The analysis of natural and sulfited commercial quebracho (Schinopsis lorentzii) and Acacia (Acacia mearnsii) proathocyanidin extracts with electrospray ionisation mass spectrometry
Jordaan, Maryam Amra
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Quebracho (Schinopsis lorentzii and Schinopsis balansae) heartwood and black wattle (Acacia mearnsii) bark extracts are important renewable industrial sources of proanthocyanidins (PACs). These extracts are used industrially in leather tanning and adhesive manufacturing. These applications are derived from their chemical properties. The poly hydroxy groups of PACs complex with proteins via hydrogen bonds and thus transforms raw skin into leather. The phloroglucinol or resorcinol type A-rings are nucleophilic and polymerise with aldehydes to form natural adhesives. The ortho hydroxy group on the B-ring form insoluble complexes with heavy metals and can be used in water purification applications. The extracts are often treated with sodium hydrogen sulphate (sulfitation) to enhance their industrial usefulness. From a literature search and discussions with role players in the black wattle and quebracho PAC extract manufacturing industry, it became evident that knowledge on the composition of commercial PACs extracts and chemical changes that takes place during sulfitation is unsatisfactory. These PAC extracts are complex due to variable hydroxylation patterns of the constituent flavan-3-ol aromatic rings, different configurations of the C-2, C-3 and C-4 stereogenic centres, different degrees of polymerisation, and the existence of angular oligomers. Gel or paper chromatography fractionations of the complex extracts are hampered by poor resolution due to their hydrophilic polyphenolic nature and efforts to isolate pure compounds have been restricted to the isolation of mainly monomers and a few dimers and trimers. PACs of the commercially important quebracho (Schinopsis lorentzii and Schinopsis balansae) and black wattle (Acacia mearnsii) extracts have a strong and stable interflavanyl bond. This stability is important from an industrial point of view as it leads to durable leather and adhesive products. It is attributed to the absence of 5-OH groups in the aromatic moieties of the extender fisetinidol and robinetinidol flavan-3-ols units. However, from an analytical point of view it is not advantageous. The high temperatures thus required to hydrolyse the interflavanyl bonds with weak acids; leads to decomposition of the intermediate monomers that renders conventional thiolysis and phloroglucinolysis based analytical methods unreliable. In this thesis we used electrospray mass spectrometry (ESI-MS) to investigate the composition of PACs in black wattle extract and the changes that takes place in the chemical composition of quebracho PACs during sulfitation. We furthermore use all the information available from literature on the phytochemistry of flavan-3-ols and PACs and the syntheses of flavan-3-ol oligomers to guide us in our ESI-MS interpretations. Previous research in our group established that quebracho PACs always consist of a catechin starter unit to which one, two or more fisetinidol extender units are attached. The first and second extender units are always attached to the relatively reactive phloroglucinol A-ring of the catechin starter unit to form predominantly dimers and angular trimers. Further extender units are attached to the relatively less reactive resorcinol A-rings of already incorporated fisetinidol extender units. This explains the relatively short degree of polymerisation of quebracho PAC extracts and their popularity as a tanning agent. Large PACs will not penetrate the spaces between skin proteins and cannot act as a tanning agent. In this thesis we established that black wattle PACs have, in addition to catechin starter units, also gallocatechin starter units and, in addition to fisetinidol extender units, also robinetinidol extender units. Acacia PACs are thus more complex combinations of catechin, gallocatechin, fisetinidol and robinetinidol monomers. This contrasts with quebracho PACs that only contain catechin and fisetinidol monomers. The higher degree of hydroxylation of gallocatechin and robinetinidol explains the higher water solubility of black wattle PACs and the less frequent need for sulfitation. We also established that during sulfitation of quebracho PACs, a sulfonic acid moiety is introduced in both the C-2 and C-4 position of the pyran heterocyclic C-ring. In the case of C-2 sulfitation, the heterocyclic ring is opened. This enhances the reactivity of the A-ring towards the reaction with formaldehyde (adhesive formation) and increases water solubility due to removal of rigidity and introduction of a polar sulfonic acid group. In the case of C-4 sulfitation, the interflavanyl bond is broken. Polarity and water solubility is thus not only increased via an additional sulfonic acid moiety, but due to the presence of shorter oligomers and a smaller average chain length. We also developed a chromatographic method to estimate the degree of sulfitation of quebracho PAC extract. We believe that we have made a valuable contribution towards a better understanding of the composition of black wattle and sulfited quebracho PAC extracts and have identified a number of misconceptions.