A solid state NMR and MS characterisation of the chemical composition of mimosa bark extract

[摘要] English: Mimosa (Acacia mearnsii) also known as black wattle, and quebracho (Schinopsis balansae, Schinopsis lorentzii) are the major commercial sources of natural condensed tannins (proanthocyanidin oligomers) used today. Mimosa bark is harvested from commercial plantations in South Africa which, according to a survey done by the Department of Water Affairs and Forestry for 2001, cover an area of about 107 000 hectares in South Africa. Quebracho is extracted from the wood of natural forests in Brazil and Argentina. Mimosa bark is extracted with water (about 50% by weight). Tara (Cæsalpinia spinosa) and Italian chestnut (Castanea sativa) are the major commercial sources of hydrolysable tannins. The ability of water soluble hydrolysable and condensed tannins (polyphenols) to react with proteins, presumably via hydrogen bonds, lies at the heart of their ability to transform raw hide into leather and their commercial application as tannin agents. It explains their existence in nature as anti-feeding agents as it renders plants indigestible to insects and herbivores. It also explains the use of milk in tea where the complexation of milk proteins with tea tannins reduces astringency. The chemistry of this process however remains uncertain. The polyphenolic nature also renders tannin extracts very susceptible to oxidation and further polymerisation and rearrangements that render the extracts even more complex. This is evident in the transformation of green tea (high flavan-3-ol content and low condensed tannin content) into Indian or black tea (low flavan-3-ol content and high condensed tannin content). The quality of red wine is to a large extent determined by the amount and composition (which changes during ageing in a poorly understood way) of its condensed tannin. The tannins react with protein receptors on the tongue to impart 'mouth feel characteristics. Wood-aged wine not only contains condensed tannins from grape skin, but also hydrolysable tannins from the wooden barrels it is aged in. The polyphenolic nature of the aromatic rings allows reaction with electrophiles. This forms the basis of adhesive manufacturing, where formaldehyde is used to polymerise tannin extracts to form adhesives. Other commercial applications of tannin extracts include the use as anti-foaming agents in oil drilling and the manufacturing of amine containing resins (via the Mannich reaction) for water purification applications (removal of heavy metals). The production of mimosa condensed tannin is a sustainable process as trees are harvested every eight years. Tannins will become a more important source of feedstock nutrients, as crude oil, which is currently used, becomes depleted. It also creates employment in rural areas. Higher oligomers of condensed tannins are built up by successive addition of flavan-3-ol monomer extension units via C-4 to C-8 or C-4 to C-6 interflavanyl bonds. Higher oligomers are impossible to purify by chromatography and other methods of analysis are required. Acid catalysed fission of the interflavanyl bonds and trapping of the monomer intermediates with toluene-α-thiol or floroglucinol followed by analysis of the trapped products with HPLC is normally used to analyse condensed tannin composition. The analysis of mimosa and quebracho tannins is however compounded by the resorcinol type A-ring in these compounds. The absence of a 5-OH group imparts stability to the interflavanyl bond against acid hydrolysis. The high temperatures thus required to hydrolyse the interflavanyl bond in mimosa and quebracho tannins leads to decomposition. Mass spectrometry and 13C NMR (nuclear magnetic resonance) spectrometry in solution have also been used with varying degrees of success. The analysis of hydrolysable tannins is even more complex than that of condensed tannins. As a result, the composition of condensed and hydrolysable tannin extracts remains uncertain, after more than 50 years of research. Of particular interest are the average chain length of tannin extracts from different sources and the composition of the constituent monomers. In this thesis the potential of solid state NMR and electrospray mass spectroscopy to solve vexing problems in tannin chemistry was investigated. Solid state NMR is particularly useful to investigate insoluble samples, overcoming problems associated with selective extraction, chemical modifications during extraction and sample preparation and uncertainty regarding compounds that are not extracted. Electrospray mass spectrometry complements MALDITOF mass spectrometry in that molecules with masses below 500 Dalton are detected. We were able to assign all the resonances in solid state NMR of hydrolysable and condensed tannins by comparing liquid and solid state spectra of pure flavonoids and tannin extract. This allowed us to distinguish unequivocally between condensed tannins and hydrolysable tannins with a simple routine experiment, avoiding laborious chemical tests. A method was developed to identify and distinguish with confidence between quebracho and mimosa condensed tannins. This method is the only available method to identify quebracho, which is of interest to oenology (quebracho tannins are added to wine) and could hitherto only be identified chemically because it tests negatively for all the available tests for tannins. We established that no insoluble higher oligomeric condensed tannins or tannins covalently bonded to other insoluble bark components remain in spent mimosa bark (after extraction of tannins). It promises an easy way for the wattle industry to investigate lower extraction temperatures and extraction time and the associated energy savings. A fingerprinting method for mimosa was developed and is already used by the industry (Annex A). As the gum resonances do not overlap with the tannin resonances, the bark can be analysed directly without the requirement of manufacturing an extract. The only sample preparation required is to grind the bark (about 100mg) finely and pack the solid state NMR rotor. As carbon is magnetised via hydrogen, less than 30 minutes NMR time is required per sample. This provides an easy way to identify the bark of quebracho, mimosa and hydrolysable tannins. A solid state NMR spectrum of the spent bark not only indicated that no condensed tannins remain, but also supports the conclusion that spent bark consists of water insoluble gums (polymers of glucose and other sugars). We believe that this method will find application in identifying novel sources of tannins from indigenous plants. We expanded our investigation into tanned leather and developed an easy method to determine whether leather was tanned with mimosa, quebracho, Italian chestnut, tara, synthetic tanning material, chromium or aluminium. We believe this method can be used by the leather industry to determine tannin loading of tanned leathers. By combining our electrospray mass spectrometry data with published MALDI-TOF mass spectrometry data we could calculate the relative composition of monomers, dimers, trimers, tetramers etc. in condensed tannin sample. These calculations were used by the mimosa and quebracho tannin industry to comply with new European Union (EU) REACH (Registration, Evaluation, Authorisation and Restriction of Chemical substances) legislation. Without compliance mimosa extract cannot be exported to the EU. Sulfitation (treating mimosa and particularly quebracho extract with bisulfite) is routinely used in industry to enhance the extract's properties (e.g. increase water solubility) and products with different levels of sulfitation are commercially available. The chemical changes associated with sulfitation remain speculation. The solid state NMR indicated that the C-ring is opened during the process. The electrospray MS conclusively demonstrated the existence of condensed tannin-sulfonate molecules for the first time. The m/e values correspond with ring opening and introduction of a sulfonate group on the C-2 position.

[发布机构] University of the Free State