| Astragalus is a traditional Chinese medicine herb with efficacies of Qi-invigorating,nourishing blood,benefiting water and dispersing edema,etc.With the gradual increase of Astragalus dosage,wild Astragalus has been in short supply,most of the commercially available Astragalus is areartificial planted.As a result,the quality of Astragalus is uneven,with fake,shoddy phenomenon occurs from time to time.In this paper,the difference of ITS sequence between genuine and common counterfeit Astragalus was used to design specific primers to identify the authenticity of Astragalus.q-PCR method was adopted based on the traditional PCR method with SYBR Green fluorescent dye to identify the authentic and counterfeit Astragalus.Meanwhile,the content of astragaloside IV was determined according to the method stipulated in Chinese Pharmacopoeia,and the accuracy of the molecular biological method was verified according to the difference of astragaloside IV content.The contents of astragaloside IV in Astragalus samples from different producing areas,dosage forms and processing methods were obtained to provide a basis for further regulating the cultivation of medicinal materials.The main results are as follows:1.ITS primers were used to amplify Astragalus samples and pseudo-Astragalus.The different regions were found by analyzing and comparing ITS sequences of the two.The Primer 5 software was used todesign a specific primer for identifying Astragalus,named HQ-F/HQ-R,with the length of 20/21 bp respectively and the amplification product length was524 bp.The primers were used to amplify the genuine and counterfeit Astragalus and its near source species,and the annealing temperature gradient was set.The results showed that when the annealing temperature was 58 ℃,the genuine Astragalus samples had a single,clear strip between500 and 700 bp,while other samples had no strip in the corresponding position.Therefore,58 ℃ was chosen as the best annealing temperature.2.Purchase 23 copies of Astragalus samples,including Astragalus membranaceus,Astragalus mongolian,Astragalus formula particles and processed Astragalus,and use primer HQ-F/HQ-R to amplify the samples with annealing temperature of 58 ℃.The specificity,sensitivity,coverage and so on were investigated by using traditional PCR method and SYBR Green dye method.The results showed that the primers of the two methods had good specificity,and there were no amplification bands(curves)except the genuine samples.For Astragalus samples from different producing areas,dosage forms and processing methods,a single amplification band(curve)can be obtained by using the two methods.The 10-fold dilution of the samples was performed with 3 replicates for each dilution level.The amplification results showed that the ordinary PCR method has a sensitivity of 10mg/kg,while the method of SYBR Green dye was 1 mg/kg,so the SYBR Green dye method had a higher sensitivity.At the same time,therelative quantification of amplified products can be realized by SYBR Green method.3.Extract astragaloside IV from samples and detecte its contents by HPLC-ELSD method according to Chinese pharmacopoeia(2015 edition).The reliability and accuracy of the early molecular biological identification method was verified according to the contents of astragaloside IV in different samples.The results showed that the contents of astragaloside IV in the genuine Astragalus were all meet the requirements except Astragalus produced in Litang county,Sichuan province.However,the content of astragaloside IV in non-Astragalus samples were lower than the minimum,and the results were consistent with the results of molecular biological identification.At the same time,it is concluded that the contents of astragaloside IV in Astragalus from different origins vary greatly,among which,the top four are Dingxi of Gansu province,Hunyuan of Shanxi province,Liupanshan of Ningxia province and Heilongjiang province,and the lowest is Astragalus from Litang county of Sichuan province. |
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