豆制何首乌中异黄酮成分的检测及对BMSCs增殖的影响  

Determination of Isoflavones and Effect on Proliferation of BMSCs in Bean-processed Polygonum multiflora Radix Praeparata

作　　者：郭千祥 史旭华 梁幼玲 刘铭超[1] 宋嬿 白俊其 黄志海[1] 丘小惠[1,3] GUO Qianxiang;SHI Xunhua;LIANG Youling;LIU mingchao;SONG Yan;BAI Junqi;HUANG Zhihai;QIU Xiaohui(The Second College of Clinic Medicine of Guangzhou University of Chinese Medicine,Guangzhou 510120,Guangdong,China;Shanghai Huayu Chinese Herbs Co.LTD,Shanghai 200002,China;Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese Medicine Syndrome,Guangzhou 510006,Guangdong,China)

机构地区：[1]广州中医药大学第二临床医学院,广东广州510120 [2]上海上药华宇药业有限公司,上海200002 [3]广东省中医症候临床研究重点实验室,广东广州510006

出　　处：《中华中医药学刊》2020年第7期157-162,I0011,共7页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金(81373967);国家发展改革委、国家中医药管理局中药标准化项目(ZYBZH-Y-SH-38);广东省科技计划(2017B030314166);广东省中医药局科研项目(20184013);广东省中医院中医药科学技术研究专项(中药精准煮散饮片标准化研究及应用启动项目)。

摘　　要：目的采用Q Exactive Plus LC-MS/MS定性鉴定豆制何首乌中微量异黄酮类成分,建立豆制何首乌与清蒸何首乌的薄层色谱(TLC)鉴别方法,并考察大豆苷元、染料木素对骨髓间充质干细胞(BMSCs)的增殖作用。方法采用UPLC-Q-Exactive高性能四极杆母离子选择性与高分辨准确质量数(HR/AM)Orbitrap检测相结合的技术定性鉴定目标成分,选用色谱柱Phenomenex C18柱(100 mm×2.1 mm,1.7μm Phenomenex公司,美国);流动相:乙腈(D)-0.1%甲酸水(A)梯度洗脱,质谱采用负离子监测模式、采用全扫描及二级质谱扫描的功能,对微量目标化学成分进行快速识别;使用硅胶GF254薄层板对豆制何首乌中黑豆异黄酮类成分进行定性鉴别;采用MTT法检测大豆苷元、染料木素对BMSCs的增殖作用。结果UPLC-Q-Exactive四级杆-静电场轨道阱高分辨质谱技术,使用少量对照品快速分析鉴定出豆制何首乌中微量的目标成分,具有高效分离及高灵敏检测优势;TLC鉴别大豆苷元斑点特征明显,专属性强、重复性好,方法简便;染料木素对BMSCs具有抑制作用,大豆苷元具有促进BMSCs增殖的作用。结论豆制何首乌中可检测到异黄酮微量组分,其中大豆苷元为LC-MS与TLC较易检测成分,可作为TLC鉴别豆制何首乌与清蒸何首乌的特征成分,可能为豆制何首乌补血功效增强的药效物质基础之一,可作为其质量控制的指标成分。Objective Q Exactive Plus LC-MS/MS was used to qualitatively identify trace isoflavones in bean-processed Polygonum multiflora radix praeparata.It is to establish a thin layer chromatography(TLC)method for the determination of Polygonum multiflora radix praeparataprocessed with steaming method or black beans,andinvestigate the proliferation of bone marrow mesenchymal stem cells(BMSCs)by daidzein and genistein.Method UPLC-Q-Exactive high-performance quadrupole precursorion selective and high-resolution accurate mass(HR/AM)Orbitrap detection combined qualitative identification of target components wereused.Phenomenex C18 column(100 mm×2.1 mm,1.7μm phenomenex company,USA)was selected.The mobile phase was acetonitrile(D)-0.1%formic acid water(A)gradient elution.Mass spectrometry used negativeion monitoring mode,full scan and two-stage mass spectrometry to quickly identify trace target chemical components.The isoflavones of Polygonum multiflora radix praeparata processed with beans were qualitatively identified by silica gel GF254 thin-layer plate.The proliferation of BMSCs by daidzein and genistein was detected by MTT assay.Result Using a small number of reference substances to quickly identify the target components,the method had the advantages of high efficiency separation and high sensitivity detection.The identification of daidzein spots by TLC was obvious,the specificity was strong,the repeatability was good,and the method was simple to operate.Genistein had an inhibitory effect on BMSCs anddaidzein had the effect of promoting the proliferation of BMSCs.Conclusion Multiple isoflavone trace components can be detected in bean-processed Polygonum multiflorum radix praeparata and daidzein can be used as a TLC method for determination of Polygonum multiflora radix praeparata processed with steaming method or black beans.It may be one of the effective components for enhancing the blood-supplying effect of Polygonum multiflora radix praeparata,and can be used as an indicator component for quality control.

关 键 词：豆制何首乌 Q-Exactive Plus LC-MS/MS 大豆苷元 染料木素 薄层色谱 BMSCS 

分 类 号：R284.1[医药卫生—中药学]