In vitro Combinatory Antimicrobial Effect of Plumbagin with Oxacillin and Tetracycline against Staphylococcus aureus

Plumbagin (5‐hydroxy‐2‐methyl‐1,4‐naphthoquinone), a bicyclic naphthoquinone naturally distributed among Plumbago species, has been reported to have antimicrobial activity against a wide range of microorganisms. In this study, plumbagin was examined for its combinatory antimicrobial effect with tetracycline or oxacillin against nine strains of Staphylococcus aureus, including its methicillin‐ and multidrug‐resistant strains. Minimum inhibitory concentrations were determined through the broth microdilution method, whereas the combinatory effect was evaluated according to the sum of fractional inhibitory concentration (ΣFIC) indices. Additive interactions were obtained for both combinations against most of the strains tested. Synergy was obtained for combination with oxacillin against two out of seven strains (ΣFIC range 0.273–0.281), both were methicillin resistant. Our results proved plumbagin as a compound suitable for anti‐Staphylococcal combinatory testing. Moreover, to the best of our knowledge, this is the first report of plumbagin synergy with oxacillin against S. aureus strains, including its resistant forms. Copyright © 2014 John Wiley & Sons, Ltd.     

胡桃醌经大鼠肠道及Caco-2细胞模型转运特征的研究

目的研究胡桃醌经大鼠肠道及Caco-2细胞的转运特征。方法应用Caco-2细胞模型和翻转肠囊法考察转运时间、药物浓度对胡桃醌吸收的影响,采用高效液相色谱法测定胡桃醌浓度,计算其表观渗透系数（Papp）。结果随着浓度增加和时间延长,胡桃醌累积通透量逐渐增加;胡桃醌浓度为100μmol L-1时,在Caco-2细胞模型表观渗透系数为1.4×10-7cm s-1;在翻转肠囊模型表观渗透系数2.0×10-7cm s-1。结论胡桃醌的转运符合被动扩散的性质,经肠道吸收率较低。     

胡桃醌-PLGA纳米粒制备及对A375恶黑细胞的体外抗肿瘤作用

目的:制备胡桃醌(juglone,Jug)聚乳酸-羟基乙酸(poly lactic-co-glycolic acid,PLGA)纳米粒(Jug-PLGA-NPs),并考察其理化性质、体外释放特征及对A375细胞的体外影响。方法:采用乳化挥发法制备Jug-PLGA-NPs,对其粒径、包封率、载药率以及体外释放特征进行考察;荧光显微镜观察PLGA-NPs在体外细胞的摄取情况,小动物活体成像仪观测PLGA-NPs在BALB/c荷瘤裸鼠尾静脉注射后体内的分布;用噻唑蓝(thiazolyl blue tetrazolium bromide,MTT)比色法检测其对A375细胞增殖抑制作用,流式细胞仪进行细胞凋亡率及细胞周期检测;蛋白免疫印迹法检测蛋白激酶B(protein kinase B,Akt),磷酸化-Akt(p-Akt),周期蛋白D1(cyclin D1)的表达情况。结果:制备的Jug-PLGA-NPs平均粒径为(149.6±21.5) nm,包封率为(68.39±2.51)%,载药率(5.07±0.98)%,具有良好的缓释特征。PLGA-NPs在体外细胞摄取和体内活体成像中具有良好的穿透和靶向性能。不同浓度Jug-PLGA-NPs均能明显抑制A375细胞增殖、促进细胞凋亡,呈明显时间浓度依赖性(P0.05),且48 h作用略优于等浓度Jug;其机制可能与调节Akt磷酸化水平,下调cyclin D1表达(P0.05),阻滞细胞于G0/G1期有关(P0.05)。结论:负载Jug的PLGA纳米微粒制备简便,具有良好的药物缓释、肿瘤靶向及抗肿瘤能力,为未来Jug的临床应用提供了一种新的药物剂型。     

青龙衣胡桃醌类成分富集

目的 以胡桃醌类成分转移率为指标,通过静态吸附和解吸附筛选最佳树脂,建立青龙衣胡桃醌类成分的富集工艺.方法 比较4种不同极性的大孔吸附树脂对青龙衣胡桃醌类成分的静态吸附与解吸性能,筛选出AB8树脂为最佳树脂,优化其工艺参数.结果 最佳工艺参数为:上样液浓度0.0760 g/ml、上样pH2.0、上样流速3BV/h,洗脱浓度70%、洗脱体积3BV、洗脱流速2BV/h,径高比1:10.工艺优化后,青龙衣中胡桃醌类成分的含量由1.026%提升到11.08%,转移率为72.08%.结论 AB8型大孔树脂能有效地富集纯化青龙衣胡桃醌类成分.     

胡桃醌通过促进c-Myc泛素化降解抑制宫颈癌细胞的生长和促进凋亡

目的 观察c-Myc蛋白在宫颈癌HeLa细胞中的表达,探讨胡桃醌通过影响c-Myc蛋白泛素化降解进而影响HeLa细胞的增殖与凋亡。方法 培养人宫颈癌HeLa细胞,分为对照组：正常培养;10、20、50 μmol/L胡桃醌组：培养液中分别加入对应浓度的胡桃醌。以Western blot方法分析胡桃醌处理后c-Myc蛋白的表达。HeLa细胞分为对照组和20 μmol/L胡桃醌组,在培养0、2、4、8 h分别用Western blot检测c-Myc蛋白表达的变化;放线菌酮（CHX）法检测c-Myc蛋白半衰期的变化;CoIP方法检测c-Myc蛋白降解影响。HeLa细胞分为空白对照组：正常培养;胡桃醌组：20 μmol/L胡桃醌培养液培养;0.5、1.0、2.0 μmol/L MG132组：分别与0、1.0、2.0 μmol/L蛋白酶体抑制剂MG132加20 μmol/L胡桃醌培养,检测c-Myc蛋白表达的水平。将si c-Myc转染入HeLa细胞后,将细胞分siNC组：空载体组;si-NC;胡桃醌组：空载体加20 μmol/L胡桃醌处理;si-cMyc组：转染Myc RNA;si-cMyc20 μmol/L胡桃醌组：转染Myc RNA加20 μmol/L胡桃醌处理。MTT及流式细胞术检测20 μmol/L胡桃醌处理后对敲除及未敲除c-Myc基因的细胞增殖及凋亡的影响。结果 与对照组相比,不同浓度胡桃醌可下调c-Myc蛋白表达且曾时间及剂量依赖性（P<0.05;P<0.01）;胡桃醌可缩短c-Myc蛋白的半衰期,加入不同浓度的MG132后,即可明显上调c-Myc蛋白水平（P<0.05; P<0.01）。未用胡桃醌组相比,胡桃醌可明显增加c-Myc蛋白的泛素降解水平。未敲除c-Myc组相比,敲除组在胡桃醌处理后吸光度值明显增加（P<0.05）和早期凋亡率明显下降（P<0.05）。结论 胡桃醌通过影响c-Myc蛋白的泛素化降解过程,进而抑制HeLa细胞增殖并促进其凋亡的发生。     

青龙衣化学成分和药理作用研究进展及质量标志物的预测分析

查阅近年来国内外相关文献,在对青龙衣化学成分及现代药理作用研究的基础上,根据中药质量标志物(Q-Marker)概念,从化学成分与亲缘关系、传统药效与药性及成分可测性、采收时期和干燥方法等方面,对青龙衣Q-Marker成分进行预测分析,发现胡桃醌、胡桃酮、绿原酸、核桃素A、核桃素B、马尾树素可作为青龙衣的Q-Marker,后续可选择以上成分作为指标,为青龙衣质量评价体系的建立提供参考。     

Discovery of natural naphthoquinones as sortase A inhibitors and potential anti-infective solutions against Staphylococcus aureus

Three natural naphthoquinones were screened to find new anti-virulence agents as inhibitors against sortase A from Staphylococcus aureus (SaSrtA) by quantifying the increase in fluorescence intensity upon substrate cleavage at various concentrations. The 5-hydroxy-1,4-naphthalenedione derivatives, juglone and plumbagin, demonstrated a potent inhibitory effect, with IC₅₀ values of 1.78 μM, respectively, 16.71 μM. The related 2-hydroxy-1,4-naphthalenedione derivative, lawsone, demonstrated the selectivity of the chemical scaffold having no significant effect on SaSrtA. The experimental assay was reinforced by molecular docking experiments, antimicrobial, and toxicological studies. Molecular docking studies and the electrophilic character analysis suggest bonding to the enzyme active cysteine residue by a Michael addition reaction. None of the compounds had a significant effect on the concentration of total thiol proteins in the Daphnia magna toxicological assay after 24 hr exposure. Juglone and plumbagin moderately inhibited biofilm formation with no significant effect on bacterial growth of S. aureus, Enterococcus faecalis, and Staphylococcus epidermidis, indicating a selective anti-virulence profile.     

胡桃醌联合顺铂对宫颈癌HeLa细胞的促凋亡作用及其机制

目的：探讨胡桃醌与顺铂联合应用对宫颈癌HeLa细胞促凋亡作用的机制,阐明其对相关信号转导通路的作用。方法：选取处于对数生长期的HeLa细胞,将其分为对照组、胡桃醌组、顺铂组和胡桃醌＋顺铂组（联合用药组）。采用MTT法观察不同时间点HeLa细胞的增殖抑制率;Hoechst 33258染色法荧光显微镜下观察细胞凋亡形态;Westernblotting法检测各组细胞凋亡相关蛋白Bcl-2、Bax和caspase-3及PI3K/AKt通路中AKt和pAKt表达水平。结果：MTT法,各用药组HeLa细胞增殖于24、48和72h均可被抑制,与对照组比较,胡桃醌组及顺铂组对细胞的抑制较明显,而联合用药组抑制更加显著;与单用药组比较,联合用药组对细胞的抑制更显著。Hoechst 33258染色,胡桃醌组和顺铂组在处理细胞12 h后,出现明显的凋亡细胞形态,联合用药组该现象更明显。Western blotting检测,与对照组比较,12 h后胡桃醌组和顺铂组HeLa细胞中Bcl-2和pAKt蛋白表达水平明显降低,而Bax、Cleave-caspase-3和AKt蛋白表达水平明显升高,联合用药组上述变化更加明显。结论：胡桃醌和顺铂联合应用可通过抑制PI3K/AKt通路,进而促进HeLa细胞凋亡。     

青龙衣中胡桃醌对S180肉瘤小鼠的抑瘤作用研究

 目的研究胡桃醌的体内抗肿瘤作用,探讨其可能的作用机制。方法建立S₁₈₀荷瘤小鼠肉瘤模型,考察胡桃醌的体内抑瘤活性及其对胸腺和脾脏指数的影响;HE染色,光镜观察各组小鼠肿瘤组织生长及病理变化;透射电镜观察肿瘤细胞超微结构改变;流式细胞仪检测肿瘤组织细胞的凋亡率及细胞周期的改变。结果在2,4和8μmol·kg^-1·d^-1剂量下,胡桃醌对S₁₈₀实体瘤的抑瘤率分别为29.04%,37.33%,52.66%,瘤重与空白对照组比较均显著降低(P<0.01);胡桃醌4,8μmol·kg^-1·d^-1剂量组脾脏指数降低(P<0.01),各剂量组胸腺指数无明显变化(P>0.05);光镜下胡桃醌各组肿瘤组织坏死程度明显增加;电镜下观察发现,胡桃醌各组均出现核染色质凝集、边聚,凋亡小体形成等典型的细胞凋亡形态学改变;胡桃醌2,4和8μmol·kg^-1·d^-1剂量下,流式细胞仪检测发现凋亡峰,凋亡率分别为(5.58±0.63)%、(6.66±0.85)%和(10.27±1.05)%。细胞周期观察发现,G₁期细胞百分率有所降低,而G₂/M期有所增加,推测胡桃醌可将细胞阻滞于G₂/M期,但还需进一步研究证实。结论胡桃醌能有效抑制小鼠S₁₈₀实体肿瘤的生长,诱导肿瘤细胞凋亡及引起G₂/M期阻滞可能是其主要作用途径。