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Title: The chemistry and ecology of British bluebells (Hyacinthoides non-scripta)
Author: Raheem, Dotsha
ISNI:       0000 0004 5993 473X
Awarding Body: Prifysgol Bangor University
Current Institution: Bangor University
Date of Award: 2016
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Bluebell chemistry was investigated in order to identify factors contributing to the success and survival of the plant in a bluebell-bracken dominated ecosystem. The plant was divided into roots, bulbs, leaves, scapes and flowers and three classes of compounds (carbohydrates, phenolics and saponins) were studied and their seasonal changes were followed. Total non-structural carbohydrates (TNC) were quantified after acid hydrolysis of the dried plant material and analysis of the reaction product by high performance liquid chromatography with diode array detection (HPLC-DAD). The bulbs contained the highest carbohydrate content compared to the other parts with TNC levels ranging from 36 – 82% of dry weight. Highest values in bulbs were reached around anthesis and remained high until the end of above ground growth. The lowest values were recorded during the underground growth phase before shoot emergence in March. The above ground parts showed low TNC contents at the start of their growth and higher values were reached during the active above ground growth. Mono- and disaccharides were studied using gas chromatography-mass spectrometry (GC-MS) after conversion into volatile oxime-trimethyl silyl derivatives. Fructose, glucose and sucrose were detected in this pool with fructose being the major sugar in the underground organs versus glucose in the above-ground parts. The bulbs showed significant increase in monosaccharides, especially fructose, during the early growth. Fructans were also investigated and their amounts were determined in the above and below ground organs following an enzymatic assay. The results showed a trend similar to the TNC especially in the bulbs. The fructan pool was further studied with matrix-assisted laser desorption ionisation – time of flight (MALDI-ToF) spectroscopy to determine the chain length of the polymer. This revealed that short chains < 10 units are more commonly found with DP3 and DP4 being the most abundant oligomers. Higher chain lengths (up to DP20) was also found, but in much lower quantities. The main trisaccharide was isolated using flash chromatography and preparative TLC in reverse phase mode. Structural elucidation was achieved using 1D and 2D NMR spectroscopy of the free and peracetylated forms and the trisaccharide was identified as 1-kestose. The phenolic profile of bluebell flowers, scapes and leaves was thoroughly studied using HPLC-UV and ESI-MS/MS analysis. The results showed that apigenin was the main flavone aglycone in the plant in addition to minor quantities of luteolin, eriodycteol and other unidentified aglycones. Phenolic acids including p-coumaric and ferulic acids were also detected. Both O- and C-glycosides were detected. The glycosidic moieties identified as hexoses, pentoses, rhamnose and glucuronic acid. The methanolic extract of bluebell flowers was investigated and four new metabolites were isolated including two phenylalkyl glycosides (benzyl-O-β-D-glucopyranosyluronic acid-(1→2)-glucopyranoside DR1 and 2-phenylethyl-O-β-D-glucopyranosyluronic acid-(1→2)-glucopyranoside DR2) and two apigenin glycosides (7-[O-β-D-glucopuranosyl-(1→3)-O-β-D-xylopyranosyl-(1→6)-O-β-D-glucopyranosyl-(1→2)-O-β-D-glucglucopyranoside uronic acid-(1→)] apigenin (DR3) and 7-[ O-β-D-xylopyranosyl-(1→6)-O-β-D-glucopyranosyl-(1→2)-O-β-D-glucglucopyranoside uronic acid-(1→)] apigenin (DR4)). The structural elucidation of these compounds was based on NMR, accurate mass and MS/MS analysis. Apigenin and p-coumaric acid were the only aglycones quantified after acid hydrolysis of the hydrolysed dried plant. The results showed that the two aglycones accumulated to a higher extent in the above ground organs with anthesis being characterised with an increase in content of both compounds to up to 15 and 0.5 mg. g-1 DM, respectively. A previously unreported saponin, (3β-[(O-β-D-glucopyranosyl-(1→3)-O- β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranosyl-(1→2)-O-α-L-arabinopyranosyl-(1→6)-O-β-D-glucopyranosyl)oxy]-17,23-epoxy-28,29-dihydroxy-27-norlanost-8-en-24-one (DR1), was isolated from bluebell flowers using RP flash chromatography. The structure was elucidated with extensive 1D and 2D NMR, acid hydrolysis and GC-MS analysis of the sugars, accurate mass and MS/MS analysis. The isolated saponin was investigated for potential anti-trypanosomal, antimicrobial, antifungal, pesticidal and schistosomicidal activities. DR1 showed high biological activities against Trypanosoma brucei and Schistosoma mansoni. Crude fractions (aqueous and 1-butanol fractions of the methanolic extracts) from bluebell seeds were also investigated for schistosomicidal activities and found to show comparable results to DR1. The metabolomics approach and multivariate analysis approach was used in order to investigate the saponins and follow their seasonal changes. The seasonal samples of different plant parts were analysed with LC-MS in positive and negative modes. The resulting spectra were processed and the data produced were compared to databases for possible identification of metabolites. Multivariate analysis highlighted the difference between bulb metabolites from the above ground organs. The LC-MS spectra obtained and peak data used to study the changes in saponin contents between the different organs throughout the study period. The bulbs were found to contain the lowest amounts of total saponins (only about 20% of the other organs) with the highest ratios being in March-April at the start of the growth then decrease afterwards. Amongst the above-ground organs, the flowers showed relatively higher contents. The LC-MS spectra from the metabolomics study were utilised in tentatively identifying a number of saponins. Two other metabolites were identified as steroidal aglycones from their HR-ESI-MS spectra. These metabolites were of 470 and 456 mass units and were characteristic of the bulbs.
Supervisor: Fitzsimmons-Thoss, Vera ; Lahmann, Martina Sponsor: Kurdish Regional Government
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available