SGMS Meeting 2000

Qualitative analysis of tannins extract by mass spectrometry

Camille Perret, Roger Pezet² and Raffaele Tabacchi¹

²Station fédérale de recherches en production végétale de Changins, Route de Duillier, CH-1260 Nyon

Botrytis cinerea produces an hydroxystilbene-oxidazing enzyme when grown on a pectin-containing medium, identified as a laccase-like stilbene oxidase [1]. Enzymatic oxidation of pterostilbene by this stilbene oxidase detoxifies this compound [2] and produces the dehydrodimer of both pterostilbene and resveratrol [3].

Polyphenolic fractions of the healthy grape berries of Gamaret cause a rapid inhibition of this laccase at low concentration. The pure tannins extract contains a mixture of proanthocyanidins and proanthocyandin gallates. These polar compounds are difficult to isolate and characterize due to their high molecular weight and their polarity.

A sensitive LC–MS method has been developed in our laboratory for the qualitative analysis of the oligomers. The potential of an IT/MS (ion trap) equipped with ES (electrospray) interface permit us to induce multiple stage fragmentation (MSⁿ), making easier the characterization of these compounds.

Polymers were fractionated according to the mean degree of polymerization (mDP), using either a divinylbenzene phase or a glass powder. The mDP is obtained by thiolysis reaction and the fractions are analysed by ESI or MALDI - FT-ICR-MS.

References:

[1] Pezet, R. (1998). Purification and characterization of a 32-KDa laccase-like stilbene oxidase produced by Botrytis cinerea Pers.:Fr. FEMS Microbiology Letters 167, 203-208.

[2] Pezet, R., Pont, V. and Hoang-Van, K. (1991). Evidence for oxidative detoxication of pterostilbene and resveratrol by a laccase-like stilbene oxidase produced by Botrytis cinerea. Physiological and Molecular Plant Pathology 39, 441-450.

[3] Breuil, A.C., Adrian, M., Pirio, N., Meunier, P., Bessis, R. and Jeandet, P. (1998). Metabolism of stilbene phytoalexins by Botrytis cinerea : 1. Characterization of a resveratrol dehydrodimer. Tetrahedron Letters 39, 537-540.