Chapter 7
Evaluation of Total Tannins and Relative Astringency in Teas 1,3
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P. J . Rider , A. Der Marderosian , and J . R. Porter 1
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Departments of Chemistry and Biology, Philadelphia College of Pharmacy and Science, 43rd Street and Woodland Avenue, Philadelphia, PA 19104 The results of our analysis of total tannin levels and relative astringency of various commercial and traditional teas is reported herewith. Our method involves complexation with BSA (bovine serum albumin), centrifugation and the assay for protein remaining in supernatant with Coomassie Blue. Tannic acid equivalence (TAE) is calculated by dividing the concentration of tannic acid required to completely precipitate the BSA by the concentration of tea required to do the same. Complete absence of supernatant BSA is verified by capillary electrophoresis (CE) using a novel buffer system. While less sensitive than more sophisticated procedures of tannin analysis, this method is simple, rapid and relatively free of opportunities for experimental error. Morton and others have long believed that ingestion of highly astringent (high tannin) foods and beverages may be strongly associated with the development of esophageal cancer (7 ). Other researchers contend that there are health benefits to be gained from dietary tannins (2 ). It may be that the deleterious effects of high tannin diets only apply when, inadvertently, carcinogens like nitrosamines are included in the diet. The purpose of this research has been to develop an assay for astringency in teas so that future studies might determine the correlation between the strength of astringency in various teas and their potential health effects. Explanation of Terminology Astringency. Astringency is defined in Medicine as the drawing together or constriction of tissue (3). Some foods and beverages taste astringent, having a "puckering" taste. According to Bate-Smith, the primary reaction whereby this astringency develops in the palate is by precipitation of glycoproteins in the mucous secretions of salivary glands (4 ). In the field of Phytochemistry, astringency has often been used as if synonymous with protein precipitation (5 ).
3
Current address: Department of Biology, LIF 136, University of South Florida, Tampa, FL 33620 0097-6156/92/0506-0093$06.00/0 © 1992 American Chemical Society Ho et al.; Phenolic Compounds in Food and Their Effects on Health I ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
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PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH I
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Tannins. Tannins are a group of polyphenols phytochemicals which are believed to be responsible for dietary astringency. Indeed, by definition, naturally occurring polyphenolic compounds must have the ability to form stable water-insoluble complexes with protein to be classified as tannins (6). Phytochemists restrict their definition of tannins to polyphenolics of 500 daltons or more (7). Astringency assays. Astringency assays are often called "total tannin" assays because no other kind of assay is believed to be better in estimating the overall amount of tannins (6 ). A standard protein is usually mixed with a test solution containing an unknown amount of tannin. After centrifugation, the amount of precipitation is calculated. The most popular protein sources are bovine serum albumin (BSA) and fresh human blood (5 ). BSA is the preferred choice because it is readily available, inexpensive and less likely to absorb light in regions where common plant constituents may interfere (8 ). Astringency assay results are usually expressed in tannic acid equivalence (TAE). Tannic Acid. Tannic acid is an amorphous mixture of compounds prepared by fermentation and extraction of galls from the young twigs of various oak trees (9). The primary contents of tannic acid are hydrolysable tannins, which are mainly polyphenolic combinations of gallic acid and glucose. High performance liquid chromatography (HPLC) of tannic acidfromvarious sources has revealed differences in composition and tanning capacity (10). Hagerman and others have stressed the need for more highly uniform standards for astringency and tannin assays (77). Methods No method for the assay of tannin in teas is currently recognized by the Association of Official Analytical Chemists (A.O.A.C.) (72 ). Of the many procedures for total tannin estimation described in the literature, we chose to investigate those utilizing protein precipitation because of their increasing promotion in recent years (6) . These procedures generally begin by mixing the astringent liquid with a protein solution like BSA (1 mg/ml in 0.20 M acetate, 0.17 M NaCl buffer at pH of 5) followed by centrifugation and analysis of the protein in either the supernatant or pellet. One method, recendy promoted in the literature, uses ninhydrin to estimate astringency via the amino acids released following a partial hydrolysis of the tannin-protein pellet (8). We examined this method but found it much more time-consuming and less reliable than measuring the residual supernatant protein with Coomassie Blue (13). Thus the Coumassie Blue or Bradford Method was integrated into the procedure outlined in Figure 1. Bovine albumin was obtained from Sigma (A-7030; lot 10H0261). Tannic acid was also obtained from Sigma (T-0125; lot 87F-0745). Coomassie Blue (Brilliant Blue G) was obtainedfromAldrich (20,139-1; lot KT02325JP). Teas were prepared by pouring 150 ml of boiling water over dry, weighed leaf material and allowing the mixture to steep for 5 minutes thus approximating the extraction efficiency of a standard cup of tea, before removing 5 ml. This was allowed to cool to room temperature before 1 ml aliquots were used to complex with BSA (Fig. 1). Tannic acid solutions were prepared in distilled water. Sephadex columns were equilibrated prior to addition of the supernatant with a 50% aqueous dilution of the same acetate buffer prepared for the BSA solution. This diluted buffer was used to wash pellets, elute BSA and provide blanks for capillary electrophoresis (CE).
Ho et al.; Phenolic Compounds in Food and Their Effects on Health I ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
Downloaded by CORNELL UNIV on August 5, 2016 | http://pubs.acs.org Publication Date: October 1, 1992 | doi: 10.1021/bk-1992-0506.ch007
7.
RIDER ET AL.
Evaluation of Total Tannins in Teas
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The Bradford assay was accomplished by mixing 100 ul of cleaned supernatant with 4 ml of Bradford reagent (14 ). After waiting 5 minutes for color stabilization, the mixtures were poured into disposable plastic cuvettes and measured at 595 nm. on a LKB Biochrome Novaspec II Model 4040-011 Spectrophotometer against blanks prepared from 100 ul of the diluted buffer instead of supernatant. Considerable time was saved in the following replication protocol. Typically, ten different concentrations of a tea were prepared for each run. Two aliquots were taken from each and, after complexation, centrifuged. Half of the samples were analyzed by the Bradford method without Sephadex cleanup of the BSA to find approximate concentrations of minimal residual BSA. Tubes were then selected for Sephadex cleanup accordingly, and based on the Bradford results of these, a limited series of tubes were employed in the CE assay. This helped keep the protein in our capillary column to a minimum.
1 ml Tea or aqueous tannin
+
1 ml BSA (2mg/ml) in the buffer: 0.20 M acetate, 0.17 M NaCl brought to pH 5.0 with NaOH
Mixed, allowed to stand for 30 min.
Centrifuged at 2,500 rpm (10 cm radius) for 30 minutes at 25°C
Supernatant applied to a 1.7 χ 5.0 cm column of Sephadex G-25 (Pharmacia Fine Chemicals, PD-10 Columns) along with a 0.2 ml wash of the pellet and eluted in the 3-5 ml fraction.
100 ul aliquots removed for Bradford Assay 300 ul aliquots removed for CE Assay
Figure 1. Outline of the procedure used to estimate the astringency or total tannin of various teas. The tannic acid curve in Figure 2 was prepared without the Sephadex clean up. It illustrates the interference at 595 nm due to nonproteins, like phenolics, which are removed by the Sephadex columns. Capillary electrophoresis verification indicated that 1.05 mg/ml is the concentration of tannic acid required for complete BSA precipitation.
Ho et al.; Phenolic Compounds in Food and Their Effects on Health I ACS Symposium Series; American Chemical Society: Washington, DC, 1992.
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PHENOLIC COMPOUNDS IN FOOD AND THEIR EFFECTS ON HEALTH I
Capillary electrophoresis was performed on an ISCO instrument model 3140. The main objective which led to our adoption of a novel buffer system for BSA estimation was our reluctance to subject our fused silica column to the derivatization normally employed to deactivate silica and make it non-adsorptive toward proteins (75). A Borate 25 mMol/ Deoxycholate 25 mMol buffer system was developed and provided by Gupta (see acknowledgments). It proved ideal for our purposes and eliminated the need for derivatization. A representative pair of electropherographs is shown in Figure 3 along with details of the operation.
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