黄芩素抗白念珠菌的作用机制研究
摘要
近年来,随着艾滋病的广泛传播,癌症的放疗、化疗,器官移植后免疫抑制剂的使用,广谱抗生素的大量使用,以及导管等越来越多的生物材料应用于人体,真菌感染率的不断增加,尤其以白念珠菌的感染最为普遍。全球很多中心检测统计资料表明,1996至2002年期间,在系统性真菌感染病例中,白念珠菌感染占50%-60%。由于长期广泛地预防性和治疗性使用唑类抗真菌药物(如氟康唑),白念珠菌的耐药现象越来越普遍,耐药程度也越来越高,日益严重的耐药现象和有限的治疗药物使白念珠菌感染成为临床上迫切需要解决的问题。
黄芩素(黄芩苷元,Baicalein,BE)是从唇形科植物黄芩的干燥根中提取的有效成份之一。黄芩是常用中药,其味苦、性寒,归肺、心、肝、胆、大肠经,具有清热燥湿、泻火解毒、止血安胎等功效。黄芩素具有抗氧化、抗炎、抗菌、抗肿瘤、抗病毒等药理活性,且对血液细胞及肝脏细胞等正常细胞无毒性。虽然有研究报道黄芩素具有抗真菌活性,但由于其活性较弱,一直未应用于临床。鉴于目前白念珠菌普遍的多药耐药现象,本课题从联合用药角度出发,探索治疗白念珠菌感染的新策略。
本课题首先采用微量液基稀释法考察黄芩素对白念珠菌的抑制作用,并以棋盘式微量稀释法测定黄芩素与氟康唑合用对白念珠菌的抑制作用。其结果显示:黄芩素单独使用时对敏感型白念珠菌具有抑制作用,其有效抗菌浓度为1μg/ml-2μg/ml,但对氟康唑耐药的白念珠菌无效。黄芩素与氟康唑合用于敏感型白念珠菌时,氟康唑的MIC80值在合用后均有降低,黄芩素的MIC80值在合用后有50%菌株呈现降低,但所有受试菌株的FIC指数均>0.5,两药相互作用表现为无关。在对氟康唑耐药的白念珠菌中,氟康唑与BE合用显示出显著的协同作用,原本对氟康唑耐药(MIC80≥64μg/ml)的菌株变得敏感,合用BE后氟康唑的MIC80值降低了32至1028倍,MIC80值分别在≤0.125至2μg/ml之间;BE的MIC80值也明显下降,受试菌株的FIC指数位于0.037-0.098之间,两药相互作用表现为协同。
应用Time-kill curves实验对临床耐药白念珠菌0603433进一步验证氟康唑与黄芩素合用的协同作用。菌液起始浓度选用体外药敏实验的1~5×103CFU/ml和时间-杀菌曲线常用的1~5×105CFU/ml。当菌液起始浓度为1~5×103CFU/ml时,药物作用24h后,16μg/ml黄芩素单用对生长曲线没有影响,未见抗菌作用,其曲线与对照组生长曲线几乎重合。10μg/ml氟康唑显示较弱的抑菌作用,合用16μg/ml黄芩素24h后则大大增强了抑菌效果,表明两药合用具有协同抑菌作用。当菌液起始浓度为1~5×105CFU/ml时,单用16μg/ml黄芩素或单用10μg/ml氟康唑对生长曲线都没有明显的抑制作用,两药合用24h后,菌浓度比单用氟康唑明显下降。
在对氟康唑耐药的白念珠菌中,以棋盘式微量稀释实验考察了BE与咪康唑、酮康唑的抗真菌相互作用。结果表明,咪康唑和酮康唑与BE合用均具有显著协同作用,合用BE后两者的MIC80值全部降到0.5μg/ml以下,FIC指数均<0.5,两药相互作用表现为协同。另外,BE与两性霉素B(Amphotericin B,AmB)合用具有协同抗真菌的作用。合用BE后两性霉素B的MIC值由1-2μg/ml降到0.062以下,明显提高了两性霉素B的杀菌作用。但对于近平滑念珠菌和光滑念珠菌中,黄芩素与氟康唑合用FIC值均在1.5以上,表现为相互作用无关。
为了比较正常的与经黄芩素处理的白念珠菌生物被膜形态差异,以FUN-1和ConA染色后,激光共聚焦显微镜观察显示:正常生物被膜中含有大量菌丝,经8μg/ml黄芩素处理的生物被膜明显受到抑制,结构疏松,菌丝成分明显减少,极易从附着物上脱落。将不同浓度的黄芩素处理白念珠菌,48小时后XTT法测定生物被膜的生长动力学,结果表明,4μg/ml至32μg/ml的黄芩素可对白念珠菌生物被膜的抑制率达70%,2μg/ml的黄芩素对白念珠菌生物被膜的抑制率为15%,1μg/ml的黄芩素对白念珠菌生物被膜生长几乎无抑制作用。
刮取经不同浓度的黄芩素处理的白念珠菌,测定CSH值,结果可见,在黄芩素浓度为32μg/ml时,白念珠菌的CSH值最小,为0.11,随着BE浓度的降低,CSH值逐渐增大,呈现出剂量依赖性,经1μg/ml BE处理的白念珠菌CSH值为0.85。采用实时定量RT-PCR法测定白念珠菌CSH相关基因CSH1的mRNA表达水平,结果表明,与对照组相比,经黄芩素处理的白念珠菌CSH1 mRNA表达水平明显下降。
将不同浓度的黄芩素处理白念珠菌SC5314,12h后以流式细胞仪测定细胞凋亡率,同时采用已知真菌细胞凋亡诱导药物两性霉素B(AmB)作为阳性对照。结果显示,与空白对照组相比,黄芩素可明显诱导白念珠菌细胞凋亡,4μg/ml、8μg/ml黄芩素分别可诱导10%及20%的细胞凋亡。DAPI染色进一步确证了白念珠菌细胞凋亡的发生。应用透射电镜技术观察8μg/ml黄芩素作用于白念珠菌后细胞超微结构的变化。在白念珠菌的正常细胞超微结构中,细胞形状规则,细胞膜和细胞壁完整、光滑、边界清晰可见,胞核形完整,染色质均匀分布。经8μg/ml黄芩素处理的白念珠菌细胞核异常,染色质固缩,向核四周凝集,细胞质中有空泡产生,另外分裂细胞多处于两细胞粘连状态。
另外,黄芩素对白念珠菌细胞内ROS产生的促进作用具有剂量依赖性,并随着时间的延长对ROS产生的促进作用也随之增强。32μg/ml黄芩素处理48h后ROS产生量是对照组的5倍。将16μg/ml的黄芩素处理白念珠菌SC5314,于不同时间点测定线粒体膜电位,同时以两性霉素B作为阳性对照。结果显示,与空白对照组相比,黄芩素可明显降低白念珠菌线粒体膜电位,经黄芩素处理12h的白念珠菌线粒体膜电位下降至未经处理组一半值。经32μg/ml黄芩素作用3h后,CAP1、SOD2及TRR1的mRNA表达水平明显上调,尤其是CAP1基因上调了近24倍,SOD2和TRR1基因的表达也分别上调了3倍和9倍。由于黄芩素可诱导白念珠菌氧化应激相关转录因子CAP1的mRNA高表达,本实验考察黄芩素对野生型菌(CAF2-1)、CAP1基因敲除菌(CJDADH)及高表达菌(CJDCAP1)细胞存活率的影响,结果显示,经32μg/ml黄芩素处理3h后,CJDADH、CAF2-1和CJDCAP1的存活率分别为35%、41.7%和54%。
基因芯片杂交实验结果表明,黄芩素作用于白念珠菌以后,可引起多种相关基因的上调或下调,差异基因功能主要涉及细胞周期、跨膜物质转运、毒力因子及热休克蛋白等。
With the wide spread of human immunodeficiency virus (HIV), misusing of tremedous antibiotic and immunosuppressive therapy to treat cancer or organ transplant, and also increasing number of biological material applying in human beings, the incidence of systemic fungal infections has been risen dramatically in recent years. Candida albicans infection is the most common isolates among them. It shows that C. albicans accounts about 50-60% in the major systemic fungal pathogen during 1996 to 2002. The triazole fluconazole is the most widely used antifungal drug to treat Candida infections. Unfortunately, wide spread uses of fluconazole have led to the rapid development of drug resistance which has severely hindered antifungal therapy.
Baicalein (BE) is a major component originally isolated from the roots of Scutellaria baicalensis Georgi. Several different functions of BE have been reported. In addition to its inhibition effect on lipoxygenase, BE was found to have antioxidant, neuroprotective, antibacterial, antiviral activities. It was reported that BE also has antifungal activities, but could not be applied in clinical therapy because of its few effect. Attempts have been made to cope with treatment failures by using combination therapy. To seek for a novel combination therapy, we investigated the in vitro interaction of fluconazole and BE against fluconazole-resistant clinical isolates of C. albicans.
In this study, we first investigated the effect of BE on C. albicans by microdilution assay and the synergistic activities between FLC and BE against FLC-resistant C. albicans by checkerboard microdilution assay. We found that the drug-sensititive C. albicans but not the fluconazole-resistant C. albicans was inhibited when baicalein was used alone,the effective concentration was 1μg/ml-2μg/ml. When used in drug-sensitive C. albicans with the combination of fluconazole and BE, each drug showed the little change comparing with drugs used alone. The fractional inhibitory concentration (FIC) index was all above 0.5,meaning no synergism in fluconazole-BE combination. However, the fluconazole-BE combination markedly reduced MIC80s, especially the MIC80 of either individual agent while used in fluconazole-resistant C. albicans. The corresponding median FIC index was 0.069 (range, 0.037 to 0.098). Synergism was observed in all isolates .
Further time-kill studies were conducted with fluconazole and BE against one chosen clinical fluconazole-resistant C. albicans 0603433. BE did not affect the growth curve at 16mg/ml after 24 h, regardless of the initial inoculum is 103 or 105 CFU/ml,the fluconazole showed a little antifungal activity for initial inoculum is 103 CFU/ml . The fungistatic activity of fluconazole was dramatically enhanced by addition of BE when used in combination for 24h.
Then we further investigated whether BE could play a same role with other antifungal agent or in other fungus. Respectively,the combination of baicalein and KCZ or MCZ reduced the MIC below to 0.5μg/ml both and showed significant synergistic activities against fluconazole-resistant C. albicans (FIC index <0.5).And the combination of BE and AmB reduced the MIC from 1-2μg/ml to 0.062μg/ml and also showed the synergistic activities .When used in Candida parapsilosis and Candida glabrata with the combination of fluconazole and BE, no synergism was observed (FIC>1.5).
In order to examine the effect of baicalein (BE) on C. albicans biofilm formation,we dyed the BE-treated biofilm and untreated biofilm with the fluorescent stains FUN-1 and concanavalin A-Alexa Fluor 488 conjugate (ConA).Confocal scanning laser microscopy (CSLM) showed that normal C. albicans biofilm exhibited a typical three-dimensional nature, composed mainly of true hyphae. When cells were treated with 8μg/mL BE, biofilm development was inhibited and growth was predominantly composed of yeast cells and pseudohyphae.True hyphae were rarely observed, a factor that contributed to the poor biofilm architectures. To provide a more quantitative assessment of the extent of cell growth of biofilms in the presence or absence of BE, the ability of cells to reduce XTT was measured after the treatment for biofilms with different concentration of BE for 48h .The inhibitory effect on biofilms appeared to be dose-related. Over 70% inhibition was observed at BE concentrations between 4μg/ml and 32μg/ml A lower concentration (2μg/ml) produced 15% inhibition and 1μg/ml BE had almost no effect on biofilm formation.
To examine the mechanism by which BE inhibits biofilm formation, the CSH of biofilm was measured by the biphasic separation method. Negative correlations were observed between BE concentration and the CSH of biofilm. High CSH was observed in normal mature biofilm. When the cells were treated with BE, CSH decreased. The relative CSH was 0.85 and 0.11 at BE concentrations of 1μg/ml and 32μg/ml, respectively.The mRNA expression levels of CSH1, which codes for the CSH-associated protein in C. albicans, were determined by real-time RT-PCR. The results showed that BE-treated cells expressed lower levels of CSH1 mRNA than the cells grown in the absence of BE, which correlated with the CSH exhibited by the cells.
Here, we describe cellular changes that accompany death in C. albicans after exposure 12h to a range of different concentrations of BE , including treatment with the antifungal agent amphotericin B (AMB) as a positive control .Flow cytometry (FACS) assay showed that 10% or 20% apoptosis of C. albicans were induced respectively at baicalein concentrations of 4μg/ml or 8μg/ml.DAPI-staining reassured the occurrence of the apoptosis. Cells dying under 8μg/ml BE treated displayed several markers characteristic of apoptosis. These include the rapid exposure of phosphatidylserine (PS) at the outer cell membrane, cellular dyskaryosis,the generation of vacuole in cytoplasm, the margination of chromatin in nuclei, nuclear fragmentation, and the degradation of DNA.
Compared to the normal cells, the apoptotic cells induced by BE showed increased endogenous reactive oxygen species (ROS) generation, and it shows the dose-dependent and time-dependent activity.The generation of ROS induced by 32μg/ml BE for 48h was 5 times higher than control . 16μg/ml BE induced cells shows less mitochondrial membrane potential and for 12h decreased 50% comparing with control. After treated with 32μg/ml BE for 3h , the mRNA expression of CAP1, SOD2 and TRR1 were apparently up-regulated.Considered the expression of CAP1 was up-regulated more than 24times than control, further experiment we did to investigate survival of wild type strains ,CAP1 knock-out strains and overexpression CAP1strains treated by BE. Reaults showed after induced by 32μg/ml BE for 3h,the survival of three strains were 35%,41.7%and54%.That means CAP1 played a role in baicalein-induced apoptosis. CAP1-deleted cells was very sensitive to baicalein.
Gene chip analysis showed that the genes affected by baicalein were related to cell cycle, drug efflux, virulence, substrate transport and heat shock.
黄芩素(黄芩苷元,Baicalein,BE)是从唇形科植物黄芩的干燥根中提取的有效成份之一。黄芩是常用中药,其味苦、性寒,归肺、心、肝、胆、大肠经,具有清热燥湿、泻火解毒、止血安胎等功效。黄芩素具有抗氧化、抗炎、抗菌、抗肿瘤、抗病毒等药理活性,且对血液细胞及肝脏细胞等正常细胞无毒性。虽然有研究报道黄芩素具有抗真菌活性,但由于其活性较弱,一直未应用于临床。鉴于目前白念珠菌普遍的多药耐药现象,本课题从联合用药角度出发,探索治疗白念珠菌感染的新策略。
本课题首先采用微量液基稀释法考察黄芩素对白念珠菌的抑制作用,并以棋盘式微量稀释法测定黄芩素与氟康唑合用对白念珠菌的抑制作用。其结果显示:黄芩素单独使用时对敏感型白念珠菌具有抑制作用,其有效抗菌浓度为1μg/ml-2μg/ml,但对氟康唑耐药的白念珠菌无效。黄芩素与氟康唑合用于敏感型白念珠菌时,氟康唑的MIC80值在合用后均有降低,黄芩素的MIC80值在合用后有50%菌株呈现降低,但所有受试菌株的FIC指数均>0.5,两药相互作用表现为无关。在对氟康唑耐药的白念珠菌中,氟康唑与BE合用显示出显著的协同作用,原本对氟康唑耐药(MIC80≥64μg/ml)的菌株变得敏感,合用BE后氟康唑的MIC80值降低了32至1028倍,MIC80值分别在≤0.125至2μg/ml之间;BE的MIC80值也明显下降,受试菌株的FIC指数位于0.037-0.098之间,两药相互作用表现为协同。
应用Time-kill curves实验对临床耐药白念珠菌0603433进一步验证氟康唑与黄芩素合用的协同作用。菌液起始浓度选用体外药敏实验的1~5×103CFU/ml和时间-杀菌曲线常用的1~5×105CFU/ml。当菌液起始浓度为1~5×103CFU/ml时,药物作用24h后,16μg/ml黄芩素单用对生长曲线没有影响,未见抗菌作用,其曲线与对照组生长曲线几乎重合。10μg/ml氟康唑显示较弱的抑菌作用,合用16μg/ml黄芩素24h后则大大增强了抑菌效果,表明两药合用具有协同抑菌作用。当菌液起始浓度为1~5×105CFU/ml时,单用16μg/ml黄芩素或单用10μg/ml氟康唑对生长曲线都没有明显的抑制作用,两药合用24h后,菌浓度比单用氟康唑明显下降。
在对氟康唑耐药的白念珠菌中,以棋盘式微量稀释实验考察了BE与咪康唑、酮康唑的抗真菌相互作用。结果表明,咪康唑和酮康唑与BE合用均具有显著协同作用,合用BE后两者的MIC80值全部降到0.5μg/ml以下,FIC指数均<0.5,两药相互作用表现为协同。另外,BE与两性霉素B(Amphotericin B,AmB)合用具有协同抗真菌的作用。合用BE后两性霉素B的MIC值由1-2μg/ml降到0.062以下,明显提高了两性霉素B的杀菌作用。但对于近平滑念珠菌和光滑念珠菌中,黄芩素与氟康唑合用FIC值均在1.5以上,表现为相互作用无关。
为了比较正常的与经黄芩素处理的白念珠菌生物被膜形态差异,以FUN-1和ConA染色后,激光共聚焦显微镜观察显示:正常生物被膜中含有大量菌丝,经8μg/ml黄芩素处理的生物被膜明显受到抑制,结构疏松,菌丝成分明显减少,极易从附着物上脱落。将不同浓度的黄芩素处理白念珠菌,48小时后XTT法测定生物被膜的生长动力学,结果表明,4μg/ml至32μg/ml的黄芩素可对白念珠菌生物被膜的抑制率达70%,2μg/ml的黄芩素对白念珠菌生物被膜的抑制率为15%,1μg/ml的黄芩素对白念珠菌生物被膜生长几乎无抑制作用。
刮取经不同浓度的黄芩素处理的白念珠菌,测定CSH值,结果可见,在黄芩素浓度为32μg/ml时,白念珠菌的CSH值最小,为0.11,随着BE浓度的降低,CSH值逐渐增大,呈现出剂量依赖性,经1μg/ml BE处理的白念珠菌CSH值为0.85。采用实时定量RT-PCR法测定白念珠菌CSH相关基因CSH1的mRNA表达水平,结果表明,与对照组相比,经黄芩素处理的白念珠菌CSH1 mRNA表达水平明显下降。
将不同浓度的黄芩素处理白念珠菌SC5314,12h后以流式细胞仪测定细胞凋亡率,同时采用已知真菌细胞凋亡诱导药物两性霉素B(AmB)作为阳性对照。结果显示,与空白对照组相比,黄芩素可明显诱导白念珠菌细胞凋亡,4μg/ml、8μg/ml黄芩素分别可诱导10%及20%的细胞凋亡。DAPI染色进一步确证了白念珠菌细胞凋亡的发生。应用透射电镜技术观察8μg/ml黄芩素作用于白念珠菌后细胞超微结构的变化。在白念珠菌的正常细胞超微结构中,细胞形状规则,细胞膜和细胞壁完整、光滑、边界清晰可见,胞核形完整,染色质均匀分布。经8μg/ml黄芩素处理的白念珠菌细胞核异常,染色质固缩,向核四周凝集,细胞质中有空泡产生,另外分裂细胞多处于两细胞粘连状态。
另外,黄芩素对白念珠菌细胞内ROS产生的促进作用具有剂量依赖性,并随着时间的延长对ROS产生的促进作用也随之增强。32μg/ml黄芩素处理48h后ROS产生量是对照组的5倍。将16μg/ml的黄芩素处理白念珠菌SC5314,于不同时间点测定线粒体膜电位,同时以两性霉素B作为阳性对照。结果显示,与空白对照组相比,黄芩素可明显降低白念珠菌线粒体膜电位,经黄芩素处理12h的白念珠菌线粒体膜电位下降至未经处理组一半值。经32μg/ml黄芩素作用3h后,CAP1、SOD2及TRR1的mRNA表达水平明显上调,尤其是CAP1基因上调了近24倍,SOD2和TRR1基因的表达也分别上调了3倍和9倍。由于黄芩素可诱导白念珠菌氧化应激相关转录因子CAP1的mRNA高表达,本实验考察黄芩素对野生型菌(CAF2-1)、CAP1基因敲除菌(CJDADH)及高表达菌(CJDCAP1)细胞存活率的影响,结果显示,经32μg/ml黄芩素处理3h后,CJDADH、CAF2-1和CJDCAP1的存活率分别为35%、41.7%和54%。
基因芯片杂交实验结果表明,黄芩素作用于白念珠菌以后,可引起多种相关基因的上调或下调,差异基因功能主要涉及细胞周期、跨膜物质转运、毒力因子及热休克蛋白等。
With the wide spread of human immunodeficiency virus (HIV), misusing of tremedous antibiotic and immunosuppressive therapy to treat cancer or organ transplant, and also increasing number of biological material applying in human beings, the incidence of systemic fungal infections has been risen dramatically in recent years. Candida albicans infection is the most common isolates among them. It shows that C. albicans accounts about 50-60% in the major systemic fungal pathogen during 1996 to 2002. The triazole fluconazole is the most widely used antifungal drug to treat Candida infections. Unfortunately, wide spread uses of fluconazole have led to the rapid development of drug resistance which has severely hindered antifungal therapy.
Baicalein (BE) is a major component originally isolated from the roots of Scutellaria baicalensis Georgi. Several different functions of BE have been reported. In addition to its inhibition effect on lipoxygenase, BE was found to have antioxidant, neuroprotective, antibacterial, antiviral activities. It was reported that BE also has antifungal activities, but could not be applied in clinical therapy because of its few effect. Attempts have been made to cope with treatment failures by using combination therapy. To seek for a novel combination therapy, we investigated the in vitro interaction of fluconazole and BE against fluconazole-resistant clinical isolates of C. albicans.
In this study, we first investigated the effect of BE on C. albicans by microdilution assay and the synergistic activities between FLC and BE against FLC-resistant C. albicans by checkerboard microdilution assay. We found that the drug-sensititive C. albicans but not the fluconazole-resistant C. albicans was inhibited when baicalein was used alone,the effective concentration was 1μg/ml-2μg/ml. When used in drug-sensitive C. albicans with the combination of fluconazole and BE, each drug showed the little change comparing with drugs used alone. The fractional inhibitory concentration (FIC) index was all above 0.5,meaning no synergism in fluconazole-BE combination. However, the fluconazole-BE combination markedly reduced MIC80s, especially the MIC80 of either individual agent while used in fluconazole-resistant C. albicans. The corresponding median FIC index was 0.069 (range, 0.037 to 0.098). Synergism was observed in all isolates .
Further time-kill studies were conducted with fluconazole and BE against one chosen clinical fluconazole-resistant C. albicans 0603433. BE did not affect the growth curve at 16mg/ml after 24 h, regardless of the initial inoculum is 103 or 105 CFU/ml,the fluconazole showed a little antifungal activity for initial inoculum is 103 CFU/ml . The fungistatic activity of fluconazole was dramatically enhanced by addition of BE when used in combination for 24h.
Then we further investigated whether BE could play a same role with other antifungal agent or in other fungus. Respectively,the combination of baicalein and KCZ or MCZ reduced the MIC below to 0.5μg/ml both and showed significant synergistic activities against fluconazole-resistant C. albicans (FIC index <0.5).And the combination of BE and AmB reduced the MIC from 1-2μg/ml to 0.062μg/ml and also showed the synergistic activities .When used in Candida parapsilosis and Candida glabrata with the combination of fluconazole and BE, no synergism was observed (FIC>1.5).
In order to examine the effect of baicalein (BE) on C. albicans biofilm formation,we dyed the BE-treated biofilm and untreated biofilm with the fluorescent stains FUN-1 and concanavalin A-Alexa Fluor 488 conjugate (ConA).Confocal scanning laser microscopy (CSLM) showed that normal C. albicans biofilm exhibited a typical three-dimensional nature, composed mainly of true hyphae. When cells were treated with 8μg/mL BE, biofilm development was inhibited and growth was predominantly composed of yeast cells and pseudohyphae.True hyphae were rarely observed, a factor that contributed to the poor biofilm architectures. To provide a more quantitative assessment of the extent of cell growth of biofilms in the presence or absence of BE, the ability of cells to reduce XTT was measured after the treatment for biofilms with different concentration of BE for 48h .The inhibitory effect on biofilms appeared to be dose-related. Over 70% inhibition was observed at BE concentrations between 4μg/ml and 32μg/ml A lower concentration (2μg/ml) produced 15% inhibition and 1μg/ml BE had almost no effect on biofilm formation.
To examine the mechanism by which BE inhibits biofilm formation, the CSH of biofilm was measured by the biphasic separation method. Negative correlations were observed between BE concentration and the CSH of biofilm. High CSH was observed in normal mature biofilm. When the cells were treated with BE, CSH decreased. The relative CSH was 0.85 and 0.11 at BE concentrations of 1μg/ml and 32μg/ml, respectively.The mRNA expression levels of CSH1, which codes for the CSH-associated protein in C. albicans, were determined by real-time RT-PCR. The results showed that BE-treated cells expressed lower levels of CSH1 mRNA than the cells grown in the absence of BE, which correlated with the CSH exhibited by the cells.
Here, we describe cellular changes that accompany death in C. albicans after exposure 12h to a range of different concentrations of BE , including treatment with the antifungal agent amphotericin B (AMB) as a positive control .Flow cytometry (FACS) assay showed that 10% or 20% apoptosis of C. albicans were induced respectively at baicalein concentrations of 4μg/ml or 8μg/ml.DAPI-staining reassured the occurrence of the apoptosis. Cells dying under 8μg/ml BE treated displayed several markers characteristic of apoptosis. These include the rapid exposure of phosphatidylserine (PS) at the outer cell membrane, cellular dyskaryosis,the generation of vacuole in cytoplasm, the margination of chromatin in nuclei, nuclear fragmentation, and the degradation of DNA.
Compared to the normal cells, the apoptotic cells induced by BE showed increased endogenous reactive oxygen species (ROS) generation, and it shows the dose-dependent and time-dependent activity.The generation of ROS induced by 32μg/ml BE for 48h was 5 times higher than control . 16μg/ml BE induced cells shows less mitochondrial membrane potential and for 12h decreased 50% comparing with control. After treated with 32μg/ml BE for 3h , the mRNA expression of CAP1, SOD2 and TRR1 were apparently up-regulated.Considered the expression of CAP1 was up-regulated more than 24times than control, further experiment we did to investigate survival of wild type strains ,CAP1 knock-out strains and overexpression CAP1strains treated by BE. Reaults showed after induced by 32μg/ml BE for 3h,the survival of three strains were 35%,41.7%and54%.That means CAP1 played a role in baicalein-induced apoptosis. CAP1-deleted cells was very sensitive to baicalein.
Gene chip analysis showed that the genes affected by baicalein were related to cell cycle, drug efflux, virulence, substrate transport and heat shock.
引文
1. Chiou C C et al. Newdrugs and novel targets for treatment of invasive fungal infections in patients with cancer. Oncologist 2000;5(2):120.
2. Yons CN et al. Transcriptional analysis of antifungal drug resistance in Canida albicans Antimicrob 2000;44(9):2296.
3. Iou CC et al. New drugs and novel targets for treatment of invasive fungal infections in patients with cancer. Oncologist.2000;5(2):120.
4.吴绍熙等.中国致病真菌10年动态流行病学研究.临床皮肤科杂志.1999;28(1):1-4.
5.张秀珍. 1986-2003年深部真菌感染及耐药趋势.抗真菌药物与真菌感染诊治研究学术会议论文集. 2003年12月厦门:67-71
6. Ruhnke M. Epidemiology of Candida albicans infections and role of non-Candida-albicans yeasts. Curr Drug Targets. 2006; 7(4):495-504.
7. Stevens DA. Therapy for opportunistic fungal infections: past, present and future. Indian J Cancer, 1995, 32(1): 1.
8. Maligie MA, Selitrennikoff CP. Cryptococcus neoformans resistance to echinocandins: (1,3)beta-glucan synthase activity is sensitive to echinocandins.Antimicrob Agents Chemother. 2005; 49(7):2851-6.
9. Onishi J, Meinz M, Thompson J, Curotto J, Dreikorn S, Rosenbach M, Douglas C, Abruzzo G, Flattery A, Kong L, Cabello A, Vicente F, Pelaez F, Diez MT, Martin I, Bills G, Giacobbe R, Dombrowski A, Schwartz R, Morris S, Harris G, Tsipouras A, Wilson K, Kurtz MB. Discovery of novel antifungal (1,3)-beta-D-glucan synthase inhibitors. Antimicrob Agents Chemother. 2000 ; 44(2):368-77.
10. Abruzzo GK, Flattery AM, Gill CJ, et al. Evaluation of the echinocandin antifungal MK-0991 (L-743,872): efficacies in mouse models of disseminated aspergillosis, candidiasis, andcryptococcosis. Antimicrob Agents Chemother, 1997, 41(11): 2333.
11. Kurtz MB, Douglas CM. Lipopeptide inhibitors of fungal glucan synthase. J Med VetMycol, 1997, 35(2): 79
12. Fujikawa K, Tsukamoto Y, Oki T, et al. Spectroscopic studies on the interaction of pradimicin BMY-28864 with mannose derivatives. Glycobiology, 1998, 8(4): 407
13. Lee TS, Khosla C, Tang Y. Engineered biosynthesis of aklanonic acid analogues. J Am Chem Soc. 2005; 127(35):12254-62.
14. Oakley KL, Moore CB, Denning DW. Activity of pradimicin BMS-181184 against Aspergillus spp. Int J Antimicrob Agents. 1999; 12(3): 267-9.
15. Walsh TJ, Giri N. Pradimicins: a novel class of broad-spectrum antifungal compounds. Eur J Clin microbiol infect Dis, 1997, 16(1): 93
16. Banks IR, Specht CA, Donlin MJ, Gerik KJ, Levitz SM, Lodge JK. A chitin synthase and its regulator protein are critical for chitosan production and growth of the fungal pathogen Cryptococcus neoformans. Eukaryot Cell. 2005; 4(11):1902-12.
17. Hector RF, Davidson AP, Johnson SM. Comparison of susceptibility of fungal isolates to lufenuron and nikkomycin Z alone or in combination with itraconazole. Am J Vet Res. 2005 Jun;66(6):1090-3.
18. Ganesan LT, Manavathu EK, Cutright JL, Alangaden GJ, Chandrasekar PH. In-vitro activity of nikkomycin Z alone and in combination with polyenes, triazoles or echinocandins against Aspergillus fumigatus. Clin Microbiol Infect. 2004 Nov;10(11):961-6.
19. Milewski S, Mignini F, Micossi L, et al. Antihistoplasmal in vitro and in vivo effect of Lys-Nva-FMDP. Med Mycol, 1998, 36(3): 177
20. Schiaffella F, Macchiarulo A, Milanese L, Vecchiarelli A, Fringuelli R. Novel ketoconazole analogues based on the replacement of 2,4-dichlorophenyl group with
1,4-benzothiazine moiety: Design, synthesis, and microbiological evaluation.Bioorg Med Chem. 2006, 28;138.
21. Fostel J, Montgomery D. Identification of the aminocatechol A-3253 as an in vitro poison of DNA topoisomerase I from Candida albicans. Antimicrob agents chemother, 1995, 39(3): 586
22.张鸿龙,顾秀玉,王蕖,等.氧代赖氨酸抗真菌作用机制的初步研究.微生物学报, 1993, 33(6): 411
23. Sendid B, Cotteau-Leroy A, Francois N, D'Haveloose A, Standaert A, Camus D, Poulain D. Candidaemia and antifungal therapy in a French University Hospital: rough trends over a decade and possible links. BMC Infect Dis. 2006; 6(1): 80.
24. Yonga G. Current drug therapy of systemic mycoses: a review. East Afr Med J, 1995, 72(6): 394
25. Galgiani JN. Fluconazole, a new antifungal agent. Ann Intern Med, 1990,113(3): 177
26. Bedini A, Venturelli C, Mussini C, Guaraldi G, Codeluppi M, Borghi V, Rumpianesi F, Barchiesi F, Esposito R. Epidemiology of candidaemia and antifungal susceptibility patterns in an Italian tertiary-care hospital. Clin Microbiol Infect. 2006; 12(1):75-80.
27. Cadle RM, Zenon GJ-3rd, Rodriguez-Barradas MC, et al. Fluconazole-induced symptomatic phenytoin toxicity. Ann Pharmacother, 1994, 28(2): 191
28. Charlier C, Hart E, Lefort A, Ribaud P, Dromer F, Denning DW, Lortholary O. Fluconazole for the management of invasive candidiasis: where do we stand after 15 years? J Antimicrob Chemother. 2006; 57(3): 384-410.
29. Terrell CL. Antifungal agents, part II: the azoles. Mayo Clin Proc, 1999, 74(1): 78
30. Scholz M, Jennrich R, Strum S, Brosman S, Johnson H, Lam R. Long-term outcome for men with androgen independent prostate cancer treated with ketoconazole and hydrocortisone. J Urol. 2005; 173(6): 1947-52.
31. Bisht GS, Awasthi AK, Dhole TN. Antimicrobial activity of Hedychium spicatum. Fitoterapia. 2006; 77(3): 240-2.
32. Kosalec I, Pepeljnjak S, Kustrak D. Acta Pharm. 2005 Dec;55(4):377-85. Antifungal activity of fluid extract and essential oil from anise fruits (Pimpinella anisum L., Apiaceae).
33.王发松,任三香.川桂叶挥发油的化学成分与抗菌活性研究.武汉植物学研究,2002, 20(2): 321.
34.王发松,任三香.香叶树果挥发油的化学成分和抗菌活性研究.天然产物研究与开发,1999, 11(6): 1.
35. Tan RX, Tang HQ, Hu J. Lignans and sesquiterpene lactones from artemisia sieversiana and Inula racemosa. Phytochemistry, 1998,49(1): 157.
36. Nicholas H.Oberlies,Jason P.Burgess,Hernan A.Navarro,et al.Novel bioactive clerodane diterpenoid from the leaves and twigs of Casearia sylvestris.J.Natural products,2002,65(2),.
37. Tania Maria de Almeida Alves, Fabiane Lacerda Ribeiro, Helmut Kloos. Polygodial, the Fungitoxic Component from the Brazilian Medicinal Plant Polygonum punctatum.Mem Inst Oswaldo Cruz, Rio de Janeiro, 2001, 96(6): 831.
38. D.E. Wedge, J.C.G. Galindo, F.A. Macias. Fungicidal activity of natural and synthetic sesquiterpene lactone analogs. Phytochemistry 2000,53: 747
39. G.Bader, M.Seibold, K.Tintelnot,et al. Cytotoxity of triterpenoid saponins. Pharmazine, 2000, 55: 72.
40. Gerald Bills,Anne Dombrowski,Sandra Aa.Morris.Hyalodendrosides A and B,antifungal Triterpenoid Glycosides from a Lignicolous Hyphomecete,Hyalodendron Species. J. Nat. Prod, 2000,63:90.
41. Hiller, K. Biologically Active Natural Products.1987, Oxford Press, Oxford, UK, 167.
42. Laura Alvarez,Maria del Carmen Perez,Jose Luis Gonzalez,et al.SC-1,an antimycotic spirostan saponin from Solanum chrysotrichum.Planta Med,2001,67:372.
43.杨得坡,胡海燕。黄芩甙元和黄芩甙对皮肤真菌与细菌抑制作用的研究。中药材,2000,23(5):272.
44.徐立春,余碧珏。银杏外种皮提取物对致病性真菌生长抑制的研究。微生物学通报,1991,18(4):225.
45. Supaporn Sawadjoon, Prasat Kittakoop, Kanyawim Kirtikara. Atropisomeric Myristinins: Selective COX-2 inhibitors and antifungal agents from Mysistica cinnamomea. J. Org. Chem, 2002, 67: 5470.
46. K.K.Atindehou,E.F.Queiroz,C.Terreaux.Three new prenylated isoflavonoids from the root bark of Erythrina vogelii.Pplanta Med,2002,68:181.
47. Lopez A, Ming DS, Towers GH.Antifungal activity of benzoic acid derivatives from Piper lanceaefolium.J Nat Prod,2002,65(1):62.
48. Zhang Z,ElSohly HN,Jacob MR,et al.Natural products inhibiting Candida albicans secreted aspartic proteases from Tovomita krukovii.Planta Med,2002,68(1):49.
49. Kyu-Ho Bang,Dong-Won Lee,Hee-Moon Park. Inhibition of fungal cell wall synthesizing. enzymes by trans-Cinnamaldehyde. Biosci. Biotechnol. Biochem, 2000, 64(5): 1061.
50. Blanca Freixa, Roser Vila, Esteban A. Antifungal principles from Piper fulvescens.Planta Med,2001,67:873.
51. Hexiang Wang, Ttzi Bun Ng. Isolation of lilin, a novel arginine- and glutamate-rich protein with potent antifungal and mitogenic activities from lily bulbs. Life Sciences,2002,70:1075.
52.刘小烛,胡忠。杜仲皮中抗真菌蛋白的分离和特性研究。云南植物研究,1994,16(4):385.
53. Hugo D. Chludil, Alicia M. Seldes, Marta S. Maier, Antifungal Steroidal Glycosides from the Patagonian Starifish Anasterias minuta. Structure-Activity Correlations. J. Nat. Prod, 2002: 65, 153.
54. Tomoo Hosoe,Hideaki Okada,Takeshi Itabashi,et al.A new pentanorlanostane derivative, Cladosporide A,as a characteristic antifungal agent against Aspergillus fumigatus,isolated from Cladosporium sp.. Chem.Pharm.Bull, 2000, 48(10): 1422.
55. Wu J.A.,Attele A.S.,Zhang L.HIV activity of medicinal herbs usage and potential devdopment. Am J Clin Med. 2001, 29:69—81
56.沈雯,陆福明,顾勇,等.BAI对高糖诱导近端肾小管上皮细胞外基质及转化生长因子岛表达的影响.中华肾脏病杂志. 2O03,19(3): 173-176.
57.董庆华,郑树,徐荣臻.黄芩苷元选择性诱导人的血病K562细胞凋亡.药学学报. 20O3, 38(11): 817-820
58. Lee H.Z.,Leung H.W.,Izi M.Y..Baiealein induced cell cycle and apoptosis in human lung squamom carcinoma CH27 cells. Antieancer Res 2005, 25(2): 959—964
59. Taniguchi H., Yoshida T., Horinaka M. Baicalein overcomes tumor necrosis factor-related apoptosis-inducing ligand resistance via two different cell-specific pathways in cancer cells but not in normal cells. Cancer Res. 2008, 68:8918-8927
60.周立剐,张颖君,蔡艳等.黄酮和甾体类化合物的抗真菌活性.天然产物研究与开发.1997, 9(3): 24—29
61.杨得坡,胡海燕,黄世亮.黄芩甙元和黄芩甙对皮肤真菌与细菌抑制作用的研究.中药材. 2000, 23(5): 272-274
62. Perea S et al. Prevalence of molecular mechanisms of resistance to azole antifungal anents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency patients. Antimicrob Agents Chemother 2001; 45;10:2676
63. D.G. Maki and P.A. Tambyah , Engineering out the risk of infection with urinary catheters. Emerg. Infect. Dis. 2001;7: 1–6.
64. R.M. Donlan and J.W. Costerton , Biofilms: survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 2002;15:167–193.
65. J.W. Costerton et al., Bacterial biofilms: a common cause of persistent infections. Science 1999;284:1318–1322.
66. Cowen LE, Sanglard D, Calabrese D et al. Evolution of drug resistance in experimental populations of Candida albicans. J Bacteriol, 2000; 182:1515-22
67. Baillie, G. S., and L. J. Douglas. Candida biofilms and their susceptibility to antifungal agents. Methods Enzymol. 1999 ;310:644-656
68. C. Potera , Forging a link between biofilms and disease. Science 1999;283:1837–1838.
69. R.M. Donlan , Biofilms and device-associated infections. Emerg. Infect. Dis. 2001;7:277–281.
70. Hawser SP, Douglas LJ. Biofilm formation by Candida species on the surface of catheter materials in vitro. Infect Imuun 1994;62:915-921
71. Kuhn, D. M., Chandra, J., Mukherjee, P. K., Ghannoum, M. A. Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces. Infect.Immun. 2002; 70: 878-888
72. Ramage G, Vande Walle K, Wickes BL, Lopez-Ribot JL. Standerdized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob. Agents Chemother 2001;45:2475-2479
73. el-Azizi M, Khardori N. Factors influencing adherence of Candida spp. to host tissues and plastic surfaces. Indian J Exp Biol 1999;37:941–951.
74. Reynolds TB,. Fink GR. Bakers’yeast, a model for fungal biofilm formation. Science 2001;291:878–881.
75. Samaranayake YH, Wu PC, Samaranayake LP, So M. Relationship between the cell surface hydrophobicity and adherence of Candida krusei and Candida albicans to epithelia and denture acrylic surfaces. APMIS 1995;103:707-713.
76. Silva TM, Glee PM, Hazen KC. Influence of cell surface hydrophobicity on attachment of Candida albicans to extracellular matrix proteins. J Med Vet Mycol 1995;33:117-122.
77. Li X, Yan Z, Xu J. Quantitative variation of biofilms among strains in natural populations of Candida albicans. Microbiology 2003;149:353-362.
78. Singleton DR, Masuoka J, Hazen KC. Cloning and analysis of a Candida albicans gene that affects cell surface hydrophobicity. J Bacteriol 2001;183: 3582-3588.
79. Madeo, F., Frohlich, E., Frohlich, K. U., 1997. A yeast mutant showing diagnostic markers of early and late apoptosis. J. Cell. Biol. 139, 729-734.
80. Madeo, F., Herker, E., Maldener, C., Wissing, S., Lachel, S., Herlan, M., Fehr, M., Lauber, K., Sigrist, S. J., Wesselborg, S., Frohlich, K. U., 2002. A caspase-related protease regulates apoptosis in yeast. Mol. Cell. 9, 911-917.
81. Madeo, F., Herker, E., Wissing, S., Jungwirth, H., Eisenberg, T., Frohlich, K. U., 2004. Apoptosis in yeast. Curr. Opin. Microbiol. 7, 655–660.
82. Silva, R. D., Sotoca, R., Johansson, B., Ludovico, P., Sansonetty, F., Silva, M. T., Peinado, J. M., Corte-Real, M., 2005. Hyperosmotic stress induces metacaspase- and mitochondria-dependent apoptosis in Saccharomyces cerevisiae. Mol. Microbiol. 58,824-834.
83. Phillips, A. J., Sudbery, I., Ramsdale, M., 2003. Apoptosis induced by environmental stresses and amphotericin B in Candida albicans. Proc. Natl. Acad. Sci. USA. 100, 14327-14332.
84. Phillips, A. J., Crowe, J. D., Ramsdale, M., 2006. Ras pathway signaling accelerates programmed cell death in the pathogenic fungus Candida albicans. Proc. Natl. Acad. Sci. USA. 103, 726-731.
85. Pereira, C., Silva, R.D., Saraiva, L., Johansson, B., Sousa, M. J., C?rte-Real, M., 2008. Mitochondria-dependent apoptosis in yeast. Biochim. Biophys. Acta. 1783, 1286-1302.
86. Perrone, G. G., Tan, S. X., Dawes, I. W., 2008. Reactive oxygen species and yeast apoptosis. Biochim. Biophys. Acta. 1783, 1354-1368.
87. Hoeberichts, F. A., ten Have, A., Woltering, E. J., 2003. A tomato metacaspase gene is upregulated during programmed cell death in Botrytis cinerea-infected leaves. Planta. 217, 517-522.
88. Alarco, A. M., and M. Raymond. 1999. The b-Zip transcription factor Cap1p is involved in multiple resistance and oxidative stress response in Candida albicans. J. Bacteriol. 181: 700-708.
89. Carmel-Hare, O., G. Storz. 2000. Roles of the glutathione-and thioredoxindependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress. Annu. Rev. Microbiol. 54: 439-461.
90. Grant, C. M. 2001. Role of the glutathione/glutaredoxin and thioredoxin systems in yeast growth and response to stress conditions. Mol. Microbiol. 39: 533-541.
91. Luk, E., M. Yang, L. T. Jensen, Y. Bourbonnais, and V. C. Culotta. 2005. Manganese activation of superoxide dismutase 2 in the mitochondria of Saccharomyces cerevisiae. J. Biol. Chem. 280: 22715-22720.
92. Pozniakovsky, A. I., D. A. Knorre, O. V. Markova, A. A. Hyman, V. P. Skulachev, and F. F. Severin. 2005. Role of mitochondria in the pheromone-and amiodarone-induced programmed cell death of yeast. J. Cell Biol. 168: 257-269.
93. Trotter, E. W., and C. M. Grant. 2003. Non-reciprocal regulation of the redox status of the glutathione-glutaredoxin and thioredoxin systems. EMBO Rep 4: 184-188.
94. Tyler, D. D. 1975. Polarographic assay and intracellular distribution of superoxide dismutase in rat liver. Biochem. J. 147: 493-504.
95. Wang, Y., Y. Y. Cao, Y. B. Cao, D. J. Wang, X. M. Jia, X. P. Fu, J. D. Zhang, Z. Xu, K. Ying, W. S. Chen, and Y. Y. Jiang. 2007. Cap1p plays regulation roles in redox, energy metabolism and substance transport: an investigation on Candida albicans under normal culture condition. Front. Biosci. 12: 145-153.
96. Wang, Y., Y. Y. Cao, X. M. Jia, Y. B. Cao, P. H. Gao, X. P. Fu, K. Ying, W. S. Chen, and Y. Y. Jiang. 2006. Cap1p is involved in multiple pathways of oxidative stress response in Candida albicans, Free Radic. Biol. Med. 40: 1201-1209.
97. Lockhart DJ, Winzeler EA. Genomics, gene expression and DNA arrays. Nature 2000; 405(6788): 827-836
98 Jones T, Federspiel NA, Chibana H, et al. The diploid genome sequence of Candida albicans. Proc Natl Acad Sci USA 2004; 101(19): 7329-7334
99徐铮,曹永兵,高平挥,et al. SMART技术构建白念珠菌全长cDNA文库.第二军医大学学报2004; 25(10): 1090-1093
100 Cao YY, Cao YB, Xu Z, et al. cDNA microarray analysis of differential gene expression in Candida albicans biofilm exposed to farnesol. Antimicrob Agents Chemother 2005; 49(2): 584-589
101. Xu Z, Cao YB, Zhang JD, et al. Differential expression change of virulence related genes during the development of resistance in Candida albicans by cDNA array analysis. Acta Biochimica et Biophysica Sinica 2005; 39(7)
102. De Backer MD, Van Dijck P. Progress in functional genomics approaches to antifungal drug target discovery. Trends Microbiol 2003; 11(10): 470-478
103. Bachewich C, Thomas DY, Whiteway M. Depletion of a polo-like kinase in Candida albicans activates cyclase-dependent hyphal-like growth. Mol Biol Cell. 2003;14:2163-80.
104. Sinha I, Wang YM, Philp R, Li CR, Yap WH, Wang Y. Cyclin-dependent kinasescontrol septin phosphorylation in Candida albicans hyphal development. Dev Cell. 2007;13:421-32.
105.Warenda AJ, Kauffman S, Sherrill TP, Becker JM, Konopka JB. Candida albicans septin mutants are defective for invasive growth and virulence. Infect Immun. 2003;71:4045-51.
106. Gonzalez-Novo A, Correa-Bordes J, Labrador L, Sanchez M, Vazquez de Aldana CR, Jimenez J. Sep7 is essential to modify septin ring dynamics and inhibit cell separation during Candida albicans hyphal growth. Mol Biol Cell. 2008 Apr;19(4):1509-18.
107. Clemente-Blanco A, Gonzalez-Novo A, Machin F, Caballero-Lima D, Aragon L, Sanchez M, de Aldana CR, Jimenez J, Correa-Bordes J. The Cdc14p phosphatase affects late cell-cycle events and morphogenesis in Candida albicans. J Cell Sci. 2006;119(Pt 6):1130-43.
108. Bruun AW, Svendsen I, Sorensen SO, Kielland-Brandt MC, Winther JR. A high-affinity inhibitor of yeast carboxypeptidase Y is encoded by TFS1 and shows homology to a family of lipid binding proteins. Biochemistry. 1998;37:3351-7.
109. Higgins, C. F. The ABC transporter channel superfamily an overview. Semin. Cell Biol. 1993, 4: 1-5.
110. Keppler, D., Y. Cui, J. Konig, I. Leier, and A. Nies. Export pumps for anionic conjugates encoded by MRP genes. Adv. Enzyme Regul. 1999, 39: 237-246.
111. Pao, S. S., I. T. Paulsen, and M. H. Saier, Jr. Major facilitator superfamily. Microbiol. Mol. Biol. Rev. 1998, 62: 1-34.
112. White TC, Marr KA, and Bowden RA. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev. 1998, 11: 382-402.
113. Rex JH, Rinaldi MG, and Pfaller MA. Resistance of Candida species to fluconazole. Antimicrob. Agents and Chemother. 1995, 39: 1-8.143: 405-416.
114. Balan, I., Alarco, AM, and Raymond, M. The Candida albicans CDR3 gene codes for an opaque-phase ABC transporter. J. Bacteriol. 1997, 79(23): 7210-7218.
115. Franz, R., Michel, S., and Morschhauser, J. A fourth gene from the Candida albicansCDR family of ABC transporters. Gene. 1998, 220: 91-98.
116. Marchetti O, Moreillon P, Entenza JM, et al. Fungicidal synergism of fluconazole and cyclosporine in Candida albicans is not dependent on multidrug efflux transporters encoded by the CDR1, CDR2, CaMDR1, and FLU1 genes. Antimicrob Agents Chemother. 2003,47,1565-1570Parks LW.Metableolism of sterols in yeast [J]. Crit.Rev. Microbiol.1978,6:301-341.
117. Parks LW, Casey WM. Physiological implications of sterol biosynthesis in yeast [J]. Annu.Rev.Microbiol.1995, 49:95-116.
118. Ashman WH, Barbuch RJ, Ulbright CE, et al.Cloning and disruption of the yeast C-8 sterol isomerase gene[J]. Lipids.1991, 26:628-632.
119. Lai MH, Bard M, Pierson CA, et al. The identification of a gene family in the Saccharomyces cerevisiae ERGosterol biosynthesis pathway [J].Gene1994, 140:41-49.
120. Gaber RF, Copple DM, Kennedy BK, et al.The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cel-lcycle-sparking sterol[J]. Mol. Cell. Biol. 1989, 9:3447-3456. 9:1177-1187.
121. Arthington BA, Bennett LG, Skatrud PL, et al. Cloning, disruption, and sequence of the gene encoding yeast C-5 sterol desaturase [J]. Gene.1991, 102:39-44.
122. Smith SJ, Parks LW. The ERG3 gene in Saccharomyces erevisiae is required for the utilization of respiratory substrates and in heme-deficient cells [J]. Yeast .1993, 9:1177-1187.
123 Wilson D, Tutulan-Cunita A, Jung W, Hauser NC, Hernandez R, Williamson T, Piekarska K, Rupp S, Young T, Stateva L. Deletion of the high-affinity cAMP phosphodiesterase encoded by PDE2 affects stress responses and virulence in Candida albicans. Mol Microbiol. 2007;65:841-56.
124. Jung WH, Warn P, Ragni E, Popolo L, Nunn CD, Turner MP, Stateva L. Deletion of PDE2, the gene encoding the high-affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans. Yeast. 2005 Mar;22(4):285-94.
125. De Backer MD, Van Dijck P. Progress in functional genomics approaches toantifungal drug target discovery. Trends Microbiol 2003; 11(10): 470-478
1.谭仁祥.植物成分分析.北京:科学出版社,2002,486-502.
2.马晓辉,葛根素的临床应用.中国实用医药,2009, 4(1):231
3. Jia G H,Jia S H.Development in pharmacological research of flavones of liquorice.Chinese Pharmaceutical Journal,1998,9,33(9):513-6.
4.王玮,张海峰,蔡源源.醋柳黄酮的研究进展.河南大学学报(医学版)2008,27(4):17-19
5.焦士蓉,黄承钰柑橘属类黄酮生物活性的研究进展.西华大学学报,2008,27(1):32
6.周燕.攀枝花苏铁叶中的一个新黄酮碳苷.植物学报.2002,44(1):101
7.何红平.小叶臭的黄皮黄酮苷成分.云南植物研究.2001,23 (2):256
8.邵伟艳,黄雄飞,祝亚非等.3个新呋喃黄酮的NMR研究.分析测试学报.2001,20 (1):8
9.杨晓虹,刘银燕,刘丽娟等.分蘖葱头中新黄酮苷的结构鉴定.药学学报,2000,35 (10):752
10.李佳,陈玉婷.夏至草中两种黄酮苷类化合物的研究.药学学报. 2002,37(3):186
11.甘露,王芬,梁鸿,等.粗叶悬钩子化学成分的分离鉴定.北京医科大学学报. 2000,32 (3):226
12.杨小风,雷海民,付宏征等.栾树种子中黄酮类化学成分.药学学报. 2000,35(3):208
13.唐于平,王颖,楼风昌等.银杏叶中的黄酮醇苷类成分.药学学报. 2000,35(5):363
14.王先荣.黄蜀葵花黄酮成分的研究.中国天然药物. 2004,2 (2):91
15.刘菊福,卢长安,廖福龙.不同产地黄芩提取物主要药效作用的比较.中国中医药信息杂志. 2001,8(3):28-30
16.周晓霞,苏佩清.野黄芩苷对人胎儿平滑肌细胞增殖的抑制作用.山东中医杂志.2003,22(2):109~110
17.曹正国,李成章,刘小平,等.中药黄芩苷对人牙周膜细胞增殖和总蛋白含量的影响.口腔医学研究. 2003,19(1):10~12
18.李成章,曹正国,樊明文.芩苷对人牙周韧带细胞超微结构的影响.牙体牙髓牙周病学杂志.2003,13(2):76~77
19.刘桦,吴晓冬,阎倩,等.黄芩苷对缺氧缺糖性心肌细胞的保护作用.中国药科大学学报.2003,34(1):55~57
20.董虎,钱微微.黄芩甙对糖尿病患者周围神经传导速度和尿微量白蛋白排泄的影响.中国中西医结合杂志. 2002,22(12):915~917
21.苏连杰,田鹤,马英丽.金莲花醇提物体内抗病毒作用的实验研究[J].中草药,2007,38(7):1062-1064.
22.杨殿福,徐泊文,郝钰.中药抗流感病毒机制研究进展[J].中国中医急症,2007,16(8):988-990
23. Zhi BY,Yan Y,Yi ZL. In vitro antioxidant and antimicrobial activities of the extract of Pericarpium Citri Reticulatae of a new Citrus cuhivar and its main flavonoids.LwT-Food Science and Technology. 2008,41(4):597.
24. Nagai T.黄芩根中抗流感A(H3N2)和B病毒的黄酮类成分5,7,4'-三羟基-8-甲氧基黄酮F36.国外医学-中医中药分册. 1996,18(3):46
25.永井隆之.生物中类黄酮药抗流感病毒活性产生的作用机制.国外医学-中医中药分册. 1995,17(6):40-42
26. Liu I X,Durham D G,Richards R M.Baicalin synergy with beta-lactam antibiotics against methicillin-resistant Staphylococcus aureus and other beta-lactam resistant strains of S.aureus.J Pharm Pharm acol. 2000,52(3):363~366
27. Hahm D H,Yeom M L,Lee E H,et a1.Effect of & tellariae radix as a novel antibacterial herb on the ppk (polyphosphate kinase) mutant of salmonella typhimurium.Microbiol. Biotech. 2001,11(6):1061~1065
28.吕小迅,周玉珍,吕华冲.黄芩、黄精抗真菌作用活性部位实验研究.广东药学院学报. 1995,11(3):180-182
29.张秀娟,李津明,霍明.黄芩水煎液黄芩甙去黄芩甙水煎液抑菌试验比较.黑龙江商学院学报(自然科学版). 1997,13(4):6~9
30.刘云波,郭丽华,邱世翠.黄芩体外抑菌作用研究.时珍国医国药. 2002,13(10):596
31.赵晶.两种抗人免疫缺陷病毒天然产物结构修饰的研究[D].北京:中国协和医科大学,1998
32.金梅花,许惠仙,金花,全吉淑,尹学哲.大豆异黄酮和皂甙对结肠癌细胞增殖和凋亡的研究.大豆科学,2008,27(6):1028-1031
33. Yoshiki Miyata,Takashi Sato,Keisuke Imada,et a1.A citrus polymethoxyfavonoid,nobiletin,is a novel MEK inhibitor [243 that exhibits antitumor metastasis in human fibrosarcoma HT-1080 cells.Biochemical and Biophysical Research Communications,2008,366 (1):168 173.
34.何晖,翟明. 4′,5,7三羟基异黄酮抗骨髓瘤细胞增殖机制的研究.天然产物研究与开发,2008,20:1067-1071
35. Shinichi Ikemoto,Kaiunobu Sugimuka,Naomasa Yoshi.et a1.Antitumor effects of scutellariae radix and its components baicalein,baicalin, and wogonin on bladder cancer cell lines.Urology,2000,55(6):951~955
36.袁榴娣,徐红,杜肇宗.黄芩甙对艾氏腹水瘤细胞影响的初步探讨.南京铁道医学院学报.1997,16(4):231~233
37.贺海平,秦菁,陈明,等.一种新的黄芩类黄酮3,5,6,7-四羟基-2,8-二甲氧基黄酮的体外免疫作用.广西医科大学学报. 2000,17(3):353~355贺海平,秦箐,陈明.黄芩类黄酮对人免疫细胞化学发光及淋巴细胞增殖的影响.中国免疫学杂志. 2000, 16(2):84~86
38. Huang Hua,Zha Xi liang.Development inresearch of antitumor efect of flavones compounds.Chin J New Clin Rem. 2002, 21(7):428-33.
39. Huynh H T.Selective induction of apoptosis in human mammary cancer cells by pycnogenol.Anticancer Res,2000,2O(4):2417-2O.
40. Ranelletfi F O,Maggiang N.Serra F G, et a1.Quercetin inhibits p21-RAS expression in human colon cancer cell lines and in primary eolorectal tumors.Int J Cancer,2O0O,85(3):438-40
41.刘新文,李欣,杨燕宁.黄芩黄酮对硒性白内障晶状体抗氧化酶表达的影响.中国生物化学与分子生物学报.2002,18(4):511~514
42.刘玉萍,Purusotsm Basnet,曹军.黄芩清除自由基活性与黄芩苷含量的相关性研究.中国中药杂志. 2002,27(8):575-579
43.谭廷华,刘爱芳,王新英,等.黄芩甙与芸香甙对·OH的清除作用.西安医科大学学报. 1997,18(1):41-43
44.张永钦,周井炎,徐辉碧.黄芩甙的抗氧化作用.华中理工大学学报.1999,27(4):111~113
45. Gao D,Sakurai K,Katoh M,et a1.Inhibition microsomal lipid peroxidation by baicalein:a possible formation of an iron-baicalein complex.Biochem Mol Biol Int,1996,39(2):215
46.胡春,丁霄霖.黄酮类化合物在不同氧化体系中的抗氧化作用研究.食品与发酵工业,1996,22(3):46-53.
47.陈时宏.黄酮类抗氧化作用及构效关系.海峡药学.1998,10(4):4-6.
48. Mora A.Structure-activity relationship of polymethoxyflavones and other flavonoids as inhibitors of non-enzymic lipid peroxidation. Biochem.Pharmacol,1990,40:79-797.
49. Wang H,Joseph J A.Structure-activity relationships of quereetin in antagonizing hydrogen pe roxide-induced cMcium dysregulation.Free Radical Biology and Medicine,1999,27(5-6):683-94.
50. Li SM,Sang SM,Pan MH,et a1.Anti-inflammatory property of the urinary metabolites of nobiletin in mouse.Bioorganic Medicinal Chemistry Letters,2007,17 (18):5177-5181.
51. Pan MH,Lai CS,Wang YJ,et a1.Polymethoxyflavonoids(PMFs) suppressed TPA-induced up-expression of iNOS and COX-2 and tumor promotion in mice.Abstracts of Papers,234th ACS National Meeting.Boston,MA,United States:August 19-23,2007.
52.范丽,董六一,江勤等.黄蜀葵花总黄酮抗炎解热作用.安徽医科大学学报.2003,38(1):25~28
53. DellaLoggia R,Sosa S,Tubaro A,et a1.Anti-inflammatory activity of Ginkgo biloba flavonoids.PlantaMed,1993,(59):A588
54.刘金霞,邓淑华,杨贺松等.黄芩茎叶总黄酮的抗炎作用机制的研究.中国药理学通报,2002,18(6):713~714
55.石新兰,凌允礼,侯健存.亚硒酸钠及抗氧化剂抑制小鼠肺泡Arthus血管炎发生的机制.中国医学科学院学报1999,(4):257~26
56.贺海平,秦箐,陈明,等.氧基黄酮的体外免疫药理作用.广西医科大学学报,2000,17(3):353~355
57.朱晓薇.国外抗炎植物药研究进展.国外医药一植物药分册. 1998.13(2):51-9.
58.许实波.杨东梅.穿心荸酮抗炎作用及毒性的初步研究.广东药学院学报,2001.17(1):35-5.
59. Hong T,Jin G B,Cho S,et a1.Evalution of the anti-inflammatory effect of baicalein on dextran sulfate sodium-induced colitis in mice.Planta Medica,2002,68(3):268-271
1. Zhang Shangtong ,Donald G. Ahearn, Carolina Mateus, et al. In vitro effects of Ag+ on planktonic and adhered cells of fluconazole-resistant and susceptible strains of Candida albicans,C.glabrata and C.krusei[J].Biomaterials,2006,27:2755–2760.
2. Paul M.J. Dennison, Mark Ramsdale, Claire L. Manson,et al.Gene disruption in Candida albicans using a synthetic, codon-optimised Cre-loxP system[J].Fungal Genetics and Biology,2005,42:737–748.
3. Oliver Reu?, Ashild Vik, Roberto Kolter, et al .The SAT1 flipper, an optimized tool for gene disruption in Candida albicans [J].Gene,2004,341:119–127.
4. Staab. Janet F and Sundstrom Paula.URA3 as a selectable marker for disruption and virulence assessment of Candida albicans genes[J].Trends in Microbiology, 2003,11:69-72.
5. Morschhauser J,Michel S,Staib P.Sequential gene disruption in Candida albicans by FLP-mediated site-specific recombineation[J].Molecular Microbiolgy,1999,3:547-556
6. R.Bryce Wilson,Dana Davis, Aaron P.Mitchell,et al. Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions[J].Journal of Bacteriology, 1999,181:1868-1874.
7. Kurtz MB and Marrinan J. Isolation of hem3 mutants from Candida albicans by sequentiall gene disruption[J]. Mol.Gen.Genet,1989,217:47-52.
8. A Negredo ,L Monteoliva , C Gil,et al. Cloning, analysis and one-step disruption of the ARG5,6 gene of Candida albicans[J].Microbiology,1997,143:297-302.
9. M.Andrew Uhl,Matt Biery, Nancy Craig,et.al.Haploinsufficiency-based large-scale forward genetic analysis of filamentous growth in the diploid human fungal pathogen Candida albicans[J].EMBO Journal,2003,22:2668-2678.
10. Dana A. Davis, Vincent M. Bruno, Lucio Loza, et al. Candida albicans Mds3p,a Conserved Regulator of pH Responses and Virulence Identified Through Insertional Mutagenesis[J]. Genetics,2002,162:1573-1581.
11. Flavia De Bernardis, Fritz A. Muhlschlegel, Antonio Cassone, et al .The pH of the HostNiche Controls Gene Expression in and Virulence of Candida albicans [J].Infect Immun,1998,66: 3317-3325.
12. Dana Davis, John E.Edwards Jr, Aaron P.Mitchell, et al .Candida albicans RIM101 pH Response Pathway Is Required for Host-Pathogen Interactions[J]. Infection and Immunity, 2000,68:5953-5959.
13. Brice Enjalbert, Andre Nantel, and Malcolm Whiteway. Stress-induced gene expression in Candida albicans:absentce of a general stresss response[J]. Molbiolcell , 2003,14:1460-1467.
14. Terry Roemer, Bo Jiang, John Davison, et al. Large-scale essential gene identifyication in Candida albicans and applications to antifungal drug discovery[J]. Molecular Microbiology,2003,50:167-181.
15. Giaever G, Shoemaker DD, Jones TW, et al. Genomic profiling of drug sensitivities via induced haploinsufficiency[J]. Nat Genet,1999,21:278-283.
16. Paula Sundstrom. Adhesion in Candida spp[J]. Cellular Microbiol,2002,4:461-469.
17. Haoping Liu. Transcriptional control of dimorphism in Candida albicans[J].Current Opinion in Microbiology,2001,4:728-735.
18. Christina M.Hull and Joseph Heitman. Fungal mating: Candida albicans flips a switch to get in the mood[J]. Current Biology,2002,12: R782-R784.
19. Shawn R.Lockhart, Karla J.Daniels,Rui Zhao,et al. Cell Biology of Mating in Candida albicans [J].Eukaryotic Cell,2003,2:49-61.
20. Douglas LJ. Candida biofilms and their role in infection[J].Trends Microbiol, 2003,11:30-36.
2. Yons CN et al. Transcriptional analysis of antifungal drug resistance in Canida albicans Antimicrob 2000;44(9):2296.
3. Iou CC et al. New drugs and novel targets for treatment of invasive fungal infections in patients with cancer. Oncologist.2000;5(2):120.
4.吴绍熙等.中国致病真菌10年动态流行病学研究.临床皮肤科杂志.1999;28(1):1-4.
5.张秀珍. 1986-2003年深部真菌感染及耐药趋势.抗真菌药物与真菌感染诊治研究学术会议论文集. 2003年12月厦门:67-71
6. Ruhnke M. Epidemiology of Candida albicans infections and role of non-Candida-albicans yeasts. Curr Drug Targets. 2006; 7(4):495-504.
7. Stevens DA. Therapy for opportunistic fungal infections: past, present and future. Indian J Cancer, 1995, 32(1): 1.
8. Maligie MA, Selitrennikoff CP. Cryptococcus neoformans resistance to echinocandins: (1,3)beta-glucan synthase activity is sensitive to echinocandins.Antimicrob Agents Chemother. 2005; 49(7):2851-6.
9. Onishi J, Meinz M, Thompson J, Curotto J, Dreikorn S, Rosenbach M, Douglas C, Abruzzo G, Flattery A, Kong L, Cabello A, Vicente F, Pelaez F, Diez MT, Martin I, Bills G, Giacobbe R, Dombrowski A, Schwartz R, Morris S, Harris G, Tsipouras A, Wilson K, Kurtz MB. Discovery of novel antifungal (1,3)-beta-D-glucan synthase inhibitors. Antimicrob Agents Chemother. 2000 ; 44(2):368-77.
10. Abruzzo GK, Flattery AM, Gill CJ, et al. Evaluation of the echinocandin antifungal MK-0991 (L-743,872): efficacies in mouse models of disseminated aspergillosis, candidiasis, andcryptococcosis. Antimicrob Agents Chemother, 1997, 41(11): 2333.
11. Kurtz MB, Douglas CM. Lipopeptide inhibitors of fungal glucan synthase. J Med VetMycol, 1997, 35(2): 79
12. Fujikawa K, Tsukamoto Y, Oki T, et al. Spectroscopic studies on the interaction of pradimicin BMY-28864 with mannose derivatives. Glycobiology, 1998, 8(4): 407
13. Lee TS, Khosla C, Tang Y. Engineered biosynthesis of aklanonic acid analogues. J Am Chem Soc. 2005; 127(35):12254-62.
14. Oakley KL, Moore CB, Denning DW. Activity of pradimicin BMS-181184 against Aspergillus spp. Int J Antimicrob Agents. 1999; 12(3): 267-9.
15. Walsh TJ, Giri N. Pradimicins: a novel class of broad-spectrum antifungal compounds. Eur J Clin microbiol infect Dis, 1997, 16(1): 93
16. Banks IR, Specht CA, Donlin MJ, Gerik KJ, Levitz SM, Lodge JK. A chitin synthase and its regulator protein are critical for chitosan production and growth of the fungal pathogen Cryptococcus neoformans. Eukaryot Cell. 2005; 4(11):1902-12.
17. Hector RF, Davidson AP, Johnson SM. Comparison of susceptibility of fungal isolates to lufenuron and nikkomycin Z alone or in combination with itraconazole. Am J Vet Res. 2005 Jun;66(6):1090-3.
18. Ganesan LT, Manavathu EK, Cutright JL, Alangaden GJ, Chandrasekar PH. In-vitro activity of nikkomycin Z alone and in combination with polyenes, triazoles or echinocandins against Aspergillus fumigatus. Clin Microbiol Infect. 2004 Nov;10(11):961-6.
19. Milewski S, Mignini F, Micossi L, et al. Antihistoplasmal in vitro and in vivo effect of Lys-Nva-FMDP. Med Mycol, 1998, 36(3): 177
20. Schiaffella F, Macchiarulo A, Milanese L, Vecchiarelli A, Fringuelli R. Novel ketoconazole analogues based on the replacement of 2,4-dichlorophenyl group with
1,4-benzothiazine moiety: Design, synthesis, and microbiological evaluation.Bioorg Med Chem. 2006, 28;138.
21. Fostel J, Montgomery D. Identification of the aminocatechol A-3253 as an in vitro poison of DNA topoisomerase I from Candida albicans. Antimicrob agents chemother, 1995, 39(3): 586
22.张鸿龙,顾秀玉,王蕖,等.氧代赖氨酸抗真菌作用机制的初步研究.微生物学报, 1993, 33(6): 411
23. Sendid B, Cotteau-Leroy A, Francois N, D'Haveloose A, Standaert A, Camus D, Poulain D. Candidaemia and antifungal therapy in a French University Hospital: rough trends over a decade and possible links. BMC Infect Dis. 2006; 6(1): 80.
24. Yonga G. Current drug therapy of systemic mycoses: a review. East Afr Med J, 1995, 72(6): 394
25. Galgiani JN. Fluconazole, a new antifungal agent. Ann Intern Med, 1990,113(3): 177
26. Bedini A, Venturelli C, Mussini C, Guaraldi G, Codeluppi M, Borghi V, Rumpianesi F, Barchiesi F, Esposito R. Epidemiology of candidaemia and antifungal susceptibility patterns in an Italian tertiary-care hospital. Clin Microbiol Infect. 2006; 12(1):75-80.
27. Cadle RM, Zenon GJ-3rd, Rodriguez-Barradas MC, et al. Fluconazole-induced symptomatic phenytoin toxicity. Ann Pharmacother, 1994, 28(2): 191
28. Charlier C, Hart E, Lefort A, Ribaud P, Dromer F, Denning DW, Lortholary O. Fluconazole for the management of invasive candidiasis: where do we stand after 15 years? J Antimicrob Chemother. 2006; 57(3): 384-410.
29. Terrell CL. Antifungal agents, part II: the azoles. Mayo Clin Proc, 1999, 74(1): 78
30. Scholz M, Jennrich R, Strum S, Brosman S, Johnson H, Lam R. Long-term outcome for men with androgen independent prostate cancer treated with ketoconazole and hydrocortisone. J Urol. 2005; 173(6): 1947-52.
31. Bisht GS, Awasthi AK, Dhole TN. Antimicrobial activity of Hedychium spicatum. Fitoterapia. 2006; 77(3): 240-2.
32. Kosalec I, Pepeljnjak S, Kustrak D. Acta Pharm. 2005 Dec;55(4):377-85. Antifungal activity of fluid extract and essential oil from anise fruits (Pimpinella anisum L., Apiaceae).
33.王发松,任三香.川桂叶挥发油的化学成分与抗菌活性研究.武汉植物学研究,2002, 20(2): 321.
34.王发松,任三香.香叶树果挥发油的化学成分和抗菌活性研究.天然产物研究与开发,1999, 11(6): 1.
35. Tan RX, Tang HQ, Hu J. Lignans and sesquiterpene lactones from artemisia sieversiana and Inula racemosa. Phytochemistry, 1998,49(1): 157.
36. Nicholas H.Oberlies,Jason P.Burgess,Hernan A.Navarro,et al.Novel bioactive clerodane diterpenoid from the leaves and twigs of Casearia sylvestris.J.Natural products,2002,65(2),.
37. Tania Maria de Almeida Alves, Fabiane Lacerda Ribeiro, Helmut Kloos. Polygodial, the Fungitoxic Component from the Brazilian Medicinal Plant Polygonum punctatum.Mem Inst Oswaldo Cruz, Rio de Janeiro, 2001, 96(6): 831.
38. D.E. Wedge, J.C.G. Galindo, F.A. Macias. Fungicidal activity of natural and synthetic sesquiterpene lactone analogs. Phytochemistry 2000,53: 747
39. G.Bader, M.Seibold, K.Tintelnot,et al. Cytotoxity of triterpenoid saponins. Pharmazine, 2000, 55: 72.
40. Gerald Bills,Anne Dombrowski,Sandra Aa.Morris.Hyalodendrosides A and B,antifungal Triterpenoid Glycosides from a Lignicolous Hyphomecete,Hyalodendron Species. J. Nat. Prod, 2000,63:90.
41. Hiller, K. Biologically Active Natural Products.1987, Oxford Press, Oxford, UK, 167.
42. Laura Alvarez,Maria del Carmen Perez,Jose Luis Gonzalez,et al.SC-1,an antimycotic spirostan saponin from Solanum chrysotrichum.Planta Med,2001,67:372.
43.杨得坡,胡海燕。黄芩甙元和黄芩甙对皮肤真菌与细菌抑制作用的研究。中药材,2000,23(5):272.
44.徐立春,余碧珏。银杏外种皮提取物对致病性真菌生长抑制的研究。微生物学通报,1991,18(4):225.
45. Supaporn Sawadjoon, Prasat Kittakoop, Kanyawim Kirtikara. Atropisomeric Myristinins: Selective COX-2 inhibitors and antifungal agents from Mysistica cinnamomea. J. Org. Chem, 2002, 67: 5470.
46. K.K.Atindehou,E.F.Queiroz,C.Terreaux.Three new prenylated isoflavonoids from the root bark of Erythrina vogelii.Pplanta Med,2002,68:181.
47. Lopez A, Ming DS, Towers GH.Antifungal activity of benzoic acid derivatives from Piper lanceaefolium.J Nat Prod,2002,65(1):62.
48. Zhang Z,ElSohly HN,Jacob MR,et al.Natural products inhibiting Candida albicans secreted aspartic proteases from Tovomita krukovii.Planta Med,2002,68(1):49.
49. Kyu-Ho Bang,Dong-Won Lee,Hee-Moon Park. Inhibition of fungal cell wall synthesizing. enzymes by trans-Cinnamaldehyde. Biosci. Biotechnol. Biochem, 2000, 64(5): 1061.
50. Blanca Freixa, Roser Vila, Esteban A. Antifungal principles from Piper fulvescens.Planta Med,2001,67:873.
51. Hexiang Wang, Ttzi Bun Ng. Isolation of lilin, a novel arginine- and glutamate-rich protein with potent antifungal and mitogenic activities from lily bulbs. Life Sciences,2002,70:1075.
52.刘小烛,胡忠。杜仲皮中抗真菌蛋白的分离和特性研究。云南植物研究,1994,16(4):385.
53. Hugo D. Chludil, Alicia M. Seldes, Marta S. Maier, Antifungal Steroidal Glycosides from the Patagonian Starifish Anasterias minuta. Structure-Activity Correlations. J. Nat. Prod, 2002: 65, 153.
54. Tomoo Hosoe,Hideaki Okada,Takeshi Itabashi,et al.A new pentanorlanostane derivative, Cladosporide A,as a characteristic antifungal agent against Aspergillus fumigatus,isolated from Cladosporium sp.. Chem.Pharm.Bull, 2000, 48(10): 1422.
55. Wu J.A.,Attele A.S.,Zhang L.HIV activity of medicinal herbs usage and potential devdopment. Am J Clin Med. 2001, 29:69—81
56.沈雯,陆福明,顾勇,等.BAI对高糖诱导近端肾小管上皮细胞外基质及转化生长因子岛表达的影响.中华肾脏病杂志. 2O03,19(3): 173-176.
57.董庆华,郑树,徐荣臻.黄芩苷元选择性诱导人的血病K562细胞凋亡.药学学报. 20O3, 38(11): 817-820
58. Lee H.Z.,Leung H.W.,Izi M.Y..Baiealein induced cell cycle and apoptosis in human lung squamom carcinoma CH27 cells. Antieancer Res 2005, 25(2): 959—964
59. Taniguchi H., Yoshida T., Horinaka M. Baicalein overcomes tumor necrosis factor-related apoptosis-inducing ligand resistance via two different cell-specific pathways in cancer cells but not in normal cells. Cancer Res. 2008, 68:8918-8927
60.周立剐,张颖君,蔡艳等.黄酮和甾体类化合物的抗真菌活性.天然产物研究与开发.1997, 9(3): 24—29
61.杨得坡,胡海燕,黄世亮.黄芩甙元和黄芩甙对皮肤真菌与细菌抑制作用的研究.中药材. 2000, 23(5): 272-274
62. Perea S et al. Prevalence of molecular mechanisms of resistance to azole antifungal anents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency patients. Antimicrob Agents Chemother 2001; 45;10:2676
63. D.G. Maki and P.A. Tambyah , Engineering out the risk of infection with urinary catheters. Emerg. Infect. Dis. 2001;7: 1–6.
64. R.M. Donlan and J.W. Costerton , Biofilms: survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 2002;15:167–193.
65. J.W. Costerton et al., Bacterial biofilms: a common cause of persistent infections. Science 1999;284:1318–1322.
66. Cowen LE, Sanglard D, Calabrese D et al. Evolution of drug resistance in experimental populations of Candida albicans. J Bacteriol, 2000; 182:1515-22
67. Baillie, G. S., and L. J. Douglas. Candida biofilms and their susceptibility to antifungal agents. Methods Enzymol. 1999 ;310:644-656
68. C. Potera , Forging a link between biofilms and disease. Science 1999;283:1837–1838.
69. R.M. Donlan , Biofilms and device-associated infections. Emerg. Infect. Dis. 2001;7:277–281.
70. Hawser SP, Douglas LJ. Biofilm formation by Candida species on the surface of catheter materials in vitro. Infect Imuun 1994;62:915-921
71. Kuhn, D. M., Chandra, J., Mukherjee, P. K., Ghannoum, M. A. Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces. Infect.Immun. 2002; 70: 878-888
72. Ramage G, Vande Walle K, Wickes BL, Lopez-Ribot JL. Standerdized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob. Agents Chemother 2001;45:2475-2479
73. el-Azizi M, Khardori N. Factors influencing adherence of Candida spp. to host tissues and plastic surfaces. Indian J Exp Biol 1999;37:941–951.
74. Reynolds TB,. Fink GR. Bakers’yeast, a model for fungal biofilm formation. Science 2001;291:878–881.
75. Samaranayake YH, Wu PC, Samaranayake LP, So M. Relationship between the cell surface hydrophobicity and adherence of Candida krusei and Candida albicans to epithelia and denture acrylic surfaces. APMIS 1995;103:707-713.
76. Silva TM, Glee PM, Hazen KC. Influence of cell surface hydrophobicity on attachment of Candida albicans to extracellular matrix proteins. J Med Vet Mycol 1995;33:117-122.
77. Li X, Yan Z, Xu J. Quantitative variation of biofilms among strains in natural populations of Candida albicans. Microbiology 2003;149:353-362.
78. Singleton DR, Masuoka J, Hazen KC. Cloning and analysis of a Candida albicans gene that affects cell surface hydrophobicity. J Bacteriol 2001;183: 3582-3588.
79. Madeo, F., Frohlich, E., Frohlich, K. U., 1997. A yeast mutant showing diagnostic markers of early and late apoptosis. J. Cell. Biol. 139, 729-734.
80. Madeo, F., Herker, E., Maldener, C., Wissing, S., Lachel, S., Herlan, M., Fehr, M., Lauber, K., Sigrist, S. J., Wesselborg, S., Frohlich, K. U., 2002. A caspase-related protease regulates apoptosis in yeast. Mol. Cell. 9, 911-917.
81. Madeo, F., Herker, E., Wissing, S., Jungwirth, H., Eisenberg, T., Frohlich, K. U., 2004. Apoptosis in yeast. Curr. Opin. Microbiol. 7, 655–660.
82. Silva, R. D., Sotoca, R., Johansson, B., Ludovico, P., Sansonetty, F., Silva, M. T., Peinado, J. M., Corte-Real, M., 2005. Hyperosmotic stress induces metacaspase- and mitochondria-dependent apoptosis in Saccharomyces cerevisiae. Mol. Microbiol. 58,824-834.
83. Phillips, A. J., Sudbery, I., Ramsdale, M., 2003. Apoptosis induced by environmental stresses and amphotericin B in Candida albicans. Proc. Natl. Acad. Sci. USA. 100, 14327-14332.
84. Phillips, A. J., Crowe, J. D., Ramsdale, M., 2006. Ras pathway signaling accelerates programmed cell death in the pathogenic fungus Candida albicans. Proc. Natl. Acad. Sci. USA. 103, 726-731.
85. Pereira, C., Silva, R.D., Saraiva, L., Johansson, B., Sousa, M. J., C?rte-Real, M., 2008. Mitochondria-dependent apoptosis in yeast. Biochim. Biophys. Acta. 1783, 1286-1302.
86. Perrone, G. G., Tan, S. X., Dawes, I. W., 2008. Reactive oxygen species and yeast apoptosis. Biochim. Biophys. Acta. 1783, 1354-1368.
87. Hoeberichts, F. A., ten Have, A., Woltering, E. J., 2003. A tomato metacaspase gene is upregulated during programmed cell death in Botrytis cinerea-infected leaves. Planta. 217, 517-522.
88. Alarco, A. M., and M. Raymond. 1999. The b-Zip transcription factor Cap1p is involved in multiple resistance and oxidative stress response in Candida albicans. J. Bacteriol. 181: 700-708.
89. Carmel-Hare, O., G. Storz. 2000. Roles of the glutathione-and thioredoxindependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress. Annu. Rev. Microbiol. 54: 439-461.
90. Grant, C. M. 2001. Role of the glutathione/glutaredoxin and thioredoxin systems in yeast growth and response to stress conditions. Mol. Microbiol. 39: 533-541.
91. Luk, E., M. Yang, L. T. Jensen, Y. Bourbonnais, and V. C. Culotta. 2005. Manganese activation of superoxide dismutase 2 in the mitochondria of Saccharomyces cerevisiae. J. Biol. Chem. 280: 22715-22720.
92. Pozniakovsky, A. I., D. A. Knorre, O. V. Markova, A. A. Hyman, V. P. Skulachev, and F. F. Severin. 2005. Role of mitochondria in the pheromone-and amiodarone-induced programmed cell death of yeast. J. Cell Biol. 168: 257-269.
93. Trotter, E. W., and C. M. Grant. 2003. Non-reciprocal regulation of the redox status of the glutathione-glutaredoxin and thioredoxin systems. EMBO Rep 4: 184-188.
94. Tyler, D. D. 1975. Polarographic assay and intracellular distribution of superoxide dismutase in rat liver. Biochem. J. 147: 493-504.
95. Wang, Y., Y. Y. Cao, Y. B. Cao, D. J. Wang, X. M. Jia, X. P. Fu, J. D. Zhang, Z. Xu, K. Ying, W. S. Chen, and Y. Y. Jiang. 2007. Cap1p plays regulation roles in redox, energy metabolism and substance transport: an investigation on Candida albicans under normal culture condition. Front. Biosci. 12: 145-153.
96. Wang, Y., Y. Y. Cao, X. M. Jia, Y. B. Cao, P. H. Gao, X. P. Fu, K. Ying, W. S. Chen, and Y. Y. Jiang. 2006. Cap1p is involved in multiple pathways of oxidative stress response in Candida albicans, Free Radic. Biol. Med. 40: 1201-1209.
97. Lockhart DJ, Winzeler EA. Genomics, gene expression and DNA arrays. Nature 2000; 405(6788): 827-836
98 Jones T, Federspiel NA, Chibana H, et al. The diploid genome sequence of Candida albicans. Proc Natl Acad Sci USA 2004; 101(19): 7329-7334
99徐铮,曹永兵,高平挥,et al. SMART技术构建白念珠菌全长cDNA文库.第二军医大学学报2004; 25(10): 1090-1093
100 Cao YY, Cao YB, Xu Z, et al. cDNA microarray analysis of differential gene expression in Candida albicans biofilm exposed to farnesol. Antimicrob Agents Chemother 2005; 49(2): 584-589
101. Xu Z, Cao YB, Zhang JD, et al. Differential expression change of virulence related genes during the development of resistance in Candida albicans by cDNA array analysis. Acta Biochimica et Biophysica Sinica 2005; 39(7)
102. De Backer MD, Van Dijck P. Progress in functional genomics approaches to antifungal drug target discovery. Trends Microbiol 2003; 11(10): 470-478
103. Bachewich C, Thomas DY, Whiteway M. Depletion of a polo-like kinase in Candida albicans activates cyclase-dependent hyphal-like growth. Mol Biol Cell. 2003;14:2163-80.
104. Sinha I, Wang YM, Philp R, Li CR, Yap WH, Wang Y. Cyclin-dependent kinasescontrol septin phosphorylation in Candida albicans hyphal development. Dev Cell. 2007;13:421-32.
105.Warenda AJ, Kauffman S, Sherrill TP, Becker JM, Konopka JB. Candida albicans septin mutants are defective for invasive growth and virulence. Infect Immun. 2003;71:4045-51.
106. Gonzalez-Novo A, Correa-Bordes J, Labrador L, Sanchez M, Vazquez de Aldana CR, Jimenez J. Sep7 is essential to modify septin ring dynamics and inhibit cell separation during Candida albicans hyphal growth. Mol Biol Cell. 2008 Apr;19(4):1509-18.
107. Clemente-Blanco A, Gonzalez-Novo A, Machin F, Caballero-Lima D, Aragon L, Sanchez M, de Aldana CR, Jimenez J, Correa-Bordes J. The Cdc14p phosphatase affects late cell-cycle events and morphogenesis in Candida albicans. J Cell Sci. 2006;119(Pt 6):1130-43.
108. Bruun AW, Svendsen I, Sorensen SO, Kielland-Brandt MC, Winther JR. A high-affinity inhibitor of yeast carboxypeptidase Y is encoded by TFS1 and shows homology to a family of lipid binding proteins. Biochemistry. 1998;37:3351-7.
109. Higgins, C. F. The ABC transporter channel superfamily an overview. Semin. Cell Biol. 1993, 4: 1-5.
110. Keppler, D., Y. Cui, J. Konig, I. Leier, and A. Nies. Export pumps for anionic conjugates encoded by MRP genes. Adv. Enzyme Regul. 1999, 39: 237-246.
111. Pao, S. S., I. T. Paulsen, and M. H. Saier, Jr. Major facilitator superfamily. Microbiol. Mol. Biol. Rev. 1998, 62: 1-34.
112. White TC, Marr KA, and Bowden RA. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev. 1998, 11: 382-402.
113. Rex JH, Rinaldi MG, and Pfaller MA. Resistance of Candida species to fluconazole. Antimicrob. Agents and Chemother. 1995, 39: 1-8.143: 405-416.
114. Balan, I., Alarco, AM, and Raymond, M. The Candida albicans CDR3 gene codes for an opaque-phase ABC transporter. J. Bacteriol. 1997, 79(23): 7210-7218.
115. Franz, R., Michel, S., and Morschhauser, J. A fourth gene from the Candida albicansCDR family of ABC transporters. Gene. 1998, 220: 91-98.
116. Marchetti O, Moreillon P, Entenza JM, et al. Fungicidal synergism of fluconazole and cyclosporine in Candida albicans is not dependent on multidrug efflux transporters encoded by the CDR1, CDR2, CaMDR1, and FLU1 genes. Antimicrob Agents Chemother. 2003,47,1565-1570Parks LW.Metableolism of sterols in yeast [J]. Crit.Rev. Microbiol.1978,6:301-341.
117. Parks LW, Casey WM. Physiological implications of sterol biosynthesis in yeast [J]. Annu.Rev.Microbiol.1995, 49:95-116.
118. Ashman WH, Barbuch RJ, Ulbright CE, et al.Cloning and disruption of the yeast C-8 sterol isomerase gene[J]. Lipids.1991, 26:628-632.
119. Lai MH, Bard M, Pierson CA, et al. The identification of a gene family in the Saccharomyces cerevisiae ERGosterol biosynthesis pathway [J].Gene1994, 140:41-49.
120. Gaber RF, Copple DM, Kennedy BK, et al.The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cel-lcycle-sparking sterol[J]. Mol. Cell. Biol. 1989, 9:3447-3456. 9:1177-1187.
121. Arthington BA, Bennett LG, Skatrud PL, et al. Cloning, disruption, and sequence of the gene encoding yeast C-5 sterol desaturase [J]. Gene.1991, 102:39-44.
122. Smith SJ, Parks LW. The ERG3 gene in Saccharomyces erevisiae is required for the utilization of respiratory substrates and in heme-deficient cells [J]. Yeast .1993, 9:1177-1187.
123 Wilson D, Tutulan-Cunita A, Jung W, Hauser NC, Hernandez R, Williamson T, Piekarska K, Rupp S, Young T, Stateva L. Deletion of the high-affinity cAMP phosphodiesterase encoded by PDE2 affects stress responses and virulence in Candida albicans. Mol Microbiol. 2007;65:841-56.
124. Jung WH, Warn P, Ragni E, Popolo L, Nunn CD, Turner MP, Stateva L. Deletion of PDE2, the gene encoding the high-affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans. Yeast. 2005 Mar;22(4):285-94.
125. De Backer MD, Van Dijck P. Progress in functional genomics approaches toantifungal drug target discovery. Trends Microbiol 2003; 11(10): 470-478
1.谭仁祥.植物成分分析.北京:科学出版社,2002,486-502.
2.马晓辉,葛根素的临床应用.中国实用医药,2009, 4(1):231
3. Jia G H,Jia S H.Development in pharmacological research of flavones of liquorice.Chinese Pharmaceutical Journal,1998,9,33(9):513-6.
4.王玮,张海峰,蔡源源.醋柳黄酮的研究进展.河南大学学报(医学版)2008,27(4):17-19
5.焦士蓉,黄承钰柑橘属类黄酮生物活性的研究进展.西华大学学报,2008,27(1):32
6.周燕.攀枝花苏铁叶中的一个新黄酮碳苷.植物学报.2002,44(1):101
7.何红平.小叶臭的黄皮黄酮苷成分.云南植物研究.2001,23 (2):256
8.邵伟艳,黄雄飞,祝亚非等.3个新呋喃黄酮的NMR研究.分析测试学报.2001,20 (1):8
9.杨晓虹,刘银燕,刘丽娟等.分蘖葱头中新黄酮苷的结构鉴定.药学学报,2000,35 (10):752
10.李佳,陈玉婷.夏至草中两种黄酮苷类化合物的研究.药学学报. 2002,37(3):186
11.甘露,王芬,梁鸿,等.粗叶悬钩子化学成分的分离鉴定.北京医科大学学报. 2000,32 (3):226
12.杨小风,雷海民,付宏征等.栾树种子中黄酮类化学成分.药学学报. 2000,35(3):208
13.唐于平,王颖,楼风昌等.银杏叶中的黄酮醇苷类成分.药学学报. 2000,35(5):363
14.王先荣.黄蜀葵花黄酮成分的研究.中国天然药物. 2004,2 (2):91
15.刘菊福,卢长安,廖福龙.不同产地黄芩提取物主要药效作用的比较.中国中医药信息杂志. 2001,8(3):28-30
16.周晓霞,苏佩清.野黄芩苷对人胎儿平滑肌细胞增殖的抑制作用.山东中医杂志.2003,22(2):109~110
17.曹正国,李成章,刘小平,等.中药黄芩苷对人牙周膜细胞增殖和总蛋白含量的影响.口腔医学研究. 2003,19(1):10~12
18.李成章,曹正国,樊明文.芩苷对人牙周韧带细胞超微结构的影响.牙体牙髓牙周病学杂志.2003,13(2):76~77
19.刘桦,吴晓冬,阎倩,等.黄芩苷对缺氧缺糖性心肌细胞的保护作用.中国药科大学学报.2003,34(1):55~57
20.董虎,钱微微.黄芩甙对糖尿病患者周围神经传导速度和尿微量白蛋白排泄的影响.中国中西医结合杂志. 2002,22(12):915~917
21.苏连杰,田鹤,马英丽.金莲花醇提物体内抗病毒作用的实验研究[J].中草药,2007,38(7):1062-1064.
22.杨殿福,徐泊文,郝钰.中药抗流感病毒机制研究进展[J].中国中医急症,2007,16(8):988-990
23. Zhi BY,Yan Y,Yi ZL. In vitro antioxidant and antimicrobial activities of the extract of Pericarpium Citri Reticulatae of a new Citrus cuhivar and its main flavonoids.LwT-Food Science and Technology. 2008,41(4):597.
24. Nagai T.黄芩根中抗流感A(H3N2)和B病毒的黄酮类成分5,7,4'-三羟基-8-甲氧基黄酮F36.国外医学-中医中药分册. 1996,18(3):46
25.永井隆之.生物中类黄酮药抗流感病毒活性产生的作用机制.国外医学-中医中药分册. 1995,17(6):40-42
26. Liu I X,Durham D G,Richards R M.Baicalin synergy with beta-lactam antibiotics against methicillin-resistant Staphylococcus aureus and other beta-lactam resistant strains of S.aureus.J Pharm Pharm acol. 2000,52(3):363~366
27. Hahm D H,Yeom M L,Lee E H,et a1.Effect of & tellariae radix as a novel antibacterial herb on the ppk (polyphosphate kinase) mutant of salmonella typhimurium.Microbiol. Biotech. 2001,11(6):1061~1065
28.吕小迅,周玉珍,吕华冲.黄芩、黄精抗真菌作用活性部位实验研究.广东药学院学报. 1995,11(3):180-182
29.张秀娟,李津明,霍明.黄芩水煎液黄芩甙去黄芩甙水煎液抑菌试验比较.黑龙江商学院学报(自然科学版). 1997,13(4):6~9
30.刘云波,郭丽华,邱世翠.黄芩体外抑菌作用研究.时珍国医国药. 2002,13(10):596
31.赵晶.两种抗人免疫缺陷病毒天然产物结构修饰的研究[D].北京:中国协和医科大学,1998
32.金梅花,许惠仙,金花,全吉淑,尹学哲.大豆异黄酮和皂甙对结肠癌细胞增殖和凋亡的研究.大豆科学,2008,27(6):1028-1031
33. Yoshiki Miyata,Takashi Sato,Keisuke Imada,et a1.A citrus polymethoxyfavonoid,nobiletin,is a novel MEK inhibitor [243 that exhibits antitumor metastasis in human fibrosarcoma HT-1080 cells.Biochemical and Biophysical Research Communications,2008,366 (1):168 173.
34.何晖,翟明. 4′,5,7三羟基异黄酮抗骨髓瘤细胞增殖机制的研究.天然产物研究与开发,2008,20:1067-1071
35. Shinichi Ikemoto,Kaiunobu Sugimuka,Naomasa Yoshi.et a1.Antitumor effects of scutellariae radix and its components baicalein,baicalin, and wogonin on bladder cancer cell lines.Urology,2000,55(6):951~955
36.袁榴娣,徐红,杜肇宗.黄芩甙对艾氏腹水瘤细胞影响的初步探讨.南京铁道医学院学报.1997,16(4):231~233
37.贺海平,秦菁,陈明,等.一种新的黄芩类黄酮3,5,6,7-四羟基-2,8-二甲氧基黄酮的体外免疫作用.广西医科大学学报. 2000,17(3):353~355贺海平,秦箐,陈明.黄芩类黄酮对人免疫细胞化学发光及淋巴细胞增殖的影响.中国免疫学杂志. 2000, 16(2):84~86
38. Huang Hua,Zha Xi liang.Development inresearch of antitumor efect of flavones compounds.Chin J New Clin Rem. 2002, 21(7):428-33.
39. Huynh H T.Selective induction of apoptosis in human mammary cancer cells by pycnogenol.Anticancer Res,2000,2O(4):2417-2O.
40. Ranelletfi F O,Maggiang N.Serra F G, et a1.Quercetin inhibits p21-RAS expression in human colon cancer cell lines and in primary eolorectal tumors.Int J Cancer,2O0O,85(3):438-40
41.刘新文,李欣,杨燕宁.黄芩黄酮对硒性白内障晶状体抗氧化酶表达的影响.中国生物化学与分子生物学报.2002,18(4):511~514
42.刘玉萍,Purusotsm Basnet,曹军.黄芩清除自由基活性与黄芩苷含量的相关性研究.中国中药杂志. 2002,27(8):575-579
43.谭廷华,刘爱芳,王新英,等.黄芩甙与芸香甙对·OH的清除作用.西安医科大学学报. 1997,18(1):41-43
44.张永钦,周井炎,徐辉碧.黄芩甙的抗氧化作用.华中理工大学学报.1999,27(4):111~113
45. Gao D,Sakurai K,Katoh M,et a1.Inhibition microsomal lipid peroxidation by baicalein:a possible formation of an iron-baicalein complex.Biochem Mol Biol Int,1996,39(2):215
46.胡春,丁霄霖.黄酮类化合物在不同氧化体系中的抗氧化作用研究.食品与发酵工业,1996,22(3):46-53.
47.陈时宏.黄酮类抗氧化作用及构效关系.海峡药学.1998,10(4):4-6.
48. Mora A.Structure-activity relationship of polymethoxyflavones and other flavonoids as inhibitors of non-enzymic lipid peroxidation. Biochem.Pharmacol,1990,40:79-797.
49. Wang H,Joseph J A.Structure-activity relationships of quereetin in antagonizing hydrogen pe roxide-induced cMcium dysregulation.Free Radical Biology and Medicine,1999,27(5-6):683-94.
50. Li SM,Sang SM,Pan MH,et a1.Anti-inflammatory property of the urinary metabolites of nobiletin in mouse.Bioorganic Medicinal Chemistry Letters,2007,17 (18):5177-5181.
51. Pan MH,Lai CS,Wang YJ,et a1.Polymethoxyflavonoids(PMFs) suppressed TPA-induced up-expression of iNOS and COX-2 and tumor promotion in mice.Abstracts of Papers,234th ACS National Meeting.Boston,MA,United States:August 19-23,2007.
52.范丽,董六一,江勤等.黄蜀葵花总黄酮抗炎解热作用.安徽医科大学学报.2003,38(1):25~28
53. DellaLoggia R,Sosa S,Tubaro A,et a1.Anti-inflammatory activity of Ginkgo biloba flavonoids.PlantaMed,1993,(59):A588
54.刘金霞,邓淑华,杨贺松等.黄芩茎叶总黄酮的抗炎作用机制的研究.中国药理学通报,2002,18(6):713~714
55.石新兰,凌允礼,侯健存.亚硒酸钠及抗氧化剂抑制小鼠肺泡Arthus血管炎发生的机制.中国医学科学院学报1999,(4):257~26
56.贺海平,秦箐,陈明,等.氧基黄酮的体外免疫药理作用.广西医科大学学报,2000,17(3):353~355
57.朱晓薇.国外抗炎植物药研究进展.国外医药一植物药分册. 1998.13(2):51-9.
58.许实波.杨东梅.穿心荸酮抗炎作用及毒性的初步研究.广东药学院学报,2001.17(1):35-5.
59. Hong T,Jin G B,Cho S,et a1.Evalution of the anti-inflammatory effect of baicalein on dextran sulfate sodium-induced colitis in mice.Planta Medica,2002,68(3):268-271
1. Zhang Shangtong ,Donald G. Ahearn, Carolina Mateus, et al. In vitro effects of Ag+ on planktonic and adhered cells of fluconazole-resistant and susceptible strains of Candida albicans,C.glabrata and C.krusei[J].Biomaterials,2006,27:2755–2760.
2. Paul M.J. Dennison, Mark Ramsdale, Claire L. Manson,et al.Gene disruption in Candida albicans using a synthetic, codon-optimised Cre-loxP system[J].Fungal Genetics and Biology,2005,42:737–748.
3. Oliver Reu?, Ashild Vik, Roberto Kolter, et al .The SAT1 flipper, an optimized tool for gene disruption in Candida albicans [J].Gene,2004,341:119–127.
4. Staab. Janet F and Sundstrom Paula.URA3 as a selectable marker for disruption and virulence assessment of Candida albicans genes[J].Trends in Microbiology, 2003,11:69-72.
5. Morschhauser J,Michel S,Staib P.Sequential gene disruption in Candida albicans by FLP-mediated site-specific recombineation[J].Molecular Microbiolgy,1999,3:547-556
6. R.Bryce Wilson,Dana Davis, Aaron P.Mitchell,et al. Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions[J].Journal of Bacteriology, 1999,181:1868-1874.
7. Kurtz MB and Marrinan J. Isolation of hem3 mutants from Candida albicans by sequentiall gene disruption[J]. Mol.Gen.Genet,1989,217:47-52.
8. A Negredo ,L Monteoliva , C Gil,et al. Cloning, analysis and one-step disruption of the ARG5,6 gene of Candida albicans[J].Microbiology,1997,143:297-302.
9. M.Andrew Uhl,Matt Biery, Nancy Craig,et.al.Haploinsufficiency-based large-scale forward genetic analysis of filamentous growth in the diploid human fungal pathogen Candida albicans[J].EMBO Journal,2003,22:2668-2678.
10. Dana A. Davis, Vincent M. Bruno, Lucio Loza, et al. Candida albicans Mds3p,a Conserved Regulator of pH Responses and Virulence Identified Through Insertional Mutagenesis[J]. Genetics,2002,162:1573-1581.
11. Flavia De Bernardis, Fritz A. Muhlschlegel, Antonio Cassone, et al .The pH of the HostNiche Controls Gene Expression in and Virulence of Candida albicans [J].Infect Immun,1998,66: 3317-3325.
12. Dana Davis, John E.Edwards Jr, Aaron P.Mitchell, et al .Candida albicans RIM101 pH Response Pathway Is Required for Host-Pathogen Interactions[J]. Infection and Immunity, 2000,68:5953-5959.
13. Brice Enjalbert, Andre Nantel, and Malcolm Whiteway. Stress-induced gene expression in Candida albicans:absentce of a general stresss response[J]. Molbiolcell , 2003,14:1460-1467.
14. Terry Roemer, Bo Jiang, John Davison, et al. Large-scale essential gene identifyication in Candida albicans and applications to antifungal drug discovery[J]. Molecular Microbiology,2003,50:167-181.
15. Giaever G, Shoemaker DD, Jones TW, et al. Genomic profiling of drug sensitivities via induced haploinsufficiency[J]. Nat Genet,1999,21:278-283.
16. Paula Sundstrom. Adhesion in Candida spp[J]. Cellular Microbiol,2002,4:461-469.
17. Haoping Liu. Transcriptional control of dimorphism in Candida albicans[J].Current Opinion in Microbiology,2001,4:728-735.
18. Christina M.Hull and Joseph Heitman. Fungal mating: Candida albicans flips a switch to get in the mood[J]. Current Biology,2002,12: R782-R784.
19. Shawn R.Lockhart, Karla J.Daniels,Rui Zhao,et al. Cell Biology of Mating in Candida albicans [J].Eukaryotic Cell,2003,2:49-61.
20. Douglas LJ. Candida biofilms and their role in infection[J].Trends Microbiol, 2003,11:30-36.