| Purpose: In this study,we took Gentianae Radix et Rhizoma and Aconiti Kusnezoff ii Radix as the research object.We first carried out herbal textual research on the twoherbs,integrated ancient and modern,traced the root cause,corrected confusion,and defined the historical evolution of medicinal herbs,laying the foundation for further re search.Secondly,based on the ITS2 and psb A-trn H sequences at the DNA molecule le vel,identified the two herbs and their original plants,to make sure about the relationsh ip,and accumulated ITS2 and psb A-trn H sequences during the experiment.The third c omponent at the level of metabolic markers was the triterpenoids of gentiopicrin,genti opicrin,and tartarin in Gentianae Radix et Rhizoma and the aconitine,hypaconitine,a nd azulenin in Aconiti Kusnezoffii Radix.The triterpenoids and alkaloids were deter mined by HPLC and HPLC fingerprints were established.Combining the results of DN A molecular identification provides a strong scientific basis for the identification of au thenticity and types of medicinal materials from the bimolecular level.Through on-site inspections of the major origins of the two medicinal herbs and the major medicinal h erbs markets in the country,data collection and information collection,combined with dual-molecule appraisal,the formulation of medicinal commodity specifications that bes t meet the current market circulation conditions was established.Materials and methods: 1 Materials: DNA molecule part: 37 leaves samples of Gentianae Radix et Rhizoma,collect from Liaoning province Baiyunshan,Qingyuan,and Hubei province Yichang,Guizhou province Liupanshui,and Heilongjiang province Daqing;8 fresh herbs from Baiyunshan and Qingyuan;49 leaves of Aconiti Kusnezoffii Radix from Baiyunshan,Qingyuan,Kuandian,and Tejinhan mountain,Yichang,Jiangyou;8 fresh herbs from Tejinhan mountain,Jiangyou;HPLC part: 65 medicinal herbs from Anhui Bozhou?Hebei Anguo?Chengdu Hehuachi?Guangxi Yulin medicinal herb markets and Qingyuan?Daqing?Yunxian;20 Kusnezoffii Radix from medicinal herb markets above and Baiyunshan?Kuandian.2 Methods: 2.1 Looking through the ancient and modern herb monographs to research the GentianaeRadix et Rhizoma and Aconiti Kusnezoffii Radix.2.2 The level of DNA molecular Extract the DNA from the medicine and leaves,and amplify the ITS2 and psb A-trn H sequences by PCR.And sequence the purified product to get the sequence messages of ITS2 and psb A-trn H.Using the software DNAMAN to joint the sequence and remove the primer and the low-quality part to get the useful sequences to analyze;then use the MEGA 7.0 software to analyze and dispose the sequences,then built the NJ-tree.2.3 The level of secondary metabolite Use the method of HPLC to measure the gentiopicrin,gentiopicrin,and tartarin content in Gentianae Radix et Rhizoma and the aconitine,hypaconitine,and azulenin content in Aconiti Kusnezoffii Radix.And set up the Fingerprint Spectrum of the seventeen batches of Gentianae Radix et Rhizoma and nineteen batches of Aconiti Kusnezoffii Radix.collect from the places above.Results: 1 Herbal research on Gentianae Radix et Rhizomagentian and Aconiti Kusnezoffii Radix shows that the mainstream of Gentianae Radix et Rhizomagentian in ancient herbs is mainly from Gentiana manshuric Kitag.and Gentiana scabra Bge.,and the medicinal cultivar of Gentiana rigesceras Franch is relatively late,and it is first divided by Wu Qijun;Aconiti Kusnezoffii Radix is often called Wutou together with Aconitum carmichaelii,and it is the same even now,because of the area differences,there is a certain degree of confusion in the name.2 The sample of two medicinal herbs have high successful rate of extracting DNA,the agarose gel electrophoresis images of the ITS2 and psb A-trn H sequences amplified in most experimental samples showed a single and bright band in the corresponding position.Sample PCR amplification successful,The K2 P genetic distance was calculated from the sequencing results,the mutation sites were compared,and the NJ cluster tree was constructed to efficiently and accurately identify the genetic relationship and species of medicinal materials.3 The contents of gentiopicroside and other indicators in Gentianae Radix et Rhizoma were higher than those in gentian,about 90% of which met the standards of Pharmacopoeia and qualified;the longdancao,a local traditional medicinal herb The content of diphenhydrin is not up to pharmacopoeia standards and is not recommended for mark et distribution.The total content of neoaconine,hypaconitine and aconitine collected fr om Aconiti Kusnezoffii Radix is about 77%,which meets the pharmacopoeia standard,but the major domestic medicinal herbs market There are many counterfeit goods in t he circulation of the Kusao products,and the content standard compliance rate is only about 14%;the HPLC fingerprints of Gentianae Radix et Rhizoma and Aconiti Kusne zoffii Radix medicinal materials are established,and the 13 fingerprint peaks of the Ge ntianae Radix et Rhizoma fingerprint spectrum are used to identify the pseudo-gentianmedicinal herbs There are altogether 8 characteristic peaks of the product and differe nt varieties of Aconiti Kusnezoffii Radix,and the similarity data can be used to identif y the counterfeit products of Aconiti Kusnezoffii Radix.4 Conducted on-the-spot inspections of the major Chinese herbal medicine markets andthe production areas of Gentianae Radix et Rhizoma and Aconiti Kusnezoffii Radix,a nd combined the research of the Pharmacopoeia literature and local standards of vario us editions;obtained the latest circulation conditions of herbal medicines,combined wi th the results of herbal medicine research and bimolecular identification,Gentianae Ra dix et Rhizoma and Aconiti Kusnezoffii Radix medicinal materials are used for standar d formulation of commodity specifications.Conclusions: 1 Through the herbal medicine research on the two medicinal materials,we can see that the mainstream of this herb is recorded as two species of Gentiana manshuric Kitag.and Gentiana scabra Bge..It is determined later that medicinal cultivar is determined by Kennedy genus later,and there are fewer counterfeits in history.In the past grass,most of the herbs and current Chuanwu were collectively referred to as Wutou,until the Ming Dynasty Li Shizhen clearly distinguished the two in the “Compendium of Materia Medica” for the first time.The phenomenon of confusion of varieties has caused market circulation obstacles and increased counterfeit goods.2 A method for the identification of DNA barcodes based on ITS2 and psb A-trn H sequencesfor the two medicinal herbs were initially established.The success rate of PCR amplification(92%)and sequencing success rate(93.77%,89.44%)can be proved.The methods for the extraction and PCR amplification of total DNA from the herbs and its primordial plants,and confusable products are accurate and feasible;the genetic distance between the experimental species is greater than the intraspecific genetic distance,and there are many mutation sites.The NJ clustering tree can clearly distinguish between different varieties and foreign ethnic groups.This method can provide accurate and powerful scientific basis for identifying sample varieties and authenticity and formulating product specification levels.3 The content of the collected Gentianae Radix et Rhizoma and Aconiti Kusnezoffii Radix were determined.The contents of the index components of Gentiana scabra Bge.and Gentiana rigesceras Franch were significantly different.The collected local gentian ingredients could not reach the level.Pharmacopoeia standards do not recommend specification;currently there are many counterfeit products of Aconiti Kusnezoffii Radix arbutus available on the market,index components are not up to standard,and it is urgent to formulate regulations to manage them;and combined with the establishment of HPLC fingerprints,it is possible to use two kinds of medicinal herbs counterfeits.A more comprehensive identification,combined with the identification of DNA molecules and the results of kinship,provides a strong scientific basis for the development of commodity specifications.4 Combining with the roots of herbal extracts and the results of two-molecule identification,the Chinese medicinal herbs were identified and classified from both genetic and secondary metabolite levels and accurately,and they were combined with visits to the major medicinal herbs markets and producing areas to combine the versions of the Pharmacopoeia and local standards.The literature research and sorting,the preliminary formulation of Gentianae Radix et Rhizoma commodity specifications are divided into two kinds of longdan and jianlongdan,the grade is divided into the goods and the selection of goods,grass Aconiti Kusnezoffii Radix has only one standard cargo,the selection of goods and the goods are two levels. |
PDF Full Text Request