Metabolomics Approach To Study On Quality Control Of Cyathula Officinalis Kuan And Sustainable Utilization Of Paris Polyphylla Smith Var.Chinensis(Franch.) Hara

As the raw materials of all the traditional Chinese medicine products,medicinal herbs play a principal role on the traditional health system.Nowadays,the medicinal herbal production is confronted two severe challenges,related to herbal quality and herbal resource respectively.For the first issue,the key question is how to establish an approach to effectively evaluate and control the herbal quality,which could well correspond to the basic thoughts of trational Chinese medicine.For the other issues,the central question is how to blance the human needs and natural supply since lots of endangered medicinal plant species have the risk of extinction due to excessive utilization by medical industry.Unlike chemical drugs,medicinal herbs are characterized by the ideas of traditional Chinese medicine and the natural of biological materials.Therefore,in order to cope with above challenges,those theories and practices that have fully reflected the significant feature of medicinal herb itself are strongly recommended,like metablomics.This emerging omics technique is aimed to quantitavivly understand all the metabolites of living systems and its dynamic responses to the changes of both endogenous factors and exogenous factors.The application of metabolomics is combinated with comprehensive analysis and feature selection,which especially fitted with the whole view and dialectical view of tradional Chinese medicine.The information revealed by metabonomics not only directly discribes the herbal quality from the point of bioactive components,but also facilitates to explain the metabolism process of plant growth and development.Therefore,metabolomics is becoming a powerful tool to deal with the challenges come from both the herbal quality and herbal resource.In the present sudy,metabolomics approach was wildly used to answer some questiones about herbal quality or resource of two typical traditional Chinese medicinal plants,Cyathula officinalis Kuan and Paris polyphylla Smith var.chinensis(Franch.)Hara.Metabolomic platforms,like HPLC,GC-MS,LC-MS,were used to obtain the information about the various metabolites in herbs.Multivariate analysie methods,like PCA,PLS-DA,HCA,and some metabolic databases,like KEGG,were used to reveal the herbal quality feature of C.officinalis and the metabolic behaviors of P.polyphylla Smith var.chinensis.The results were expected to provide some practical guidance to produce herbal products of C.officinalis,and explore a road to sustainably use the rescouse of P.polyphylla Smith var.chinensis.The main results were listed as below:For the herbal quality of Cyathula officinalis,We demonstrated that the herbal quality of C.officinalis was significantly affected by herbal varieties,processed methods and growth years.We established a metablic fingerprint method to authenticate C.officinalis and evaluate its herbal quality.We pointed out some doubts about the current quality control standards of C.officinalis.We also screened out some potential chemical markers to use for quality control.1)The metabolic profile of C.officinalis significantly differed with its adulterated herb,C,capitata,which may support the differentiation of clinical use.Their difference could be well illustrated by metabolic fingerprint throuth comparison for the overall feature of metabolic profile or the content of sveral important bioactive compounds.Those significantly different metabolites between C.officinalis and C.capitata were speculated come from metabolic pathways,like butanoate metabolism,alanine,aspartate and glutamate metabolism,glutathione metabolism,phenylalanine metabolism.The method established by our study based on HPLC/LC-MS platform with the aid of muativariate analysis was a powerful tool to identify C.ofcinalis and C.capitata,Several known bioactive metabolites such as 4-Aminobutyric acid,scopoletin,donepezil and betaine have highly possibility to choose as quality markers of C.officinalis.2)The chemical profile of C.officinalis could be quite different when collected in different growth years.Gluconic acid,xylitol,glutaric acid,pipecolinic acid,ribonic acid,mannose,oxalic acid,digalacturonic acid,lactic acid,2-deoxyerythritol,acetol,3-hydroxybutyric acid,citramalic acid,N-carbamylglutamate,and cellobiose are the main 15 discrimination metabolites between different growth years.With the aim to boost the consistency of herbal quality,C.officinalis is recommended to harvest in 4th growth year based on the information provided by GC-MS.The method of GC-MS combined with multivariate analysis is a powerful tool to discriminate the different herbal growth age.3)The two processing techniques,stir-frying with wine and salt,both had significant effect on herbal chemical profiles.At least 17 components were significantly varied after stir-frying with wine,including 3 newly detected components,2 content-increasing components and 12 content-decreasing components.While at least 11 components were significantly vared after stir-flying with salt,including 2 newly detected components,6 content-increasing components and 3 content-decreasing components.The contents of three known active components,puerarin,cyasterone,and daidzein,were not observed expected increasing after the crude herbs were processed,except a little increasing of the content of puerarin after the crude herbs were processed with salt.More precise active components of the processed products of C.officialis need to future study to improve the current quality standard of C.officinalis in Chinese Pharmacopoeia.The method provided by this study is a powerful tool to identify and quality control between crude and processed C.officinalis because of its quantificational and visual evaluation system.For the herbal resourse of paris polyphylla Smith var.chinensis,To solve the imbalance between supply and demand of P.polyphylla Smith var.chinensis,the possibility and potential risk of three different solutions were discussed,including seeking alternative medicinal resourse,breeding high yield cultivated varities and accelerating seed germination.We established a metablic fingerprint method to identify and quality control of P.polyphylla Smith var.chinensis and its sibling species.We pointed out some important morphological traits that closely related herbal yield.We also described the massive metabolic variation during the seeed sprouting process and screened out some key metabolites and pathways that may participate in the regulation of seed germination.1)No significant difference of P.polyphylla saponins was observed between P.polyphylla Smith var.chinensis and its two sbling species,tentatively named as SY and DY.So does the similarity coefficient of chemical fimgeqprints between different herbal varities.These facts could be viewed as the explains that support SY and DY to be used as the alternative herbs of P.polyphylla Smith var.chinensis.However,all the samples have low content of P.polyphylla saponins that not achieved the current standard of Chinese Pharmacopoeia.The similarity coefficient of chemical fingerprints showed a large variation among samples in same herbal varity.Therefore,except the effects of genetic varities and environmental conditions,there must be have other important factors that have great influence on herbal quality of P.polyphylla Smith var,chinensis,such as herb age.The method which was combinated HPLC fingerprint and multivariate analysis is a powerful tool to evaluate the consistency of herbal quality.In the fingerprint,eight important peaks were highlighted due to the highly effect to indentifcation between different herbal varities.2)An important approach to increase herbal production is to breed high-yield cultivated variety,which characterized ideal plant morphology.There was ample morphological diversity in wild P.polyphylla,whose variation of agronomic traits reduced according to followed order:content of polyphyllin,weight of dry rhizome,petiole length,stem length,petal length,pedicel length,sepal length,leaf width,leaf length,sepal width,leaf number,stamen number,petal number.Most of those traits were significantly correlated to each other and generally represented the characterization of photosynthetic organs or reproductive organ.The total content of polyphyllin ??????? varied between 0.02%and 0.87%and averaged at 0.13%,which showed no significant correlation with any agronomic trait.Plant breeders should play more attention on those germplasm resources with large leaves,large sepals and high stem.3)Using seed to propagate seedling has obvious advantages,such as expected over 200 propagation coefficient to thoroughly solve the imbalance between supply and demand of P.polyphylla Smith var.chinensis.The difficulty in seed germination of P.polyphylla mainly caused by embryo after-ripening.Under the conditions provided by the present study,the seed of P.polyphylla Smith var.chinensis was able to germinated and developmeted to be a nomal seedlings within about 160 days.Accordint to the variation of the whole metabolic prifine,the seed sprouting process coulbe be divided into three sub-processes,after-ripening process,root-growth process and seedling establishment process.Over 150 metabolites were observed significantly changed during germination process,and many of them exhibited obvious trend to accumulate in a certain stage.Pathway analysis revealed that those metabolites related to 15 metabolic pathways,named aminoacyl-tRNA biosynthesiss,alanine,aspartate and glutamate metabolism,glycine,serine and threonine metabolism,cyanoamino acid metabolism,cysteine and methionine metabolism,sulfur metabolism,arginine and proline metabolism,carbon fixation in photosynthetic organisms,lysine biosynthesis,glutathione metabolism,valine,leucine and isoleucine biosynthesis,citrate cycle(TCA cycle),glyoxylate and dicarboxylate metabolism,pantothenate and CoA biosynthesis,and pyrimidine metabolism.