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The Molecular Weight Characterisation of Pinus radiata Bark Condensed Tannins

Crude condensed tannins extracted from Pinus radiata bark (sourced from Tokoroa) with various solvents (Hot water (HW), methanol/water/acetic acid (MWA), acetone/water (AW) and hot aqueous 2% sulphite/urea solution (SFU)) were subjected to Sephadex LH-20 column chromatography to purify the condensed tannins by removing carbohydrate and flavonoid components (crude extracts were approximately 70% condensed tannins). The condensed tannins from the HW and MWA extractions were then fractionated on a Sephadex LH-20 column using a stepwise gradient of methanol, water and acetone to separate the condensed tannins according to molecular weight (MW). Fractions were collected and pooled to give eight final fractions (HW 1-8 and MWA 1-8). Purified and fractionated condensed tannin fractions were then analysed with a range of techniques including 1H nuclear magnetic resonance (NMR) spectroscopy, acid/phloroglucinol depolymerisation coupled with high performance liquid chromatography (HPLC) with UV and MS detection, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS), electrospray ionisation mass spectrometry (ESI-MS) and gel permeation chromatography (GPC). In the MALDI-TOF MS spectrum of the crude extracts the most intense signal was typically the tetramer peak and the decamer was typically the largest oligomer detected. In the spectra of late-eluting column fractions (i.e. HW and MWA 5-7) oligomers greater than the nonamer (DP up to 14) were detected and the most intense signal was shifted from the tetramer to higher MW oligomers. ESI MS showed itself to be better at analysing small MW condensed tannins. The MS results demonstrated that prior separation of the condensed tannins was effective in improving MS detection of higher MW oligomers. The mDP of the crude and fractionated fractions was obtained by depolymerisation/HPLC. The mean degree of polymerisation (mDP) of the purified condensed tannins ranged between 7.3 (AW) and 9.2 (SFU). The mDP of the fractionated condensed tannins ranged between 2 and 18. The depolymerisation/HPLC results of the fractionated condensed tannins were used to construct a GPC calibration curve of condensed tannin MW versus GPC retention time. To identify the GPC dimer peak a procyanidin dimer was synthesised by reacting condensed tannins with catechin under acidic conditions. Through the use of various NMR techniques (DEPT135, COSY, HSQC and HMBC experiments) the synthetic dimer was characterised and the 1H and 13C NMR spectra fully assigned. The GPC calibration curve enabled MW profiles for the HW and MWA 2-8 fractions and crude extracts to be obtained. GPC analysis confirmed the presence of larger oligomers that were not detected by MALDI-TOF MS and ESI MS. GPC number average MW results were in agreement with the average MW results obtained from depolymerisation/HPLC results. Pinus radiata bark condensed tannins from two different geographical locations (Golden Downs (Nelson) and Waimate (South Canterbury)) and different tree heights (0, 10 and 20 m) were analysed using the techniques described above to discover if there was any variability between samples. Trends that were observed between the different condensed tannins were decreases in condensed tannin yield and mDP as bark was obtained from higher up a tree. Extracts from New Zealand native bark (totara, rimu and kauri) were also analysed, showing that Rimu was the only bark of these species that contained significant amounts of condensed tannins. Overall this research has provided additional information on the structure and MW of P. radiata condensed tannins.
Type of thesis
Bogun, B. R. (2007). The Molecular Weight Characterisation of Pinus radiata Bark Condensed Tannins (Thesis, Master of Science (Technology) (MSc(Tech))). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/3937
The University of Waikato
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