Research On The Quality Evaluation Of Mongolian Medicine Compound Dedu Guri Gumu-7 Based On High-throughput Sequencing Technology

Objective:In recent years,the demand for natural medicines in the global markets has continued to increase,and the herbal medicine industry has become one of the most distinctive traditional industries in China.As an important part of traditional Chinese medicine,ethnic medicine has attracted more and more attention.One of them,Mongolian medicine absorbed relevant skills and theories in practice by other ethnic groups such as Tibetan medicine and traditional Chinese medicine.There are thousands of traditional Mongolian medicine prescriptions which have been approved down and have positive effects,besides,there are over 400 frequently-used preparations.Mongolian compound preparation Gurigumu-7(G-7)is an effective medicine for treating liver disease,which was documented in the Drug standards of Ministry of public health of China(Mongolian medicine Fascicule)(1998 edition),due to lacking of comprehensive qualitative and quantitative indicators contained in the original standard,it is very important to establish reliable and accurate experimental methods to control the intrinsic quality and ensure the quality of G-7 effectively.In order to address the single detection method in the local standard,this present study identified the species composition of Mongolian medicine compound G-7 and evaluated its quality,with a view to establishing a comprehensive and effective quality evaluation method for complex pharmaceutical preparations and advancing research on the quality standards of compound preparations.Methods:First of all,collecting of raw materials of Mongolian medicine compound preparation G-7 as a control,which all identified by morphological identification and DNA barcoding molecular identification method to ensure the accuracy.Besides,two reference G-7 samples(RS01 and RS02)were prepared according to the Drug standards of Ministry of public health of China(Mongolian medicine Fascicule)(1998 edition)in the laboratory,and RS02 was added with Panax ginseng as a positive control.In addition,collecting three commercial G-7 samples(SS01-SS03),and twelve hospital preparations of G-7 samples(YN01-YN12)from inner Mongolia.Using four different gene regions ITS,ITS2,rbcL and matK as molecular markers,two references,three commercial and twelve hospital preparations of G-7 samples were sequenced based on Illumina NovaSeq 6000 platform.Filter clean data obtained by sequencing to remove low-quality sequences,high-quality sequences through assembly and sequence evaluation,and then calculate the sequencing depth and coverage to remove unreliable sequences with sequencing depth less than three and coverage less than 95%.Using of TCM-BOL,GenBank and BOLD database to identify the material medicines.Results:This present study gained about 120 G raw data,and about 800 million reads based on high-throughput sequencing technology,and more than 500,000 high-quality sequences were obtained after processing.The analysis results show that combing four different kinds of DNA barcoding ITS,ITS2,rbcL and matK could detect all the biological components specified in the two reference samples,and RS02 could detect the positive control Panax ginseng,which can indicate that the method has good accuracy,stability and reproducibility,and at the same time,the identification efficiency of ITS and ITS2 regions is higher than rbcL and matK regions.Combined with ITS2?rbcL and matK sequences,we detected four labeled herbal materials in three commercial G-7 samples.In addition,the number of labeled herbal species was different in twelve hospital preparation G-7 samples,YN04 was the sample with the largest number of species detected,a total five labeled herbal materials,YN11 was the sample with the least number of species,only 2 labeled herbal materials.In addition,only the sample of YN12 was detected Akebia quinata,the rest samples were found Clematis armandii and Clematis chinensis which are the adulterants of Akebia quinata to some extent,and we even found Aristolochia manshuriensis in YN02.The composition of HSYA in Carthami flos and GA in Chebulae fructus were determined based on high performance liquid chromatography(HPLC).The results showed that the two components had good linearity in their respective linear ranges,in which the regression equation of HSYA is:y=2×107x-293586,r2=0.999,and the regression equation of GA is:y=3×107x-64310,r2=0.999.Contents of HSYA and GA were similar in three commercial G-7 samples,indicating that there were no significant differences among of them.There were significant differences in the contents of the two components in twelve hospital preparations G-7 samples,YN09 contained the highest HSYA and the lowest is YN02,YN08 contained the highest GA and the lowest is YN07.Conclusion:High-throughput sequencing technology can effectively identify the biological components in Mongolian medicine compound preparation G-7.The quality evaluation of compound preparations should include the identification of prescription ingredients and determination of the content of active ingredients.Therefore,combining high-throughput sequencing technology with HPLC provides new idea and method for the quality evaluation of compound preparations.