Tannin assays

In this assay, the interaction of tannins with protein in an agar gel is quantified. The insolnble precipitates form rings around an origin. The diameter of the rings is proportional to the tannin amonnts present. It needs to be added that not all tannins bind to proteins, and not all precipitates are insoluble. The method of this exercise follows the Tannin Assay as described by Hagerman (1987). 

Figure 2. Absence of a correlation between the background absorbance in the tannin assay and the amount of polymeric pigment in 12 wines as determined in the standard Somers assay at wine pH. The analysis was performed twice about eight months apart. 2003 American Society for Enology and Viticulture.

The tannins in red wine are made up of chains of more-or-less polymerized fiavanols (procyanidins). These procyanidins are either homogeneous, with regular linking, or heterogeneous, linked by different bonds (Section 6.3.7). In both cases, certain bonds are broken when these molecules are heated in an acid medium, and the resulting carbocations are partially converted into cyanidin if the medium is sufficiently conducive to oxidation. This property has been used in tannin assays (FA method) for many years (Ribereau-Gayon and Stonestreet, 1966). 

Table 6.8. Comparison of evolutions in the results of tannin assays in wine aged under different conditions for 15 months (a, b and c are oak barrels of different origins) (Glories, 1992, unpublished)...

Martin, J. S. and M. M. Martin, Tannin assays in ecological studies Lack of correlation between phenolics, proanthocyanidins, and protein precipitating constituents in mature foliage of six oak species, Oecologia (Berlin), 54, 205-211 (1982). 

Because of the time and expense involved, biological assays are used primarily for research purposes. The first chemical method for assaying L-ascorbic acid was the titration with 2,6-dichlorophenolindophenol solution (76). This method is not appHcable in the presence of a variety of interfering substances, eg, reduced metal ions, sulfites, tannins, or colored dyes. This 2,6-dichlorophenolindophenol method and other chemical and physiochemical methods are based on the reducing character of L-ascorbic acid (77). Colorimetric reactions with metal ions as weU as other redox systems, eg, potassium hexacyanoferrate(III), methylene blue, chloramine, etc, have been used for the assay, but they are unspecific because of interferences from a large number of reducing substances contained in foods and natural products (78). These methods have been used extensively in fish research (79). A specific photometric method for the assay of vitamin C in biological samples is based on the oxidation of ascorbic acid to dehydroascorbic acid with 2,4-dinitrophenylhydrazine (80). In the microfluorometric method, ascorbic acid is oxidized to dehydroascorbic acid in the presence of charcoal. The oxidized form is reacted with o-phenylenediamine to produce a fluorescent compound that is detected with an excitation maximum of ca 350 nm and an emission maximum of ca 430 nm (81). 

Hplc techniques are used to routinely separate and quantify less volatile compounds. The hplc columns used to affect this separation are selected based on the constituents of interest. They are typically reverse phase or anion exchange in nature. The constituents routinely assayed in this type of analysis are those high in molecular weight or low in volatility. Specific compounds of interest include wood sugars, vanillin, and tannin complexes. The most common types of hplc detectors employed in the analysis of distilled spirits are the refractive index detector and the ultraviolet detector. Additionally, the recent introduction of the photodiode array detector is making a significant impact in the analysis of distilled spirits. 

The proanthocyanidin assay is carried out in a solution of butanol-concentrated hydrochloric acid, where proanthocyanidins (condensed tannins) are converted to antho-cyanidins (products of autoxidation of carbocations formed by cleavage of interfla-vanoid bonds) (Matus-Cadiz and others 2008). 

Other analytical assays proposed for the quantification of hydrolyzable tannins in plant materials include the rhodanine assay for the estimation of gallotannins (Berardini and others 2004) and sodium nitrate for the quantitative determination of ellagic acid (Wilson and Flagerman 1990). 

The platelet hist UIline release assay demonstrated that cotton mill dust extract, cotton bract extract, cotton leaf extract, dialyzed CMD extract, polyphenols, compound 48/80, rutin, trimethylamine HCl, quercetin, catechin, tannic acid, ellagic acid and sodium metasilicate all release histamine directly (48). Thus not only do tannin compounds induce histamine release, but they may also form higher molecular weight polymers and contain components that survive acid hydrolytic conditions (48). Tannins are widely distributed in the plant kingdom. 

We have employed two different protocols for the chemical fractionation of GSE obtained from MegaNatural-AZ based on the amounts needed for bioactivity-based assays. Batches of GSE (50 g) were extracted in acetone/water (7 3) under N2 with mechanical agitation for 12 h. The acetone was removed on a rotary evaporator and the aqueous phase was freeze-dried to yield 48 g of tannin crude extract (TCE). TCE was further fractionated following two different methods. 

Butler LG, Price ML, Brotherton JE (1982) VaniUin assay for proanthocyanidins (condensed tannins) modification of the solvent for estimation of the degree of polymerization. J Agric Food Chem 30 1087-1089... 

PHARMACOLOGICAL ACTIVITIES AND CLINICAL TRIALS Antibacterial activity. Alcohol extract of black tea, assayed on Salmonella typhi and Salmonella paratyphi A, was active on all strains of Salmonella paratyphi A, and only 42.19% of Salmonella typhi strains were inhibited hy the extract ". Ffot water extract of the dried entire plant and the tannin fraction, on agar plate, were active on Escherichia coli. Pseudomonas aeruginosa, and Staphylococcus aureus. ... 

Bacon, J.R. and Rhodes, M.J.C., Development of a competition assay for the evaluation of binding of human parotid salivary proteins to dietary complex phenols and tannins using a peroxidase-labeled tannin, J. Agric. Food Chem., 46, 5083, 1998. 

Scalbert et al. (1989) described several methods to determine the concentration of hydrolysable and condensed tannins (proanthocyanidins see Chapter 1, section 3.13.1) in wood extracts. Tannins are complex and heterogeneous In addition to the distinction between the flavonoid-based condensed tannins and the gallic acid-based hydrolysable tannins, each group can display a large degree of chemical variability that can affect the efficacy of the different assays. Interference of chemically related nontannin compounds, such as flavonoids, can in certain cases bias the results. 

An alternative colorimetric method relies on the reaction with vanillin under acidic conditions. A 2-mL aliquot of a freshly prepared solution of vanillin (1 g/100 mL) in 70% sulfuric acid is added to 1 mL of aqueous plant extract. The mixture is incubated in a 20°C-waterbath and after exactly 15 min. the absorbance at 500 nm read. The concentration of proanthocyanidins is expressed as (+)-catechin equivalents (used for the standard curve). This assay is specific for flavonols. As a consequence, when using this assay to determine the concentration of condensed tannins, widely distributed monomeric flavonols, such as catechin (1.39) and epicatechin (1.90), can interfere (Hagerman and Butler, 1989). 

The contents of the ampule are diluted in water to a final volume of 50 mL. A 1-mL sample is then taken for the assay. To this sample 1.5 mL 0.667% (w/v) rhodanine in methanol is added. After exactly 5 min. 1 mL 0.5N KOH is added. After 2.5 min. water is added to a final volume of 25 mL. The absorbance is read at 520 nm after a 5-10 min. incubation. A standard curve is made by reaction of gallic acid in 0.2N sulfuric acid with the rhodanine solution. Hagerman and Butler (1989) argued that this assay is more suitable than the potassium iodate assay for the determination of hydrolysable tannins, although it has to be kept in mind that the rhodanine assay is sensitive to any gallic acid ester, including those in non-tannin compounds. 

Hagerman, A., And Butler, L. G., 1989, Choosing appropriate methods and standards for assaying tannin, J. Chem. Ecol. 15 1795-1810. 

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