基于UHPLC-Q-TOF/MS技术分析肿节风总黄酮提取物促进巨核细胞分化的效应成分  

UHPLC-Q-TOF/MS Analysis of the Active Components of Total Flavonoids Extracts from Sarcandra glabra in Promoting Megakaryocyte Differentiation

作　　者：张钟康 卢晓南 卢震 胡佳[1,2] 刘慧珍 卢婷 尚广彬 ZHANG Zhongkang;LU Xiaonan;LU Zhen;HU Jia;LIU Huizhen;LU Ting;SHANG Guangbin(Research Center for Differentiation and Development of Basic Theory of TCM,Jiangxi University of Chinese Medicine,Nanchang 330004 Jiangxi,China;Jiangxi Province Key Laboratory of TCM Etiopathogenisis,Nanchang 330004 Jiangxi,China;School of Traditional Chinese Medicine,Jiangxi University of Chinese Medicine,Nanchang 330004 Jiangxi,China;Clinical Medical College,Jiangxi University of Chinese Medicine,Nanchang 330004 Jiangxi,China)

机构地区：[1]江西中医药大学中医基础理论分化发展中心,江西南昌330004 [2]江西省中医病因生物学重点实验室,江西南昌330004 [3]江西中医药大学中医学院,江西南昌330004 [4]江西中医药大学临床医学院,江西南昌330004

出　　处：《中药新药与临床药理》2024年第1期56-64,共9页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(81960743,82260798);江西省自然科学基金面上项目(20192BAB205108);江西中医药大学中西医结合一级学科(江西省双一流学科,zxyylxk20220103)。

摘　　要：目的筛选肿节风总黄酮提取物促进巨核细胞分化的效应成分。方法(1)以人巨核细胞白血病细胞(Dami)与人骨髓基质细胞(HS-5)共培养的方式建立巨核细胞分化障碍模型作为评价体系,实验分组:Dami组(Dami)、对照组(Dami+HS-5)、PMA组[Dami+HS-5+5 ng·mL-1佛波醇12-十四酸酯13-乙酸酯(PMA)]、模型组[Dami+HS-5+1%兔抗大鼠血小板血清(APS)+5 ng·mL-1PMA],培养48 h。采用流式细胞术检测巨核细胞分化成熟表面标记分子CD41a、CD61的表达情况。(2)将49只SD雄性大鼠随机分为空白血浆组、15 min组、30 min组、60 min组、90 min组、120 min组、240 min组,每组7只。各给药组大鼠灌胃肿节风总黄酮提取物1.26 g·kg^(-1),在6个设定时间点(15、30、60、90、120、240 min)采血制备肿节风总黄酮提取物经时含药血浆。(3)采用超高效液相色谱-四极杆串联飞行时间质谱法(UHPLC-Q-TOF/MS)对肿节风总黄酮提取物经时含药血浆进行分析,以峰面积构建肿节风总黄酮提取物经时含药血浆中的化学成分随时间变化量矩阵(X矩阵)。将所采集的6个不同时间点的肿节风总黄酮经时含药血浆对巨核细胞分化成熟障碍模型进行干预,采用流式细胞术检测细胞表面分子CD41a、CD61的表达水平,构建肿节风总黄酮提取物经时含药血浆效应矩阵(Y矩阵)。(4)将X矩阵和Y矩阵数据标准化处理后,采用偏最小二乘法(Partial least squares,PLS)计算分析量效关系,以变量重要性投影(Variable importance for projection,VIP)>1为阈值,筛选与细胞表面分子CD41a、CD61变化相关的效应成分,并进行化学成分鉴定,作为肿节风总黄酮提取物中促进巨核细胞分化的潜在效应成分,最后回归评价体系验证其药效。结果(1)与Dami组比较,对照组Dami细胞表面的CD41a表达水平明显升高(P<0.05)。与对照组比较,PMA组Dami细胞表面的CD41a、CD61表达水平均显著升高(P<0.01)。与PMA组比较,模型组Dami细胞表面的CObjective To screen the active components of total flavonoid extracts of Sarcandra glabra to promote megakaryocyte differentiation.Methods(1)A model of megakaryocyte differentiation disorder was established by co-culturing human megakaryocytic leukaemia cells(Dami)with human bone marrow stromal cells(HS-5)as an evaluation system,and the experimental groupings were as follows:the Dami group(Dami),the control group(Dami+HS-5),and the PMA group[Dami+HS-5+5 ng·mL-1foprolol 12-tetradecanoate 13-acetate(PMA)],and model group[Dami+HS-5+1%rabbit anti-rat platelet serum(APS)+5 ng·mL-1PMA]were cultured for 48 hours.The expressions of megakaryocyte differentiation and maturation surface marker molecules,CD41a and CD61 were detected by flow cytometry.(2)Forty-nine SD male rats were randomly divided into blank plasma group,15-minute group,30-minute group,60-minute group,90-minute group,120-minute group,and 240-minute group,with 7 rats in each group.The rats in each administration group were gavaged with 1.26 g·kg^(-1)of total flavonoids extracts of Sarcandra glabra,and blood was collected at six set time points(15,30,60,90,120,240 minutes)for the preparation of time-dependent serum-containing plasma of total flavonoids extracts of Sarcandra glabra.(3)Ultra-high performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry(UHPLCQ-TOF/MS)was used to analyze the plasma of the time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra,and the peak area was used to construct a matrix(X-matrix)of the amount of chemical composition change over time in the time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra.The collected time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra at six different time points was used to intervene in the model of megakaryocyte differentiation and maturation disorder,and the expression of cell surface molecules CD41a and CD61 was detected by flow cytometry to construct the

关 键 词：肿节风总黄酮提取物 免疫性血小板减少症 巨核细胞分化障碍模型 人巨核白血病细胞 人骨髓基质细胞 超高效液相色谱-四极杆串联飞行时间质谱法 偏最小二乘法 胡萝卜苷 迷迭香酸-4-O-β-D-葡萄糖 

分 类 号：R284.1[医药卫生—中药学] R285.5[医药卫生—中医学]