| Objective: Through the study of morphological markers,chemical markers and biological markers,the quality markers of Aconiti Kusnezoffii were determined,which provided a basis for the in-depth study of Aconiti Kusnezoffii and the establishment of scientific quality evaluation system.Methods: 1.The shape,size,color,surface characteristics,texture,broken surface,s-mell and taste of Aconiti Kusnezoffii were identified by eye observation,touch,smell,taste.and the macroscopic trait Q-marker of Aconiti Kusnezoffii were established.To observe the shape,size(length,diameter),color,cell inclusion and cell wall of Aconiti Kusnezoffii powder by microscope,and measure and photograph the record to clarify the microscopic trait Q-marker of Aconiti Kusnezoffii.2.The acute toxicity test of mice was used to determine the 0% and100% estimated lethal dose(Dmin,Dmax),LD50 and its 95% confidence interval.The mouse hot plate test and xylene-induced mouse ear swelling test were used to study the anti-inflammatory and analgesic pharmacodynamics of Aconiti Kusnezoffii.And then the UPLC-Q-TOF-MS technique was used to detect the blood components of the anti-inflammatory and analgesic test of Aconiti Kusnezoffii and establish the chemical Q-marker of Aconiti Kusnezoffii.3.DNA was extracted,PCR amplified,DNA quality detected by electrophoresis,purified and sequenced using DNA barcoding technique and plant genome extraction kit,and then data were analyzed using Codon Code Aligner17.0 and MEGA 7.0software,and construct a phylogenetic tree using the Neighbor-joining method and establish the biological Q-marker of Aconitum kusnezoffii.Results: 1.In the study of the morphological markers of Aconitum kusnezoffii,it has an irregular long conical shape,slightly curved,1.5～8.1cm long and 0.4～2.5cm in diameter.The surface color is gray brown and black brown.The surface is wrinkled,with longitudinal wrinkles,punctate fibrous root marks and several nodular lateral roots.There are residual stems and a small amount of adventitious root residues at the top.There are withered buds on one side of the top and round or oblate adventitious root residues on the other side.It is rigid,with gray-white,dark gray or yellow-white cross-section,fissured,polygonal,round,and large or hollow pith.The gas is micro,the taste is spicy,and the tongue is numb,its characteristics can be formulated as a macro-trait Q-marker of Aconiti Kusnezoffii;Through microscopic identification and analysis,the stone cells were colorless or pale yellow,and the cells connected with the metacortex were brown,with avariety of shapes,such as square,long strip,rectangle,triangle,someof which were extended toform fibrous,with a diameter of 18～139μm,and the length is 115～274μm.The wall thicknessis different,the layer is obvious,and some containbrown matter.It is bright yellow and white under the polarizing microscope.These characteristics can be used as micro-trait Q-marker of Aconitum kusnezoffii.2.The acute toxicity test in mice showed that Dmin was 0.38g/kg,Dmax was 1.60g/kg,LD50 was0.681g/kg,and the 95% confidence interval was 0.510～0.863g/kg.According to the acute toxicity experiment,the anti-inflammatory and analgesic pharmacodynamics experiments of Aconiti Kusnezoffii were carried out by hot plate method and auricular swelling method.The components of Aconiti Kusnezoffii entering blood were analyzed,and the chemical Q-marker of the efficacy of Aconiti Kusnezoffii were preliminarily determined to be aconitine,hypaconitine and mesaconine.3.DNA Barcoding analysis showed that the samples could successfully extract DNA.The five sequences of ITS,ITS2,psb A-trn H,mat K and rbc L could amplify the target fragment and obtain high-quality sequencing peak map.From the results of sequencealignment and genetic distance,there was almost no variation in Aconitum.The averageintraspecific genetic distances of ITS,ITS2,psb A-trn H,mat K and rbc L sequences were0.000,0.2528,0.000,0.000 and 0.000,The average interspecific genetic distance was0.8270,4.741,1.425,5.9150 and 1.4257,Referring to the existing Aconitum medicinal sequences and related sequences in the Genbank database,NJ phylogenetic tree analysis showed that in the phylogenetic tree of matk sequence,Aconiti Kusnezoffii was divided into one branch,which could be distinguished from other Aconitum sequences.Conclusion: 1.Through the study of macroscopic trait markers and microscopic trait markers,the traits and microscopic characteristics of Aconitum kusnezoffii were sortedout,which provided a reference for the formulation of Aconitum kusnezoffii standards.2.From the study of chemical markers of Aconiti Kusnezoffii,the chemical components of the traditional efficacy of Aconiti Kusnezoffii are aconitine,hypaconitine and mesaconitine,which clarifies the material basis of efficacy and provides a theoretical basisfor the quality evaluation of Aconiti Kusnezoffii.3.From the study of DNA Barcoding of Aconiti Kusnezoffii,it was determined at the sequence identified between Aconiti Kusnezoffii and other Aconitum herb was matk sequence.which provided a scientific basis for the identification of Aconiti Kusnezoffii and its related species from the DNA molecular level. |
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