不同煎煮时间的银翘散对小鼠脾脏淋巴细胞体外增殖的影响
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摘要
目的:为分析不同煎煮时间银翘散对小鼠脾脏淋巴细胞体外增殖的影响。方法:首先建立不同煎煮时间(0、1.5、3、4.5、6min)银翘散中三种主要成份(绿原酸、连翘苷、牛蒡子苷)的高效液相色谱,使用Innoval ODS-2柱(4.6mm×150mm;5μm),以甲醇(A)-0.05%磷酸水(B)为流动相梯度洗脱,在波长237nm检测不同煎煮时间银翘散中三种主要成份,流速1.0mL/min。其次将小鼠连续15天腹腔注射环磷酰胺(60 mg/kg),在无菌操作下分离小鼠脾脏,制备脾脏细胞并用含10%胎牛血清蛋白的RPMI 1640培养,调整细胞浓度(2.0×10~6)分别加入诱导剂刀豆蛋白(ConA)和脂多糖(LPS)以及不同煎煮时间银翘散,经24h培养后,采用MTT法检测T和B淋巴细胞增殖。银翘散与ConA共同影响免疫抑制小鼠脾脏T淋巴细胞的增殖,在1.5min时最佳;与LPS共同影响免疫抑制小鼠脾脏B淋巴细胞的增殖,在3min时最佳。结果表明:银翘散煎煮时间不同所含成分含量有所差异,对T、B淋巴细胞影响不同。
Objective: To investigate effect of Yinqiao powder in different decocted time on proliferation of splenic lymphocytes in immunosuppressed mouse models. Methods: Firstly,high performance liquid chromatography(HPLC) was established for the three main components(Chlorogenic acid、Forsythin and Arctium) of Yinqiao Powder in Different Decocted Time(0、1.5、3、4.5 and 6 min).The analysis was carried out by HPLC with column of Innoval ODS-2(4.6 mm×150 mm,5 μm),and eluted with mobile phase containing of methanol(A)-0.05% phosphoric acid(B) in a gradient mode,the detection wavelength was 237 nm,andthe flow rate was 1.0 mL/min.Then,the mice received intraperitoneal injection of cyclophosphamide(60 mg/kg) once a day,for 15 days.Mouse spleen cells were prepared by aseptic separation and in vitro culturedin RPMI 1640 containing 10% fetal blood serum,with the adjusted cell concentration of(2.0×10~6),and stimulant concanavalin(ConA)、lipopolysaccharide(LPS) and Yinqiao Powder in different decocted time. After cells were cultured for 24 h,MTT assay the proliferations of T and Blymphocytes. Yinqiao Powder and ConA stimulated the proliferation of spleen T lymphocyte in immunosuppressed mice,which was the best at 1.5 minutes, while Yinqiao Powder and LPS stimulated the proliferation of spleen B lymphocyte in immunosuppressed mice at 3 min.Results The contents of components in Yinqiao Powder differ with decocting time,and have different effects on T and B lymphocyte.
Objective: To investigate effect of Yinqiao powder in different decocted time on proliferation of splenic lymphocytes in immunosuppressed mouse models. Methods: Firstly,high performance liquid chromatography(HPLC) was established for the three main components(Chlorogenic acid、Forsythin and Arctium) of Yinqiao Powder in Different Decocted Time(0、1.5、3、4.5 and 6 min).The analysis was carried out by HPLC with column of Innoval ODS-2(4.6 mm×150 mm,5 μm),and eluted with mobile phase containing of methanol(A)-0.05% phosphoric acid(B) in a gradient mode,the detection wavelength was 237 nm,andthe flow rate was 1.0 mL/min.Then,the mice received intraperitoneal injection of cyclophosphamide(60 mg/kg) once a day,for 15 days.Mouse spleen cells were prepared by aseptic separation and in vitro culturedin RPMI 1640 containing 10% fetal blood serum,with the adjusted cell concentration of(2.0×10~6),and stimulant concanavalin(ConA)、lipopolysaccharide(LPS) and Yinqiao Powder in different decocted time. After cells were cultured for 24 h,MTT assay the proliferations of T and Blymphocytes. Yinqiao Powder and ConA stimulated the proliferation of spleen T lymphocyte in immunosuppressed mice,which was the best at 1.5 minutes, while Yinqiao Powder and LPS stimulated the proliferation of spleen B lymphocyte in immunosuppressed mice at 3 min.Results The contents of components in Yinqiao Powder differ with decocting time,and have different effects on T and B lymphocyte.
引文
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[2] 霍炳杰,刘亚娴,常靓,等.银翘散在不同煎煮时间下对免疫抑制小鼠外周血T细胞亚群及免疫器官重量的影响[J].江苏中医药,2010,42(3):72-73.
[3] 霍炳杰,常靓,吕雅蕾,等.银翘散不同煎煮时间对甲型流感病毒感染小鼠肺组织病毒载量的影响[J].中医杂志2017,58(2):147-150.
[4] 霍炳杰,常靓,刘羽,等.银翘散在不同煎煮时间下体外抑制流感病毒及对小鼠巨噬细胞Toll样受体及其下游信号转导通路的影响[J].中华中医药学刊,2016,34(7):1581-1584.
[5] 康乐,姚东璧,胡庭俊,等.天门冬多糖对小鼠脾脏淋巴细胞体外增殖的影响[J].动物营养与饲料科学,2010,37(10):15-17.
[6] 官佳懿,张永红,崔德凤,等.绿原酸对小鼠脾脏淋巴细胞体外增殖和分泌功能的影响[J].中国农学通报,2013,29(29):27-31.
[7] 印酬,龙厚宁,刘莉,等.银翘散的 UPLC 指纹图谱研究[J].中药材,2017,40(2):379-383.
[8] 刘险峰.传统煎煮法对银翘散汤剂抗炎解热作用的影响[J].世界最新医学信息文摘,2015,15(3):133.
[9] 束雅春,秦昆明,陈亚军,等.不同后下时间银翘散汤剂解热抗炎作用比较研究[J].中华中医药杂志(原中国医药学报),2014,29(5):1459-1464.
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