Study On The Processing Mechanisms And Quality Control Of Aconiti Lateralis Radix Praeparata

Aconiti Lateralis Radix Praeparata is the processed product of the lateral roots of medicinal plant Aconitum carmichaelii Debx.It possesses a variety of pharmacological activities including cardiotonic,antiarrhythmic,anti-inflammatory and analgesic,anti-tumor,etc.,on top of the toxicities in cardiovascular and nervous systems.The main toxic and active components have been deemed to be diterpenoid alkaloids.In the 2020edition of the"Chinese Pharmacopoeia",there are four processed products that can be directly used in clinical practice,named“Heishunpian”,“Baifupian”,“Paofupian”,and“Danfupian”.And there are more than ten different processed products in different provincial processing specifications.However,the chemical basis of Aconiti Lateralis Radix Praeparata and the detoxification mechanisms of the dynamic changes of toxic and active components under different processing conditions still need further exploration and improvement.Due to the variety and instability of the processing procedures,the subtypes and contents of the differential chemical components between different processed products are still indistinct,and there is also a lack of suitable quality evaluation and quality control methods that can comprehensively monitor the relative contents of different subtypes of components.In view of the aforementioned problems,we systematically elucidated the chemical basis of Aconiti Lateralis Radix Praeparata by isolation of the chemical components and establishment of an efficient and sensitive targeted characterization approach based on liquid chromatography-mass spectrometry;established a quantitative analysis tactics for multiple components and a pseudotargeted metabolomics method to realize the comparison and quality evaluation of different processed products;unraveled and verified the detoxification mechanisms of Aconiti Lateralis Radix Praeparata during processing from both chemical and pharmacological aspects,and further optimized the processing procedures.The detailed research works mainly include:1.Chemical constituents and their antitumor activities from Aconiti Lateralis Radix PraeparataBy reviewing the phytochemical literatures,a database of known diterpenoid alkaloids from Aconiti Lateralis Radix Praeparata was constructed based on Compound Discoverer software,which included the information of 160 diterpenoid alkaloids isolated from the plant Aconitum carmichaelii Debx.prior to 2022.By means of LC-MS techniques,targeted diterpenoid alkaloids of different subtypes in Aconiti Lateralis Radix Praeparata were identified.A total of 46 compounds were obtained by a comprehensive use of column chromatography,high-speed counter-current chromatography and semi-synthesis,and their structures were elucidated by analysis of their HR-ESI-MS,1D and 2D NMR spectra.Finally,43 diterpenoid alkaloids with diverse structures were identified,including 4 new compounds,and the first diterpenoid alkaloid glucoside with a C20-type skeleton.Among these compounds,37 diterpenoid alkaloids were systematically evaluated for their in vitro antitumor activities,and the structure-activity relationships of C19-type diterpenoid alkaloids were also discussed,indicating the active sites of C-8,C-10 and C-14 positions and nitrogen atom.2.Targeted characterization of diterpenoid alkaloids in Aconiti Lateralis Radix PraeparataThe MSⁿ（n=2,3,4）fragments of the isolated 43 diterpenoid alkaloids with diverse structures were obtained by the CID mode of linear ion trap-Orbitrap（LTQ-Orbitrap）mass spectrometry,and their fragmentation behaviors were summarized.An offline two-dimensional chromatographic separation system based on ultra performance convergence chromatography（UPC²）and reversed phase（RP）chromatography was constructed with satisfactory orthogonality,which enables the comprehensively exposure of more diterpenoid alkaloids in Aconiti Lateralis Radix Praeparata through two different orthogonal separation methods,especially the co-eluting trace components and isomers,etc.According to the principle of molecular design,the prediction database of diterpenoid alkaloids based on Compound Discoverer software were also constructed,and a specific precursor ion list used for targeted data acquisition of diterpenoid alkaloids was obtained by mass defect filtering technique.A comprehensive analytical strategy was proposed to systematically characterize the diterpenoid alkaloids in Aconiti Lateralis Radix Praeparata,including various data acquisition techniques of mass spectrometry such as precursor ion list and dynamic exclusion,as well as various data post-processing techniques such as elemental composition analysis,unsaturated degree analysis,diagnostic ion filtering,neutral loss filtering,database matching,etc.A total of 659 components were characterized,526 of which were the potential new compounds in Aconiti Lateralis Radix Praeparata.3.Comparison and quality evaluation of different processed products of Aconiti Lateralis Radix PraeparataA quantitative analysis method with good specificity,linearity,repeatability,precision,stability and recovery was developed based on the dynamic MRM mode of triple quadrupole mass spectrometry for the simultaneous determination and comparison of 15 key components in different processed products of Aconiti Lateralis Radix Praeparata.And a pseudotargeted metabolomics analysis method that could simultaneously monitor 324 ion transitions was also established for the semi-quantification and comparison of different subtypes of components in different processed products,which displayed good linearity,repeatability and stability.By combining with different multivariate statistical analysis methods such as principal component analysis,orthogonal partial least square-discriminate analysis,clustering heatmap,etc.,different processed products of Aconiti Lateralis Radix Praeparata were compared to screen out arrays of significantly up-regulated or down-regulated differential chemical components,and their differences in chemical constituents were further validated by determination results.4.Detoxification mechanisms of processing and the optimization of processing procedures of Aconiti Lateralis Radix PraeparataDifferent impact factors during processing including boiling time,steaming pressure and time,and p H value of the solution,etc.had been systemically investigated and confirmed to exert great influence on the contents of different chemical constituents in Aconiti Lateralis Radix Praeparata,such as diester-type diterpenoid alkaloids,diester-type diterpenoid alkaloids and aminoalcohol-type diterpenoid alkaloids,etc.Different chemical parts of Aconiti Lateralis Radix Praeparata were prepared,and their acute toxicity and cardiotonic activity were preliminarily evaluated,the results indicated that diester-type diterpenoid alkaloids were the main toxic components,and the cardiotonic activity may be exerted by a variety of components.From both the chemical and pharmacological angle,the detoxification mechanisms were revealed and verified that toxic diester-type diterpenoid alkaloids could be decomposed into monoester-type diterpenoid alkaloids and aminoalcohol-type diterpenoid alkaloids with reduced toxicity and retained activity.According to the detoxification mechanisms of Aconiti Lateralis Radix Praeparata during processing,the processing procedures were further optimized in terms of traditional processing method by extending the boiling time to more than 3 hours,adjusting the p H value to 12 with the saturated Ca（OH）₂solution,or steaming at pressure of 70 k Pa for more than 2 hours,etc.to obtain the processed products of which the contents of the toxic diester-type diterpenoid alkaloids were reduced to 10-100 times.In addition,by calculating the relative contents of the three main diester-type diterpenoid alkaloids and monoester-type diterpenoid alkaloids（DDA/MDA）,as well as the relative contents of the three aminoalcohol-type diterpenoid alkaloids and monoester-type diterpenoid alkaloids（ADA/MDA）,the comprehensive quality evaluation of multiple batches of dispensing granules and Chinese patent medicines was achieved based on the processing degree of Aconiti Lateralis Radix Praeparata.The results showed that dispensing granules may possess low toxicity,thus suitable for clinical use,and the DDA/MDA and ADA/MDA values of most of the Chinese patent medicines lied in 4.44-22.79%and 0.09-24.86%respectively,indicating their good quality consistency.In conclusion,by means of various LC-MS techniques,we clarified the chemical basis of Aconiti Lateralis Radix Praeparata through the targeted separation and characterization of the diterpenoid alkaloids by mass spectrometry;systematically investigated the influence of different impact factors during processing on the chemical constituents of Aconiti Lateralis Radix Praeparata,and prepared different chemical parts for evaluation of toxicities and bioactivities,revealing the detoxification mechanisms during processing from both the chemical and pharmacological perspectives,and further optimized the processing procedures;established the quantification method of 15 key components and pseudotargeted metabolomic method that simultaneously monitored 324 ion pairs for the comprehensive quality evaluation and quality control of decoction slices and prescriptions of Aconiti Lateralis Radix Praeparata.The development of a variety of highly sensitive and selective methods based on mass spectrometry provides a valuable reference for interpretation of the chemical constituents and establishment of the quality evaluation methods of TCM complex systems.