Identification Of 4 Species Ephedra Using Molecular Markers Combined With Metabolomics Method And Evaluation Of Drug-related Activities

Ephedra is one species of gymnosperm,which used in Chinese medicine and Tibetan medicine.The Ephedra plant species located in the Qinghai Tibet plateau were similar on morphology,especially between E.gerardiana and E.saxatilis,which affected the further study in deep and leaded to inconsistent quality of traditional medicine.In this research,DNA barcoding technology and metabolomics are used to develop the method for identification of E.saxatilis,E.gerardiana,E.monosperma,E.intermedia and E.sinica;establish a method for determination of ephedrine and pseudoephedrine in Ephedra species from the Qinghai Tibet Plateau;analyze the antioxidant capacity and complement resistance of Ephedra species.The main research contents and results are as follows:(1)141 samples of 16 populations of E.saxatilis,E.gerardiana,E.monosperma,E.intermedia and E.sinica,were collected for DNA analysis and prepared sample for metabonomics analysis;(2)The DNA were extracted by kit extraction method,ITS2 and trnT-trnF sequences were used primers for amplification and sequencing.The phylogenetic tree of Ephedra species was constructed by NJ and ML method,and the intraspecific and interspecific genetic distances ofEphedra species were calculated based on K2 P model.The results show that: The amplification success rates using ITS2 and trn T-trnF sequences were 70.21% and 93.62%,respectively.Using these two markers can separate E.monosperma and E.sinica from other species of Ephedra except but it useless for separate between E.saxatilis and E.gerardiana.The genetic distance between inter-species of Ephedra using markers above showed that the smallest genetic distance between E.gerardiana and E.monosperma,and largest between E.monosperma and E.intermedia.(3)The HPLC method for determination content of ephedrine and pseudoephedrine was established.The result showed that the ephedrine content is the highest in E.intermedia,but lowest in E.gerardiana.There was no significant difference in the contents of ephedrine and pseudoephedrine among species.The clusted results of ephedrine and pseudoephedrine were similar to the molecular phylogeny;(4)UPLC-MS and GC-MS methods were used to study metabolomics of E.saxatilis,E.gerardiana,E.monosperma and E.intermedia.Multivariate statistical analysis such as PCA principal component analysis,orthogonal partial least squares-discriminant analysis(OPLS-DA)were performed using ropls Version 1.6.2 software.The metabolites of Ephedra species were annotated by KEGG database and HMDB database.According to the variable weight value(VIP)> 1and significant difference test(P <0.05)to screen differential metabolitesbetween different species of Ephedra species.The results show that: 212 compounds were identified by UPLC-MS,37 by KEGG database and 199 by HMDB database.11 kinds of Ephedra differential metabolites were screened,respectively: Osmanthuside A,Ergosterol peroxide,Kinetensin1-3,Tafluprost,Tanacetol B,9(Z),11(E)-Conjugated Linoleic Acid,N-Arachidonoyl glycine,Gibberellin A4 glucosyl ester,Repaglinide aromatic amine,5-Hydroxybuspirone,Exemestane.A total of 213 compounds were identified by GC-MS,149 metabolites were annotated in the KEGG database,and 95 metabolites were annotated in the HMDB database.14 kinds of Ephedra species differential metabolites were screened,respectively:Ketoleucine,trans-4-Hydroxy-L-proline,L-Serine,L-Homocarnosine,Acetylsalicylic acid,Methyl succinic acid,glucose-1-phosphate,rac-Glycerol 3-phosphoate,Ethyl-N-propylamine,Butylbis(trimethylsilyl)amine,D-(-)-Fructose,N-Acetyl-D-glucosamine,palatinitol,Catechol.GC-MS has a good identification effect on E.saxatilis and E.monosperma,but it has a poor identification effect on E.gerardiana and E.intermedia.UPLC-MS has a good identification ability on E.saxatilis,E.gerardiana,E.monosperma and E.intermedia,and can identify different kinds of Ephedra species.(5)DPPH method and classical method were used to determine the antioxidant capacity and anti complement capacity in different Ephedra species.The results showed that the content of ephedrine hydrochloride from high to little were in E.intermedia,E.saxatilis,E.monosperma,E.sinica and E.gerardiana.The content of pseudoephedrine hydrochloride from high to little were in E.monosperma,E.sinica,E.intermedia,E.saxatilis,E.gerardiana.The antioxidant capacity of different species of Ephedra from high to little were in E.gerardiana,E.monosperma,E.saxatilis,E.intermedia.The anticomplement ability of different species of Ephedra from high to little were in E.monosperma,E.saxatilis,E.intermedia and E.gerardiana.The antioxidative and anticomplementary abilities of E.monosperma and E.saxatilis were better than in E.intermedia.The results not only provide methods and technology for identification of Ephedra species distributed in the Qinghai Tibet Plateau,but also provide data support and theoretical basis for the discovering effective components in Ephedra.