A total of 12 775 SSRs were identified from Scutellaria baicalensis Georgi genomic database, accounting for 2.56% of the total genomic sequences. The result showed that S. baicalensis SSRs were based on 68.32% dinucleotide and 18.63% trinucleotide repeats; CT/GA and TTC/GAA were predominant in the dinucleotide motifs and the trinucleotide motifs respectively. Nine primers were selected to produce highly reproducible SSR bands and were used in studying the genetic diversity of S. baicalensis, 50 individuals from ten populations. 68 SSR polymorphic loci were detected, these loci were polymorphic and displayed 4 to 12 alleles per locus with a mean number of 7; the effect number of alleles was 3. Expected heterozygosities were 0.6 and were far more greater than the average in dicotyledonous plants. PIC (polymorphism information content) was 0.72, Shannon's information index was 1.32, these all proved that S. baicalensis had a high genetic diversity in general. Genetic differentiation among population Gst was 0.131, genetic variation among population accounted for 13.1% and genetic variation within population accounted for 86.9%. The cluster analysis showed that 10 populations S. Baicalensis were classified into 2 groups, but it was not associated with geographical distribution.

High resolution melting (HRM), based on melting curve analysis, requires not only saturating dyes that fluoresce in the presence of double-stranded DNA, but also higher resolution detection equipment. The melting curve is a novel method for sequence matching, genotyping and mutation scanning. The technology is simple, accurate, rapid, closed-tube, low-cost, and high-throughput, which make it gain more and more applications. This review article presents the basic principles, key factors and both the advantage and limitations of HRM. The potential application is discussed in the study of molecular identity of traditional Chinese medicine.
DNA Mutational Analysis ; methods ; Drugs, Chinese Herbal ; classification ; Genotyping Techniques ; methods ; Medicine, Chinese Traditional ; Nucleic Acid Denaturation

A total of 12 775 SSRs were identified from Scutellaria baicalensis Georgi genomic database, accounting for 2.56% of the total genomic sequences. The result showed that S. baicalensis SSRs were based on 68.32% dinucleotide and 18.63% trinucleotide repeats; CT/GA and TTC/GAA were predominant in the dinucleotide motifs and the trinucleotide motifs respectively. Nine primers were selected to produce highly reproducible SSR bands and were used in studying the genetic diversity of S. baicalensis, 50 individuals from ten populations. 68 SSR polymorphic loci were detected, these loci were polymorphic and displayed 4 to 12 alleles per locus with a mean number of 7; the effect number of alleles was 3. Expected heterozygosities were 0.6 and were far more greater than the average in dicotyledonous plants. PIC (polymorphism information content) was 0.72, Shannon's information index was 1.32, these all proved that S. baicalensis had a high genetic diversity in general. Genetic differentiation among population Gst was 0.131, genetic variation among population accounted for 13.1% and genetic variation within population accounted for 86.9%. The cluster analysis showed that 10 populations S. Baicalensis were classified into 2 groups, but it was not associated with geographical distribution.
Alleles ; Cluster Analysis ; Genetic Variation ; Genomics ; Microsatellite Repeats ; Scutellaria baicalensis ; genetics ; Trinucleotide Repeats

In this paper, Liuwei Dihuang pill was used to study the identification of Chinese patent medicine by fluorescence sequencing typing technology. The DNA of Paeonia suffruticosa was used as template to amplify by five pair of FAM fluorescence labeling primers. Then, the amplified products were sequenced. The sequencing results were analyzed by GeneMarker V1.80 to screen the best fluorescence labeling primers. As a result, psbA-trnH fluorescence labeling primer was used to identify the raw materials of Liuwei Dihuang pill. The results showed that three kinds of raw plant medicinal materials in Liuwei Dihuang pill were able to be correctly identified by psbA-trnH fluorescence labeling primer. The fluorescence sequencing typing technology can stably and accurately distinguish raw medicinal materials in Chinese patent medicine.
DNA Primers ; chemistry ; genetics ; DNA, Plant ; chemistry ; genetics ; Drugs, Chinese Herbal ; chemistry ; standards ; Fluorescent Dyes ; chemistry ; Plants, Medicinal ; chemistry ; genetics ; Polymerase Chain Reaction ; instrumentation ; methods ; Quality Control ; Staining and Labeling

To obtained an accurate, rapid and efficient method for authenticate medicinal snakes listed in Chinese Pharmacopoeia (Zaocysd humnades, Bungarus multicinctus, Agkistrodon acutus), a rapid PCR method for authenticate snakes and its adulterants was established based on the classic molecular authentication methods. DNA was extracted by alkaline lysis and the specific primers were amplified by two-steps PCR amplification method. The denatured and annealing temperature and cycle numbers were optimized. When 100 x SYBR Green I was added in the PCR product, strong green fluorescence was visualized under 365 nm UV whereas adulterants without. The whole process can complete in 30-45 minutes. The established method provides the technical support for authentication of the snakes on field.
Animals ; China ; DNA Primers ; genetics ; Polymerase Chain Reaction ; methods ; Reptilian Proteins ; genetics ; Snakes ; classification ; genetics

To simply and rapid authenticate Lonicera japanica. Rapid allele-specific PCR primer was designed base on trnL-trnF 625 G/T Single nucleotide polymorphism and the PCR reaction systems including annealing temperature was optimized; optimized results were performed to authenticate L. japanica and its 9 adulterants. When 100 x SYBR Green I was added in the PCR product of 87 degrees C initial denatured 1 min; 87 degrees C denatured 5 s, 68 degrees C annealing 5 s, 30 cycle; L. japanica visualize strong green fluorescence under 365 nm UV lamp whereas adulterants without. The results indicate rapid allele-specific PCR could authenticate L. japanica and its adulterants rapidly and simply.
Alleles ; DNA Primers ; genetics ; Drug Contamination ; prevention & control ; Lonicera ; classification ; genetics ; Polymerase Chain Reaction ; methods ; Quality Control

High resolution melting (HRM) , an important technology for genotyping and mutation scanning, has broad prospects in the authenticity of traditional Chinese medicine. This paper selected universal CO I primers and used HRM to establish a new method for authenticity of Hairy Antler. PCR was conducted at the annealing temperature of 60 °C and 45 cycles. The range of the DNA template concentration, the primer concentration and the Mg2+ ion concentration were further optimized. The results showed that the Tm values of Cervus nippon were (81.96 ± 0.07), (84.51 ± 0.03) °C and Cervus elaphus was(82.58 ± 0.13), (85.95 ± 0.05) °C with 10-100 mg · L(-1) DNA template, 0.2 µLmol · L(-1) primer, 2.0 mmol · L(-1) Mg2+. This method can authenticate of hairy antler and is simple, fast, high-throughput, visualization.
Animals ; Antlers ; chemistry ; DNA ; chemistry ; genetics ; DNA Primers ; genetics ; Deer ; classification ; genetics ; Genotype ; Medicine, Chinese Traditional ; standards ; Polymerase Chain Reaction ; Transition Temperature

The study is aimed to explore a rapid method to extract DNA from fried Chinese medicinal products. The alkaline lysis buffer was made of sodium hydroxide, 1% PVP and 1% TritonX-100 and Tris-HCl solution was neutralized, through heat cracking and neutralization two step to extract DNA from processed and prepared products of traditional Chinese medicine. Then universal primes were used to amplify PCR products for fired Chinese medicinal materials. The results indicated the optimized alkaline lysis method for extracting DNA is quick and easy. Extracting of the different processed Sophora japonica of DNA concentration was (420.61 ± 123.91) g x L(-1). Using 5% Chelex-100 resin purification can improve the DNA concentration. Our results showed that the optimized alkaline lysis method is suitable for Chinese medicinal materials for quickly DNA extraction.
Alkalies ; chemistry ; Chemical Fractionation ; methods ; DNA, Plant ; genetics ; isolation & purification ; Hydrolysis ; Plants, Medicinal ; chemistry ; classification ; genetics ; Polymerase Chain Reaction ; Sophora ; chemistry ; classification ; genetics

Molecular identification of Chinese traditional medicine has come from laboratory research into application, but there are some misunderstandings and problems emerging after rapid development. In this paper, we discuss the usage principle, hot field and technology innovation in molecular identification of Chinese traditional medicine. And molecular identification of traditional Chinese medicine has scientific and objective basis, follows the certain systematic research background, and adopts practical principles to establish case by case multi-class identification system. In order to achieve rapid, on-site, high throughput, low cost of traditional Chinese medicine identification purpose, molecular identification technology is further developing for meet the actual needs and the laboratory results further transformation in the service of traditional Chinese medicine industry.
China ; Drugs, Chinese Herbal ; chemistry ; standards ; Medicine, Chinese Traditional ; Plants, Medicinal ; chemistry ; classification ; genetics ; Quality Control

OBJECTIVETo observe the anti-inflammatory effect of total saponins of Panax japonicus on LPS-induced RAW264. 7 macrophages.

METHODThe effect of total saponins of P. japonicus of different concentrations on RAW264. 7 cell viability was determined with the MTT method. The NO kit assay was adopted to detect the NO release of total saponins of P. japonicus to LPS-induced RAW264. 7 cells. The enzyme linked immunosorbent assay (ELISA) was used to detect the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin 1-beta (IL-1beta). The reverse transeriptase-polymerase chain reaction (RT-PCR) was used to determine the expression of inducible nitric oxide synthase (iNOS) ,TNF-alpha,IL-1beta. The protein expression of nuclear transcription factor-kappaB p65 (NF-kappaB p65) was tested by Western blot.

RESULTThe safe medication range of total saponins of P. japonicus was less than 80 mg x L(-1). Compared with the LPS model group, total saponins of P. japonicus high, middle and low dose groups (0.1, 1, 10, 40 mg x L(-1)) could significantly reduce the secretion of NO, TNF-alpha, IL-1beta of LPS-induced RAW264. 7 cells, and inhibit the expressions of iNOS, TNF-alpha and IL-1beta mRNA and the protein expression of NF-kappaB p65.

CONCLUSIONThis study preliminarily proves the protective effect of total saponins of P. japonicus on LPS-induced RAW264.7 macrophages. Its action mechanism may be related to NF-kappaB signal pathway.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Inflammation ; drug therapy ; genetics ; immunology ; Interleukin-1beta ; genetics ; immunology ; Lipopolysaccharides ; adverse effects ; Macrophages ; drug effects ; immunology ; Mice ; NF-kappa B ; genetics ; immunology ; Nitric Oxide ; immunology ; Nitric Oxide Synthase Type II ; genetics ; immunology ; Panax ; chemistry ; Protective Agents ; pharmacology ; Saponins ; pharmacology