Study On The Difference Of Isoquinoline Alkaloids Before And After Processing Of Radix Linderae Based On Metabonomics

Lindera radix is the dry root of Lindera aggregatea（SIMS）kosm term.Its main functions are to move Qi to relieve pain,to warm kidney and to disperse cold.In the Chinese Pharmacopoeia（2020 Edition）,isonorboldine was used as the component for the determination of the content of Linderae radix,but the single content determination was not enough to reflect the overall quality of Linderae radix.The research on processed products of Radix Linderae mainly focuses on vinegar processing.The research on the differences of material basis of Radix Linderae and its processed products generally involves only a few effective components,which is not systematic and comprehensive.The research on the processing mechanism and methods of Radix Linderae is even less.Therefore,it is important to study the differences of isoquinoline alkaloids before and after processing of Radix Linderae based on metabonomics technology,It is of great significance to improve the quality standard and expand the clinical application.Objective:1.To study the fragment pathway of isoquinoline alkaloids in Radix Linderae.2.To screen the different metabolites before and after processing.3.Objective to study the difference of isoquinoline alkaloids before and after processing.Methods:Use Waters ACQUITY UPLC REH C₁₈ chromatographic column（1.7um,2.1×100mm）for liquid phase,mass spectrometry was conducted by electrospray ion source,Waters Xevo G2-XS QTOF time of flight mass spectrometer.The isoquinoline alkaloids were analyzed by time-dependent mass spectrometry scanning mode（MS^E）.Combined with references and databases,the fragment pathway of isoquinoline alkaloids was studied based on the accurate mass charge ratio of primary mass spectrometry and secondary mass spectrometry fragments.2.Stir frying,vinegar making,wine making and salt making were carried out,and each method was repeated for 10 times.The samples were analyzed by mass spectrometry respectively.The molecular ion peak was extracted by markerlynx.After matching and normalization,the table with retention time and peak intensity after molecular ion peak normalization was obtained,Then the data was imported into Simca-p 14.1,and principal component analysis（PCA）and orthogonal partial least squares discriminant analysis（OPLS-DA）were used for pattern recognition and analysis.3.Combined with the pathway of fragment summarized in 1,two kinds of isoquinoline alkaloids in the mixed samples were identified.The peak areas of the two kinds of alkaloids in raw and processed products were extracted by the mass spectrometry software quanlynx,and the content differences between raw and processed products were compared.Results:1.According to the primary and secondary mass spectra,combined with references,the main aporphine alkaloids in Radix Lindera can be divided into three types:isonorboldine type,laurotetanine type and corydine type.Because they contain the same structural framework,they can produce 177.0704 fragment ions,and fragment ions 297.1227,311.1283,295.0970 can be used to identify the three main aporphine alkaloids.Benzylisoquinoline type alkaloids can be divided into three types:reticular type,coclaurine type,karakorine type and N-methylhigenamine-7-O-β-glucopyranoside type.The partial cleavage of benzylisoquinoline alkaloids is similar to that of aporphine alkaloids,because ofα-cleavage,The characteristic ion of 143.0497 can be produced.Combined with fragment ions 299.1289,269.1178,391.1545and 255.1021,these four main types of benzylisoquinoline alkaloids can be identified quickly.2.Using nontarget metabonomics,principal component analysis showed that processed products and raw products were well differentiated.Using orthogonal partial least squares discriminant analysis,set VIP value greater than 1.5,a total of 48different compounds were identified,including 39 isoquinoline alkaloids,accounting for the vast majority.3.Combined with fragment pathway,A total of 68 compounds were identified by target metabonomics.Through experiments and literatures,the cleavage law of 7isoquinoline type in Radix Linderae was summarized.For the first time,UPLC-QTOF-MS/MS technology,non-target metabonomics technology and target metabonomics strategy were used to identify 48 different metabolites before and after processing,in which isoquinoline alkaloids accounted for the majority.The characteristic profiles of isoquinoline alkaloids in Radix Linderae and its processed products were established for the first time.The isoquinoline alkaloids in Radix Linderae and its processed products were characterized comprehensively.The differences of isoquinoline alkaloids between Radix Linderae and its processed products were compared qualitatively and quantitatively for the first time,It provides a scientific basis for the study of processing mechanism and quality control of lindreae radix and preparations containing linderae radix,and lays a foundation for the study of pharmacodynamic differences caused by different processing methods of lindreae radix.