三七的18S rRNA,matK基因序列和HPLC化学指纹图谱分析研究

目的分析中药三七Panaxnotoginseng的18SrRNA和matK基因的分子特征和三七的化学指纹特征,为三七的正品药材基原鉴定提供分子和化学依据。方法采用PCR直接测序技术测定三七及其7种伪品的18SrRNA和matK基因部分核苷酸序列以及不同产地三七的DNA分子特征。利用HPLC的化学分析技术,明确产地对三七化学成分的影响,以及三七不同部位的化学指纹特征。结果(1)三七及其7种常见伪品的核糖体18SrRNA基因序列存在很大的差异。(2)不同产地的三七的核糖体18SrRNA和叶绿体matK基因序列特征完全一致,分别与GenBank上已报道的R1型(D85171)和M1型(AB027526)序列吻合。(3)不同产地的三七HPLC指纹图谱相似。(4)三七不同部位均具有其相对稳定的HPLC指纹特征,其中花、叶具有特有的指纹区,根、须根、剪口、筋条等不同商品规格的HPLC指纹图谱比较相似。结论基因序列标记能从分子水平定性分辨三七及其伪品的遗传背景差异,为中药品种标准化提供了先进可行、稳定可靠的分子标准;HPLC指纹图谱分析可以直观地为三七的化学成分定性,三七不同商品规格的特征性指纹有望成为以其为原材料的各种产品的质控标准,而三七不同部位(尤其是花和叶)的HPLC指纹图谱将有望成为制定三七花、三七叶新药用资源质控标准的依据。

Study on molecular and chemical identification of Panax notoginseng by 18S rRNA gene and matK gene sequencing and by HPLC fingerprinting

Objective To identify the origin of famous Sanqi (Panax notoginseng) on the basis of its sequence of nuclear 18S rRNA and chloroplast genes and chemical fingerprint. Method Applying molecular approaches such as PCR amplification and sequencing of nuclear 18S rRNA and chloroplast genes to identify Panax notoginseng and its seven adulterants, also to analyze Panax notoginseng in different localities. Using HPLC fingerprinting to figure out the relation between chemical compositions and its geographical distribution of Panax notoginseng, and to define particular fingerprint of different parts-used of Panax notoginseng as well. Result 1 There are much diversity in two genes among Panax notoginseng and seven adulterants. 2 The sequences of 18S rRNA and matK genes of Panax notoginseng from different localities are identified to genotype R1 and genotype M1 in GenBank (D85171 and AB027526), respectively. 3 The HPLC profiles of methanol extract of Panax notoginseng leaves from different localities revealed that there are different chemical fingerprints. 4 Various parts-used (including main root, lateral root, fibrous root, cutted rhizome) of Radix possess similar HPLC profiles, while the commercial products containing Folium Notoginseng and Flos Notoginseng have organ-specific HPLC profiles. Conclusion DNA molecular markers can be applied to solve identification problems of easily-confused species, even the tracing of geographical origin in the standardization of quality control. HPLC fingerprinting can be qualitatively analyzed commercial Notoginseng samples without genomic DNA, which provide an alternative tool for the standardization of quality control. Otherwise, it is suggested that specific peaks in HPLC profiles among leaf, flower and root of Notoginseng can be used for inspecting parameters of quality control in GAP production