【作者】 孙健；

【导师】 周秋丽；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 本实验首先建立HPLC-UV/MS测定方法,对黄连解毒汤整体定性,多指标定量分析,确定各色谱峰的来源,并在此基础上对所制备含药血清进行多成分的定性、定量分析,血药浓度-时间曲线分析;测定黄连解毒汤对小鼠移植性肿瘤H22、S180、MFC的抑制作用,大鼠含药血清对10种人瘤株的抑制作用;判断吸收入血的发挥抗肿瘤作用的主要有效成分;多指标检测主要有效成分的抗肿瘤作用。以HPLC-UV归属了黄连解毒汤21个色谱峰的药材来源,通过HPLC-UV/ MS对其中11个主要色谱峰进行了指认,其中10种成分吸收入血,各药材合煎后的血药浓度-时间曲线发生改变;黄连解毒汤对小鼠移植性肿瘤H22、S180、MFC的确有抑制作用,高剂量组抑制率均超过30%,含药血清对10种人瘤株有不同程度的抑制作用,原形入血成分中,汉黄芩素为抑制人肺腺癌SPC-A1增殖的主要有效成分,通过集落形成实验、周期分析、hoechst33342/PI、AO/EB双染、电镜,结合琼脂糖凝胶电泳检测阶梯状条带,Annexin-V/PI、线粒体膜电位等检测方法确定汉黄芩素诱导人肺腺癌SPC-A1凋亡,抑制其分裂和集落形成能力,阻滞周期等作用。实验的创造性成果在于:系统的研究了黄连解毒汤及其含药血清抗肿瘤作用的物质基础,结合多成分血药浓度-时间曲线判断黄连解毒汤体内抗肿瘤主要有效成分,从体内实验与体外实验两个方面,整体水平、细胞水平与分子水平三个层次,阐述了黄连解毒汤的抗肿瘤作用及汉黄芩素抗肿瘤作用机制。更多还原

【Abstract】 Huanglianjiedu decoction is the sutra recipe of defevering anddetoxifying. It consists of four medicines herbs: Rhizoma Coptidis,Radix Scutellariae, Cortex Phellodendri Chinensis, Fructus Gardeniae.No papers reported reseach on antitumor effect of HuanglianjieduDecoction, but we found antitumor effect of Huanglianjiedu Decoctionin vivo.First, we established a comprehensive high-performance liquidchromatographic (HPLC) analysis method of HuanglianjieduDecoction . Which was performed by HPLC-UV/MS technology forthe chemical constituents of mixed and divided “HuanglianjieduDecoction”. The chromatogram of Huanglianjiedu Decoction show21 main peaks, 1、2、5、18 from Fructus Gardeniae, 8、13、14、15、16、17、19、21 from Radix Scutellariae. While 10 from RhizomaCoptidis, and 20 from Cortex Phellodendri Chinensis, 3、4、6、9、11、12 came from them together. By comparison of the retention time,the on-line UV spectra and MS spectra, 11 peaks were identified as5(geniposide), 9(jatrorrhizine), 10(coptisine), 11(palmatine),12(berberine), 13(baicalin), 14(oroxin A), 17(wogonoside),19(baicalein), 20(obaculactone), 21(wogonin), then 8 of them werequantified by LC-UV. The method could represent the characteristicsof Huanglianjiedu Decoction, and it could be used to evaluate qualityand quantity of Huanglianjiedu Decoction. It distinguished betweenRhizoma Coptidis,and Phellodendri Chinensis by HPLC for the firsttime.To investigate the parameters of serum pharmacochemistry ofHuanglianjiedu Decoction. Based on the established HPLCchromatogram analysis,we compared the relation of the peaks amongcontrol serum,serum containing drug,Huanglianjiedu Decoction andthe ingredients of crude drug.Simultaneously, many compoundswere detected,ten of them were the original compounds contained inHuanglianjiedu Decoction.Three of them were metabolites. Theoriginal conpounds consists of 5(geniposide), 13(baicalin), 14(oroxinA), 17(wogonoside), 19(baicalein), 21(wogonin) and some others . Onthe other hand, 9(jatrorrhizine), 11(palmatine), 12(berberine) weren’tdetected. The compounds are absorbed into blood and theirmetabolites are the effective constituents.the original crude drugs arethe main ingredient of the prescriptions.Its serum pharmacochemistryshould be subject to complete investigation so as to illuminate thepharmacology and active mechanism of Huanglianjiedu Decoction.Antitumor activities were tested in mice with experimental tumorH22, S180, MFC in vivo, and their thymus index, spleen index, tumorinhibitory rate were evaluated. The effects on cancer cells from humanwere investigated in vitro by serum pharmacological approach.SPC-A1, SGC-7901, Bel-7402, NCI-H446, Swillc, Hela , Mcf-7 ,U251, MGC-803, SMMC-7721 cancer cells were incubated in culturemedia containing serum from rats medicated with HLJDT. Theinhibitory effects of HLJDT serum was observed by MTT assay.HLJDT showed a significant Antitumor activities on H22, S180 inmice. All of the HLJDT serum in different dosage groups cound inhibithighly the proliferation of 10 cancer cells from human. The HLJDTcould significantly inhibit the tumor H22, S180 in micedose-dependetly.Huanglianjiedu Decoction has obvious anticancer effects :IC50values of SPC-A1 is 103.49 mg·L-1, SGC-7901 153.32 mg·L-1,Bel-7402 185.34 mg·L-1, NCI-H446 264.68 mg·L-1, the mainactive ingredient of Huanglianjiedu Decoction is berberine. At thesame time, the drug serum has obvious anticancer effects againstSPC-A1 63.57%, SGC-7901 49.37%, Bel-7402 36.41%, NCI-H44635.24%, Swillc 30.08%, Hela 25.56%, Mcf-7 23.64%, U25123.05%, MGC-803 16.75%, SMMC 12.15%. The main activeingredient of the drug serum is wogonin.The morphological changes of SPC-A1 cells treated withwogonin were observed by light microscope, inverted fluorescencemicroscope and transmission electronic microscope. The effects ofwogonin on the cell cycle distribution and apoptosis were determinedusing propidium iodide staining and through flow cytometry.Apoptotic SPC-A1 cells were determined using an ApoAlert AnnexinV-FITC Apoptosis Kit. DNA fragmentation of SPC-A1 cells wasassayed by agarose gel electrophoresis. Changes in mitochondrialmembrane potential (△Ψm) in SPC-A1 cells after treatment withwogonin were assessed using 3,3,-dihexyloxacarbocyanine iodide[DiOC6(3)] and through flow cytometry.Analysis of SPC-A1 cell cycle was performed through flowcytometry. In the presence of different concentration of wogonin , thecells were blocked at G0/ G1 phase, then blocked at s, and the effectwas strengthened with the increase of the concentration. These resultsindicate that an alteration and/or redistribution of cell cycle, whichoccurred during the cell proliferation by the addition of wogonin. Theresults from flow cytometry analysis showed the presence of a sub-G1peak characteristic of potential apoptotic cells.Wogonin-induced apoptosis was stained by Annexin V in aconcentration–dependent manner. Approximately 11.33% of the cellswere apoptotic after the treatment with 5μg/ml wogonin, 16.63%10μg/ml, 25.58%20mg/L, 25.71%40mg/L.DNA fragmentation was increased in a time-dependent manner.The typical ladder bar were observerd when SPC-A1 cells were treatedwith 20 μg/ml wogonin.In our experiment, we used the ampholytic cationicfluorochrome DiOC6(3) to monitor the changes in △Ψm induced bywogonin. SPC-A1 cells treated with wogonin exhibited a significantreduction in cellular uptake of the fluorochrome. It was observed that△Ψm decreased concentration–dependent manner after exposure towogonin.更多还原