Analysis of hydrolysable tannins

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Abstract

This review deals with the main issues relating to the analysis of hydrolysable tannins (HTs). It covers briefly their distribution in the plant kingdom and describes their main structural features. HTs have often been ignored because they are apparently more difficult to analyse than condensed tannins. Meaningful analytical data are critically dependent on appropriate sample preparation, storage and extraction techniques. This requires some understanding of the reactivities of hydrolysable tannins. Mixtures of HTs have been measured by general tannin assays, such as precipitation with metals or proteins, and by colorimetric assays for total phenols. Some HTs can also be measured by more specific colorimetric tests. Although colorimetric assays are widely used for tannin analyses, they generally do not provide accurate quantitative data. At best, they provide data for comparative purposes. Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) are useful tools to screen samples for the different types of tannins, hydrolysable or condensed tannins. In addition, there are physicochemical techniques, such as nuclear magnetic resonance (NMR) and mass spectrometry (MS), which are used to identify pure tannin compounds. More recently, other MS techniques have been developed that are capable of yielding molecular weights of crude tannin mixtures.

Section snippets

Occurrence and structures of hydrolysable tannins (HTs)

Table 1 illustrates that hydrolysable tannins are synthesized by a wide variety of plants and trees (Haslam, 1981; Okuda et al., 1990; Kumar and Vaithiyanathan, 1990) and several of these have been used as animal feeds (Le Houerou, 1980). They can occur in wood, bark, leaves, fruits and galls. It is worth noting that some species produce either gallotannins or ellagitannins, whilst others produce complex mixtures containing gallo-, ellagi- and condensed tannins. For example Acacia, Acer and

Extraction of HTs

There has been much debate about whether fresh, freeze-dried or air-dried samples should be analysed. The general recommendations are that fresh samples are the preferred option for extracting tannins. However, this is often not practical or feasible. The next best option is to transport the samples cold (e.g. on ice or carbon dioxide) before freeze-drying. However, if samples are to be air-dried, it is recommended that they be dried in the shade. Okuda et al. (1989) demonstrated that some

Stability and reactions of HTs

HTs are more likely to react with the extracting solvent than condensed tannins. For example, methanol cleaves the depside bonds in gallotannins (Fig. 6) at neutral pH and RT (Tedder et al., 1972; Porter, 1989), but acidified methanol (pH<3) will not cleave these bonds (Haslam et al., 1961). Large and complex tannins are easily degraded into smaller tannins by water or dilute acids especially at elevated temperatures in just 30 min (Beasley et al., 1977; Okuda et al., 1989, Okuda et al., 1990).

Assays for the determination of total phenols

There are several methods to measure the contents of total phenols in plant materials (Hagerman and Butler, 1989). Most of these assays suffer a major defect despite the fact that they are very widely used: every phenolic compound produces a different colour yield per unit mass in the colorimetric assays. As the quantitative composition of an unknown mixture of phenolics is by definition not known, it is clearly impossible to use a single ‘standard phenol’ on which to base such assays.

KIO3-reagent for gallo- and ellagitannins

Bate-Smith (1977) described a procedure for measuring gallo- and ellagitannins in Acer species, which yield a pink reaction product with the KIO3-reagent. Hagerman et al. (1997) noted, however, that this assay was not suitable for complex mixtures of tannins as they tended to give brown rather than pink products. Furthermore, this is not a robust assay as development of the colour is critically dependent on the temperature and duration of the reaction. Hagerman et al. (1997) suggests that

Surveys for HTs and condensed tannins (CTs) by thin layer chromatography (TLC)

TLC is a relatively cheap but powerful technique to screen plant extracts for the presence of different types of tannins. Extracts are spotted onto layers of cellulose or silica gel that are attached to glass plates or plastic sheets. The bottom of the plate is then placed into a solvent. As the solvent migrates up the plate, the various phenolic compounds are separated. When the solvent has reached the top, the plate is removed and the solvent is dried off. The plate can then be turned by 90°

Surveys for HTs and CTs by high performance liquid chromatography (HPLC)

Mixtures of tannins can be separated on two types of HPLC columns. In normal phase HPLC, the stainless steel columns are tightly packed with polar silica particles (<5 μm). In reverse phase HPLC, the silica particles are coated with non-polar alkane chains, e.g. C8 or C18 chains. As a first approximation in reverse phase HPLC, CTs often appear as poorly resolved ‘humps’ eluting between 10 and 20 min, whereas mixtures of HTs appeared in later eluting ‘humps’ under the conditions employed by

Nuclear magnetic resonance (NMR) for identification of tannins

NMR and mass spectrometry is the main technique for identifying individual compounds and requires ca. 1–5 mg of pure tannin compounds. The compound is then dissolved in 1–2 ml of a deuterated solvent such as water, methanol, acetone or dimethyl sulfoxide before the NMR spectrum is recorded. The absorption of protons (1H NMR) or carbons (13C NMR) yields chemical shifts which provide detailed information about the chemical environment of each individual atom. This allows the recognition of chemical

Mass spectrometry (MS) of HTs and CTs

Several different types of MS techniques exist and significant progress has been achieved in the past 10–15 years in this field (Okuda et al., 1989). Modern MS techniques can be applied to microgram quantities of labile, polar and large molecular weight compounds without prior derivatisation. Briefly, the compounds of interest are ionised and then yield significant structural information such as molecular ions or characteristic fragment ions. Fast atom bombardment MS in the negative ion mode was

Which method to choose for tannin analysis?

Unfortunately this question does not have an easy answer — yet answers are needed in order to assess the nutritional value of different plants or tree fodders or for screening new varieties in breeding programmes. Our lack of understanding as far as tannin structure/activity relationships are concerned is a serious obstacle to recommending any particular analytical method at the present. Therefore, it is suggested that wherever possible an array of methods be used to characterise tanniniferous

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