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Yang, Yicheng (2011) Quantitative analysis and reaction of pyrrolizidine alkaloids and its metabolites by nuclear magnetic resonance spectroscopy. Doctoral thesis, London Metropolitan University.
Hepatotoxic pyrrolizidine alkaloids (PAs) are widely distributed in plants throughout the world. The toxic effects of PAs are known to present a health risk to humans when they are accidentally exposed to them. Analytical techniques used for identification and quantitation of PAs in plants are TLC, HPLC, GC, GC - MS and LC - MS, which involve chromatographic separation prior to quantitation. NMR shows its advantage in not needing any separation procedure in quantifying PAs in plants. However, the use of NMR in quantitation of PAs has never been reported. In toxicity-related metabolism, PAs are oxidised by cytochrome P450 in the liver to generate dehydro-PAs which are considered to be the primary toxic metabolite. Both dehydro-PAs and its hydrolysed product, dehydroretronecine (DHR) are responsible for toxic actions in humans and animals.
Chapter 1 presents a review of structure classification, chemistry, analytical techniques, metabolism and toxicity of PAs in respect of the three aspects in this thesis. They are i) quantitation of PAs in natural plants; ii) interactions of monocrotaline (MCT) and its metabolites in human blood by using 1H-NMR and iii) chemical modification of dehydro-metabolites of MCT.
In chapter 2 a 1H-NMR quantitative methodology for PAs in PAs-containing plants was developed and validated by quantifying clivorine (OTO-type PA) in Ligularia fischeri (Ledeb.). Two reported data analysis methods (calibration plots and internal standard methods) were compared with a new method which involved the use of the Chenomx software. The concentration of clivorine (CLI) in Ligularia fischeri (Ledeb.) was ea 1.89 mg/g of the plant on average. Results from the 1H-NMR calibration plots and the Chenomx methods showed no statistical significance, as well as when an independent method, LC-MS, was adopted. The 1H-NMR limits of detection of CLI in the plant extract were 3.12 µg/mL (D20) and 3.51 µg/mL (CDCl3), which were about 2000-fold higher than that of LC - MS, 1.50 ng/mL (D20). Furthermore, 1H-NMR technique was employed in quantitation of isoline (RET-type PA) in Senecio Va/garis L. and senecionine (RET-type PA) in Ligularia duciformis (C. Winkl) Hand.-Mazz, Senecio arguensis Turcz and Senecio eannabifolius Less, which were ca 0.12, 0.05, 0.02 and 0.02 mg/g of the plant on average, respectively.
MCT is a naturally occurring hepatotoxic pyrrolizidine alkaloid found in plants. The investigation in chapter 3 is aimed at furthering the understanding of the role of blood in mediating the transport of MCT and its reactive metabolites in humans. Reactions of MCT and its metabolites, dehydromonocrotaline (DHM), retronecine (RET) and DHR with human blood plasma, red blood cells (RBCs), and whole blood were studied in vitro by 1H-NMR. In plasma, MCT remained intact and weakly associated with plasma proteins; and DHM was rapidly hydrolysed releasing the necic and lactone acids, and the reactive pyrrolic metabolite. MCT and its metabolite DHM entered RBCs at 46.0% and 48.9% respectively in 30 min. No polymerisation of DHR was observed when incubated with plasma and RBCs. The data clearly showed that both human plasma and RBCs could be the carriers for the transportation of MCT and its metabolites, DHM, RET and DHR between organs and could stabilise the reactive MCT metabolite DHR.
In chapter 4, with the aim of preventing PA dehydrometabolites from polymerisation in aqueous solution and possessing potential binding abilities with nucleophilic macromolecules, DHM and DHR were chemically modified at C-3, C-7 and C-9 sites. However, derivatives from the chemical modification on C-3 of DHM and DHR were either unstable and polymerised rapidly. Therefore, a new polymerisation pathway was proposed as only C-7 and C-9 sites of dehydro-metabolites were involved. Chemical modifications on C-7 and C-9 sites of DHM were carried out by reacting with methanol, ethanol and t-butanol to give 7-methoxy-1-methoxylmethyl-6,7-dihydro-SH-pyrrolizidine (Compound 9), 7-ethoxy-1-methoxylmethyl-6,7-dihydro-SH-pyrrolizidine (Compound 13) and 1-methyl-6,7-dihydro-SH-pyrrolizidine (Compound 14). Reactions of the nucleoside, deoxyguanosine (dG) with DHM and DHR in methanol, respectively, gave the mono-conjugated dG-DHM adduct and bi-conjugated dG-DHR-dG adducts, indicating DHR acted as a DNA crosslinker in PA-induced genotoxicity. No binding was observed between compounds 9, 13, 14 and dG.
Chapter 5 presents a conclusion of the work carried out including a discussion and an innovation for the quantitation and reaction of PAs and its metabolites. Suggestions for further work are also proposed.
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