咳尔康颗粒对肺腺癌细胞A549的体外抗肿瘤作用
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摘要
目的制备咳尔康颗粒并观察其对人肺腺癌细胞A549的体外抗肿瘤作用。方法将处方药材经水提取,减压浓缩,用淀粉和糊精为辅料制备咳尔康颗粒。以黄芩苷为指认成分,采用高效液相色谱(HPLC)法建立咳尔康颗粒质量控制标准。以不同浓度的咳尔康颗粒溶液作用于肿瘤细胞A549,光学显微镜观察细胞形态,4,6-联脒-2-苯基吲哚(DAPI)染色后荧光显微镜观察细胞核固缩的凋亡过程,噻唑蓝(MTT)实验测定细胞增殖,实时定量聚合酶链反应(PCR)测定c-myc、c-fos、bax/bcl-2和caspase-9基因mRNA表达。结果制得咳尔康颗粒并建立HPLC质量控制标准,线性回归系数(r)为0.9929,平均加样回收率为98.03%,RSD为2.571%,指认成分黄芩苷含量为0.3498‰。咳尔康颗粒作用于肿瘤细胞A549后,细胞形态出现体积缩小,与周围的细胞脱离,核质浓缩,核膜破碎,最终形成凋亡小体;细胞增殖受到剂量依赖性抑制,半数抑制浓度(IC50)为74.07 g·L~(-1);原癌基因c-myc和c-fos的mRNA表达量降低,凋亡基因bax/bcl-2和caspase-9的mRNA表达量增加。结论咳尔康颗粒具有体外抗肿瘤作用。
Objective To prepare Keerkang granule and observe its anti-tumor effects in vitro on tumor cell A549.Methods Crude drugs in prescription were extracted with water and concentrated under reduced pressure. Granule was made from the concentrate with starch and dextrin which served as excipients. HPLC was used for the quality control of the granule and baicalin served as the recognized ingredient. After treated with different concentrations of Keerkang granule,cell morphology and karyon morphology of A549 tumor cells were observed by optical microscope and fluorescence microscope with 4',6-diamidino-2-phenylindole( DAPI) staining,respectively. Cell proliferation was measured with MTT test. The genes mRNA expressions of cmyc,c-fos,bax/bcl-2,and caspase-9 were detected with real-time PCR. Results Keerkang granule was prepared and its HPLC quality control was set up. The linear coefficient( r) was 0.9929; Average recovery was 98.03%; RSD was 2.571% and the content of the recognized ingredient baicalin was 0. 3498‰. Compared with negative control,after cultured with different concentrations of Keerkang granule,tumor cells A549 appeared shrinkage and nuclear chromatin condensation; Cell proliferation was inhibited in concentration-dependent manner and its IC50 was 74.07 g·L~(-1); proto-oncogenes mRNA expressions of c-myc and c-fos were decreased; the apoptosis genes mRNA expressions of bax/bcl-2 and caspase-9 were increased. Conclusion Keerkang granule is demonstrated to have the anti-tumor effects in vitro.
Objective To prepare Keerkang granule and observe its anti-tumor effects in vitro on tumor cell A549.Methods Crude drugs in prescription were extracted with water and concentrated under reduced pressure. Granule was made from the concentrate with starch and dextrin which served as excipients. HPLC was used for the quality control of the granule and baicalin served as the recognized ingredient. After treated with different concentrations of Keerkang granule,cell morphology and karyon morphology of A549 tumor cells were observed by optical microscope and fluorescence microscope with 4',6-diamidino-2-phenylindole( DAPI) staining,respectively. Cell proliferation was measured with MTT test. The genes mRNA expressions of cmyc,c-fos,bax/bcl-2,and caspase-9 were detected with real-time PCR. Results Keerkang granule was prepared and its HPLC quality control was set up. The linear coefficient( r) was 0.9929; Average recovery was 98.03%; RSD was 2.571% and the content of the recognized ingredient baicalin was 0. 3498‰. Compared with negative control,after cultured with different concentrations of Keerkang granule,tumor cells A549 appeared shrinkage and nuclear chromatin condensation; Cell proliferation was inhibited in concentration-dependent manner and its IC50 was 74.07 g·L~(-1); proto-oncogenes mRNA expressions of c-myc and c-fos were decreased; the apoptosis genes mRNA expressions of bax/bcl-2 and caspase-9 were increased. Conclusion Keerkang granule is demonstrated to have the anti-tumor effects in vitro.
引文
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[16]秦沛怡,李慧杰.齐元富.运用二陈汤治疗恶性肿瘤经验[J].湖南中医杂志,2017,33(4):27-28.
[17]赵柏庆,尹海燕,周开创,等.二陈汤改善晚期肺癌(痰湿证)化疗后生存质量观察[J].新中医,2015,47(1):182-194.
[2]张彬.恶性肿瘤治疗的现状决定了中西医结合治疗恶性肿瘤的重要性[J].世界最新医学信息文摘,2015,15(26):156-127.
[3]刘雪丽,周学锋,王君瑜,等.中药抗肿瘤作用机制研究进展[J].中国药师,2016,19(6):1158-1162.
[4]孙展鹏,李孟全,王元奕,等.咳尔康口服液止咳祛痰平喘的实验研究[J].医药导报,2009,28(3):29-31.
[5]李厚忠,张羽飞,齐敏,等.咳尔康口服液治疗哮喘的机制研究[J].中医药信息,2014,31(1):33-36.
[6]黄永存,孙立群,黄波,等.咳尔康口服液体外抑菌作用的研究[J].国际中医中药杂志,2009,31(3):205-206.
[7]黄永存,孙立群,黄波,等.咳尔康口服液体外抗病毒作用的研究[J].国际中医中药杂志,2009,31(5):397-398.
[8]才玉婷,李孟全,梁忆红,等.咳尔康口服液对小白鼠碳粒廓清功能的影响[J].牡丹江医学院学报,2009,30(5):16-17.
[9]肖琦,阳文武,张德伟,等.半夏总生物碱含量影响因素及药理作用研究进展[J].中国药业,2016,25(3):123-126.
[10]曹慧娟,鄢云彪,戴建业,等.黄芩苷对人肺腺癌A549细胞的体内外研究[J].中国实验方剂学杂志,2013,19(13):216-220.
[11]侯玮婷,罗佳波.复方茯苓多糖口服液抗肿瘤作用和免疫调节功能的初步研究[J].中药药理与临床,2017,33(2):78-81.
[12]李彦坤,李鑫,彭洪,等.甘草提取物(GL-1)对Hela细胞增殖抑制及促凋亡作用[J].中国公共卫生,2016,32(9):1186-1188.
[13]李雪飞,江洪.橙皮苷药理学作用机制及研究进展[M].海南医学,2016,27(14):2337-2340.
[14]国家药典委员会.中华人民共和国药典(四部)[M].北京:中国医药科技出版社,2015:59-61.
[15]姚海强,王济,李玲孺,等.痰湿体质与肿瘤的相关性探讨[J].环球中医药,2016,9(5):596-598.
[16]秦沛怡,李慧杰.齐元富.运用二陈汤治疗恶性肿瘤经验[J].湖南中医杂志,2017,33(4):27-28.
[17]赵柏庆,尹海燕,周开创,等.二陈汤改善晚期肺癌(痰湿证)化疗后生存质量观察[J].新中医,2015,47(1):182-194.