Discovery And Identification Of Quality Markers Of Gardenia Fructus By An Omics Discriminant-grey Correlation-biological Activity Strategy

Objective:Gardenia Fructus(GF)is a commonly used Chinese medicinal material for both food and medicine in China,and a large variety of Chinese medicinal material in the big health industry.To study and establish the bioactivity markers closely related to efficacy is the core of building a quality evaluation system with "Quality Markers" as the core,and it is also an urgent scientific problem to be solved in gardenia industry.Methods:Liver transgenic zebrafish Tg(l-fabp :EGFP)labled by green fluorescence,neutrophils transgenic zebrafish Tg(lyz:EGFP)labled by green fluorescence and wild AB line zebrafish developing until 3 dpf(days post fertilization,dpf)were used to establish liver injury,inflammation and thrombosis models of zebrafish,and to evaluate the activity differences of gardenia from different habitats.Samples from two areas with the largest difference in bioactivity were selected for downstream analysis.High-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(HPLC-Q-TOF/MS)was applied to detect compounds information from the GF samples and R software was used to extract ion compounds from the raw MS data.Herbal metabolomics and grey correlation analysis(GCA)were used to identify compounds with potential antithrombotic,anti-inflammatory and liver protective activity.The compounds confirmed to have antithrombotic,anti-inflammatory and liver protective activity by a bioassay conducted in zebrafish were regarded as antithrombotic,anti-inflammatory and liver protective quality markers.Results:1.Among the samples of GF,those from Fuding City,Fujian Province and Jiujiang City,Jiangxi Province significantly differed in bioactivity.HPLC-Q-TOF/MS and R software discovered a total of 1056 ion compounds.Orthogonal partial least squares discriminant analysis(OPLS-DA)obtained 27 compounds from GF as potential bioactive compounds.Further,5 compounds were found to be positively correlated with the liver protective bioactivity of GF through a GCA,and genipin-1-?-D-gentiobioside,geniposide and crocin-1 were identified.Of these,2 compounds,geniposide and crocin-1,were confirmed as responsible for the liver protective action of GF.2.Among the samples of GF,those from Panan City,Zhejiang Province and Jichun City,Hubei Province significantly differed in bioactivity.HPLC-Q-TOF/MS and R software discovered a total of 926 ion compounds.Orthogonal partial least squares discriminant analysis(OPLS-DA)obtained 21 compounds from GF as potential bioactive compounds.Further,15 compounds were found to be positively correlated with the anti-inflammatory bioactivity of GF through a GCA,and genipin-1-?-D-gentiobioside,quinic acid,gardenoside,D-glucuronic acid,L-Malic acid,Mannitol,Rutin,Chlorogenic acid,shanzhiside and D-glucaric acid were identified.Of these,9 compounds,genipin-1-?-D-gentiobioside,quinic acid,gardenoside,D-glucuronic acid,L-Malic acid,Mannitol,Rutin,Chlorogenic acid were confirmed as responsible for the anti-inflammatory action of GF.3.Among the samples of GF,those from Huoshan City,Anhui Province and Jichun City,Hubei Province significantly differed in bioactivity.HPLC-Q-TOF/MS and R software discovered a total of 614 ion compounds.Orthogonal partial least squares discriminant analysis(OPLS-DA)obtained 19 compounds from GF as potential bioactive compounds.Further,10 compounds were found to be positively correlated with the antithrombotic bioactivity of GF through a GCA,and genipin-1-?-D-gentiobioside,geniposide,citric acid,and quinic acid were identified.Of these,three compounds,geniposide,citric acid,and quinic acid,were confirmed as responsible for the antithrombotic action of GF.4.The bioactivity markers of GF were analyzed,and the quality markers of GF were identified as genipin-1-?-D-gentiobioside,geniposide,and gardenoside.Conclusion:This research have succeeded to discover and identify the possible representative antithrombotic,anti-inflammatory and liver protective compounds of GF using an efficient integrated research strategy we established,an Omics Discriminant-Grey Correlation-Biological Activity strategy.The quality makers we found could also contribute to provided more accurate index components for comprehensive quality control of GF.