Metabolic Fingerprinting Investigation Of Tussilago Farfara L. By GC-MS And UPLC

Ethnopharmacology:Tussilago farfara. L.(Compositae) is a perennial herb that is widely spread in China, North Africa, Siberia, and Europe as an antitussive herbal medicine. According to the theory of TCM, the flower buds of T. farfara are harvested in the winter or spring before the flower buds unearth and blossom in spring. It is so difficult to observe the whole development process that appropriate harvest time of the flower buds became harder and it is lack of objective and reasonable scientific guidance. In Europe, the plant has been used as herbal remedies for virtually all the same applications, but the leaves are preferred to flower buds. The similarities and differences between the two medicinal parts are necessary to be researched and analysed. Some Tussilago farfara L. on the market which mixed with much pedicel and rhizomes sell seconds at best quality prices. Plant metabolomics, a rapidly growing and valuable nontargeted analytical technology, provides a global picture of plant metabolic profiles at the whole metabolite level. In addition, there exist two hepatotoxic pyrrolizidine akaloids(HPAs):senkirkine and senecionine. For safety evaluation and quality control, the study of HPAs content difference in various Tussilago farfara L. samples is particularly important.Aim of the study:(1) To establish GC-MS and UPLC methods coupled with multivariate analysis for the metabolic fingerprinting of T. farfara, and to characterize the metabolic difference between various samples. This work will not only expand our knowledge on the metabolic composition of T. farfara, but also help us to understand the basis of the reasonable harvest time and different traditional use of T. farfara in Europe and China.(2) To establish UPLC analysis method to determinate the content of HPAs in various T. farfara samples for evaluating the safty of the medicine herb and investigating the relationship between the hepatotoxic and efficacy action of T. farfara.Material and methods:(1) All plant samples, included different places, different developmental stages and different parts of T. farfara, were extracted by solvents to obtain the whole metabolites, and then the extractions were detected by GC-MS and UPLC. The identification of GC-MS metabolites was based on searching the mass spectral database library NIST2005or comparison with the corresponding standards and that of UPLC was based on the corresponding standards and references. After data preprocessing, SIMCA-P software was used. PCA was run for obtaining a general overview of the variance of metabolites, and partial least-squares discriminant analysis (PLS-DA) and orthogonal discriminant partial least squares analysis (OPLS-DA) was performed to obtain the marker metabolites.(2) Using UPLC analysis to detect the content of HPAs in various T. farfara samples and compare the high or low level of these samples.Results:(1) GC-MS and UPLC metabolomics results:successfully established GC-MS and UPLC analysis methods, overall identified54primary metabolites and18secondary metabolites. The results of different places:cluster analysis showed that there divided into two categories, Shanxi origin and Hebei origin; similarity results indicated that compared with control fingerprint, the similarity of all origin T. farfara were above0.9; PCA analysis were as same as the cluster analysis results. The results of different developmental stages: The analysis showed clear separation of the different development stages and a trend of gradual change. Samples from early development and after flowering were far away from others, and find out9primary metabolites and7secondary metabolites as biomarkers. The results of different parts:roots, leaves, flower buds and pedicels in the PCA score plot can be obviously divided.(2) Through extraction and separation, we get pure senkirkine compouds. And then its content determination were analysed by UPLC. In different places of Shanxi, Hebei and Gansu, the results showed senkirkine content were over90ug/g, other places were lower, and the content of honey roast samples were lower than80ug/g; different developmental stages the results showed that the senkirkine content increased gradually to the highest in December, and decreased after blossom; the results of different parts:of showed the highest content of senkirkine in leaves and flower buds, the content of senkirkine were higher, and it was lower in pedicel and roots. There were not senkirkine in the proprietary Chinese medicines which including T. farfara.Conclusions:(1) This work successfully established an effective analysis based on GC-MS and UPLC technology combined with metabolomics approach. It can evaluate the quality of T. farfara and provided a new idea and method for us.(2) This job also successfully established UPLC analysis to determinate the content of HPAs in various T. farfara samples for evaluating the safty of the medicine herb and investigating the relationship between the hepatotoxic and efficacy action of T. farfaraprovides the scientific basis and guidance.