Synergistic Antimicrobial Activity of Berberine Hydrochloride, Baicalein and Borneol against Candida albicans

Objective To determine the synergistic effects of berberine hydrochloride, baicalein,and borneol in different combinations on Candida albicans. Methods The broth microdilution method was used to determine the minimal inhibitory concentration(MIC) and minimal bactericidal concentration(MBC) of the three agents, and the checkerboard method was simultaneously used to determine the MIC and fractional inhibitory concentration index(FICI) of the combination of three antimicrobial agents to study their extracorporeal effects. Results Berberine hydrochloride was the most potent inhibitor of C. albicans(MIC and MBC of 0.160 and 0.640 mg/mL), followed by borneol(MIC and MBC of 0.320 and 0.640 mg/mL) and baicalein(MIC and MBC of 1.28 and 20.48 mg/mL). Moreover, the antifungal effect of the combination was significantly stronger than that tested alone. Further in vivo study showed that the mortality rate of tainted mice reduced over 50% compared with the control group. Conclusion The results of experiments in vitro and in vivo indicate the synergistic effect of the combination of three antimicrobial agents on C. albicans, which can make reference for the future clinical treatment.     

Synergistic activity of lawsone methyl ether in combination with some antibiotics and artocarpin against methicillin-resistant Staphylococcus aureus, Candida albicans, and Trychophyton rubrum

Objective: One appealing strategy to overcome and prevent resistant problem is the use of combined two or more antibacterial substances. Lawsone methyl ether(LME) is the naphthoquinone found in the leaves of Impatiens balsamina. The objective of this study is to determine the interaction of LME with some antibiotics(ampicillin, tetracycline, norfloxacin, and clotrimazole) and a natural compound, artocarpin against methicillin-resistant Staphylococcus aureus(MRSA), Candida albicans, and Trychophyton rubrum.Methods: A broth microdilution method was used to determine the minimum inhibition concentration(MIC). Synergistic effects were evaluated at their own MIC using the checkerboard method and time-kill assay.Results: LME showed moderate antibacterial activity against MRSA with MIC value of 15.6 μg/mL, and exhibited strong antifungal activities against T. rubrum and C. albicans with MIC values of 7.8 and 3.9 μg/m L,respectively. The interaction of LME with the natural compound artocarpin against MRSA produced a synergy with fractional inhibitory concentration index(FICI) value of 0.31, while the combination of LME and clotrimazole exhibited synergy against C. albicans and T. rubrum with FICI values of 0.38 and 0.24, respectively. The time-kill assays confirmed that the compounds in combination enhanced their antimicrobial activities against the resistant microorganisms with different degrees.Conclusion: LME in combination with clotrimazole exhibited synergy effect against C. albicans and T.rubrum. In combination with artocarpin, it showed synergy effect against MRSA.     

Antimicrobial effect of sodium houttuyfonate on Staphylococcus epidermidis and Candida albicans biofilms

Objective

To study antimicrobial effect of Sodium houttuyfonate (SH) on Staphylococcus epidermidis (SE) and Candida albicans (CA).

Methods

The prepared strain broths (OD₆₀₀=0.05) containing SE and CA were firstly used to test the minimal inhibitory concentrations (MICs) of SH, azithromycin (AZM) and fluconazole (FLU) by micro-dilution method. Then the biofilms of SE and CA were matured in 96-well plates, and co-cultured with SH, AZM and FLU for 1, 2 and 3 days to assess the antibiofilm efficacies of the agents with different concentrations by crystal violet staining method. At last, the treated biofilms of SE and CA by 2×MIC agents were observed by scanning electronic microscope.

Results

The MICs of SE and CA were 256 and 1024 µg/mL, respectively. After the 1st, 2nd and 3rd day of medications, the suppressions of biofilm were about 60% (P<0.01), 76% (P=0.000) and 75% (P=0.000) by 2×MIC SH, the suppressions of biofilm were about 90% (P=0.000), 88% (P=0.000) and 90% (P=0.000) by 2×MIC SH, which could be testified by scanning electron microscope results. However, the inhibitions of biofilm attachment had no significant difference for SE by SH and azithromycin and CA by SH and fluconazole.

Conclusion

SH had widely anti-pathogenic effect on pathogenic biofilm formation of either bacteria or fungus, had more influence on enclosed cells of SE and CA than the traditional antibiotics, revealing its target might be the extracellular polymeric substances, and was more active to inhibit the growth of CA than SE.     

Antimicrobial effect of sodium houttuyfonate on Staphylococcus epidermidis and Candida albicans biofilms

OBJECTIVE： To study antimicrobial effect of Sodi- um houttuyfonate （SH） on Staphylococcus epider- midis （SE） and Candida albicans （CA）. METHODS： The prepared strain broths （OD600=0.05） containing SE and CA were firstly used to test the minimal inhibitory concentrations （MICs） of SH, azithromycin （AZM） and fluconazole （FLU） by mi- cro-dilution method. Then the biofilms of SE and CA were matured in 96-well plates, and co-cultured with SH, AZM and FLU for 1, 2 and 3 days to assess the antibiofilm efficacies of the agents with differ- ent concentrations by crystal violet staining meth- od. At last, the treated biofilms of SE and CA by 2× MIC agents were observed by scanning electronic microscope. RESULTS： The MlCs of SE and CA were 256 and 1024 μg/mL, respectively. After the 1st, 2nd and3rd day of medications, the suppressions of biofilm were about 60% （P〈0.01）, 76% （P=0.000） and 75% （P=0.000） by 2×MIC SH, the suppressions of biofilm were about 90% （P=0.000）, 88% （P=0.000） and 90% （P=0.000） by 2×MIC SH, which could be testified by scanning electron microscope results. However, the inhibitions of biofilm attachment had no significant difference for SE by SH and azithromycin and CA by SH and fluconazole. CONCLUSION： SH had widely anti-pathogenic ef- fect on pathogenic biofilm formation of either bac- teria or fungus, had more influence on enclosed cells of SE and CA than the traditional antibiotics, revealing its target might be the extracellular poly- meric substances, and was more active to inhibit the growth of CA than SE.     

黄芩苷对白色念珠菌酶活性的影响

目的运用分光光度法检测黄芩苷作用白色念珠菌后,其琥珀酸脱氢酶(SDH)、细胞色素C氧化酶(CCO)、Ca2 -Mg2 -ATPase活性的改变,以探讨黄芩苷抗白色念珠菌作用靶位,为探索中药活性成分抗真菌作用机理提供依据。方法收集经黄芩苷作用了相应时间的白色念珠菌,制备菌悬液,反复冻融研磨,差速离心法提取白色念珠菌线粒体;Lowry法检测线粒体蛋白质含量;分光光度法检测SDH酶、CCO酶、Ca2 -Mg2 -ATPase活力。结果①0.25,0.5,1mg/ml黄芩苷浓度组白色念珠菌SDH活力明显低于0mg/ml黄芩苷浓度组(P<0.05),且出现黄芩苷浓度越高SDH活力越低的趋势。不同作用时间组间白色念珠菌SDH活力差异无统计学意义(P>0.05)。②白色念珠菌经不同浓度黄芩苷作用不同时间后,其CCO酶活力均无明显差异(P>0.05)。③0.25,0.5,1mg/ml黄芩苷浓度组与0mg/ml黄芩苷浓度组比较,Ca2 -Mg2 -ATPase活力差异显著,黄芩苷浓度越高,Ca2 -Mg2 -ATPase活力越低,作用时间对Ca2 -Mg2 -AT-Pase活力没有明显影响(P>0.05)。结论①黄芩苷具有明显的抗白色念珠菌作用;②黄芩苷可降低白色念珠菌SDH酶活力;③黄芩苷对白色念珠菌CCO酶活力未见明显影响;④黄芩苷可降低白色念珠菌Ca2 -Mg2 -ATPase活力。     

扶正祛毒方联合盐酸小檗碱对乳腺癌细胞生长抑制作用的临床观察

目的:在传统使用盐酸小檗碱抑制乳腺癌细胞生长的基础上,以中医扶正祛邪理论为指导,联合使用扶正祛毒方及盐酸小檗碱治疗乳腺癌患者,以观察其对乳腺癌细胞生长的抑制作用,并进行讨论,以求为临床使用中西药联合治疗乳腺癌提供新思路、新方法。方法:选取2014年3月—2015年12月在医院进行治疗的乳腺癌患者59例,将其随机按数字表法分为对照组和观察组,对照组患者(29例)给予盐酸小檗碱进行治疗,观察组(30例)患者给予扶正祛毒方联合盐酸小檗碱进行治疗,观察比较两组患者的治疗效果。结果:治疗后,相比于对照组,观察组患者总有效率明显较高,统计差异具有明显的意义(χ2=4.584 4,P0.05);治疗前,相比于对照组,观察组患者的肿瘤坏死因子、白细胞介素-12水平无意义(P0.05)。治疗后,相比于对照组,观察组患者的肿瘤坏死因子、白细胞介素-12水平相对优于对照组,统计差异有意义(P0.05)。结论:在中医扶正祛邪理论的指导下,采用扶正祛毒方联合盐酸小檗碱治疗乳腺癌优于单纯使用盐酸小檗碱,能够更加有效地抑制乳腺癌细胞生长,促进癌细胞凋亡,减少乳腺癌患者的痛苦,提高其生活质量,是中西药联合抑制乳腺癌细胞生长的有效方法之一,值得推广。     

高效液相色谱法测定二妙丸中盐酸药根碱、盐酸巴马汀和盐酸小檗碱的含量

 目的建立同时测定二妙丸中盐酸药根碱、盐酸巴马汀和盐酸小檗碱含量的高效液相色谱方法。方法采用AgilentExtend C₁₈色谱柱(4.6 mm×150 mm,5μm),以水-乙腈(55∶45,每100 mL中含磷酸二氢钾0.34 g,十二烷基硫酸钠0.17 g)为流动相,紫外检测波长345 nm,柱温35℃,流速1 mL·min^-1。结果盐酸药根碱、盐酸巴马汀和盐酸小檗碱的理论塔板数分别为6 085,6 995和6 970。盐酸药根碱回归方程为Y=141 547X-7 456.9,r=0.999 1,线性范围6.1～122 ng;盐酸巴马汀回归方程为Y=93 047X+47 140,r=0.999 5,线性范围0.128～1.28μg;盐酸小檗碱回归方程为Y=86 106X+87 243,r=0.999 6,线性范围0.12～1.2μg。3组分的平均回收率分别为99.69%,100.23%和99.32%,RSD分别为3.97%,2.04%和2.25%(n=6)。结论该法操作简单,可以同时测定盐酸药根碱、盐酸巴马汀和盐酸小檗碱的含量,精密度高,重复性好,检测快速,定量准确,可用于二妙丸的质量控制。     

HPLC法测定苦柏洗剂中盐酸巴马汀、蛇床子素和盐酸小檗碱含量

目的：建立以高效液相色谱法同时测定苦柏洗剂中盐酸巴马汀、蛇床子素和盐酸小檗碱含量的方法。方法：采用高效液相色谱法,以盐酸巴马汀、蛇床子素和盐酸小檗碱作为指标成分,采用Zafex Acutfex PA-C1s(4.6 mm x 250mm,5μm）色谱柱,柱温：25℃,流动相：0.1%磷酸溶液（含15%乙腈和0.2%十二烷基硫酸钠）-乙腈（63:37）,流速：1.0mL·min-1,PDA检测器,检测波长：322nm。结果：盐酸巴马汀在0.6530~9.7955μg·mL-1范围内线性关系良好,r=0.9999,方法的平均回收率为97.13%,RSD为0.89%（n=6）。蛇床子素在0.3260~4.8903μg·mL范围内线性关系良好,r=0.9998,方法的平均回收率为95.86%,RSD为0.69%（n=6）。盐酸小檗碱在16.3554~245.3304μg·mL=1范围内线性关系良好,r=0.9999,方法的平均回收率为98.81%,RSD为0.80%（n=6）。结论：建立的苦柏洗剂中盐酸巴马汀、蛇床子素和盐酸小檗碱的含量测定方法,结果准确,重复性和分离度好,可用于同时测定苦柏洗剂中盐酸巴马汀、蛇床子素和盐酸小檗碱的含量,为更好地控制苦柏洗剂质量稳定和可靠提供科学依据。     

黄芩甙元和黄芩甙对皮肤真菌与细菌抑制作用的研究

本文研究了黄芩甙和黄芩甙元对十七种皮肤致病真菌与十六种皮肤细菌的抑制活性。在琼脂药物培养基上,黄芩甙元表现出了对酵母型真菌高度的选择抑制作用,其最低抑制浓度ＭＩＣ在７０～１００μｇ／ｍｌ之间,而对皮肤癣菌和丝状霉菌没有活性;黄芩甙则对所有的供试真菌几乎没有作用。另外,与脚气和腋臭有关的负责菌如何固着微球菌、表皮葡萄球菌和干燥棒杆菌等。在黄芩甙元浓度为２５０μｇ／ｍｌ的条件下,其生长繁殖也能完全抑制     

胡桃楸提取物抗白念珠菌作用的机制研究

目的：探讨胡桃楸提取物的抗白念珠菌作用机制。方法：应用同位素参入技术测定不同浓度的胡桃楸提取物对[3↑H]-TdR、[3↑H]-UdR、[3↑H]-Leu3种同位素前体参入白念珠菌的抑制率。结果：10、20和40μg/ml胡桃楸提取物对[3↑H]-TdR、[3↑H]-UdR、[3↑H]-Leu参入白念珠菌均具有显著的抑制作用，且呈剂量依赖性。各剂量的胡桃楸提取物作用后[3↑H]-UdR的参入抑制率均显著高于[3↑H]-TdR的参入抑制率、[3↑H]-Leu的参入抑制率；而[3↑H]-TdR的参入抑制率与[3↑H]-Leu的参入抑制率无显著性差异。结论：胡桃楸提取物抗白念珠菌的作用机制可能是通过抑制白念珠菌的DNA、RNA、蛋白质的生物合成导致菌细胞死亡而起到杀菌作用．     

没食子酸抑制白念珠菌生物膜作用的研究

目的:研究没食子酸对体外白念珠菌生物膜的影响.方法:采用XTT减低法评价没食子酸对白念珠菌的生物膜及黏附性的影响;镜下观察没食子酸对白念珠菌生物膜的形态学影响;细胞毒试验检测该药的毒副作用.结果:没食子酸抑制白念珠菌生物膜最低药物浓度SMIC_(50),SMIC_(80)分别是500,1 000 mg·L~(-1);100,1 000 mg·L~(-1) 的没食子酸对白念珠菌的早期黏附及菌丝生长有抑制作用;没食子酸对人细胞毒性较弱.结论:没食子酸对体外白念珠菌生物膜有较强的抑制作用.     

杜仲抗菌肽EuCHIT1对白色念珠菌的抑菌作用及机制研究

目的：研究杜仲抗菌肽EuCHIT1对白色念珠菌的抗菌活性及机制。方法：通过聚合酶链式反应（PCR）扩增EuCHIT1蛋白的编码区,构建原核表达质粒并转化到大肠杆菌Escherichia coli BL21（DE3）,经异丙基硫代半乳糖苷（IPTG）诱导,采用镍亲和色谱法纯化得到可溶性的EuCHIT1;采用二倍最小抑菌浓度（MIC）法稀释EuCHIT1并测定其对白色念珠菌的抑菌圈直径;采用荧光分光光度计和酶标仪分别检测EuCHIT1对白色念珠菌细胞膜的去极化作用及对细胞膜通透性的影响;通过鲎试验动态浊度法和高效液相色谱法分别测定白色念珠菌中（1,3）-β-D-葡聚糖和麦角甾醇含量;酶联免疫吸附法（ELISA）检测EuCHIT1对白色念珠菌感染的SD大鼠肺上皮WTRL1细胞中白细胞介素-1β（IL-1β）、IL-6及肿瘤坏死因子-α（TNF-α）表达的影响。结果：纯化得到的杜仲抗菌肽EuCHIT1相对分子质量为77 210,对白色念珠菌MIC值为32 μg·mL-1,EuCHIT1质量浓度与抑菌活性之间具有量效关系;EuCHIT1可增加白色念珠菌细胞壁的去极化及通透性,降低白色念珠菌细胞壁（1,3）-β-D-葡聚糖及麦角甾醇的含量,同时EuCHIT1可降低白色念珠菌感染的WTRL1细胞中IL-6及TNF-α水平（P<0.05）。结论：EuCHIT1对白色念珠菌具有抑菌作用,主要机制可能是破坏细胞壁,导致细胞膜去极化、通透性增加,并降低白色念珠菌感染的肺上皮细胞IL-6及TNF-α水平。     

HPLC测定岩黄连生物总碱中脱氢卡维丁、盐酸巴马汀和盐酸小檗碱

目的:建立高效液相色谱法测定岩黄连生物总碱中脱氢卡维丁、盐酸巴马汀及盐酸小檗碱含量的方法,为完善岩黄连生物总碱的质量评价提供依据。方法:采用高效液相色谱法,以Agilent XDB C18(4.6 mm×150 mm,5μm)为色谱柱,以乙腈-0.01 mol.L-1磷酸二氢钾水溶液(18.5∶81.5)为流动相,流速1.0 mL.min-1,柱温30℃,检测波长347 nm。结果:脱氢卡维丁进样量在15.375～92.25 mg.L-1(r=0.999 9)呈良好的线性关系,平均加样回收率96.43%,RSD 1.13%;盐酸巴马汀进样量在14.91～89.46 g.L-1(r=0.999 9)呈良好的线性关系,平均加样回收率96.28%,RSD 1.22%;盐酸小檗碱进样量在3.525～28.2 mg.L-1(r=0.999 7)呈良好的线性关系,平均加样回收率为103.02%,RSD 1.35%。结论:所建立的方法简便、准确、重复性好,可以用于控制岩黄连生物总碱的质量。     

盐酸小檗碱脱苦三元复合物的制备及其表征

目的：对制备的三元复合物脱苦效果进行评价,并对三元复合物进行红外光谱（IR）和差示扫描量热（DSC）表征分析。方法：采用共沉淀法,并利用富含β-乳球蛋白（β-LG）的乳清分离蛋白WPI90和盐酸小檗碱（BH）、β-环糊精（β-CD）制备β-CD/BH/WPI90三元复合物。结果：乳清分离蛋白（WPI90）、盐酸小檗碱和β-环糊精形成了三元复合物（β-CD/BH/WPI90）,盐酸小檗碱三元复合物（β-CD/BH/WPI90）脱苦效果优于盐酸小檗碱二元复合物（BH/β-CD）。且三元复合物中的盐酸小檗碱含量为3.20%。结论：乳清分离蛋白WPI90和盐酸小檗碱（BH）、β-环糊精（β-CD）形成的三元复合物对BH的苦味具有明显的降低效果。β-CD/BH/WPI90三元复合物的红外光谱（IR）和差示扫描量热（DSC）表征不同于3种物质简单的物理混合。     

峨眉黄连与三角叶黄连不同居群盐酸小檗碱和盐酸巴马汀的含量测定

通过峨眉黄连、三角叶黄连不同海拔高度的居群中小檗碱类生物碱含量的比较,拟证实生态环境和栽培管理对其药材质量影响较大;黄连药材种质资源不同,质量存在较大的差异,峨眉黄连根、根茎、叶的小檗碱含量均较高,间接说明古代雅连药材质量优于其它黄连(同在野生状态下)与基源种质有关。     

Enhanced antifungal activity of ketoconazole by Euphorbia characias latex against Candida albicans

The in vitro suseptibility of Candida albicans to ketoconazole and Euphorbia characias latex alone or in combination was tested using the macrobroth dilution method. The MIC 80% of crude latex and ketoconazole are respectively 159 microg protein/ml and 0.3901 microg/ml. This method permits us to determine an affinity constant K(aff) for crude latex (0.015 microg(-1) protein ml) and ketoconazole (23.828 microg(-1) ml). The utilization of a mixture of latex at several concentrations (7.8-15.62-31.25-62.5 and 125 microg protein/ml) and ketoconazole indicates a synergistic effect between latex and ketoconazole. For latex concentrations of 31.25 and 62.5 microg protein/ml the MIC 80% of ketoconazole were inferior (0.194 and 0.183 microg/ml respectively) to that obtained with ketoconazole alone (0.390 microg/ml). A synergistic effect is therefore obtained between ketoconazole on the one hand and two concentrations of Euphorbia characias latex.     

吴茱萸碱和盐酸小檗碱对大肠癌HT29细胞端粒酶活性的影响

目的:探讨左金丸的主要成分吴茱萸碱和盐酸小檗碱对结肠癌HT29细胞凋亡及端粒酶催化亚基(hTERT)表达的影响,为临床应用左金丸治疗大肠癌提供实验依据。方法:体外培养人结肠癌HT29细胞,加入不同浓度的吴茱萸碱及盐酸小檗碱进行培养,采用活细胞计数试剂盒(CCK-8)检测细胞增殖,TRAP法检测端粒酶活性,RT-PCR法检测hTERT的表达。结果:吴茱萸碱和盐酸小檗碱作用HT29细胞后,增殖明显受到抑制,呈剂量和时间依赖性;能够降低HT29端粒酶活性,但二者无协同作用;并且端粒酶hTERT表达降低,具有剂量依赖关系。结论:吴茱萸碱和盐酸小檗碱对结肠癌HT29细胞具有显著生长抑制作用,该抑制作用可能通过下调端粒酶hTERT基因表达,降低端粒酶的活性,为临床治疗大肠癌提供实验依据。     

高效液相色谱法测定痔疮洗剂中盐酸小檗碱和盐酸巴马汀的含量

目的采用高效液相色谱法对痔疮洗剂中盐酸小檗碱和盐酸巴马汀的含量进行测定,以控制本品的质量。方法采用Kromasil100—5C18（150mm×4．6mm,5um）色谱柱,以0．1％磷酸溶液-乙腈（50：50,每100mL含十二烷基磺酸钠0．1g）为流动相;检测波长：265nm。结果盐酸小檗碱和盐酸巴马汀分别在0．02～1．0ug和0．0172～0．861ug范围内线性关系良好。盐酸小檗碱的平均回收率为97．79％,盐酸巴马汀的平均回收率为99．22％。结论该法准确、稳定性好,可用于本品的质量控制。     

冰片与羟苯乙酯合用的体外溶血效应

目的 观察冰片和羟苯乙酯单用及合用对红细胞的溶血作用。方法 兔耳背中动脉取血,制成2 %的红细胞悬液,用不同浓度的冰片和羟苯乙酯与其反应,32 ℃孵育30 min,离心,检测吸光度,给出溶血曲线,并用等辐射分析法和响应面法进行分析。结果 冰片的半数溶血浓度（HC50）95 %的置信区间为263.39~393.27 μg·mL-1,羟苯乙酯的HC50 95 %的置信区间为867.93~916.65 μg·mL-1,冰片和羟苯乙酯合用后,溶血曲线左移,合用的HC50点位于两药物单用HC50的95 %置信区间内,响应面法验证了其为相加效应。结论 冰片和羟苯乙酯合用后溶血效应为相加作用,临床浓度时羟苯乙酯的溶血率很低,溶血效应主要源于冰片。     

黄连等6种中草药对都柏林念珠菌抗菌活性的比较研究

目的比较黄连、知母、七叶一枝花、黄芩、大黄、五倍子对都柏林念珠菌的体外抗菌活性。方法采用出芽实验法、肉汤微量稀释法和传统的琼脂扩散法。结果都柏林念珠菌对黄连、知母、七叶一枝花、黄芩敏感，抑菌固直径在23～28mm之间，MIC分别是：0．06～0．125，0．06～0．125，0．125～0．25，0．25～0．5g/ml。黄连、知母、黄芩、大黄抑制出芽效果更好。结论黄连、知母、黄芩对都柏林念珠菌具有较好的抑菌效果，且抑制都柏林念珠菌出芽效果也较好。