Based On Metabonomics And Network Pharmacology, Research On The Toxicity And Drying Attenuation Principle Of Mongolian Herb Aconite

Aconiti kusnezoffii radix.Is one of the most commonly used traditional Chinese medicine and Mongolian medicine.In order to develop the application of Aconitum kusnezoffii radix.in a more comprehensive way,a new method of drying Aconitum kusnezoffii radix.was developed on the basis of a traditional method of processing and fumigation,in order to reduce the toxicity of crude drugs.In order to explore the mechanism of detoxification by raw Aconitum kusnezoffii radix.,baking processed Aconitum kusnezoffii radix.and processed Aconitum kusnezoffii radix were given to Wistar rats by gavage,and the effects of endogenous metabolites and metabolic pathways were studied by metabonomics methods of liver,serum and urine.The effects of Aconitum kusnezoffii radix on pain,rheumatism,inflammation and other symptoms were studied by network pharmacology method " The mechanism of multi-component,multi-target and multi-channel "is an attempt to explain the toxicity of Mongolian medicine kusnezoffii radix and the mechanism of reducing toxicity by baking processed.Objective:1.To select the representative Mongolian medicine Aconitum kusnezoffii radix,which conforms to the Chinese Pharmacopoeia and local standards.2.The toxicity of raw Aconitum kusnezoffii radix.,baking processed Aconitum kusnezoffii radix.and processed Aconitum kusnezoffii radix to various tissues was observed by histopathological sections.3.To collect the liver,urine and serum samples of the rats in raw Aconitum kusnezoffii radix.,baking processed Aconitum kusnezoffii radix and processed Aconitum kusnezoffii radix and blank control groups for non-target metabonomics test,select the different metabolites,determine the possible biomarkers in the baked group,and make the relative content analysis.4.Based on the platform of traditional Chinese medicine network pharmacology analysis,the main active components,targets and related diseases of Aconitum kusnezoffii radix were screened,and the component target disease direct action network was constructed by using the software of Cytoscape.Methods:1.HPLC was used to determine the six main alkaloids of aconitine,hypaconitine,neoaconitine,benzoylaconitine,benzoylhypaconitine and benzoylneoaconitine in the groups of rawAconitum kusnezoffii radix.,baking processed Aconitum kusnezoffii radix.and processed Aconitum kusnezoffii radix;2.40 male Wistar rats were randomly divided into four groups 10,namely:control group,raw group,baked group,processed group.After one week of adaptive feeding and 21 days of administration,the changes of physical signs and the pathological changes of nine tissues including heart,liver,spleen,lung,kidney,spinal cord,brain nerve,sciatic nerve and stomach were observed in each group.3.40 male Wistar rats were randomly divided into four groups,10 rats in each group,i.e.control group,raw group,baked group and processed group.After one week of adaptive feeding and 21 days of drug administration,urine,serum and liver samples were collected from rats in the raw,baked,processed and control groups,and non-target metabonomics was detected by UPLC-QTOF-MS technology,metabonalyst was used to analyze the data after pretreatment by progenesis Qi software.To screen the different metabolites of urine,serum and liver of the model group and the blank control group,which si:multaneously meet the requirements of the multiple of difference(FC)≥1.2 or ≤0.8,Q-value<0.05 and VIP)1.0 under the OPLS-DA mode,through searching the database such as HMDB,thermogram clustering analysis and so on Venn map analysis showed that there were different metabolites in the control group compared with the raw,baked and processed groups,and the related metabolic pathways and relative content changes of the selected different metabolites were analyzed.The urine,serum and urine samples of rats in blank control group,raw group,baked group and baked processed group were analyzed to screen the differential metabolites and target metabolic pathway of processed treatment and other diseases from the differential metabolites of serum and liver;4.The main active components,target points and related diseases ofAconitum kusnezoffii radix.were screened based on the Chinese medicine network pharmacology analysis platform The interaction network of protein and protein was constructed by using the software of Cytoscape and String.The enrichment analysis of Gene Ontology(GO)and action pathway(KEGG)was carried out by using the software of David.Results:1.According to the HPLC method,3.2217mg·g-1,0.4981mg·g-1,0.3271mg·g-1,4.3883mg·g-1 and 0.6046mg · g-1 were determined.They are all in accordance with Chinese Pharmacopoeia and local standards.2.The pathological changes of heart,liver,spleen,lung,kidney,spinal nerve,brain,sciatic nerve and stomach were observed 21 days after administration in control group,raw group,baked group and processed group.no pathological changes were found in brain and sciatic nerve;no pathological changes were found in heart,liver,lung,spleen,stomach and spinal cord except for a few rats in raw group In addition to the control group.3.33 potential metabolic markers(15 in liver,9 in serum and 9 in urine)were screened from liver,serum and urine samples,which may be related to the mechanism of detoxification and pharmacodynamics.Compared with the control group,the difference metabolites were selected for relative content analysis.L-leucine,acetic acid,L-glutamine,L-histidine,L-arginine,α-linolenic acid,taurocholate,pyruvic acid,benzoic acid and acetone aldehyde 15 different metabolites were selected from the liver,such as dihydrouracil,fucose-1-phosphate,linoleic acid,L-methionine and aconitic acid,were used as biomarkers of liver;The mean value normalized by metabonomics data was used to express the change of the relative content of biomarkers in the non target metabolizing group.Compared with the control group,the relative content of L-arginine,α-linolenic acid,benzoic acid,dihydrouracil and linoleic acid in the baked group significantly up-regulated(P<0.05).The relative contents of L-leucine,acetic acid,L-glutamine,L-histidine,taurocholate,pyruvate,glyoxal,fucose-1-phosphate,L-methionine and CIS aconitic acid were significantly down-regulated(P<0.05).By analyzing the related metabolic pathways,the results showed that taurine and low calcium amino acid metabolism,nitrogen metabolism,histidine metabolism,ketone synthesis and degradation,α-linolenic acid metabolism,valine,leucine and isoleucine biosynthesis,riboflavin metabolism,cysteine and methionine metabolism,pantothenic acid and coenzyme A biosynthesis,pyruvate metabolism,linoleic acid and vitamin B6 metabolism were involved,There are 13 metabolic pathways of glyoxylic acid and dicarboxylic acid metabolism;9 Differential metabolites such as L-proline,L-methionine,L-phenylalanine,stearic acid,L-asparagine,L-glutamine,aconitic acid,L-fucose and sorbitol are selected from serum as serum biomarkers;the mean value normalized by metabonomics data is used to represent the biomarkers selected from non target metabolism group Compared with the control group,the relative contents of stearic acid,L-glutamine,aconitic acid and L-fucose in the baked group significantly up-regulated(P<0.05),while the relative contents of L-proline,L-methionine,L-phenylalanine,L-asparagine and sorbitol significantly down-regulated(P<0.05).The results showed that the metabolism of Aconitum involved in nicotinic acid and nicotinamide metabolism and fatty acid biosynthesis,phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism,tryptophan metabolism,cysteine and methionine metabolism,histidine metabolism,unsaturated fatty acid biosynthesis,fructose and mannose metabolism,alanine,aspartic acid and glutamic acid metabolism,D-glutamine and D-glutamic acid metabolism,arginine and proline metabolism,glyoxylic acid and binary acid metabolism 13 metabolic pathways,isonicotinic acid,9-cis-retinol,n-acetyl-1-aspartic acid,L-valine,3-dehydrologin,2-deoxyepinephrine,3-methylthiopropionic acid,1-pyrroline-2-carboxylic acid,deoxycytidine and other 9-cis-retinol,n-acetyl-acetyl metabolites were selected as urine biomarkers;compared with the control group,isonicotinic acid,9-cis-retinol,n-acetyl-acetyl were selected as urine biomarkers-L-aspartic acid,L-valine and 3-dehydrologin were significantly up-regulated(P<0.05),2-deoxyepinephrine,3-methylthiopropionic acid,1-pyrroline-2-carboxylic acid and deoxycytidine were significantly down-regulated(P<0.05).By analyzing the related metabolic pathways,the results showed that there were four metabolic pathways involved in arginine and proline metabolism,cysteine and methionine metabolism,histidine metabolism,tyrosine metabolism,etc.;the comprehensive analysis of liver,serum and urine related metabolic pathways showed that dried Aconitum was mainly through regulating the body a total of 30 metabolic pathways to detoxify and play a pharmacodynamic role.4.There are 8 active components,82 targets and 113 diseases."Component target disease" network analysis showed that the Aconitum kusnezoffii radix acted on prostaglandin G/H synthase 1,muscarinic acylcholine Receptor M3,muscarinic acylcholine receptor M1,Android receptor,medium channel protection type 5 subunit alpha,muscarinic acylcholine Receptor M5,prostaglandin G/H synthase mainly through izoteolin active components 2,Muscarinic acetylcholine receptor M4 Retinoic acid receptor RXR-alpha,Delta-type opioid receptor,Acetylcholinesterase,Alpha-1B adrenergic receptor,Sodium-dependent dopamine transporter,Beta-2 adrenergic receptor,Alpha-1D adrenergic receptor,Sodium-dependent serotonin transporter,Mu-type opioid receptor,Alpha-1A adrenergic Key targets such as receptor play a role in many diseases.GO annotation and KEGG pathway enrichment analysis showed that Aconitum kusnezoffii radix played an analgesic and analgesic role by regulating ptgs1,chrm3,chrm1,AR,SCN5A,PTGS2,rxra,OPRD1,ache,adralb,SLC6A3,ADRB2,adrald,SLC6A4,OPRM1,ADRA1A and other targets.By regulating PTGSI,CHRM3,AR,PTGS2,OPRD1,SLC6A4,OPRM1 and other targets,it can act on the rheumatic pathway.By regulating ptgs1,chrm3,chrm1,AR,SCN5A,chrm5,PTGS2,rxra,OPRD1,ache,adralb,SLC6A3,ADRB2,adrald,SLC6A4,OPRM1,ADRAIA and other targets,it acts on the inflammatory pathway.The results show that Aconitum kusnezoffii radix can play a role in the treatment of diseases through the mechanism of "multi-component,multi-target and multi-channel".Conclusion:1.Raw Aconitum kusnezoffii radix was collected from Wula mountain of Baotou,Inner Mongolia,and the baked Aconitum kusnezoffii radix was also in accordance with the Chinese Pharmacopoeia and local standards;2.The three drugs did not cause toxicity to brain tissue and sciatic nerve 21 days after the administration of raw Aconitum kusnezoffii radix,baked Processed Aconitum kusnezoffii radix and Processed Aconitum kusnezoffii radix to rats;raw Aconitum kusnezoffii radix to heart,liver,lung,spleen There are a small amount of toxicity in stomach and spinal cord;there are some toxicity in kidney in raw,baked and Processed Aconitum;3.33 potential metabolic markers screened from liver,serum and urine samples may be related to the detoxification and efficacy mechanism of baked Aconitum kusnezoffii radix.The metabolic pathways related to liver,serum and urine showed that the main metabolic pathways ofAconitum kusnezoffii radix were detoxification and pharmacodynamics by regulating 30 metabolic pathways in the body;4.Aconitum kusnezoffii radix played an analgesic role by regulating targets such as CACNA1A,CHRM2,chrm4,CNR1,KCNJ10,PTGS2,scnlla,SCN5A and tacr2.By regulating the targets of CACNA1A,CHRM2,chrm4,CNR1,PTGS2 and scnl 1a,the pain pathway was activated.To lay a theoretical foundation for revealing the mechanism of biological effect of"multi-component,multi-target and multi-channel" in Aconitum kusnezoffii radix.