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Welbeck, Welbeck, Edward William (2010) Separation, identification, and standardisation of plant metabolites in traditional Chinese medicines by chromatographic, spectroscopic and multivariate analysis processes. Doctoral thesis, London Metropolitan University.
Quality control of Chinese medicinal herbal extracts is an important criteria used to assess the characteristics of traditionally prepared samples under analysis. From a chemical perspective, the quality assessment of medicinal herbal extracts are analysed by their chemical profiles as metabolic fingerprints highlighting one or two phytochemical marker compounds existing within samples. Generally comparisons are made with a standardised reference monograph to decide on whether a medicinal herb is authentic or counterfeit. Standardisation tools used for the evaluation of medicinal herbs include a chromatographic element to separate these often complex mixtures, (e.g. HPTLC, HPLC, GC-MS and LC-MS), followed by structural characterisation using MS or NMR. Adding to the complexity of Chinese medicinal herbs is the preparations used prior to administering the drug. These drugs are often prescribed as a mixture of several herbs in a definitive composition as a herbal formulation with a multitude of metabolites in them, claiming to have multiple efficacies. The chapters presented in this thesis describe method developments and their applications in plant metabolomics of Chinese herbal prescriptions. Chapter 1 presents a short review of the flavonoid plant metabolites, plant metabolomics and the technologies used for metabolomics with the perspective for compound identification, lanthanide complexes as NMR shift reagents, and also the Chinese medicinal herbal prescriptions and the need for quality control.
In Chapter 2, a new approach for the evaluation of Chinese medicinal herbal prescriptions involves the use of online LC-NMR, which potentially has -the possibility of becoming a powerful standardisation tool for assessing herbal quality in an efficient manner. Often overlooked because of its lower sensitivity compared to the other hyphenated MS methodologies, the potential of LC-NMR was evaluated using the 10-component Chinese medicinal herbal formulation "Longdan Xiegan (LX) Decoction". A total of 5 metabolites were unambiguously described in the formulation supporting LC-MS findings and another 7 were tentatively explained based on chemical shift assignments and coupling constant data; three metabolites being defined for the first time. The LX-Decoction formulation was used as a system to exemplify a method such as LC-NMR as a credible instrument for standardisation and quality control investigations of this type.
The work presented in chapter 3 is a metabolomics investigation on the LC-NMR spectra obtained from the LX-Decoction, and was described for the first time using multivariate analysis models. Principal component analysis (PCA) in conjunction with Soft Independent Modelling of Class Analogy (SIMCA) classification was performed on LX-Decoction spectra and also pure reference metabolites analysed by the same LC-NMR method. The reference metabolite spectra generated formed an LC-NMR library that was used in correlation with LX-Decoction spectra using PCA models. Correlations were observed between both LX-Decoction spectra and reference metabolite spectra using 2D and 3D PCA models, speeding up the efficiency of spectral classification of LX-Decoction fractions. In addition to this, SIMCA classification was performed as a confirmation aid for correlations observed within models generated. A metabolic profiling investigation was also carried out on the whole prescription of the LX-Decoction without the need for fractionation using Chemonx NMR Suite metabolic profiling software, with a custom built metabolite library. The 5 metabolites described in the first investigation were successfully profiled in the whole decoction formulation and their concentrations determined.
Chapter 4 describes the application of the lanthanides complexes [Eu(EDTAH)(H2O)2] and (Eu(D03A)(H2O)2] as NMR shift reagents (LSRs) were applied to an investigation of a one herb prescription the Danggui Decoction (DG), and a two herb formulation Danggui Buxue Tang (DBT) in conjunction with multivariate analysis statistical models for the first time. Changes observed between pre- and post-additions of the LSRs were evaluated based on the displacement distances herbal extracts shifted from their original positions to their residing locations in PCA models. The lanthanide complex [Eu(EDTAH)(H20h] was shown to shift five different samples of DG originating from various sources by varying amounts; governed predominantly by the amount of carbohydrates found within the extracts. Displacement distances were calculated for selective regions of the NMR spectra for DG extracts to explain the chemistry behind the nature of the shifts and the interactions observed. The reference metabolite calycosin was subjected to successive [Eu(D03A)(H20h] additions. The chemical shift movement upon additions was calculated for individual protons in calycosin with protons closest to electron withdrawing oxygen atoms being most affected by LSR interactions.
Chapter 5 proposes a summation of the presented work with discussion and concluding remarks for applications in a quality control and standardisation. Suggestions for further work for future studies are also proposed.
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