Quality Assessement Of Three Swertia Species Based On Characteristic Chemical Fingerprints

Today,Traditional Chinese Medicines(TCMs)show growing market demands,urging the development of tissue-cultured seedlings'industrial production.It is challenging to evaluate TCMs'qualities that are associated with hundreds of thousands of chemical components,in particular because many environmental factors may exert their effects on the synthesis and accumulation of secondary metabolites,such as temperature,light,carbon source,nitrogen source,plant growth regulators(PGRs),and so forth.In addition to conventional methods depended on the morphology and microscopic characteristics,quantification analysis of important active chemicals has been widely credited as playing a big part in the TCMs'quality assessment.Providing good performances on both comprehensively qualitative analysis and quantification of active markers,the multi-components fingerprint has increasingly become the workhorse of quality assessment of TCMs.Furthermore,discriminant,classification and prediction of samples could be achieved by the pattern recognition method for the protocol establishment of TCMs'quality assessment,a consequence of the multiple dimensions information handling by interpreting the nonlinear complex system which is characterized by multiple different ingredients.Swertia species belonging to Gentianaceae family,with a wide distribution at southwestern China,has been recognized as agents that can nourish liver and stomach,clear heat-toxin,and disperse stagnated liver qi for promoting bile flow from ancient times,especially in Asian countries such as China,Japan,India and Pakistan,etc.In present paper,characteristic fingerprints of tissue-cultured seedlings including S.mileensis,S.nervosa and S.kouitchensis were established based on high performance liquid chromatography(HPLC)and ultraviolet spectroscopy(UV)for the qualitative and quantitative analysis.Then,the multivariate statistical analysis was employed to interpret chemical fingerprints information for investigating effects of PGRs,growth periods and organogenesis on the accumulation of characteristic components,developing foundation for the cultivation and reasonable application of herbal medicines.The paper comprises three sections.The UPLC and UV fingerprints of S.mileensis were established for screening active markers and analyzing the inter-species similarity in section 1(chapter 1).Effects of PGRs,growth periods and organogenesis on the accumulation of characteristic components were studied concomitantly developed rapid quality assessment method of three medicinal plants from Swertia genus in the section 2(chapter 2-4).The section 3(chapter 5)is the review that covers 30 species and 419 phytochemical compounds in Swertia based on173 references reported from 1966 to 2016.(1)Four Swertia species were collected from natural setting to perform the qualitative and quantitative analysis using the UPLC and UV combined with principal component analysis(PCA).The common peak of swertiamarin,which showed a good baseline separation,has been recommended as the characteristic ingredient for the quality assessment,according to the chromatographic fingerprint.The highest content of swertiamarin was found in leaf of S.dividii(159.82 mg/g),followed by stem of S.mileensis(25.91 mg/g).The content in S.angustifolia was the lowest,even less than the limit of detection(LOD)(6.53?g/mL).The UV fingerprint showed that characteristic absorption peaks of samples were aggregated at the area of 204-290 nm,in which the high absorbance–meaning abundant secoiridoids existed in extractions–was observed at 240 nm,while the resonating structure resulted from C=O and benzene ring instead by that at 270 nm.The UV data that suffered from the 4 point smoothing for eliminating the interference of baseline drift and noise,was applied to perform the PCA using the SIMCA-P~+software,indicating the significant difference between stem and leaf as well as different species.According to the similarity analysis based on the UPLC data,remarkable resemblance(>0.92)was found in S.dividii and S.mileensis which was different(<0.50)with other two species.What's more,S.dividii and S.mileensis,S.punicea and S.angustifolia were collected from low-and high-altitude area,respectively,implying that altitude might be one of environmental factors resulting in the chemical difference among four Swertia species.(2)Effects of 6-benzyladenine(6-BA)and?-naphthaleneacetic acid(NAA)on the accumulation of chemical components in S.mileensis were explored using the UPLC combined with the multivariate statistical analysis.From the quantitative result,the combination of 2.0 mg/L 6-BA and 0.05 mg/L NAA was temporarily considered as the optimal formulation for the accumulation of swertiamarin(100.11±0.25 mg/g)rather than extremely high or low concentrations of PGRs.On the other hand,the swertiamarin level was significantly lower in the wild samples than that of all the tissue-cultured ones,suggesting that high yields of secondary metabolites could be obtained through the tissue culture technology.Results stemmed from partial least squares discriminant analysis(PLS-DA),orthogonal partial least squares discriminant analysis(OPLS-DA)and hierarchical clustering analysis(HCA)demonstrated that the most important roles in recognizing wild WPSMs from tissue-cultured samples were variables across the area with the first 5 minutes where the dead time of column was deleted.The poor separation was obtained for all but SMT 5 samples,which indicated that the combination of 2.0 mg/L 6-BA+0.01 mg/L NAA significantly(p<0.05)prompted the accumulation of swertiamarin.(3)The aim of this study is to discuss the diversities of metabolites(loganic acid,sweroside,swertiamarin and gentiopicroside)at different growth stages and organs of Swertia nervosa using the UPLC/UV coupled with chemometric method.The chromatogram showed that swertiamarin,sweroside,gentiopicroside and loganic acid have been regarded as common peaks,with good baseline seperations.According to quantitative analysis,the aerial part was the main site to accumulate these four secoiridoid glycosides,in which high concentrations appeared in stem(2.19±0.04–7.29±4.82 mg/g)at proliferative stage,whereas the rooted stage instead by the leaf(1.00±0.26–19.57±5.34 mg/g).Furthermore,rooting process remarkably(p<0.05)increase swertiamarin content concomitantly reduced(p<0.05)that of loganic acid,sweroside and gentiopocroside,implying that the biosynthesis and transport mechanisms might be initiated by the root formation.That remarkable variation was found in different parts by PLS-DA and HCA indicated an obvious changing of accumulation of secondary metabolites at two growth periods.(4)In this study,the chromatography-spetrum fingerprint were established,respectively,for exploring accumulations of S.kouitchensis stemmed from two different organogenesis ways using HPLC and UV coupled with low data fusion strategy in conjunction with PLS-DA method.The chromatogram showed that swertiamarin,sweroside,gentiopicroside and mangiferin have been regarded as common peaks,with good baseline seperations.The aerial part was prominent in accumulating these four characteristic compounds,in which the highest level(~30.54±0.2 mg/g)was observed in swertiamarin whose contents in indirect organogenesis samples were higher than that in direct ones,indicating that the biosynthesis of secoiridoids has initiated since the callus satge.The content of sweroside was lowest,even unable to determine in root.Additionally,the root mean square error of estimation(RMSEE)and root mean square error of cross validation(RMSECV)of data fusion model of HPLC(246 nm)+HPLC(258 nm)were less than 0.07 and 0.1,respectively.The accuracy of classification was 100%.This study provided an efficient and reasonable method to recognize different materials of in vitro regenerative S.kouitchensis.