Educación Ambiental (EA)

AND ANTIVIRAL ACTIVITY OF POLYFLAVONOIDS

A.Pizzi

ENSTIB-LERMAB, University Henry Poincare – Nancy 1, Epinal, France

A

The determination by Matrix-Assisted Laser Desorption/Ionization time–of-flight (MALDI-TOF) mass spectroscopy of the oligomeric nature of the two major industrial polyflvonoid tannins which exist, namely mimosa and quebracho tannins, and some of their modified derivatives indicates that: (i) mimosa tannin is predominantly composed of prorobinetinidins while quebracho is predominantly composed of profisetinidins, that (ii) mimosa tannin is heavily branched due to the presence of considerable proportions of "angular" units in its structure while quebracho tannin is almost completely linear. These structural differences also contribute to the considerable differences in viscoity of water solutions of the two tannins. (iii) the interflavonoid link is more easily hydrolysable, and does appear to sometime hydrolyse in quebracho tannin and profisetinidins, partly due to the linear structure of this tannin, and confirming NMR findings that this tannin is subject to polymerisation/depolymerisation equilibria. This tannin hydrolysis does not appear to occur in mimosa tannin in which the interflavonoid link is completely stable to hydrolysis. (iv) Sulphitation has been shown to influence the detachment of catechol B- rings much more than pyrogallol-type B-rings. (vi) The distribution of tannin oligomers, and the tannins number average degree of polymerisation obtained by MALDI-TOF, up to nonamers and decamers, appear to compare well with the results obtained by other techniques. As regards procyanidin tannins, it has been possible to determine for mangrove polyflavonoid tannins that: (i) procyanidins oligomers formed by catechin/epicatechin, epigallocatechin and epicatechin gallate monomers are present in great proportions. (ii) oligomers, up to nonamers, in which the repeating unit at 528-529 Da is a catechin gallate dimer that has lost both the gallic acid residues and an hydroxy group are the predominant species. (iii) oligomers of the two types covalently linked to each other also occur.

Water solution of non-purified polyflavonoid extracts appear to be incolloidal state, this being due mainly to the hydrocolloid gums extracted with the tannin as well as to the tannin itself.

Commercial, industrially produced mimosa, quebracho, pine and pecan polyflavonoid tannin extracts water solutions of different concentrations behave mainly as viscous liquids at the concentrations which are generally used for their main industrial applications. Clear indications of viscoelastic response are also noticeable, among these the cross-over of the elastic and viscous moduli curves at the lower concentrations of the range investigated, with some differences being noticeable between each tannin and the others, pine and quebracho tannin extracts showing the more marked viscoelastic behaviour. Other than pH dependence (and related structural considerations), the parameters which were found to be of interest as regards the noticeable viscoelastic behaviour of the tannin extracts were the existence in the solutions of labile microstructures which can be broken by applied shear. This is supported by the well known thixotropic behaviour of concentrated, commercial polyflavonoid tannin extracts water solutions.

Such microstructures appear to be due or (i) to the known colloidal interactions of these materials, or (ii) to other types of secondary interactions between tannin oligomers and particularly between tannin and carbohydrate oligomers. The latter is supported by the dependence of this effect from both the average molecular masses of the tannin and of the carbohydrate oligomers.

The behaviour of polyflavonoid tannins as regards their antioxydant capacity and radical scavenging ability has been examined. Radical formation and radical decay reactions of some polyflavonoid and hydrolysable tannins has been followed, and comparative kinetics determined, for both light induced radicals and by radical transfer from a less stable chemical species to the tannin as part of an investigation of the role of tannin as antioxidants.

The five parameters which appear to have a bearing on the very complex pattern of the rates of tannin radical formation and radical decay were found to be (i) the extent of the colloidal state of the tannin in solution (ii) the stereochemical structure at the interflavonoid units linkage (iii) the ease of heterocyclic pyran ring opening, (iv) the relative numbers of A- and B-rings hydroxy groups and (v) solvation effects when the tannin is in solution. It is the combination of these five factors which appears to determine the behaviour as an antioxidant of a particular tannin under a set of application conditions.

The chapter ends with some recent results on the antiviral activity of polyflavonoid tannins for a great variety of viruses.

I

Polyflavonoids also called condensed tannins are natural polyphenolic materials. Industrial polyflavonoid tannin extracts are mostly composed of flavan-3-ols repeating units, and smaller fractions of polysaccharides and simple sugars. Two types of phenolic rings having different reactivities are present on each flavan-3-ol repeating unit, namely A-rings and B-rings, with each repeating unit being linked 4,6 or 4,8 with the units which precede and follow it.

O

OH

OH

OH

(OH)

HO

(OH)

1'

2

3

4

5

6

7

8

A

B

Recently, the radical and ionic mechanisms of the reaction of autocondensation and networking of polyflavonoid tannins induced by bases and by weak Lewis acids has been described [2-8]. Different polyflavonoid tannins however present different structures and different average molecular masses, and as a consequence often present peculiarly different behaviour in their application [10]. The most common method of examination of the relative structures of polyflavonoid tannins, and of their differences, is by 13C NMR [10].

T

O

N

P

Since its introduction by Karas and Hillenkamp in 1987 [11], Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry has greatly expanded the use of mass spectrometry towards large molecules and has revealed itself to be a powerful method for the characterization of both synthetic and natural polymers [12-17]. Fragmentation of analyte molecules upon laser irradiation can be substantially reduced by embedding them in a light absorbing matrix. As a result intact analyte molecules are desorbed and ionized along with the matrix and can be analysed in a mass spectrometer. This soft ionization technique is mostly combined with time-of-flight (TOF) mass analysers. This is so as TOF-MS present the advantage of being capable to provide a complete mass spectrum per event, for its virtually unlimited mass range, for the small amount of analyte necessary and the relatively low cost of the equipment.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry (MS) is a technique that has revealed itself to be a very useful tool in defining the oligomeric structure of polyflavonoids, much more pointed than other techniques used before. As an example even oligomers up to and even of more than 10 flavonoid repeating units have been clearly detected in commercial polyflavonoid extracts by using such a technique. The technique was used to compare the structures of the most common industrial polyflavonoid condensed tannins.