紫背万年青调控非小细胞肺癌A549细胞凋亡活性成分筛选和含量测定标准研究

Screening and Determination of The Active Ingredient of Rhoeo Spathacea in Regulating Apoptosis of A549 Cells in Non-Small Cell Lung Cancer

目的 对紫背万年青中的化学成分进行非小细胞肺癌A549细胞凋亡活性测定,并以筛选出的活性成分为指标建立含量测定方法,用以评价该植物的功效。方法 将A549细胞和不同浓度的紫草素、木犀草素、补骨脂素(8、16、32μg·mL^(-1))孵育后,使用Western-Blot法检测caspase-3蛋白表达,对所得结果进行统计分析,研究各成分对A549细胞凋亡的调控活性;选择紫草素为含量测定指标,使用Agilent TC C_(18)(柱长25 cm,内径0.46 cm,填料直径5μm)色谱柱,乙腈(A)-0.1%磷酸(B)为流动相,梯度洗脱,程序为0~10 min, 20%~20%A;10~20 min, 20%~80%A;20~30 min, 80%~20%A;30~35 min, 20%~20%A;流速1.0 mL·min^(-1),检测波长274 nm,柱温25℃,进样量10μL;测定10批紫背万年青样品中补骨脂素的含量。结果 3个待测成分中,补骨脂素对caspase-3蛋白表达存在明显的调控效果,提示该成分具有较强的促进A549细胞凋亡活性;10批样品含量测定结果显示,补骨脂素的含量为0.873 7~1.065 4 mg·g^(-1)。结论 本文对紫背万年青中的化学成分进行了A549细胞凋亡活性进行了研究,并基于此建立了含量测定标准,能够较为科学、准确地对紫背万年青的活性进行可量化评价。

OBJECTIVE To determine the apoptotic activity of non-small cell lung cancer A549 cells by the chemical constituents of Rhoeo spathacea,and to establish a method to determine the content of the selected active constituents to evaluate the efficacy of the plant.METHODS A549 cells were incubated with different concentrations of shikonin,luteolin and psoralen(8,16,32μg·mL^(-1)),and the expression of caspase-3 protein was detected by Western-Blot method.The results were analyzed statistically,and the regulatory activities of each component on apoptosis of A549 cells were investigated.Agilent TC C_(18)(column length:25 cm,inner diameter:0.46 cm,packing diameter:5μm)column was used.Acetonitrile(A)-0.1%phosphoric acid(B)was used as mobile phase with gradient elute.The time program was set as follow:0-10 min,20%-20%A,10-20 min,20%-80%A,20-30 min,80%-20%A,30-35 min,20%-20%A.The flow rate was 1.0 mL·min^(-1),the detection wavelength was 274 nm,the column temperature was 25℃,and the sample size was 10μL.The content of psoralen in 10 batches of Rhoeo spathacea samples were determined.RESULTS Among the three components to be tested,psoralen had obvious regulation effect on the expression of caspase-3 protein,suggesting that this component had strong activity in promoting apoptosis of A549 cells.The contents of 10 batches of samples showed that the content of psoralen was 0.8737-1.0654 mg·g^(-1).CONCLUSION In this paper,A549 cells apoptosis activity of the chemical constituents of Dieffenbachia purpura is studied,and based on this,the content determination standard is established,which can be more scientific and accurate to quantify the activity of Rhoeo spathacea.