Direct analysis of complex tannin mixtures.

Tannins occur naturally in plants and can produce many benefits.

• Agriculture: Ruminant protein absorption1

• Medicines: Herbal remedies

• Health: anti cancer properties of green tea

• Industry: inks, dyes, adhesives and leather

• Food: clarifying agents, antioxidants 2

• Difficult to analyse conventionally 4

Classification of Tannins

1) Ellagitannin - Hydrolysable

Ellagitannins differ from gallotannins in that at least 2 gallic acid units surrounding the core are linked through carbon-carbon bonds.

2) Gallotannin - Hydrolysable

Gallotannins are constructed of depsidically connected units of gallic acid surrounding a polyolic core

3) Condensed Tannin

Condensed tannins are polymers of flavan-3-ol (catechin) units, where each monomer unit is linked 4,8 or 4,6 with an adjacent subunit

4) Complex Tannin

Complex tannins are constructed of catechin units linked glucosidically to gallotannin or ellagitannin at least 2 gallic acid units surrounding the core are linked through carbon-carbon

Methods

Source of Tannins: The tannins investigated originate from the leather industry and include: Tara tannins (Peru), Sumac tannins (unknown origin) and three samples of chestnut tannins from Slovenia, France and Italy.

Sample Preparation: The tannins were used as received. Tannins extracts were dissolved at 18 mg/ml (or as a saturated solution) in acetone-water (4:1 v/v). Five parts of a 0.4 mmol/ml solution of trans-3-indole acrylic acid was subsequently mixed with 1 part of the

sample solution. Where applicable, the samples were doped with sodium (NaCl, 18 mg/ml in 4:1 acetone/water) or potassium (KCl, saturated solution in 4:1 acetone/water) ions to facilitate increased ion formation. The sample platen was loaded with 0.4 ml of the final sample mixture and the tannin samples were analysed using an SAI LT3 LaserTof mass spectrometer in linear mode.

Results

MALDI TOF mass spectra of a range of hydrolysable tannins

Discussion

MALDI-TOF mass spectrometric analysis has been employed previously to analyse heterogeneous mixtures of condensed tannins 5, 6 , a difficult task by conventional analysis.

MALDI-TOF spectra of gallotannins were readily assigned. In the case of the Tara and Sumac tannins, a certain degree of regularity was observed in the MALDI-TOF mass spectrum and notably these tannins possess very similar mass distributions but different average masses (see Figure 1). In both spectra two sets of peaks that are separated by 152 amu are evident and have been assigned to a unit of gallic acid (C6H2(OH)2COO) using modelling correlations.

Structural assignment of these tannins advocates that Tara tannins are composed of gallic acid units centred upon a core of quinic acid whereas, in contrast, the core in the Sumac tannins is a glucose unit. Chestnut tannins are also hydrolysable tannins, but without the structural dispersity evident in the Tara and Sumac tannins. Our research confirms the general classification of the chestnut tannins as ellagitannins, i.e. consisting of a glucose core surrounded by gallic acid and ellagic acid units. Interesting to note is that the origin of the chestnut tannins seems to have little influence on their tannin composition (data not shown).

Conclusion

For the first time, compositional analysis of hydrolysable-tannins from heterogeneous, biological, sources using MALDI-TOFMS has been successfully demonstrated.

References

1. Caygill, J.C. and Mueller-Harvey, I.. Secondary Plant Products - Antinutritional and beneficial actions in animal feeding. Nottingham University Press, Nottingham, UK. 1999, pp129.

2. Porter, M.L., Krueger, C.G., Wiebe, D.A., Cunningham, D.G., Reed, J.D., J. Sci. Food Agric., 2001, 81, 1306.

3. Collingborn, F.M.B., Gowen, S.R. and Mueller-Harvey, I., J. Agric. Food Chem., 2000, 48, 5297.

4. Mueller-Harvey, I., Animal Feed Science and Technology, 2001, 91, 3.

5. Hedqvist, H., Mueller-Harvey, I., Reed, J.D., Krueger, C.G. and Murphy, M., Animal Feed Science and Technology, 2000, 87, 41.

6. Pasch, H., Pizzi, A., Rode, K., Polymer, 2001, 42, 7531. 6b. Krueger, C.G., Dopke, N.C., Treichel, P.M., Folts, J., Reed, J.D., J. Agric. Food Chem., 2000, 48, 1663. 6c. Ohnishi-Kameyama, M., Yanagida, A., Kanda, T., Nagata, T., Rapid Commun. Mass Spectrom., 1997, 11, 31.