Dna Molecular Markers In The Identification Of Chinese Herbal Medicines

The standardization and modernization of Traditional Chinese Medicines hinge on the authentication of their botanical identities. The purpose of authentication is identify the adulterants from the trully Meteria Medica. The straight method is to rely on Morphological characteristics for the integrity, moreover the way is depentent on experiential conclusions. At present, It is difficult to identify precious and not integrity medicinal materials, animals medicinal materials, processed botanicals in slice, several sources botanicals, et al.DNA is the basic component of a living organism whereas chemical and phenotypic expression is controlled by the arrangement and expression of genes in the DNA. Since the identification based on genotype is not influenced by growth stage and enviornmental condition of plants, it provides a promising alternative to conventional methods by phenotype. DNA Molecular Gentic Markers has offered an additional tool for the quality control of Chinese herbal drugs over morphological and chemical markers for authentication of species-origin. In the light of the advances in molecular biotechnology in the past few decades, genetic tools are now considered to provide more standardized and reliable methods for authentication of herbal materials at the DNA level.The main content and results of this study is described as followed:1. The study to isolate DNA from Traditional Chinese HerbsDNA extraction is difficult in many plants because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. This is particularly true with Traditional Chinese Herbs , which are rich in polysaccharides, starch, or other substances that are difficult to remove from DNA preparations and are known to interfere with the enzymatic manipulation of DNA. Here we describe a modified procedure based on the hexadecyltrimethylammonium bromide (CTAB) method to isolate DNA from botanicals containing high levels of polysaccharides and starch. The procedure is applicable to both dry and fresh Traditional Chinese Herbals as well as was tested on Panax Ginseng.C.A.Meyer. Panax quinquefolius. Linn.. Trichosanthes kirilowii Maxim. Pinellia ternata (Thunb.)Breit. Glycyrrhiza uralensis Fisch. This method solved the problems of DNA degradation, and low yield due to binding and coprecipitation with starches and polysaccharides. The isolated DNA proved amenable to PCR amplification.2. Application of Multiplex Allele-Specific PCR for Authentication of Traditional Chinese Materia MedicaThe definition of Allele, a site at which DNA-either coding or noncoding—differs among genomes, is a coding locus(region). Single-nucleotide polymorphism(SNP) can be suitable used as molecular markers because of the advantages of abundance, stability and easily detected from genome. Based on the SNP in genome by searching for the GenBank database in NCBI, the AS-PCR primers were designed with specific mismatches at the 3' end that allow preferential amplification of one allele relative to another on account of the primers being complementary to the SNP site. The SNP can be detected according to the presence and absence of the PCR final products on gel electrophoresis. Using this technology, We identified successfully Panax Ginseng.CA.Meyer, and Panax quinquefolius. Linn.;Pinellia ternata(Thunb.)Breit. and its adulterants;Pinellia pedatisecta Schott. and its adulterants;Panthera tigris Linnaeus, and its adulterants;Pantherapardus Linnaeus, and its adulterants.3. Application of Inter-Simple Sequence Repeat for Authentication of several sources of Traditional Chinese HerbsThe first studies employing Inter-Simple Sequence Repeat (ISSR) markers were published in 1994 (Zietkiewicz et al.1994;). In this paper, We apply ISSR marker to authenticate three species of Traditional Chinese Herbs: three sources of Rheum palmatum L.. Rheum tanguticum Max//w.ex.Balf.or Rheum officinale Baill.;three sources of Glycyrrhiza uralensis Fisch.> Glycyrrhiza inflata Bator Glycyrrhiza glabra L.;two sources of Astragalus membranaceus(Fisch.)Bge.var.mongholicus (Bge.)Hsiao. or Astragalus membranaceus(Fisch.)Bge.Generally speaking, according this study, we can draw some conclusions and as follows:1. A key step in our protocol is the precipitation of DNA in a high concentration of salt (2.5 M NaCl) in the presence of isopropanol, immediately following phenol and chloroform extractions. Employing high salt concentrations to remove polysaccharides. The common procedure is to grind plant tissue in liquid nitrogen and transfer it to a preheated extraction buffer. Liquid nitrogen can be difficult to procure in some locations;thus, This method not requiring its use would be helpful. Our procedure produced a good yiele of DNA with an A26o/2so ratio in the range of 1.7-1.9. This method is rapid, simple, and efficient for isolating DNA from botanicals rich in polysaccharides and starch.2. The AS-PCR was applied to the identification of 2 Panax species\Panax Ginseng C.A.Meyer, and P. quinquefolius Linn.. By using 2 primers, PCR amplifications were performed with total DNA extracted from Panax Ginseng and Panax quinquefolius as template under 66 °C annealing temperatures in the same PCR reaction, and resulting product was detected by agarose gel electrophoreses. The result showed that two expected fragments one from nuclear 18S rRNA gene of Panax ginseng 249 bp fragment and other from chloroplast matK gene of Panax quinquefolius 1049 bp fragment, were observed simultaneously when the set of species-specific primers encountered template DNA of the corresponding species.3. AS-PCR was applied to the identification of Pinellia ternata (Thunb.)Breit. and its adulterants. A specific primer was designed according to the genomic ribosomal internal transcribed spacer region for Pinellia ternata (Thunb.)Breit. PCR amplifications were performed with total DNA extracted from Pinellia ternata (Thunb.)Breit. and its adulterants as template under 62°C annealing temperatures, and resulting product was detected by agarose gel electrophoreses. The result showed that one 395 bp expected fragment from Pinellia ternata (Thunb.)Breit. and others no fragment, were observed simultaneously.A specific primer was designed according to chloroplast rpl 20 gene for Pinellia pedatisecta Schott.. PCR amplifications were performed with total DNA extracted from Pinellia pedatisecta Schott. and its adulterants as template under 65°C annealing temperatures, and resulting product was detected by agarose gel electrophoreses. The result showed that one 354 bp expected fragment from Pinellia pedatisecta Schott. and others no fragment, were observed.4. AS-PCR was applied to the identification of Panthera tigris L. and its adulterants. A specific primer (PtlF&PtlR) was designed according to the mitochondrion cytochrome b for Panthera tigris L.. PCRamplifications were performed with total DNA extracted from Panthera tigris L. and its adulterants as template under 52°C annealing temperatures, and resulting product was detected by agarose gel electrophoreses. The result showed that one 326 bp expected fragment from Panthera tigris L. and no others, were observed simultaneously.A specific primer (Pp3F&Pp3R) was designed according to the cytochrome b for Panthera pardus L.. PCR amplifications were performed with total DNA extracted from Panthera pardus L. and its adulterants as template under 59°C annealing temperatures, and resulting product was detected by agarose gel electrophoreses. The result showed that one 374 bp expected fragment from Panthera pardus L. and no others, were observed simultaneously.5. Genomic DNA of three species were extracted as the ISSR template, and the influencing factors of ISSR were studied and the experiment parameters were optimized. By adjusting template DNA concentration, Mg2+concentration, dNTP and Taq polymerase contents, and annealing temperature, the PCR amplification conditions were optimized. Forty ISSR primers were used to screen the suitable primers for authentication. The conclusion is that one species-specific primer UBC818 has been obtained to identify three sources of Rheum palmatum L.? Rheum tanguticum Max/m.ex.Balf.or Rheum officinale Baill.;also, that one species-specific primer UBC826 is able to validate three sources of Glycyrrhiza uralensis Fisch.> Glycyrrhiza inflata Bat.or Glycyrrhiza glabra L.;that one species-specific primer UBC866 was employed to authenticate two sources of Astragalus mew&ra?acew.s(Fisch.)Bge.var.mongholicus> and Astragalus membranaceus(Fisch.)BgQ..Conclusions:The accurate identification of herbal drugs is the basis and prerequisite of clinical application as well as pharmacological research.Qualiry control for Chinese herbal drugs not only is a prerequisite for the modernization of Chinese medicine, but also is an important component of standardization of Chinese herbal drugs. We have concluded all literatures from 1994 to present which Panax ginseng C.A.Meyer and Panax quinquefolius Linn, have been represented as a leading role in the expanding field to establish an objective authenti-cation method by molecular techniques, as well as compared characteristics and applicability of many molecular markers. The times that RAPD played a primary role as a representation in the field of molecular genetic markers has finished gradully. The third molecular genetic marker—Single Nuleotide Polymorphism has come into being in the actualizing process of Human Genome Project. Going with the maturity of method of sequencing analysis, The purpose of authentication in Traditional Chinese Herbs was achieved by utilizing the specific—SNP locus that discovered by directly DNA sequencing analysis. Accordingly, Specific-primer PCR is gradually in the highest flight in the field of identification of Chinese Materia Medica. AS-PCR marker has become a rapid, simple, low cost, reliable and high throughput method for SNP distinguish. ISSR marker is usually highly polymorphic and informative. A unique fingerprint of an individual can be obtained by PCR amplification of these marker loci. Moreover, we reviewed the advantanges and limitations of the molecular techniques in TCM authentication.