The Study Of Innovative Techniques On Quality Control Of Traditional Chinese Medicine

Quality control(QC)is the precondition for the application of traditional Chinese medicine(TCM).Some negative quality issues,such as illegal adulteration or substitution,products from protected animals within Chinese patent medicine,lack of uniform standards for different forms' decoction pieces,are still hindering the development of TCM,as it continues making contributions to the world.To guarantee the quality of TCM products,some innovative techniques for quality control have been studied based on the production practice.And quality traceability system for the whole industry chain were discussed,in a bid to build a quality evaluation system related to therapeutic effects,which could promote process optimization and form a higher level of whole range quality control system for TCM.1.Identification of herbal compound through Combination of Full-length DNA Barcodes(CFDB)Long reads of full-length DNA barcode sequences were obtained from Single-Molecule Real-Time(SMRT)sequencing,a third generation sequencing(TGS)technique on Pacific Biosciences(PacBio)RS II platform,and the analysis of circular consensus sequence(CCS)was applied to the acquisition of high quality and accuracy result reads.While different kinds of DNA barcodes were combined for complementation.The identification procedure was as follows:DNA extraction,PCR amplification,magnetic beads purification,long reads sequencing,data CCS and Reads extraction,result reads clustering and species identification.The formula of Shengmai Powder(Sheng Mai San,SMS)was used as an example,that SMS powder and its raw materials,Ginseng Radix et Rhizoma(Panax ginseng C.A.Mey.),Ophiopogonis Radix(Ophiopogon japonicus(L.f)Ker-Gwal.),Schisandrae Chinensis Fructus(Scisandra chinensis(Turcz.)Baill.),along with their DNA mixture(HSM,ratio as 3:3:2)were used to the preparation of ITS2 and psbA-trnH library samples,respectively,each triple as parallel.Then the library samples were sequenced and data analyzed.And clones from PCR product of SMS ITS2 were sequenced by means of Sanger sequencing,for comparison analysis.The formula of Sanweijili powder(sanweijilisan,SJ),composed by Malvae Fructus(Malva veriticillata L.),Tribuli Fructus(Tribulus terrestris L.)and Chinese crab(Eriocheir sinensis H.Miline-Edwalds,Fanghai in Chinese),along with their DNA mixture(SH),were used as methodology validation,while CO1 added to identify the animal material Fanghai.81 clones of ITS2 sequence from four PCR products of SMS were pick out and identified,of which 76 for P.ginseng,5 for S.chinensis,and none for O.japonicus.The results of raw materials from TGS showed as expected,except the ITS2 library samples of O.japonicus,which showed a lot of exogenous sequences due to its awful PCR amplification efficiency.A total of 25874 result reads,which came from 58147 sequencing read by Pacbio,were extracted for SMS and HSM,that 2523 ITS2 result reads and 10065 psbA-trnH result reads for SMS,while 2225 and 11061 for HSM,respectively.As a result,ITS2 library samples of SMS and HSM showed the result reads of P.ginseng and S.chinensis,while psbA-trnH showed Panax sp.,S.chinensis,and O.japonicus.For one portion of SMS as example,there were 840 and 50 ITS2 result reads of P.ginseng and S.chinensis,3429 psbA-trnH result reads of Panax sp.,986 and 28 psbA-trnH result reads of S.chinensis and O.japonicus,respectively.The ITS2 result reads of O.japonicus nearly could not be found in all SMS and HSM,while the psbA-trnH result reads of P.ginseng could hardly be distinguished from other Panax species such as P.quinquefolius and P.japonicus.All the raw materials of Sanweijili powder had been identified while several other biological sources were checked out by the combination of CO1 and ITS2,including 5 exogenous plant species,2 exogenous animal species and 2 microorganism species.As the deeper the sequencing,the more species were likely to be detected.Simultaneously,no exogenous species had been found in the 10 library samples of the DNA mixtures(HSM and SH),led to the conclusion that the contaminations which were likely from the unpurified raw materials,indeed exist in the powders.The identification method is workable and stable,for total effective rate of all result reads was 99.93%,while most samples(39/46)reached 100%.Almost all the result reads could be explained and the results of parallel samples remain consistent.The sensitivity of this method is extremely high that the very litter exogenous material incorporated into raw materials,no matter plant or animal origin,had been checked out.The identification results were accurate and reliable,because no ITS2 result reads of P.quinquefolius was found in source of P.ginseng,though their ITS2 sequences are extremely similar.The combination of barcodes makes identification more accurately and completely.At the same time,the extension ability of this method had been verified,that if appropriate barcodes selected,the method could be used in other biometric identification fields.Compared with existing identification methods,such as Sanger sequencing of clones and the second generation sequencing,the method researched can be easily handled,and its identification result is more reliable owe to its high throughput with higher accuracy and sensitivity.2 The study on the Standard Decoction of decoction pieces and its quality controlQuality control problems,including identification difficulty,short of unified standard and equivalence,exist in different types of Chinese herbal pieces,as the convenience they provide for clinical application.And the standard decoction of medicinal slices,which refers to clinical application of traditional herbal pieces and plays a role of standard reference for quality control.is expected to solve these problems.For a standard material and standard system,the standard decoction of medicinal slices can provide standards for new types of medicinal slices,especially for the dispensing granule which was widely used in clinical.To establish a standard for different dosage forms of Ginseng Radix et Rhizoma and provide reference for production practice,12 batches of standard decoction of Ginseng Radix et Rhizoma(SDRG)were prepared through standard process from representative samples.Then the paste-forming rate,index components content as well as their transfer rate,and high performance liquid chromatographic(HPLC)fingerprints similarity were analyzed.And the differences among the slices,the standard decoction and the granule,all from the same batch,were comparatively analyzed to reveal the mass transfer rule in the process.The extract rates of 12 batches SDRG were in the range of 40.5%-58.8%(47.2%± 5.6%,x±s);transfer rates of ginsenoside Rg1+Re and Rb1 were in the range of 57.5%-88.5%(76.0%±9.2%),68.7%-94.8%(80.3%±9.1%),respectively;HPLC fingerprint chromatogram similarity between batches were in the range of 0.905-0.997(0.970±0.022),and 0.966-0.996(0.986±0.009)compared with the reference fingerprint.And an admittance standard for SDRG had been put forward:the content of ginsenoside Rb1 and Rg1+Re shall not be less than 0.282 g·L-1 and 0.529 g·L-1 respectively,extract rate shall not be less than 40.5%,the fingerprint similarity between batches shall not be less than 0.905,and 0.966 when compared with the reference fingerprint.10 kinds of saponins were identified from 12 common peaks.And ginsenoside Rg1+Re content per equivalent(CpE)maybe a good parameter for quality control of the extract or the granule.The process from slices to standard decoction was qualitative transfer,while standard decoction to granule was the quantities'change.The standard decoction could act as standard reference object in the production process of formula granule,to guarantee the consistent of final productions through batch charging and process optimization.As to different forms of Chinese herbal pieces,whether traditional forms or modern ones such as ultramicro decoction piece and formula granule,are almost all dispersed in water in clinical practice;in consideration of the same material basis between the water liquid used and the standard decoction,the standard decoction and its concentration can act as reference material in the aspect of identification and quality control.3 The study of quality control system for Chinese medicinal materialsThere are so many factors,such as germplasm and producing area,which could influence the quality of Chinese medicinal materials,that a quality traceability system and some technologies for key point control were needed.Comparing with the existing traceability system,the obstructions that Chinese medicinal materials traceability system faced should be analyzed.And as this traceability system chain was incomplete nowadays,some key technologies,such as the analysis of producing area,cultivation management,remain to be studied,in order to contribute to the traceability system from the aspects of both industrial structure and critical control point.The intensification of production and incomplete of material transfer system are impeding the application of the traceability system.The relationship between the quality of Schisandrae Chinensis Fructus and the ecology of its growing area were analyzed.The suitable producing areas in global range for S.chinensis were analyzed by the global geographic information system for medicinal plants(GMPGIS),and different suggestions were given according to the genuine producing area and the non-authentic producing areas.Finally,a primary quality control system for Schisandrae Chinensis Fructus was established based on hazard analysis critical control point system(HACCP).4 SummeryThe identification method of combination of full-length DNA barcodes had provided a novel method for completely biotic identification for Chinese medicinal compound formula.The standard decoction could act as standard material for different types of decoction slices that a new idea for quality control emerged.The industrial structure of traditional Chinese medicine will be streamlined and optimized under the police of good,along with the breakthrough of key technologies in each link node of the industry chain.At the same time,the development of modern testing technologies will provide quality evaluation measures for all TCM products,such as raw materials,decoction pieces and herbal compound,and promote the process design for the quality formation.Thus,a high-tech level and whole range quality control system in the industry chain of TCM will be built,through quality control and information traceability at every note.