ITS2序列鉴定甘草混淆伪品的研究
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摘要
为了对中药材甘草及其混伪品进行分子鉴定,以区分正品甘草与混伪品。从兰州市不同商家收集9种甘草,提取DNA,扩增ITS2序列并进行双向测序,在GenBank数据库收集甘草混伪品苦豆子、苦参、黄芪和山豆根的序列,应用MEGA 7.0进行序列变异分析,以Kimura 2-Parameter(K2P)模型计算遗传距离,并构建邻近(NJ)树。基于ITS2序列的序列变异、最近距离法和系统邻近树分析结果表明,该方法可将2015版《中华人民共和国药典》中规定的正品甘草与其混伪品进行区分。说明ITS2序列可有效区分5种甘草混伪品,为中兽医和中医临床应用提供保障依据。
Radix Glycyrrhizae is the most commonly used TCM or TCVM clinic,but the phenomenon of Radix Glycyrrhizae confounding products had caused great confusion to the clinical safety of TCM and TCVM,and it was difficult to satisfy the clinical drug use only by experience identification. In the study,molecular identification of Radix Glycyrrhizae and its adulteration was carried out to distinguish genuine Radix Glycyrrhizae from adulterates. In this experiment,9 kinds of Radix Glycyrrhizae were collected from different merchants in Lanzhou,ITS2 sequence was amplified and bidirectional sequencing was carried out after DNA was extracted,and the results were provided by sequencing company. Radix Glycyrrhizae,Radix Sophorae Flavescentis,Radix Astragali and Radix Sophorae Tonkinensis,which were mixed with Radix Glycyrrhizae. The sequence source of Radix Glycyrrhizae adulterant were collected from GenBank. The sequence of astragalus root and soybean root was analyzed by MEGA7.0. The genetic distance was calculated by Kimura 2-Parameterus K2 P model,and the adjacent tree was constructed. Based on the sequence variation of ITS2 sequence,the results of nearest distance method and phylogenetic proximity tree analysis showed that licorice,as a product of Chinese Pharmacopoeia,could be distinguished from its adulterates. 5 kinds of licorice adulterants can be distinguished effectively by the sequence of ITS2,which can provide evidence for traditional Chinese veterinary medicine and TCM clinic.
Radix Glycyrrhizae is the most commonly used TCM or TCVM clinic,but the phenomenon of Radix Glycyrrhizae confounding products had caused great confusion to the clinical safety of TCM and TCVM,and it was difficult to satisfy the clinical drug use only by experience identification. In the study,molecular identification of Radix Glycyrrhizae and its adulteration was carried out to distinguish genuine Radix Glycyrrhizae from adulterates. In this experiment,9 kinds of Radix Glycyrrhizae were collected from different merchants in Lanzhou,ITS2 sequence was amplified and bidirectional sequencing was carried out after DNA was extracted,and the results were provided by sequencing company. Radix Glycyrrhizae,Radix Sophorae Flavescentis,Radix Astragali and Radix Sophorae Tonkinensis,which were mixed with Radix Glycyrrhizae. The sequence source of Radix Glycyrrhizae adulterant were collected from GenBank. The sequence of astragalus root and soybean root was analyzed by MEGA7.0. The genetic distance was calculated by Kimura 2-Parameterus K2 P model,and the adjacent tree was constructed. Based on the sequence variation of ITS2 sequence,the results of nearest distance method and phylogenetic proximity tree analysis showed that licorice,as a product of Chinese Pharmacopoeia,could be distinguished from its adulterates. 5 kinds of licorice adulterants can be distinguished effectively by the sequence of ITS2,which can provide evidence for traditional Chinese veterinary medicine and TCM clinic.
引文
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[5]底迎亚,卢亚品,于贺,等.甘草泻心汤治疗化疗后口腔溃疡的应用[J].中国中医药现代远程教育,2018(2):88-89.
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[14]许玲,张申.炙甘草,黄芪,党参对心气虚患者心脏功能和血管状况影响比较研究[J].中医研究,1997(2):31-32.
[15]乔留卷,李平凡,郭金帅,等.加味芍药甘草汤治疗马属动物脾胃虚弱[J].中国兽医杂志,2000(5):40.
[16]王思文.炙甘草汤的临床应用及实验研究[J].吉林中医药,2018,38(1):96-98.
[17]刘鸿杰,黎杰轩,梁延昌,等.炙甘草汤对慢性疲劳综合征大鼠行为学免疫球蛋白和干扰素水平的影响[J].中国兽医杂志,2018,54(3):95-97+103.
[18]纪云飞,王瑞君,李晓波.复方四君子汤的化学成分和药理作用研究进展[J].中草药,2016,47(5):837-843.
[19]孟良艳,陈秀琴,石达友,等.四君子汤对脾虚大鼠肠道菌群多样性的影响[J].畜牧兽医学报,2013,44(12):2029-2035.
[20]姚金彤,马莹莹.二陈汤的临床应用研究进展[J].世界最新医学信息文摘,2018,18(68):58-60.
[21]张国士.加减二陈汤在兽医临床上的应用[J].河南畜牧兽医(综合版),2001,22(6):33-33.
[22]杨宋琪,唐小龙,陈海燕,等.基于ITS2条形码的贯叶金丝桃及其混伪品鉴别研究[J].中国药业,2018,27(11):1-4.
[2]Li J Y,Cao H Y,Liu P,et al.Glycyrrhizic acid in the treatment of liver diseases:literature review[J].Biomed Research International,2014,2014(8):872139.
[3]Methlie P,Husebye E E,Hustad S,et al.Grapefruit juice and licorice increase cortisol availability in patients with Addison apos;disease[J].European Journal of Endocrinology,2011,165(5):761-769.
[4]李秋莉.炙甘草汤加减治疗冠心病心律失常的疗效评价[J].临床医药文献电子杂志,2018,5(1):165+167.
[5]底迎亚,卢亚品,于贺,等.甘草泻心汤治疗化疗后口腔溃疡的应用[J].中国中医药现代远程教育,2018(2):88-89.
[6]朱飞叶,杜羽,谢冠群.芍药甘草汤治疗便秘的文献及应用研究[J].浙江中医药大学学报,2017(3):186-188.
[7]朱美玲,王林娜,胡培,等.分子鉴定技术在南药研究中的应用进展[J].世界中医药,2018,13(2):522-526.
[8]Zhang J,Hu X,Wang P,et al.Investigation on species authenticity for herbal products of Celastrus Orbiculatus,and Tripterygum Wilfordii,from Markets Using ITS2 Barcoding[J].Molecules,2018,23(4):967.
[9]Mei Q,Chen X,Xiang L,et al.DNA barcode for identifying folium artemisiae argyi from counterfeits[J].Biological&Pharmaceutical Bulletin,2016,39(9):1531-1537.
[10]Lv T,Teng R,Shao Q,et al.DNA barcodes for the identification of Anoectochilus roxburghii and its adulterants[J].Planta,2015,242(5):1167-1174.
[11]尹艳,刘逊,王兵,等.中药穿山甲的DNA分子鉴定研究[J].中国中药杂志,2017,42(11):2078-2084.
[12]袁伯川.柴胡属药用植物的分子鉴定及柴胡药材的质量考察[D].北京:北京中医药大学,2017.
[13]黄辉庆,黄志海,黄娟,等.应用ITS2条形码鉴定岭南药材余甘子及其混淆中药品种[J].世界科学技术:中医药现代化,2016,18(8):1419-1423.
[14]许玲,张申.炙甘草,黄芪,党参对心气虚患者心脏功能和血管状况影响比较研究[J].中医研究,1997(2):31-32.
[15]乔留卷,李平凡,郭金帅,等.加味芍药甘草汤治疗马属动物脾胃虚弱[J].中国兽医杂志,2000(5):40.
[16]王思文.炙甘草汤的临床应用及实验研究[J].吉林中医药,2018,38(1):96-98.
[17]刘鸿杰,黎杰轩,梁延昌,等.炙甘草汤对慢性疲劳综合征大鼠行为学免疫球蛋白和干扰素水平的影响[J].中国兽医杂志,2018,54(3):95-97+103.
[18]纪云飞,王瑞君,李晓波.复方四君子汤的化学成分和药理作用研究进展[J].中草药,2016,47(5):837-843.
[19]孟良艳,陈秀琴,石达友,等.四君子汤对脾虚大鼠肠道菌群多样性的影响[J].畜牧兽医学报,2013,44(12):2029-2035.
[20]姚金彤,马莹莹.二陈汤的临床应用研究进展[J].世界最新医学信息文摘,2018,18(68):58-60.
[21]张国士.加减二陈汤在兽医临床上的应用[J].河南畜牧兽医(综合版),2001,22(6):33-33.
[22]杨宋琪,唐小龙,陈海燕,等.基于ITS2条形码的贯叶金丝桃及其混伪品鉴别研究[J].中国药业,2018,27(11):1-4.