蒙药蜀葵花HPLC指纹图谱及rbcL序列分子鉴定研究

Fingerprint and molecular identification of rbcL sequence of Mongolian medicine Althaeae Roseae Flos by HPLC

目的建立蜀葵花HPLC指纹图谱及rbcL序列DNA条形码(barcoding)分子鉴定方法。方法色谱柱为Welchrom Column C18(300 mm×4.6 mm,5μm),以乙腈-0.1%甲酸溶液为流动相、梯度洗脱,体积流量1.0 mL/min,柱温35℃,检测波长365 nm,进样量10μL;采用“中药色谱指纹图谱相似度评价软件(2012版A版)”进行相似度评价;同时以rbcL为引物,对11份蜀葵花进行PCR扩增并测定序列,运用DNDMAN和MEGA7.0及CodonCode Aligner17.0软件进行分析。结果建立了11份蜀葵花的HPLC指纹图谱,得到21个共有峰,相似度均大于0.88,并标定了金丝桃苷(7号峰)、槲皮素(15号峰)、芹菜素(19号峰)、山柰素(20号峰)4个成分;rbcL序列扩增和测序成功率100%,测序到的长度500 bp,GC含量为44.10%~44.40%,种内遗传距离0~0.0040,平均遗传距离0.0014,变异位点数10个,相似度为99.00%。结论建立的HPLC指纹图谱方法稳定可靠、rbcL序列DNA barcoding分子鉴定方法重复性好,可用于蜀葵花的质量控制。

Objective To establish the fingerprint of Althaeae Roseae Flos by HPLC and the molecular identification method of DNA barcode of rbcL sequence.Methods The fingerprint establishment of Althaeae Roseae Flos was performed on Welchrom Column C18(300 mm×4.6 mm,5μm)with acetonitrile-0.1%formic acid solution as mobile phase for gradient elution,with flow rate of 1.0 mL/min,column temperature of 35℃,detection wavelength of 365 nm,injection volume of 10μL.DNA barcode molecular identification method was used for PCR amplification and determination of rbcL sequence.Results The fingerprints of 11 samples were established,21 common peaks were obtained,their similarities were calculated,and four components(hyperoside,quercetin,apigenin and kaempferide)were determined.The total length of rbcL sequence of 11 samples was measured,and the G+C content was 44.10%—44.40%and genetic distance(K2P)was 0.0014.There were 10 ectopic points and the similarity was 99.00%.Conclusion The two methods are stable and reliable,which can provide basis for the identification and quality control of A.rosea.