Methods Of On-site,Quickly Authentication Of Chinese Herbal Medicine

Flos lonicerae is commonly used and a large amount of Chinese herbal medicines, and they possess biological and pharmaceutical properties such as anti-bacterial, anti-inflammatory, anti-viral, liver protectant, anti-angiogenic, and anti-nociceptive activities. Accoding to the Chinese Pharmacopoeia (Version2010), the only botanical original of flos lonicerae is L. japonica Thunb. However, in previous version of Chinese Pharmacopoeia, L. hypoglauca Miq., L.confusa (Sweet) DC., L. dasystyla Rehd. were also treated as flos lonicerae, that make it misuse widely in folk. Because of the flower morphology and chemical constituents possesses high similarity, it hard to application of morphological characteristics or chemical analysis to distinguishing flos lonicerae from its adulterants. Rapid, accurate and convenient authentication methods were greatly needed.Although many molecular authentication methods such as ISSR、PCR-RFLP、 or DNA barcoding were developed to distinguishing flos lonicerae and its adulterants, the bad accuracy or time-cosuming disadvantages limited it application in routine authtication.In this paper, we presentation4molecular methods to authenticate flos lonicerae and its adulterants, explore rapid molecular authentication method, on site authentication method, mixture adulterants authentication method and Chinese patent medicine authentication methods. The presentation research includes the following5aspects:1. Establishment of flos lonicerae molecular identification method and its applicationFristly, an EST-SSR molecular marker was used to authenticate flos Ionicerae and its adulterants. We searched form EST database and obtained3705EST-SSRs of L. japonica and2818EST-SSRs of L japonica var. chinensis. And we also obtained87EST-SSRs which repeat numbers had significant difference between L. japonica and L japonica var. chinensis using BlastN analysis.15EST-SSRs of different repeat numbers more than6bp were selected and analysis in45samples of honeysuckle.13pairs of EST-SSR primers had PCR products in all samples and jp.ssr4, jp.ssr64and jp.ssr65were used to identify L.japonica, L.japonica var. chinensis and its adulterants.Secondly, a PCR-RFLP method was build to authenticate flos lonicerae and its adulterants.When used ClustalW to alignment flos lonicerae and its adulterants’ nucleotide sequences, a190G/T SNP mutation was found in PsbA-TrnH sequences and a625C/A SNP mutation in TrnL-TrnF sequences. The mutation made flos lonicerae PsbA-TrnH fragment can be digested by Hinf I endonuclease (restriction site GANTC) and TrnL-TrnF fragment digested by Nal IV endonuclease (restriction site GGNN△CC).96flos lonicerae samples and69adulterant samples was amplification by PsbA-TrnH and TrnL-TrnF primer, the PCR products was digested by Hinf I endonuclease and Nal IV endonuclease, respectively. All flos loniceraes were distinguish from its adulterants by agarose gel electrophoresis fragments polymorphism.Thirdly, an allele-specific PCR method was build to authenticate flos lonicerae and its adulterants. An allele-specific PCR primer was designed by Primer Premier5.0software and according to625C/A SNP mutation in TrnL-TrnF sequences. When the annealing temperature was61℃, DNA from L. japonica would be amplified468bp whereas PCR products from all of the9adulterants were324bp.Fourthly, a loop-mediated isothermal amplification method was build to authenticate flos lonicerae and its adulterants. LAMP amplification primers were designed according to625C/A SNP mutation in TrnL-TrnF sequences and tested in21honeysuckles and50other Chinese medicinal medica. When incubation with63℃for1hours, honeysuckles will distinguished with other Chinese medicinal medica by SYBR Green I fluorescence detection and incubation with65℃for1hours can distinguish flos lonicerae and its adulterants.Allele-specific PCR was selected to authentication flos lonicerae on mixture adulterant and Chinese patent medicine, and loop-mediated isothermal amplification was selected to on-site authenticate by the advantages of simple and convenient. When amplification by allele-specific PCR primers LJ1F,/LJ1R, LJ2F/LJ2R, the established method can detect5%of intentional adulteration DNA into L. japonica. Allele-specific PCR was also used to authenticate Chinese patent medicine FuFangYuXingCaoPian (FFYXCP), LingYangGanMaoPian (LYGMP), YinQiaoJieDuPian (YQJDP), the sesult show that all of the three are containing both flos lonicerae and honeysuckle-like adulterants. Verified by PCR-RFLP and TrnL-TrnF cloning and sequencing show that except L. japonica. FFYXCP, LYGMP, YQJDP may also contain L. confuse, L. hypoglauca, L. confuse, L. hypoglauca and L. cranthoides respectively.Loop-mediated isothermal amplification method was selected to rapid detect the toxic Duanchangcao (Gelsmium elegans). The visualisation of the DNA amplification during LAMP was achieved through the inspection of the reaction in daylight on a white background after incubation in63℃with hydroxynaphthol blue reagent. The G. elegans samples were distinguished from Lonicera by changed from violet to sky blue, whereas the Lonicera samples remained violet.2. Rapid extraction of DNA from Chinese medicinal materials by alkaline lysis methodAn alkaline lysis method was improved for rapid extract DNA from TCM and its formulation was optimized. DNA of144Chinese medicinal materials were extraction using the optimized DNA extraction buffer, and as the template to amplify a PCR production using PsbA-TrnH primers for plant materials and CO1primers for animal materials. The results showed that0.5M sodium hydroxide,1%PVP and1%triton X-100in buffer have the optimum effect on DNA extraction of TCM. Use these DNA from144Chinese medicinal materials as templates as DNA temple for PCR amplification, about123Chinese medicinal materials obtain their PCR products with expected size. Flos lonicerae and its adulterants’ DNA were also extracted by alkaline lysis method and PCR amplification with5universal primers ITS2, PsbA-TrnH, rbcL, matK, TrnL-TrnF and all obtain their expected size PCR products.This method was successfully used in molecular identification of Zaocys, Bungarus Parvus and Lonicera samples with specific primers respectively. The whole process of this method could be finished in10minutes and the new method also avoided using toxic reagents such as chloroform or phenol.3. Effect of PCR Enhancer in Molecular Authenticate of Chinese Herbal Medicine and used in flos lonicerae identification. Genomic DNA from180kinds of Chinese herbal medicine was extract by CTAB method and alkaline lysis method, respectively. The DNA temples were amplification with5universal primers (ITS2、PsbA-TrnH、 rbcL、marK、TrnL-TrnF) in a PCR system with and without BSA and PVP, the result show that PCR success rate have a great improve when0.5%BSA and1%PVP as PCR enhancer. Tested in a G/A SNP site on flos lonicerae lipoxygenase gene by allele-specific PCR, show that BSA and PVP can improve genotyping accuracy in TCM molecular authentication.4. Rapid allele-specific PCR to authentication flos loniceraeA rapid flos lonicerae molecular authentication system was build with extraction of DNA by alkaline lysis method and authentication by rapid allele-specific PCR method. When the PCR program was set as:87℃lmin;87℃denature0s,68℃anneal and extend5s for30cycles; and detection by SYBR Green I fluorescence, the PCR amplification time can reduce to26min in9700PCR instrument and genotyping in40min.5. On-site inspection of flos loniceraeSelf-made alkaline lysis DNA extracted kits and isothermal temperature heating tank, hand-held grinding apparatus and ultraviolet lamp was equipped to on-site inspection of flos lonicerae. The result was accord with morphological identification when applied in authentication Duanchangcao on site.Conclusion:The presentation research compared4molecular methods to authentication flos lonicerae; selected allele-specific PCR and loop-mediated isothermal amplification to rapid, accurate and convenient authenticate flos lonicerae and its adulterants. In the paper, we explore rapid molecular authentication method, on site authentication method, mixture adulterants authentication method and Chinese patent medicine authentication methods. We also develop an alkaline lysis method to rapid extract DNA from Chinese herbal medince, combine with PCR enhancer and rapid AS-PCR, the PCR amplification time can reduce to26min in9700PCR instrument and genotyping in40min.In the paper, we develop rapid AS-PCR for rapid authentication system and LAMP method for on-site inspection, success solves rapid, accurate and convenient methods for authenticate flos lonicerae.