| The safety of Chinese medicine has always been an important issue affecting the development of the industry.From the Aristolochia incident to the Heshouwu incident,the safety problems caused by some toxic medicinal materials have had a serious impact on the development and promotion of Chinese medicine.How to prevent Chinese medicine The safety issues of toxic medicinal materials,especially for toxic medicinal materials,the establishment of risk prevention and control measures for toxic medicinal materials is of great significance to improving the safety of traditional Chinese medicine.Aconite,sweet and hot,returning to the heart,kidney and spleen,has the functions of rejuvenating the yang,replenishing the fire,replenishing the yang,dispelling cold and relieving pain.It has been used by physicians in the past and is irreplaceable in clinical effectiveness.However,due to its toxicity,the safety of aconite and its compound preparation has become an important obstacle to restrict its clinical use.Among the toxicity of aconite,its cardiotoxicity is the most common and the most lethal.The mechanism of aconite cardiotoxicity involves ion channels,mitochondrial function,oxidative damage of cell membranes and gap junction channels.The gap junction channel is a special channel that is mainly responsible for connecting adjacent cardiomyocytes and participating in the regulation of the exchange of chemical information and electrical coupling between them.It is an important guarantee for maintaining normal cardiac electrical activity.At present,there are few studies on the gap junction pathway of aconite cardiotoxicity,and the specific mechanism of action has not yet been clarified.Based on this,This research is based on the national major scientific engineering construction project plan(the construction of a technical platform for clinical evaluation of new drugs for major diseases that reflects the characteristics of traditional Chinese medicine).this study uses aconite cardiotoxicity as the research entry point,and uses gap junction channels as the mechanism to explore,from the aspects of"components-pathways-targets-clinical evaluation indicators" to carry out"aconite alkaloid monomers-medicinal materials-compounds," etc.The integrated,full-chain research on the toxicity of aconite,mechanism exploration,and risk prevention and control provide a reference for building a safety evaluation system for toxic traditional Chinese medicine.This research includes the following four parts:Part One Literature ResearchPurposeBased on previous literature research,focusing on the toxicity of aconite,gradually sort out the research entry points of the subject,and provide theoretical basis and guidance for further development of aconite cardiotoxicity,construction of aconite safety rating system,and risk prevention and control research.MethodFirstly,based on the Citespace software,carry out a bibliometric analysis of the related studies on the adverse reactions of aconite;secondly,based on the previous literature research to review the cardiotoxicity of aconite;finally,with the help of Pubchem,PDB database,PharmMapper database,etc.,the chemical structure of aconite diester alkaloids,And the protein structure of gap junction channel-related regulatory proteins(CX40,CX43,SCN5A),and import the gene targets corresponding to this regulatory protein into FunRich software for gene information interaction and biological process enrichment analysis.ResultChapter 1:Bibliometrics:245 research documents were included.The earliest research can be traced back to 1980.It can be roughly divided into the budding period(1980 to 2004)and the rapid growth period(2004 to present);548 authors are involved,and the units involved There are 110 institutions,and the units with the most publications are related units under Chengdu University of Traditional Chinese Medicine(44 articles,17.96%).The rapid growth period involves 428 keywords,23 of which have a frequency of?5,and "cardiotoxicity" has a frequency of 11 times.This is a relatively new keyword in the field of research.based on this,we review on the cardiotoxicity of aconite in the next step.Chapter 2 Literature review:Aconite cardiotoxicity manifestations include palpitations,arrhythmia and abnormal blood pressure,etc.,and even some cases may be accompanied by cardiogenic shock and even cause death.The combing chemical components include alkaloids and non-alkaloid components such as sugars and lipids.Cardiotoxic substances are mainly based on diester alkaloids.The mechanism of toxicity involves ion channels,mitochondrial function,cell membrane oxidative damage,and gap junction channels.Among them,gap junction channels have not been clearly studied,involving CX40,CX43,SCN5A,etc.,so the next step is to carry out the next step of aconite cardiotoxic gap junction channel biological process regulation the study.Chapter 3 Biological Process:The database retrieves the chemical composition structure of aconite,the 2D and 3D structure of the gap junction channel regulatory protein,and imports the FunRich software to analyze the interactive information of each gene.It is found that these 3 genes and 33 genes such as SRC and CSK are produced.Interaction,biological processes involve oligomerization of connexins into connexons,transport of connexons to the palsma membrane,regulation of gap junction activity,c-src mediated regulation of Cx43 function,and closure of gap junctions,Transport of connexins along the secretory pathway,Microtubule-dependent Trafficking of connexons from Golgi to the plasma membrane,etc.summary There are many adverse reactions to aconite,and cardiotoxicity has gradually become a hot spot for scholars.The mechanism of aconite cardiotoxicity involves ion channels,mitochondrial function,oxidative damage of cell membranes,and gap junction channels.Gap junction channels are important pathways for information exchange and electrical coupling between cardiomyocytes.They may affect cardiac electrophysiology and cause cardiotoxicity by participating in the regulation of ion channels.The gene targets corresponding to the gap junction channel-related regulatory proteins(CX40,CX43,SCN5A)interact with multiple genes in the human body and participate in multiple biological processes in the human body.Part ? Experimental research on aconite alkaloidsPurposeStarting from the toxic substances of aconite,explore the toxicity difference,toxicity mechanism,and toxic metabolism characteristics of the two kinds of aconite mono-and di-ester alkaloids,and lay the foundation for the construction of aconite safety rating system and risk prevention and control research.MethodFirst,the cardiomyocyte cytotoxicity experiment was carried out on the main toxic alkaloid of the aconite-diester alkaloid(aconitine)through rat H9C2 cardiomyocytes.The technical methods involved in the experiment include:cell incubation,cell viability detection,growth curve drawing,ELISA detection,calculation of cell leakage rate,etc.;Secondly,the rat cardiotoxicity verification and comparison of the two alkaloids of aconite mono and diester were carried out.The technical methods involved in the experiment include:rat gavage,orbital plexus venous blood sampling,electrocardiogram detection,and ELISA detection of related cardiotoxicity indicators Wait.Finally,an exploratory study was carried out on the metabolic characteristics of the two alkaloids of aconite mono-and di-esters in rats.The study used UPLC technology to perform qualitative and quantitative analysis on blood samples collected at various time points after intragastric administration of rats.ResultCell experiment:The minimum toxic concentration of aconitine is 0.03125%,IC50 is 2.882mmol/l,low concentration(0.0250mol/l,0.05040mol/l,0.10090mol/l)presents a positive excitation effect when the concentration is between 0.2018-1.6144mol It shows concentration and time dependence at 1/1;within 4h,with the prolongation of the drug action time,the cell morphology is gradually distorted,the cell nucleus gradually shrinks,the boundary gradually becomes blurred,the cell gap gradually increases,and the cell arrangement is irregular;conventional indicators LDH,In the detection of c-TNI and CK,the differences between the concentration and time groups showed statistical significance(P<0.05);warning indicators:there were no statistically significant differences in SCN5A and CX43 between the control group,and the concentration and time groups.(P value>0.05),the difference of CX40 is statistically significant(P value<0.05).Animal experiment:The minimum toxic dose of aconitine is 0.3mg/kg.The toxicity verification and comparison of the two alkaloids at the same dose was carried out at this dose.Results:1)ECG:Heart rate:AC group 90min,120min and KB group Differences(P values are 0.0293,0.0093,respectively),the BAC group is always indistinguishable from the KB group(P value>0.05);QTc:There is a difference in the AC group at 5 min and 10 min(P values are 0.0097,0.0255,respectively),the BAC group Except for 20 min,there was no difference(P=0.0593),and the other time points were different(P values were 0.0080,0.0426,0.0200,0.0007,0.0007,0.0007,0.0413).2)Conventional indicators:CK:There are differences between the AC group and the BAC group(P values are<0.0001,0.0097,respectively);c-TNI:There are differences between the AC group and the BAC group(P values are 0.0043,0.0005,respectively);LDH:There were differences between the AC group and the BAC group(both P values<0.0001).3)Early warning indicators:SCN5A:AC group has a difference(P=0.0126),BAC group has no difference(P=0.6407);CX40:AC group has a difference(P<0.0001),BAC group has no difference(P=0.0828);CX43:There are differences between the AC group and the BAC group(P values are 0.0011,0.0107,respectively).4)Other indicators:H-FABP:There are differences between the AC and BAC groups(both P values<0.0001).Pharmacokinetic study:AC plasma peak time is about 45 minutes,BAC is about 20 minutes;AC and BAC pharmacokinetic parameters are significantly different,Cmax,Tmax,AUC0-t,AUMC0-t,MRT0-t The P values of<0.0001,0.0151,0.0013,0.0132,0.0177,respectively.Summary1)A lower concentration of AC(0.03125%)can cause changes in H9C2 cardiomyocyte cytotoxicity,and the dose of AC that causes changes in rat toxicity without causing death in rats is 0.3mg/kg;2)The ECG center rate may not change significantly under the toxic dose of BAC.It is necessary to combine further QTc,conventional cardiotoxicity indicators(CK,LDH,c-TNI)and slit channel-related indicators(CX40,CX43,SCN5A)and other indicators to investigate comprehensively;4)The peak time of AC rats in vivo is about 45 minutes,and BAC is about 20 minutes,but the presence of its components can still be detected at 24 hours,indicating that its toxicity is an acute reaction,but it takes a certain time for degradation and absorption in the body.5)There is a one-way and irreversible degradation process between AC and BAC,and AC can be converted into BAC,but BAC cannot be converted into AC,and given the same dose,AC is more toxic than BAC.Part Three Experimental Research on AconitePurposeCarry out "toxicity verification-toxicity comparison-toxic substance basic exploration-toxicity metabolism characteristics study" of "different batches,different formulations,and different decoction times" around the medicinal materials of aconite,explore the best medication plan of aconite,and build an integrated,full chain Aconite cardiotoxicity research and risk prevention and control system,and provide reference for clinical medication.MethodFirst of all,starting from the basic material level,using UPLC detection technology to perform qualitative and quantitative analysis of two alkaloids in different batches,different dosage forms,and different decoction times of Aconite medicinal materials.The techniques and methods involved in the experimental process are liquid medicine Preparation and extraction,qualitative and quantitative UPLC detection of drugs;Secondly,from the perspective of toxicity verification,the cardiotoxicity of Aconite medicinal materials in different dosage forms and different decoction times was verified by animal experiments.The technical methods involved in the experimental process include drug preparation,intragastric administration of rats,and intravenous injection from the orbit of rats.Blood collection,storage,and processing of blood samples,ELISA testing of samples,etc.;Finally,starting from the characteristics of toxic metabolism,rats were administered aconite medicinal materials with different dosage forms and different decoction times to explore the metabolic characteristics of the two alkaloids in the rats in each aconite administration group.The experimental process involved The technical methods includes preparation of drugs,intragastric administration of rats,blood collection from the vein of the orbit of rats,storage and processing of blood samples,and UPLC detection of samples.ResultUPLC detection of aconite medicinal materials:construct a linear equation:BAC:Y=3980x+489,r=0.9993;AC:Y=20600x+101,r=0.9992.Aconite powder AC content 125.78,534.94,314.34ng/g,BAC content 39.52,45.24,45.80;Aconite decoction for 30min AC content 0.3056,0.26765,0.168ng/ml,BAC content 1796,1352,2516 ng/ml;Aconite decoction 120min AC content 0.3056,0.444,0.3532,BAC content 2360,2812,2212Animal experiment:Aconite decoction was administered twice the maximum clinical dose,and powder was administered at 1/10 of the water decoction.Under these conditions,the toxicity verification results of different dosage forms and different decoction times of aconite were carried out.The results indicate:1)ECG:Heart rate:there is a difference between F0 and FB groups at 90 min and 120 min(P values are both<0.05),and there is no difference at other time points(P values are both>0.05),and there is no difference in the FB group;QTc:F0 group The QTc value at each time point was compared with the KB group,and the difference was not statistically significant(P values were 0.0931,0.5877,0.0736,0.0649,0.1212,0.4848,0.3939,0.6277);FA group at 5min,10min,20min,30min,The QTc values of 45min,60min,and 90min are different(P values are 0.0007,0.0047,0.0007,0.0007,0.0007,0.0007,0.0007);the FB group is at 5min,20min,30min,45min,60min,90min,120min,etc.QTc values are different(P values are 0.0293,0.0386,0.0143,0.0007,0.0007,0.0007,0.0413,respectively).2)Conventional indicators:CK:F0 group and FA group are different(P value is 0.0013,0.0104,respectively),FB group has no difference(P=0.2824);c-TNI:F0 group,FA group is different(P value is respectively 0.0245,0.0266),there was no difference in the FB group(P=0.0813),LDH:there was a difference in the F0 group and the FA group(P values were 0.0245,0.0266,respectively),and there was no difference in the FB group(P=0.0813).3)Early warning indicators:SCN5A:There is a difference between the F0 group(P=0.0395),and there is no difference between the FA and FB groups(P>0.05);CX40:There is a difference between the F0 and FA groups(P values are 0.0088,0.0050,respectively),FB There was no difference between the two groups(P=0.1091);CX43:There was a difference between the F0 and FA groups(P values were 0.0280,0.0111,respectively),and there was no difference between the FB group(P=0.1535).Pharmacokinetics:Construct a linear equation:BAC:Y=0.85x-0.00127,r=0.9978;AC:Y=2.14x+0.0909,r=0.9993.The AC content of rats in each administration group of Aconite is below the lower limit of quantification,BAC content can be detected in F0 group and FB group,and the BAC content of FA group is also below the lower limit of quantification.Comparing the AC pharmacokinetic parameters of each group(F0 group VS FA group,FA group VS FB group,F0 group VS FB group),the P values of Cmax,Tmax,AUC0-t,AUMC0-t,MRT0-t are(0.5333,0.7032,0.1905),(0.2000,0.5317,0.0952),(0.5000,0.7302,0.0530),(>0.9999,0.7032,>0.9999),(0.5333,0.5000,0.1905),(0.2667,0.5317,0.0952),each There was no statistically significant difference in group parameters(all P values>0.05).Comparing the BAC pharmacokinetic parameters of each group(F0 group VS FA group,FA group VS FB group,F0 group VS FB group),the P values of Cmax,Tmax,AUC0-t,AUMC0-t,MRT0-t are(0.2222,0.3333,0.6200),(0.5000,>0.9999,0.1981),(0.5000,0.6667,0.0530),(0.5000,0.5000,0.0379?),(0.8889,0.3333,0.0262 ?),whether there are differences in the parameters of each group Statistically significant is the comparison of AUMC0-t and MRT0-t in the F0 group VS FB group(all P>0.05),and the differences in the parameters of the other groups were not statistically significant(all P>0.05).Summary1)There are differences in the alkaloid content in aconite between different batches.After 30 minutes of decoction,the difference can be effectively reduced,and the alkaloid content in the aconite can be controlled;2)The toxic alkaloids in aconite are not only diester alkaloids represented by aconitine,but monoester alkaloids represented by benzoylaconitine are also toxic;3)Normal decoction can control the content of diester alkaloids(aconitine)in the medicinal materials of aconite,but cannot reduce its toxicity,so long decoction is still needed in clinical use.Part Four Clinical Research of Fuzi Compound Prescription PurposeStarting from the compound level of aconite,carry out observational research on the clinical safety of aconite-containing preparations and comprehensively explain the clinical safety of aconite after processing and modern pharmaceutical technology.MethodCarrying out the clinical trials(single-center,open,randomized,parallel,double-blind,single-dose,and dose-increasing research design)carried out in Phase I clinical ward of Xiyuan Hospital of the Chinese Academy of Chinese Medical Sciences to develop aconite compound(with Qiguben ointment)Clinical safety observation research.ResultThe study enrolled 8 healthy subjects,half males and half males,and administered 25g aconite-containing preparations orally.Observe the vital signs 30min before,30min,1h,2h,4h,8h,24h,48h,72h,96h,120h after the drug.(Body temperature,heart rate,respiration,blood pressure),there is no significant difference between each time point after administration than before administration(P value>0.05);ECG:there is no significant difference in dynamic ECG indicators at each time point within 24h,within 24h The PR interval,QRS interval,QT interval,and QTC interval at each time point were 0.0781,0.5528,0.1406,0.1563,and 0.1096 compared with the P value before administration.1-5d PR interval,QRS interval,QT The P values of the interval and QTC interval before administration were:PR interval:0.4453,0.4844,0.1250,0.4219,0.7422;QRS interval:>0.9999,0.5859,0.4375,0.0625,0.2188;QT interval:0.6406,0.1953,0.1719,0.5703,0.0156*;QTC interval:0.6875,0.7969,0.5469,0.3516,0.0625.Markers of cardiac injury:Compared with the pre-administration,the P values of CTNT were>0.9999,0.5000,>0.9999;the P values of Pro-BNP were 0.7422,0.5469,>0.9999,respectively,which were not statistically significant.SummaryThe aconite-containing preparation is a compound Chinese patent ointment strictly processed and used at a clinically safe dose.It has not been found to cause abnormal changes in related laboratory tests and toxicity indicators.After proper processing and processing,it can effectively control the toxicity of aconite,so the actual clinical use of medicine.In this case,it is necessary to control the medicine dosage,carry out the necessary processing and processing,and consider the appropriate treatment course and individual differences in the human body.Conclusion1)Construction of aconite safety evaluation system:the use of"components-pathways-targets-clinical evaluation indicators" integrated and a whole-chain research strategy has carried out the "alkaloid monomer-medicinal-compound""toxic body" of Aconite Internal and external verification-the basis of toxic substances-the characteristics of toxic metabolism"research,constructing the safety evaluation system of aconite cardiotoxicity;2)Mechanism exploration and early warning of Aconite cardiotoxicity:Gap junction channel is an important pathway responsible for information exchange and electrical coupling located on cardiomyocytes.It plays an important role in the mechanism of Aconite cardiotoxicity and may issue early warning signals of toxicity.,The establishment of early warning of Aconite cardiotoxicity will help build its risk prevention and control system;3)Risk prevention and control of Aconite cardiotoxicity:From the level of Aconite"alkaloid monomer-medicinal material-compound" to clarify its "toxic substance basis-toxicity mechanism-toxic metabolism characteristics," and control its toxicity from various links.Prevent find and treat it early to prevent the disease. |
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