The identification of the national drug Zidansan and its proximity based on DNA bar code technology.

Abstract: Objective To explore the application of DNA bar code technology in the identification of the national medicine zidanginseng and its near-species, and to provide reference for the identification of zidan ginseng medicinal herbs.
method to establish the DNA bar code reference database of Zidan ginseng and its mixed fakes, and analyze the data by comparing sequence, analyzing the variation site, calculating the genetic distance and constructing adjacent trees, and using the database to identify the market medicinal materials.
results DNA bar code technology can effectively identify zidan ginseng and its mixed artifacts.
Conclusion ITS2 sequence can be used as the identification bar code of Zidan ginseng and its mixed counterfeits, and the technology has important application value in the practical application of national drug seed identification, standard-setting and market supervision.
Zidansan, also known as Yunnan sage, yan dansan, etc., is the dry root of the lip-shaped plant Yunnan sage Salvia Yunnanensis C.H. Wright, mainly produced in Yunnan, Sichuan and other places, for the Yunnan Province Chinese Herbal Medicine Standard, with varieties, with Pain relief, live blood, the effect of mind removal, for menstruation, intercessal syllation,accumulation, chest and abdominal tingling, cerebral palsy pain, sores, restlessness, liver and spleen, angina and other asymptomatic.
it has a long history of drug use in Yunnan folk, originally seen in the "Nanherbs" recorded "one taste of four things can be offset to make up blood", the formation of preparations dandenong women's and kang frying paste, purple dan live blood tablets and other drugs for its junices , with a high value of development and utilization.
its main components are dansorone I, IIA, IIB, pentonabinol, dansanol, vitamin E, etc., with a variety of pharmacological effects to inhibit platelet aggregation, increase coronary blood flow and other pharmacological effects.
in recent years, due to over-mining, the resources of Zidan ginseng are increasingly scarce, and even face endangered.
there are more concrites on the market at present, after literature and market research, in addition to other medicinal herbs cattle are easy to confuse, there are a variety of sage plants in the folk as purple danginseng use, mainly related to dansan S.miltiorrhiza Bunge, leaves salet s. bulleyana Diels, trijudl s. trijugals, mlyning Sage S.przewalskii Maxim., buckwheat sage s.kiaometiensis L?vl., hairy weasel S.digitaloids Diels, orange sage S.aerea L?vl., maroon sage S.castanea Diels, etc.
there are currently applied characteristics, microscopy, physical and chemical (infrared spectra, thin layer chromatography) and fingerprint mapping methods to identify the same plant Dansin and its mixed artifacts of the report, because of the complexity of the mixed pseudo-counterfeiting of zidanginseng, these methods are vulnerable to biological growth and development, external environmental changes and subjective factors, such as the impact of 11, can not achieve effective identification, clinical drug safety is a great threat.
DNA barcode technology is one of the hot topics of current biological research, has been widely used in chinese medicinal herbs (excluding minerals) of the base identification and the identification of its mixed artifacts, and achieved good results.
Chen Shilin and others have shown that the ITS2 sequence is an ideal barcode for the identification of medicinal plants and their mixtures.
, some scholars have used DNA bar code technology to study the identification of Dansan, Gansu Dansan and yak, indicating that the technology can effectively identify 3 types of medicinal herbs, because the study only selected 3 types of samples, and did not contain purple Dansan samples, can not be used for the identification of zidan ginseng and its mixed counterfeits.
in view of this, the study used DNA bar code technology to study the identification of zidan ginseng and its proximity.
on this basis, 45 batches of commercially available medicinal herbs labeled Zidansan and Dansan were identified, with a view to providing scientific reference for the safety and quality control of zidansan medicine.
1 Instrument s1.1 Instrument 1-14K High-Speed Refrigeration Centrifuge (Sigma Company); 6670 Plant Tissue Grinder (SPEX Sample Prep Company); Biometra Tone PCR Meter; DYY-8C Type Electric Phoine Meter (Beijing 61 Instrument Factory); JY04S-3C Uv Gel Imaging Analyzer (Beijing Junyi Oriental Electrophorede Equipment Co., Ltd.);
1.2 Reagent Plant Genome Extraction Kit (Tiangen Biotech Co., China) ;D L2000 DNA marker was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.;
27 plant samples of 1.3 materials, including Yunnan sage Salviayunnanensis C.H. Wright, Dansan S.miltirhiza Bunge, saster sage sesame sesames S. bulleyana Diels, clover sage S. trijuga Diels 4 species, identified by the author of the plant characteristics.
also downloaded 19 IT2 sequences of common counterfeits from GenBank, including Gansi S. przewalskii Maxim., buckwheat sage S.kiaometiensis L?vl., And Yukio Weasel S.digitaloids Diels, orange sage S.aerea L?vl., maroon sage S.castanea Diels, lychee s.plebea R.Br. and 6 other species, see table 1.
a total of 46 ITS2 sequences from the 10 species mentioned above are used to construct a standard barcode database for zitan ginseng and proximity species.
23 batches of Zidanginseng and 22 batches of Dansan medicinal herbs purchased in the market, by Dali University Professor Xia From long and the author according to the characteristics of identification, the two identification results are consistent to retain the sample, inconsistent when discarded (Table 2).
2 method 2.1 DNA extraction of medicinal herbs using 75% ethanol to clean the surface, placed in 75% ethanol soaked 3 min, dried after taking 40 to 50 mg of liquid nitrogen grinding;
total DNA extraction is carried out according to the plant tissue extraction kit operation procedure, part of the step is adjusted, after tissue grinding, using nuclear separation buffer (100 mmol/L Tris-HCl, pH8.0; 20 mmol/L EDTA, pH 8.0; 0.0 3mmol/L NaCl;2% PVP40; 2% beta-pyridox ethanol) was pumped 2 times, water bath temperature was 56 degrees C, water bath time was 10h, and chloroform-isosterol (24:1) was pumped 2 times after a water bath.
DNA precipitation, the pre-cooled isopropanol was changed to a pre-cooled isopropanol at .20 oC.
2.2 PCR amplification and sequencing OF2 sequence amplification primers, as well as PCR conditions for reference to the literature, primer synthesis by bioengineering bioengineering (Shanghai) Co., Ltd.
striped PCR products sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for two-way sequencing.
2.3 Data processing DNA sequence data is stitched together by CodonCode Aligner 5.1.5 (CodonCode Co., United States).
HMMer annotation method based on the Hidden Malv model, the resulting sequence and GenBank sequence were removed from the 5.8 S and 28S segments, and the ITS2 sequence was obtained.
the sheared sequence is submitted to the National Biotechnology Information Center (NCBI, ) and the Chinese Medicine DNA Barcode Database (TCM Barcode, ) for comparison and verification.
uses MEGA 6.0 (Molecular Genetic SR. Analysis) to build the NJ tree, Kimura 2-Parameter (K2P) method to calculate the genetic distance.
3 results and analysis 3.1 sequencing results analysis 27 batch of plant samples including 4 sage species, the leaf DNA amplification and sequencing success rate are 100%, 23 batches of purple dansin herbs, 18 batches of successful amplification, sequencing success rate of 100%.
after the failure to expand the 5 batches of medicinal herbs analysis, the failure may be due to the zifan ginseng herbs are mainly wild resources, mostly for the private small amount of collection, its drying methods are diversified, the 5 batches of medicinal herbs in the drying process, may be used a higher temperature for baking, destroying the DNA of the herbs.
and 22 batches of dansan herbs its sequencing and amplification success rate are 100%, because dansan is planted artificially, its processing method specifications, but also from the side to confirm the above speculation.
3.2 ITS2 series interspecies genetic distance analysis used to construct reference database 46 ITS2 sequences (27 plant sample sequences, 19 GenBank download sequences) length range of 228 to 230 bp, GC content of 62.28% to 68.85%. The genetic distance between 13 samples in the
Zidan ginseng species is 0 to 0.018, and the K2P distance between other close proximity species is 0.027 to 0.132.
the 7 samples in the Dansan species were 0 to 0.009, and the K2P distance between the other proximity species was 0.027 to 0.132.
the intra-species genetic distance of zidan sansand and dansan are less than the genetic distance between species, indicating that the ITS2 sequence can be used for the identification of zidanginseng and Dansan and its mixed artifacts.
3.3 cluster analysis based on 46 ITS2 sequences with 10 species, the nJ system cluster tree (Figure 1) showed that 10 species of sage plants were divided into 4, of which Yunnan sage (purple dansan) and dansan, lychee grass 3 species are clustered separately, can be with other near- There was a clear distinction between the other 7 species, gansicil, buckwheat, yass, orange sage, maroon sage, clover, and cess, which showed that the seven species were close lying close, and the USE of ITS2 sequence could not distinguish between the above species.
can be seen through the NJ tree, ITS2 bar code can effectively identify zitan ginseng, dansand and its mixed artifacts.
3.4 Zidan ginseng medicinal identification ability analysis will be 45 batches of two experts in accordance with the characteristics of the identification of medicinal herbs (number HMS-01 to 45), after the extraction of DNA sequencing, the will be THE ITS2 sequence in the NCBI database (NCBI,) and Chinese herbal DNA barcode identification system (TCM-DBS, " similarity search, DNA bar code comparison see table 3.
can be seen from Table 3, 40 batches of zitan ginseng and dansan medicinal DNA identification results and expert character identification results are consistent.
build nJ tree (Figure 2) with 40 batch of herbs IT2 sequence and the 46 reference sample IT2 sequences mentioned above.
can be seen from Figure 2, Zidansan and Dan participate in reference plant samples into one, in which samples HMS-02 to 12 and HMS-16, HMS-17, HMS-19 to 23 and Zidan ginseng polymer as one, sample HSM24 to 45 and Dansan together as one, showing a clear monoline.
mixed goods are gathered together as one, can be distinguished from Zidanginseng and Dansan ginseng.
, the REFORE, THE ITS2 sequence can be used as a zitan ginseng and its mixed counterfeit identification, is the ideal DNA bar code for zidan ginseng identification.
3.5 ZidansDNA DNA barcode standard sequence analysis refers to the 2015 version of the Chinese Pharmacopoeia DNA barcode guidelines, selected a total of 30 sequences of Zidan ginseng herbs and plant samples, compared to the length of 228 bp, there are 4 variation sites, respectively, 31-point A-T mutation, 38-point G-T variation, 170-point T-C variation, 204-point C-T variation.
dominant haplist sequence features are shown in Figure 3.
the code based on open source PHP QR Code encodes the Latin name and ITS2-dominated hapnotype sequence of the Zidan ginseng original species, and obtains a 2D DNA barcode image of Zidansan (Figure 3).
compare the genetic distance of the above 40 batch of medicinal samples with the standard sequence of zitans DNA barcode (YN1825MT27) to calculate the genetic distance, it can be seen that the maximum genetic distance in the zimdenaut species is 0.018 as the limit, which can clearly distinguish Dan's participation in zitan ginseng herbs, the results are shown in Figure 4.
4 Discussion 4.1 ITS2 can be used as zitan ginseng and its mixed counterfeit DNA bar code DNA identification technology for the identification of the original species of medicinal herbs provides a strong scientific and technological support, which is not limited by environmental factors, sample morphology and material site, has become an effective means of identification of Chinese medicinal plants and medicinal varieties.
the study used the method for the identification of the national medicine zidanginseng and its mixed counterfeits, the results showed that the average K2P genetic distance within the zidan ginseng species is less than the average K2P genetic distance between species with the proximity species;
the results of the identification of 40 batches of medicinal materials showed that the DNA bar code results were consistent with the results of expert character identification.
, the ITS2 sequence can be used as a DNA barcode for the identification of zitan ginseng-based plants and proximity species.
4.2 The meaning of THE standard sequence of DNA bar code for the identification of ethnic medicine is the premise of ensuring the quality and efficacy of medicinal herbs.
the identification method of medicinal materials runs through all aspects of planting, processing, production, and the traditional characteristics, microscopy and physical and chemical in the identification of chinese medicinal herbs have certain limitations, it is difficult to meet the hospital, customs and other industries fast, standard identification needs.
the 2015 edition of the Chinese Pharmacopoeia has included DNA barcodes ITS2 and psbA-trnH sequences as candidate sequences.
the study constructed a standard DNA bar code series of zidan ginseng and used it for the identification of 40 batches of medicinal herbs, the results showed that the identification effect was good.
compared with Chinese herbal medicine, ethnic medicine is mostly derived from the folk trade, and the identification can be limited reference, clinical application is not easy to distinguish, has potential hidden dangers.
to build common ethnic medicine.