Chapter 19
Proanthocyanidins, Bisflavanols, and Hydrolyzable Tannins in Green and Black Teas 1
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Ulrich H. Engelhardt , Christiane Lakenbrink , and Olaf Pokorny
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Institut fuer Lebensmittelchemie, Schleinitzstrasse 20, Technical University, 38106 Braunschweig, Germany Zuckerinstitut e. V., Langer Kamp 5, Braunschweig, Germany
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The content of proanthocyanidins and bisflavonols in green and black tea samples was determined. The average content of proanthocyanidins (sum of 16 different compounds) in green tea was 0.84 % while that in black tea was considerably lower (0.5 %). However, the reverse was true for the bisflavanol (2 compounds) content, which was 0.05 % in green teas and 0.65 % in black teas. The hydrolyzable tannin strictinin (1-O -galloyl-4,6-(-)-hexahydroxydiphenoyl-β-D-glucose) was determined using an HPLC-ESI-MS/MS procedure and alternatively by HPLC-UV after polyamide clean-up. Strictinin contents in tea samples varied between 0.2 and 1.5 g/kg. Analysis of a fermentation series (green tea, 30, 60, 90 and 120 min fermentation of the same batch of tea leaves) showed a decrease with increasing fermentation time. The in-vitro antioxidant activity of selected tea proanthocyanidins (ABTS -method) was between 4.38 (EGC-EGC: epigallocatechin4β 8epigallocatechin) and 7.66 (EGCG-ECG: epigallocetechin gallate-4β8-epicatechin gallate) mmol/mmol Trolox.
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© 2004 American Chemical Society In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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Flavonoids from green and black tea and other sources have attracted much interest. Catechins, especially epigallocatechin gallate (EGCG), theaflavins and flavonol glycosides are thought to be responsible for antioxidative properties in tea (e.g. 7-5). Flavonol glycosides (FOG) have activities against myocardial infarction, stroke and several types of cancers. The properties of the compounds, including their absorption characteristics, have recently been reviewed (7-5). Data for the contents of catechins and other groups of flavonoids in tea are readily available (e.g. 7, 6-8). However, there is little information available on the contents of proanthocyanidins, bisflavanols and hydrolyzable tannins in tea.
Determination of Proanthocyanidins Proanthocyanidins and bisflavanols are oligomericflavanolswhich might consist of the same monomeric units. The difference is the type of the interflavonoid bond, which is usually 4-8 or 4-6 for proanthocyanidins and 2'-2' in case of bisflavanols as shown infigure1.
Figure 7. Bisflavanols and proanthocyanidins
A number of proanthocyanidins and bisflavanols have been isolatedfromfresh tea leaves, green tea and oolong tea samples (9-12), but data on their contents are scarce. In one study the amounts of four proanthocyanidins and bisflavanols A and Β have been detected in fresh tea leaves and their changes during fermentation studied (75). The amounts of individual compounds were between 0.8 and 2.1 g/kg fresh weight. Galloylated compounds fast degraded during fermentation while the amounts of non-esterified compounds were about the same after 12 h of fermentation. The amounts of bisflavanols A and Β were 1.4 and 0.6 g/kg, respectively (13).
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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Methodology The method used for separation and structural elucidation of compounds has been described elsewhere (8, 14, 15). Briefly, proanthocyanidins were extracted from ground leaves (2.5 g) twice with 100 mL 75 % acetone (aq.) in case of exhaustive extractions. After removing the organic solvent the residue was made up to 50 mL with water. The extract (5 mL) was brought to a conditioned polyamide cartridge (1 g polyamide). After washing (methanol, acidified methanol, 20 mL resp.) the compounds were eluted using 75 % actecone (aq.). This extract was dried, made up to 10 mL with 5 % acetonitrile and separated by RP-HPLC. For calibration purposes self-isolated and characterized proanthocyanidins were employed (75). The specifics of methods used were as follows: recovery of GC-4oc-8-EGCG (gallocatechin-4a-8-epigallocatechin gallate) was 86 % and that for EGCG-4P8-ECG was 81 %. The RSD was between 2.1 and 10.7 % depending on the compound.
Table I. Proanthocyanidins and bisflavanols in green and black tea samples
Proanthocyanidins Bisflavanols Sum proportion of bisflavanols
Green tea 0.13 -1.89 0.01 -0.11 0.9- 12.7*
Average 0.84 0.05 0.90 5.9*
Black tea 0.10-0.98 0.33-0.81 40.277.1*
Average 0.50 0.65 1.15 59.7*
Note: Units are g/100 g; * = %fromthe sum of both groups of compounds Table I gives an overview of the contents of proanthocyanidins and bisflavonols in green and black teas (no. of samples was 29 green and 9 black teas).The average amounts of proanthocyanidins and bisflavanols in green and black teas was around 1 % dmb which is surprisingly high. The ratio of proanthocyanidins and bisflavanols might serve as an analytical citerion for die discrimination between green and black tea samples. In black tea samples the proportion of bisflavanols determined to the sum of both groups of compounds was around 60 percent. In green tea this proportion was usually below 10 % which supports the statement that the content of proanthocyanidins does decrease during fermentation while the content of bisflavanols is increased. To give more detailed information on the contents of individual compounds average contents are shown in Figure 2. As can be seen in black tea the major compound was bisflavanol A while in green teas EGC-4B-8-EGCG and GC-4B-8-EGCG were most abundant.
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
Figure 2. Average contents of bisflavanols and proanthocyanidins in green and black tea samples
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258 Extraction of proanthocyanidins in tea brews
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To assess the extraction efficiency green tea beverages were prepared in the following matter: 1.5 g of tea were brewed with 100 mL of water (80 °C) for 3 min, thenfiltered.Figure 3 gives the extraction efficiencies of proanthocyanidins in % compared with "total extract" (extraction with aq. acetone, see above). As can be deduced from the figure the extraction of the 4-6 compounds was in general lower compared with the 4-8 compounds. Figure 3 shows the extraction yield of Chinese green tea samples.
Determination of strictinin (1 -O-galloyl-4,6-(-)~ hexahydroxydiphenoyl-P-D-glucose) Strictinin was identified as a constituent of green and black teas by Japanese groups (9,13). We also found strictinin along with l-0-digalloyl-4,6-(-)-hexahydroxydiphenoyl-P-D-glucose in green and black tea samples after the clean-up procedure described above. It was not possible to determine strictinin along with the proanthocyanidins as it was present in 2fractionsof the polyamide clean-up. Consequently, a different method of determination had to be developed. The first approach was a HPLC-ESI-MS/MS procedure which had the advantage that no sample clean-up was necessary. Briefly, ώβ instrument was run in the negative mode and the mass 633 [M-H]" was isolated andfragmentedto yield a fragment at m/z 301 ([ellagic acid-Η]'). The corresponding mass track was used for quantification purposes. Alternatively, the clean-up procedure by polyamide was modified (bothfractionswere pooled and analyzed). The latter has the drawback that a lengthy gradient system has to be employed, which is time-consuming. A study with a fermentation series showed that the amount of strictinin decreased during fermentation; strictinin takes part in the thearubigin formation (i) or that the compound is irreversibly bound to proteins during the firing process (ii). Figure 5 shows the decrease of the strictinin content with increasing fermentation time. The determination of the digallate was not possible due to the fact that the standard isolated was not pure enough, moreover, there are probably chemical reactions in solution leading to the formation of at least 2 peaks in the chromatogram. A method to isolate a pure standard of strictinin-3'-gallate via HSCCC is in progress. This pure standard will also be useful in establishing the behavior of the compound in solution.
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
Figure 3. Extraction of proanthocyanidins and bisflavanols in tea brews as compared with an exhaustive extraction, details see text
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Figure 4. Strictinin (R=H) and its 3 '- gallate (R = galloyl)
Table IL Strictinin in selected green and black teas strictinin* (LC-MS/MS) 1285 876 694 1027 277 220 219 209 203 177
Sample origin type Darjeeling Assam Indonesia China SC 12/28** CHM 61/60** SF 186** S 15/10** CG 28 U 864** CG 28 V 929**
black black black green green green green green green green
strictinin* (LC-UV) 1525 690 574 1271 n.d. n.d. n.d. n.d. n.d. n.d.
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* Data given in mg · kg" ; ** Kenyan samples
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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8-ECG EGC-4p->8-EGCG GC-48-EGCG EGCG-4p-^8-ECG EGCG-4p->8-EGCG bisflavanol A strictinin quercetin rutin
TEAC (literature data) TEAC [mtnol Trolox/mmol] fmmol Trolox/mmol] 1.0+/-0.02 n.b. 1.0+/- 0.03" n.b. 2.5 +/- 0.02 2.97 2.70 2.82 3.8 +/- 0.06 3.46 4.8+/-0.06" 4.11 4.9 +/- 0.02" 3.84 4.26 4.79 4.38 4.90 5.60 7.66 6.68 6.33 5.50 3.84 3.88 2.4 +/- 0.06" 2.82 17
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In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
263 data from the literature. Taking those data into account it is likely that the contribution of the dimeric and trimeric proanthocyanidins and bisflavanols to the antioxidant capacity of tea beverages is the same order of magnitude as the flavonol glycosides. Strictinin has also a pretty high antioxidant capacity but the amounts in tea sample are usually low compared with the proanthocyanidins/bisflavanols.
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Acknowledgements This research project was supported by the FEI (Forschungskreis der Ernàhrungsindustrie e.V, Bonn), the AIF and the Ministry of Economics and Technology (AIF-FV: 10805 N). The support of the German Tea Trade Association is gratefully acknowledged.
References 1
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