Isolation And Analysis Of The Diterpenoid Alkaloids In Tibetan Medicine Ponka

BackgroundsPonka is a commonly used medicinal material in Tibetan medicine,originally listed in the "Yue Wang Medicine Diagnosis".It has the effect of clearing heat and detoxifying.It is often used in Tibetan prescription for the treatment of hepatitis,pneumonia,enteritis,gastritis,cholecystitis and influenza.The plant is derived from the dried whole grass of Aconitum tanguticum(Maxim.)Stapf or Aconitum naviculare(Bruhl.)Stapf in the Buttercup family.Modern studies have shown that diterpenoid alkaloids are the main active components of Ponka,which have anti-inflammatory,antibacterial and antiviral effects.The chemical composition of Ponka has been studied extensively,but little attention has been paid to its activity.Based on literature mining,this paper intends to preliminarily analyze the clinical application characteristics of Ponka and its potential mechanism of action in treating the primary symptoms,and further study the pharmacodynamic substance basis and quality evaluation of Ponka under the guidance of activity.PurposesStarting from literature mining,the main indications of Ponka were identified.On this basis,the chemical component separation strategy under activity tracking was implemented to separate and analyze the chemical components of Ponka,and the material basis of the drug effect of Ponka was clarified.Methods(1)The cluster analysis algorithm of TCM inheritance computing platform was used to analyze the common drug pairs and association rules of Ponka clinical drugs with three different support degrees of 20%,25%and 30%,and different cluster numbers were set to extract the core drug combinations of Ponka prescriptions.The prescription of Ponka was divided according to the type of disease,and the disease types and rules of clinical application of Ponka were analyzed.The first two categories of Ponka attending diseases were analyzed,and the common drug pairs and drug association rules were extracted by setting different support numbers.The core combination of prescriptions was analyzed by clustering the prescriptions.(2)Through the Swiss Target Prediction platform,SIB database and UniProtKB database,the active compounds in Ponka were collected and the active ingredient targets were predicted using the network pharmacological method and molecular docking technology.Disease targets of hepatitis and cholecystitis were collected using OMIM database,GeneCards database,PharmGKB database of genetic pharmacology and pharmacogenomics database,TTD database of drug targets,and disease target genes of compound and disease intersection were obtained by venny map.Cytoscape 3.9.1 software is used to map the "component-disease-key target" network to screen key compounds for disease treatment.Cytoscape 3.9.1 software was used to acquire the PPI network map of intersection gene and Cytoscape 3.9.1 to obtain the key targets of Ponka therapy.Through the enrichment analysis of intersection targets through the DAVID database,the biological processes,molecular functions,cell components and action pathways involved in Ponka treatment of diseases were obtained.The potential mechanism of action of Ponka treatment of diseases was comprehensively analyzed.The predicted targets and core compounds were verified by using AutoDocktools1.5.6 software.(3)Liver injury model of cholestasis rats was induced by intragastric administration of 1%ANIT solution,and UDCA(60mg/kg),Ponka alkaline extractions(4.96mg/kg),acid extractions(54.25mg/kg)and water extractions(682mg/kg)were given to explore the effect.(4)Using atmospheric silica gel,reverse silica gel,MCI column chromatography,high pressure liquid chromatography,recrystallization and other methods to separate and purify the lipid soluble alkaloid parts of Aconitum tanguticum(Maxim.)Stapf,combined with thin layer chromatography,mass spectrometry,spectrum(1H-NMR,13CNMR,1H-1H-COSY,HSQC,HMBC)and other technical means,The structure of the compound was analyzed and the quality of Ponka was preliminarily analyzed by high efficiency thin layer technique.Results(1)Association rule analysis of Ponka decoction indicated that Ponka was commonly used with swertia rotica,Poileng melon seeds,safflower,Honglian,Xylosa,Chekhoa,Chekhoa,keranise,bashaga,Yuangzi,antidiarrhea wood and musk.Disease analysis showed that Ponkadol was used in the treatment of cholecystitis and different types of hepatitis.(2)Through literature and database search,a total of 179 monomer compounds isolated and identified in Ponka were collected.The results of network pharmacological analysis showed that there were 26 key compounds and 13 core genes involved in the anti-hepatitis action of Ponka,27 key components and 9 core genes involved in the treatment of cholecystitis,and 159 biological processes,160 molecular functions and 160 cell compositions of Ponka anti-hepatitis were obtained by GO enrichment analysis of the obtained key targets.KEGG pathway was enriched and 221 anti-hepatitis pathways were obtained.GO enrichment analysis showed that Ponka was involved in the treatment of cholecystitis through 28 biological processes,molecular function and cell composition,and 21 related pathways were obtained by enrichment of KEGG pathway.The molecular coupling results suggested that alkaloids had strong binding ability.(3)An animal model of cholestasis in rats was established successfully.Compared with the model group,there was no significant difference between the drug administration groups,but the acidic and alkaline parts of Ponka showed some improvement.(4)The alkaloid components of Aconitum tanguticum(Maxim.)Stapf,one of the original plants of Ponka,were studied,and 15 compounds were obtained.The structures of 12 of these compounds were identified by four spectra and the methods that were comparable with those in the literature.The 12 compounds were diterpenoid alkaloids,Azitine A*(1),Naviculine C*(2),Tangirine(3),Tanguticinine E(4).Tanguticinine B(5),Tangutimine(6),Hetisinone(7),Atisine(8),Dihydroatisine(9),Heteratisine(10),6-benzoylheteratisine(11)and 6-dehydroheteratisine(12),where 1 and 2 are new compounds.ConclusionsPonka is the main drug used in Tibetan medicine to treat hepatobiliary diseases,and diterpenoid alkaloids are the main active components of Ponka.