0.30 0.62 %) compared with the Assams (1.08 - 1.69 % ). Very high amounts were found in African tea samples (1.75 - 2.41 %). The minor theaflavins constituted 5 - 10% of the total found. Flavone C glycosides (FCG) The methodology consists of an extraction, polyamide column chromatography I, enzyme hydrolysis of interfering F O G , polyamide column chromatography II and H P L C (19). The amounts of F C G in are given in table I. There is no obvious difference in the F C G pattern of teas from different origins.

Behaviour of tea polyphenols during decaffeination In an experimental study we compared the total polyphenols, the contents of catechins and relative amounts of theaflavins in 5 teas each consisting of 4 samples (1 untreated, 1 decaffeinated using either C 0 , ethylacetate or dichloromethane). It turned out that ethylacetate extraction gave samples lower in total polyphenols, catechins and theaflavins than both the other decaffeination techniques (23). Details of the technology applied except the solvents used were not available. 2

Polyphenol contents in tea, coffee, and cocoa beverages In a study carried out in cooperation with Unilever Research at Colworth House (UK) we analyzed consumer brews of tea (e.g. tea bags containing 3.125 g + 235 m L of water, brewing times between 25 and 240 sec), instant coffee, coffee (different degrees of roast, 20 or 40 g ground coffee χ L" ), cocoa mixes and dry mixes on cereal base. To get reproducible results, the brewing procedures were standardized. We used 4 types of tea bags (U.K., U.S. and two international type tea bags). The tea bags were placed in a mug, the initially boiling water was added and stirred for 5 sec. Right before the end of the brewing time the bag was removed from the brew and squeezed against the beakers wall. This procedure was repeated six times and the brews combined to form an analytical sample. The methods used were as described above, for chlorogenic acids determination see (24). Chlorogenic acids are the main polyphenolic compounds in coffee and those are also present in tea samples. The data will be published in more detail (25). Selected results from the U . K . tea bags are compiled in table III. Please note that in this investigation, the data for individual 1


117 polyphenols have also been determined. In table III e.g. only the sum of flavonol glycosides is given, in fact 13 individual flavonol glycosides have been analyzed. The 70 % methanol extract data give the absolute composition of the tea bag product as sold.


Table III, Results of the brewing studies using the U . K . tea bag Brew time 40sec* 120sec* M e O H ext***

TSS 2480 2990 n.d.

TPP 675 841 16.0

TotalFlavs** 584 729 14.9

Catechins 39 56 1.34

FOG 50 63 0.863

FCG 5.23 6.65 0.089

TF 55 77 1.54

NOTE: * Data on aqueous brews are given in mg/liter of brew; ** Totalflavonoids= TPP (gallic acid + theogallin + chlorogenic acids) *** Data on MeOH extract are given in % on weight of leaf. TSS - total soluble solids; n.d. = not determined. The results for the different types of tea bags were similar. A t an average adult daily tea consumption of 600 ml (26), tea contributes (as aglycones) 23.8 mg flavonols and flavones (2 min brew) to the average U K total daily intake of 29.8 mg (27). Tea is the dominant dietary source of these flavonoids (more than 80 % for the 2 min brew) in the U K . - In case of coffee 3 types of coffee (low, medium and dark roast) were brewed in a percolator. The amount of coffee was 20 g per Liter of water, in case of the medium roast sample also a sample with 40 g/L was analyzed. Compared with the total polyphenols in tea brews the figures were higher in coffee. However, no catechins, flavonol or flavone glycosides or other flavonoids could be detected in coffee brews. The group of chlorogenic acids ( C G A ; 6 main compounds) were the main polyphenols detected in coffee (cf. Table IV).

Table I V . Coffee-data Type Light-roast (LR) Medium-roast (MR) Dark-roast (DR) L R : 20 g/L M R : 20 g/L M R : 40 g/L DR: 20 g/L

TSS n.d. n.d. n.d. 5.64 g/L 5.74 g/L 12.11 g/L 6.08 g/L

TPP 5.41 % 5.25 % 5.70 % 0.96 g/L 1.06 g/L 2.27 g/L 1.06 g/L

CGA 1.69% 1.92% 1.55% 0.375 g/L 0.403 g/L 0.859 g/L 0.342 g/L

Catechins, FOG n. det. n. det. n. det. n. det. n. det. n. det. n. det.

Note: n.d.: not determined; n. det: not detected In the cocoa drinks (methanol extracts) 6 % of total polyphenols could be detected along with small amounts of chlorogenic acids, catechins and flavonol glycosides. The amounts of total polyphenols in cereal based dry mixes (containing wheat flour, barley among others) was below 1 %. Neither the catechins nor other flavonoids analyzed were detectable.


118 This research project was supported by the FEI (Forschungskreis der Emâhrungsindustrie e.V, Bonn), the A I F and the Ministry o f Economics and Technology (AIF-FV: 10805 N). The support of the German Tea Trade Association is gratefully acknowledged. - The brewing studies were supported by Unilever Research, Colworth, U K . Special thanks are due to Dr Peter Collier, Clive Dacombe, Paul Quinlan and Conrad Astill for their advice.


References 1 2 3 4 5 6 7 8 9 10 11 12 13

Blot, W.J.; McLaughlin, J.K. Crit Rev Food Sci Nutr 1997 37 (8), 739-760 Balentine, D . A . ; Wiseman, S.A.; Bouwens, L . C . M . Crit Rev Food Sci Nutr 1997 37 (8), 693-704 Tijburg, L.B.M.; Mattern, T.; Folts, J.D.; Weisgerber, U . M . ; Katan, M . B . Crit Rev Food Sci Nutr 1997 37(8), 771-785 Nonaka, G.; Saka, R.; Nishioka, I. Phytochemistry 1984 23, 1753-5 Nonaka, G.; Kawahara, O.; Nishioka, I. Chem Pharm Bull 1983 31, 3906-14 Hashimoto, F.; Nonaka, G . ; Nishioka, I. Chem Pharm Bull 1989 37, 3255-63 Roeder, Α.; Lam T . M . L . ; Galensa, R. Monatszeitschrift für Brauwissenschaft 1995 11/12, 390-6. McMurrough, I. J Chromatogr 1981 218, 683-93. Kiehne, Α.; Lakenbrink, C.; Engelhardt, U . H . Ζ Lebensm Unters Forsch 1997 205, 153 - 7 Lakenbrink C.; Engelhardt, U.H. Lebensmittelchemie 1999 53, 20 Lakenbrink, C.; Engelhardt, U . H . ; Wray, V. Unpublished Lakenbrink, C.; Lam T.M.L.; Engelhardt, U.H.; Wray, V. Nat Prod Lett (in press) Engelhardt, U.H.; Finger, Α.; Herzig, B.; Kuhr, S. Dtsch Lebensm Rdschau 1992 88, 69-73

14 15 16 17

Engelhardt, U.H.; Lakenbrink, C.; Lapczynski, S.; Maiwald, B. 1999 unpublished Kiehne, Α.; Engelhardt, U . H . Ζ Lebensm Unters Forsch 1996 202, 48-54 Kiehne, Α.; Engelhardt, U.H. Ζ Lebensm Unters Forsch 1996 202, 299-302 Engelhardt, U.H (1998): In: COST 916: Polyphenols in Food; Amado, R.; Andersson, H.; Bardocz, S.; Serra, F. Eds, Office for Official Publications of the European Communities, Luxembourg, pp 49-55 18 Kuhr, S.; Engelhardt, U.H. Ζ Lebensm Unters Forsch 1991 192, 526-529 19 Engelhardt, U.H.; Finger, Α.; Kuhr, S. Ζ Lebensm Unters Forsch 1993 197, 239244 20 Singleton, V.L.; Rossi, J.A.J. Am J Enol Vitic 1965 16, 144-153 21 Hollman, P.C.H.; Tijburg, L.B.M.; Yang, C.S. Crit Rev Food Sci Nutr 1997 37(8), 719-738 22 Lapczynski, S.; Engelhardt, U.H. 1999 unpublished 23 Humpa, Α.; Engelhardt, U.H 1996 unpublished 24 Engelhardt, U. H.; Finger, Α.; Herzig, B. Lebensmittelchemie 1989 43, 58-59. 25 Lakenbrink, C.; Lapczynski, S.; Maiwald, B.; Engelhardt, U.H. 1999 unpublished 26 MAFF Total Diet Study 1987 27 M A F F Total Diet Study 1995


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