2009 Sukanda T

Application of metabolomics techniques for the quality assessment of oriental folk medicine Angelica acutiloba roots

Sukanda Tianniam

Laboratory of Bioresource Engineering (Fukusaki Laboratory)

ABSTRACT

In this study, metabolomics technique was applied to evaluate the quality differences in dried Angelica acutiloba roots (toki in Japanese) in the attempt to establish an alternative, automated and standardized QC method for commercial markets. The global metabolic profiles of different toki samples were analyzed through three different high-throughput analytical platforms in order to investigate its potential as a rapid analytical protocol in analyzing various kinds of compounds found in toki samples. Gas chromatography mass spectrometry (GC-MS) was utilized to investigate the hydrophilic, low molecular weight compounds which include primary metabolites. Hydrophobic, low molecular weight metabolites were evaluated by ultra performance liquid chromatography (UPLC) MS, whereas pyrolyser (PY) GC-MS was applied to analyze all volatiles, low and high molecular weight compounds. The quality differences in toki were distinctly observed with chemometric techniques by principal component analysis (PCA), in which additional information to the entire chemical constituents in relations to differentiation was acquired. Sample separations were predominantly affected by cultivation area in which it was identified as the major influential factor for dictating quality discrimination. Sustainable classification model by partial least square discriminant analysis (PLS-DA) were successfully constructed based on the total chemical pattern. Ultimately, by adapting the aspects of quality assessment to metabolomics, a legitimate and useful quality assessment tool was established possessing superior capabilities in analysis, efficiency and practicality.

SUMMARY OF THE WORK

Chapter 1. General Introduction

Treatment by traditional herbal medicine has been widely accepted for its effective therapeutic properties in remedying diverse diseases. One of the important traditional herbal materials is the dried roots of Angelica acutiloba known for treating women’s gynecological disorders, and for its potent immunomodulatory activities utilized in combination with conventional cancer therapy. Currently, the existing quality assessment method for its commercial product has become insufficient because a high-throughput protocol that adapts a holistic analysis of the entire complex chemical composition of the herbal material is ignored. In the commerce of toki, although herbal products are known for its medicinal attributes, quality is determined through the examination of the root’s morphological properties, together with fragrance and flavor by professional herbalists without the knowledge of its bioactive constituents (traditional method). Another method involves the targeted analyses of bioactive compounds in toki in order to measure its therapeutic efficacy. However, the applications of both procedures to global markets have created some practical difficulties. Therefore, it is important to develop an efficient QC procedure for the commercial product of toki that is able to interpret and integrate the aspects of quality determination. Metabolomics is an interdisciplinary tool that includes a quantitative exhaustive profiling of all metabolites contained in a target organism through the use of high throughput machines. Hence a non-targeted global analysis of the chemical or chromatographic patterns of various metabolites could be used to discriminate between samples of different status or origin via computational multivariate analysis (chemometrics), and interpret the trends and variables that have a dominant affect in dissociating diverse samples. The application of metabolomics to the quality assessment of toki should prove to be a simple, fast and sufficient method.

Chapter 2. Metabolite profiling of hydrophilic, low molecular compounds in dried Angelica acutiloba roots for quality assessment through the utilization of gas chromatography mass spectrometry

GC-MS is considered the most mature technology for analytical chromatography possessing high through-put capability, sensitivity, reproducibility and repeatability. Hence, GC-MS was utilized to elucidate the profiling of hydrophilic, low molecular weight compounds which included primary metabolites. The differences between Yamato-toki with respect to quality determined by traditional method were rapidly processed and evaluated through principal component analysis (PCA). Distinct discrimination between good, moderate and bad quality were shown where separation was influenced by the differences in cultivars and cultivation areas; the latter being the primary component for sample separation. This implicates the importance of cultivation area in affecting quality discrimination, and the degree of influences it possesses in dictating the global metabolic variations of Yamato-toki. Furthermore, PCA results signified two reducing sugars as being the most accumulated in the bad quality, whereas significant quantities of specific organic and amino acids were found in good and moderate qualities.

Chapter 3. Metabolic fingerprinting of hydrophobic, low molecular weight compounds in dried Angelica acutiloba roots for quality assessment through the utilization of ultra performance LC-MS

UPLC-MS provides an analytical system pertaining higher throughput and better separation efficiency with higher resolution in analyzing hydrophobic, low molecular weight compounds in comparison with conventional HPLC. Accurate mass chromatographic fingerprints of positive and negative ion modes were collected simultaneously, and compared through PCA analysis. Distinct partitioning of root samples was effectively observed, in which cultivation area was the major influential factor for sample disassociation. The affect of species differences in toki samples was diminutive to quality discrimination, and differences in rank could only be achieved when variability in cultivation area was eliminated. Henceforth, classification models by partial least square discriminant analysis (PLS-DA) were constructed in predicting quality based on cultivation area in which the results was reliable and accurate in categorizing test set samples. Further insights to the chemical constituents in relations to quality discrimination were attained where some ion markers significantly linked to dissociation of toki were tentatively identified as some secondary metabolites.

Chapter 4. Metabolic fingerprinting of volatiles, low and high molecular weight compounds in dried Angelica acutiloba roots for quality assessment through the utilization of pyrolyser coupled to GC-MS

The implementation of the PY-GC-MS was made in order to procure fast analysis of a broader spectrum of compounds, such as volatiles, low and high molecular weight compounds. Metabolic fingerprinting was employed making data analysis less tedious, less complex and less time-consuming. Furthermore, the combination of PY to the GC-MS analytical system has eliminated sample preparation procedures, as raw solid samples of toki could be directly injected to the PY for extraction, and consecutively be analyzed through the GC-MS. Hence, the chromatographic pattern attained from PY-GC-MS represents a more universal view of the global metabolic profile. With the introduction of metabolic fingerprinting to PY-GC-MS analysis, optimization to its functionality (analytical, practical and systematical aspects) was obtained, establishing its application as one of the useful QC method.

Chapter 4. General Conclusion

The establishment of reliable, efficient and standardized quality assessment method for toki was successfully developed. Through the application of metabolomics technique, the influence of cultivation area to quality assessment was conceptualized and applied to the development of a legitimate QC method. The implementations of the three analytical platforms investigated have shown to have high robustness and reliability. In conclusion, a combination between GC-MS to pyrolyser amplified the sufficiency of the system as a powerful quality assessment tool for industrial applications in the global commercial markets. This is because PY-GC-MS maintains the GC-MS high-throughput capability, sensitivity and reproducibility while omitting sample preparation procedures and maximizing chemical universality. Hence, a more complete overview of the global metabolic fingerprints could be acquired in one single step where trained technician is not mandatory. The development of GC-MS-based metabolite profiling is well-suited for exploring the biological pathways of the herbal plant in the attempt to discover the optimal cultivation condition for yielding the best quality of toki in agriculture. UPLC-based metabolomics would be most appropriate for pharmaceutical investigations where further elucidation to the therapeutic efficacy of toki roots and its correlation to quality assessment could be attained based on the fingerprints of secondary metabolites.

RELATED PUBLICATIONS:

1.) Tianniam, S., Tarachiwin, L., Bamba, T., Kobayashi, A., and Fukusaki, E. (2008) Metabolic profiling of Angelica acutiloba roots utilizing gas chromatography time-of-flight mass spectrometry for quality assessment based on cultivation area and cultivar via multivariate pattern recognition. J. Biosci. Bioeng. 105, 111-999.

2.) Tianniam, S., Bamba, T., and Fukusaki, E. (2009) Non-targeted metabolic fingerprinting of oriental folk medicine Angelica acutiloba roots by ultra performance liquid chromatography time-of-flight mass spectrometry. J. Sep. Sci. (Accepted)

OTHER PUBLICATION:

1.) Tianniam, S., Bamba, T., and Fukusaki, E. (2009) Pyrolysis GC-MS-based metabolic fingerprinting for the quality evaluation of commercial Angelica acutiloba roots. J. Biosci. Bioeng. (Revision)