Reserach On Quality Control And DNA Barcoding Identification Of Arnebia Euchroma

Arnebia euchroma is a perennial herb of the genus Boraginaceae.It is used as a root medicine and has various pharmacological activities such as anti-inflammatory and anti-tumor.With the continuous reduction of comfrey resources,there are gradually appearing in the market such as false and true,and second-rate charge.Therefore,Arnebia euchroma of Xinjiang native medicine was used as the experimental material.First,the active ingredient was determined and the extraction process of flavonoids in comfrey leaves was optimized,quality research was performed again,and DNA barcodes suitable for purple grass identification were selected to further control Arnebia euchroma.The quality of medicinal materials and standardized production provide a basis,and at the same time,it is of great significance to ensure the safety and effectiveness of clinical medication.The main research contents and results are as following.(1)Screen the best exogenous hormones for soaking seeds of Arnebia euchroma.Use the ultrasonic extraction process to optimize the extraction process of total flavonoids in Arnebia euchrom a leaves.This indicated that the best exogenous hormone for seed soaking was 150 mg/L chlormequat.The optimal process conditions are:ultrasonic extraction times 4 times,ultrasonic extraction temperature 45℃,ethanol concentration 70%,material-liquid ratio 1:30.(2)Ultraviolet spectrophotometry(UV)was used to determine the content of total flavonoids.polysaccharides and total hydroxynaphthoquinone in wild Arnebia euchroma from different origins and growth years.The 14 batches of artificially cultivated Amebia euchroma in different growth periods were collected and high-performance liquid chromatography Method(HPLC)and UV method to determine the content of its active ingredients.It shows that the active ingredient content in Arnebia euchroma is different in different places and growing years.In May,the 2-year-old Arnebia euchroma was harvested with the highest total levosporin,acetylshikonin,and hydroxynaphthoquino ne,with total pigment content of 0.109%,1.388%,and 1.906%,respectively.β’-dimethy’lacryloyl akanin is the highest at 0.535%.(3)Qualitative identification and quantitative analysis of 34 batches of Arnebia euchroma by thin layer chromatography(TLC)and HPLC.To establishe HPLC fingerprints,combined with stoichiometry to study the characteristics and classification of its chemical components,the content of naphthoquinones was determined,and the differential markers were screened.As a result,the Arnebia euchroma TLC identification method had strong specificity and clear spots.The HPLC fingerprint of Arnebia euchroma was obtained,12 common peaks were identified.The similarity of other samples were more than 0.86,except that the three batches of medicinal herbs on the market were less than 0.72.These 34 batches of samples could be classified into two categoriess by HCA.The PCA result was similar with HCA.The wild,cultivated and market of Arnebia euchroma was completely distinguished by OPLS-DA.Six constituents,such as shikonin,acetylshikonin and β,β’-dimethylacrylshikonin were screened as biomarkers,representing the major differences of Arnebia euchroma.There was some difference of the content of three active components in samples.(4)Three barcode sequences of ITS,ITS2,and psbA-trnH were used to identify and analyze the purple grass and its fakes,and the accuracy and stability of the three barcodes for the identification of the purple herbal medicine and its fakes were examined and compared.The results show that the sequence of identification success rate of each sequence obtained by the two methods is ITS2,ITS,psbA-trnH.The sequence with the smallest interspecies variation greater than the largest intraspecies variation is ITS2,and the average intraspecies variation is the smallest.In the barcoding gap test,there is no overlapping area within and between species of ITS2 sequence,followed by the smaller overlapping area of ITS sequence.The NJ tree results showed that the ITS and ITS2 sequences had a good separation effect on the samples,both of which separated the soft comfrey from other genera,and the psbA-trnH sequence did not separate the purple herbs and their mixed fakes.The comprehensive results show that the ITS2 sequence is an ideal barcode for identifying Arnebia euchroma and their fakes.