摘要
目的:基于超高效液相色谱-四极杆-飞行时间质谱法(UPLC-Q-TOF-MS)、解吸电喷雾电离质谱成像(DESI-MSI)、高光谱成像技术对天元芷通方入脑成分及其脑内分布进行探究。方法:10只BALB/c小鼠随机分为空白组(n=3)和给药组(n=7),给药组灌胃生药质量浓度约5 g·mL-1的天元芷通方药液0.3 mL,空白组灌胃等体积生理盐水,取小鼠全脑分别制备组织匀浆及冰冻切片。组织匀浆采用UPLC-Q-TOF-MS技术分别在正、负离子模式下进行入脑成分的定性分析。冰冻切片采用DESI-MSI观察入脑成分的脑内分布。选用暗场增强显微镜对各组小鼠脑切片进行高光谱成像扫描,收集微观视场中至少1000个像素点可见-近红外(400~1000 nm)波段的散射光数据并创建平均光谱,对空白及给药小鼠脑组织中成分进行比对。结果:UPLC-Q-TOF-MS共鉴定出天元芷通方入脑成分27个,包括有机酸类10个、苷类5个、生物碱类4个、酚类1个、黄酮类4个、苯酞类2个、其他类1个,主要来源于天麻、川芎、醋延胡索、酸枣仁和制巴戟天;质谱成像分析得到14个成分,其中阿魏酸、延胡索乙素、N-甲基去氢小檗碱主要分布在大脑皮层区域,维生素B5、斑鸠菊酸、蓖麻油酸主要分布在下丘脑区域,榄香脂素、十八烯酸、十八碳酸主要分布在皮层及下丘脑区域,洋川芎内酯B、藁本内酯、亚油酸、9,12-十八碳二烯酰乙酯、斯皮诺素则在脑组织大部分区域均有分布;高光谱成像显示,在可见-近红外波段范围内,给药组小鼠脑组织平均光谱发生明显红移,表明空白组和给药组小鼠间脑内化学成分的物理性质或含量存在差异,进一步验证了质谱成像的结果。结论:通过UPLC-Q-TOF-MS技术与质谱成像、高光谱成像技术的结合,明确了天元芷通方入脑治疗头痛的药物成分及在脑组织中的分布特征,为复方质量标准研究指标成分的选择和药效作用机制研究提供参考。
Objective:To investigated the components and distribution of Tianyuan Zhitong prescription into the brain based on ultra-high performance liquid chromatography-Quadrupole time-of-flight mass spectrometry(UPLCQ-TOF-MS),mass spectrometry and hyperspectral imaging techniques.Method:10 BALB/c mice were randomly divided into blank group and administration group.The blank group was intragastric with normal saline,and the administration group was intragastric with Tianyuan Zhitong formula.The whole brain was prepared with tissue homogenate and frozen section respectively.The tissue homogenates were qualitatively analyzed in positive and negative ion mode by UPLC-Q-TOF-MS technique,gradient elution of methanol(A)-0.1%formic acid aqueous solution(B),electrospray ionization source(ESI),and m/z 100-1800 scanning.Frozen sections were used for imaging to observe the distribution of components in the brain.The image parameters of mass spectrometry were electrospray solvent 95%methanol,flow rate 5μL·min-1,Sprayer incidence Angle 60°,collection Angle 10°,the positive and negative scanning rate is 300μL·min-1,the mass spectrometry analysis mode is sensitive mode,m/z50~1200 full scanning.Cytoviva darkfield enhancement microscope was used to optically observe the blank and administered mouse brain sections,×20 objective lens immersed in oil for hyperspectral imaging,the hyperspectral system scanned the slices,and collected the scattered light data of at least 1000 pixels in the visible-near-infrared(VNIR,400~1000 nm)band in the microscopic field of view and created an average spectrum.Result:A total of 27 components were identified by UPLC-Q-TOF-MS,including 10 organic acids,5 glycosides,4 alkaloids,1 phenols,4 flavonoids,2 phthalides and 1 other compounds.14 components were identified by mass spectrometry,including ferulic acid,ligusticolactone B,ligustilide,linoleic acid,9,12-octadecadienoyl ethyl ester,elietin,fumaricin,nmethyldehydroberberine,spinotin,vitamin B5,octadecenoic acid,octadecanoic acid,cuculinic acid and ricinoleic acid,which were mainly distributed in the cerebral cortex and hypothalamus.Hyperspectral imaging showed that in visible-near infrared band range,the average spectrum of mice brain tissue in administration group was significantly redshifted,indicating that there were differences in physical properties or contents of chemical components in brain between the blank group and the administration group.Conclusion:Through the combination of UPLC-Q-TOF-MS technique and imaging technique,the pharmacodynamic components of Tianyuan Zhitong prescription in the treatment of headache and the regional characteristics in brain tissue were clarified,which provided reference for the establishment of the quality standard of the compound and the study of the pharmacodynamic material basis.
作者
程怡
兰群
吴冰雨
王锦玉
刘德文
仝燕
CHENG Yi;LAN Qun;WU Bingyu;WANG Jinyu;LIU Dewen;TONG Yan(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第12期166-172,共7页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(82003951)。
