Tannins: chemical analysis

The amount and type of tannins synthesized by plants varies considerably depending on plant species, cultivars, tissues, stage of development, and environmental conditions. Therefore, the study of the nutritional effects of tannins on animals requires quantification of the tannins present in a particular diet.. Due to the complexity of tannins, several methods have been developed for their quantification. None of them, however, is completely satisfactory.

Sample preparation

The first factor to consider is how the forage or the feed is consumed by the animal - feeds should be analyzed in the form eaten by the animals

If the samples are collected fresh and they have to be stored, freeze-drying is the gentlest method of preservation and is recommended instead of freezing, air or oven-drying.

• If freeze drying is too expensive or the equipment is not available, freezing without thawing of the sample before extraction is suggested.
• If drying is the only means available for preserving the material, drying temperature should be higher than 40° C (to avoid oxidation by the still active enzymes) and lower than 60° C (to avoid heat damage and polymerization).

Sample handling

After cutting the sample should be -

• Stored in a cold, dark container,
• Cut in small pieces and freeze with liquid nitrogen,
• Pulverized with a mortar and a pestle, and
• Immediately extracted or freeze dried and stored at -4° C.

Sample extraction

Tannins are extracted with an aqueous organic solvent.

• 70% acetone and 30% water is a more effective extractant than alcoholic solvents.
  • Acetone inhibits tannin-protein interaction. This is a limitation in protein precipitation assays.
• In many plants, there is a large fraction (sometimes >50%) of the tannins that cannot be extracted (insoluble tannins).
  • This unextractable fraction cannot be ignored because of its nutritional effects.

Sample purification and isolation

Tannins need to be purified from low molecular weight phenolics and pigments that are present in crude plants extracts.

• Purification is essential for the preparation of suitable standards.
• Purification of large quantities of tannins can be done by taking advantage of their absorption by Sephadex LH-20. Reference: Hagerman A.E., Klucher K.M., 1986 - Tannin-protein interaction. In:Plant flavanoids in biology and medicine: biochemical, pharmacological, and structure-activity relationships. Ed. Cody V., Middleton E. Jr., Harborne J. - Alan R. Liss, New York, pp 67-76.

• An alternative and faster isolation method has been developed by Giner-Chavez, 1996 (see "mixed assays").

Chemical assays

Tannin assays can be divided into

• Colorimetric
• Gravimetric
• Protein precipitation
• Mixed

Colorimetric assays

Folin-Dennis method and its modifications (Folin-Ciocalteau method)

• The reaction is based on the reduction of phosphomolybdic acid by phenols in aqueous alkali.
• The method determines the total free phenolic groups and is therefore a method to determine total soluble phenolics (either HT and PA).
  • Problem: It does not differentiate between tannins and many phenolics that are not tannins. Interfering compounds such as ascorbic acid, tyrosine and possibly glucose are also measured.

Vanillin-HCl assay

• Specific for PAs or condensed tannins.
• Exo-type reaction - vanillin reacts with the meta-substituted A-ring of flavanols to form a chromophore; the number of flavanols is proportional to the absorbance of the solution.
• Problems:
  • Low molecular weight flavanols overreact and large polymers underreact,
  • Catechin is used as standard. This monomer gives the maximum optical density leading to underestimation of large polymers.

Butanol-HCl assay

• Specific for PAs or condensed tannins
• Endo-type reaction - the method involves the HCl catalyzed depolimerization of condensed tannins in butanol to yield a red anthocyanidin product that can be detected spectrophotometrically.
• Problem: Tannin polymers are cleaved into dimers or trimers instead of monomers and this leads to underestimation.
• The degree of polymerization of the PAs can be estimated by combining the butanol-HCl assay with the vanillin assay
  • The acid butanol assay measures the total number of flavanoid residues present and the vanillin assay measures the number of molecules.
• The butanol-HCl assay is also used to estimate the amount of insoluble tannins from extraction residues or from NDF.
  • Problem: Not all red pigments dissolve, resulting in tannin underestimation.

Rhodanine assay

• Specific to gallotannins ( one type of HT)
• The sample is subjected to hydrolysis to release gallic acid. The reaction between gallic acid and the dye rhodanine produces an intense color that is measured spectrophotometrically.

Wilson and Hagerman assay

• Specific for ellagitannins ( another HT)
• The sample is subjected to hydrolysis to release ellagic acid. The reaction between ellagic acid and the sodium nitrite produce a colored solution that is measured spectrophotometrically.

All the above mentioned colorimetric methods are described in:Waterman P.G., Mole S. (1994) - Analysis of phenolic plant metabolites. Blackwell Scientific Publications, Oxford, UK

Gravimetric assays

Gravimetric method with Ytterbium (Reed et al., 1985)

• Determines only soluble tannins present in plant extracts; insoluble tannins are not measured.
• Based on the ability of trivalent ytterbium to selectively precipitate polyphenols from plant extracts.
• Advantages:
  • Standards are not needed,
  • The precipitate can be easily dissolved with oxalic acid to yield a solution of polyphenolics and insoluble Yb-oxalate. The solution can be used for further analysis (colorimetric analysis, chromatography, inhibition studies).
• Problems:
  • Not all polyphenols are precipitated.
  • Low repeatability in plants with low levels of tannins.

Reference: Reed J.D., Horvath P.J., Allen M.S., Van Soest P.J. (1985) - Gravimetric determination of soluble phenolics including tannins from leaves by precipitation with trivalent ytterbium. J. Sci. Food Agric., 36:255-261

Gravimetric method with PVP (Makkar et al., 1995)

• Determines only soluble tannins present in plant extracts; insoluble tannins are not measured.
• PVP irreversibly binds tannins.
• This method is not very sensitive and tends to underestimate tannins.

Reference:Makkar H.P.S., Blümmel M., Becker K. (1995) - Formation of complexes between polyvinil pyrrolidones or polyethylene glycols and tannins and their implication in gas production and true digestibility in in vitro techniques. Br. J. Nutrition, 73:897-913

Gravimetric method based on the detergent system (Horvarth et al., 1981)

• Includes soluble and insoluble tannins.
• Steps -
  • Measurement of the acid-detergent residue of the NDF (NAD) and the neutral-detergent residue of the ADF (AND),
  • The difference NAD-AND is used to estimate tannins. This value has been successfully used in the summative equation of Van Soest to estimate the fraction of the feeds that is indigestible due to the action of tannins.
• Problems: many soluble tannins are not measured.

Reference:Horvath, P.J. (1981) - The nutritional and ecological significance of acer-tannins and related polyphenols. M.S. Thesis. Cornell University, Ithaca, NY, USA

Protein precipitation assays

These methods are more closely related to the biological effects of tannins.

Radial diffusion assay (Hagerman, 1987)

• This method depends on the formation of complexes between tannins and bovine serum albumin embedded in agar.
• Plant extracts are placed in a well in the agar. They diffuse in the agar and precipitate the albumin if tannins are present. In this case a opaque circle forms.
  • The diameter of the circle is proportional to the amount of tannins in the extract
  • Suitable standards are necessary to estimate the amount of tannins.
  • The most commonly used standard is tannic acid and the results are expressed in tannic acid equivalents.
  • This method allows determination of large number of samples with limited laboratory facilities.
  • Problem: less useful for quantification than the colorimetric procedures.

Reference: Hagerman A.E. (1987) - Radial diffusion method for determining tannins in plant extracts. J. Chem. Ecol.,13: 437-449

Mixed assays

Method of Giner-Chavez, 1996

This a method for condensed tannins or PAs that combines some previous methods in an attempt to eliminate their major problems and reduce the time required for the analysis.
The method consists of -

• Extraction of tannins from plant samples using 70% aqueous acetone (traditional method).
• Isolation of plant condensed tannins using trivalent ytterbium to prepare the standard.
• Analysis of the condensed tannins using the butanol-HCl method (traditional method).

The main innovation in this method is that instead of using an external standard like quebracho (as suggested for the butanol assay), internal standards (tannins from the same plant under analysis) are used.

• External standards have the serious limitation that the extinction coefficients for the chromophores produced with them usually are different from those obtained from the plant extracts
  • In other words, each gram of external standard (i.e. cyanidin or quebracho) has a different absorption than each gram of tannin from the plant extract. Moreover, the absorption varies with plant species because of the wide variety of tannin types present in nature.
• In Giner-Chavez's method, the internal standard is obtained by precipitating tannins with ytterbium trivalent such as in Reed's method.
  • In this way, even though not all tannins present in the plants extracts are precipitated by Yb, it is possible to isolate and quantify tannins for each plant that can then be used for the standard curve.
• The same goal could have been achieved by isolating the tannins with Sephadex (see "Sample purification") but it takes twice as much time (4 days vs. 2 days).
• The use of internal standards provides a more realistic estimation than with external standards.
  • Using quebracho, the tannin content of Desmodium ovalifolium was found to be over 200% !! However, the value was about ten times smaller when an internal standard was used.

Reference: Giner-Chavez B.I. (1996) - Condensed tannins in tropical forages - Ph. D. Thesis. Cornell University, Ithaca, NY, USA