Biochemical mechanism of modulation of human P-glycoprotein (ABCB1) by curcumin I, II, and III purified from Turmeric powder

P-glycoprotein (Pgp, ABCB1) is an ATP-dependent drug efflux pump linked to development of multidrug resistance (MDR) in cancer cells. Previously [Biochem Pharmacol 2002;64:573-82], we reported that a curcumin mixture could modulate both function and expression of Pgp. This study focuses on the effect of three major curcuminoids--curcumin I, II and III purified from a curcumin mixture--on modulation of Pgp function in a multidrug resistant human cervical carcinoma cell line (KB-V1). The similar IC(50) values for cytotoxicity of curcuminoids of KB-V1, and KB-3-1 (parental drug sensitive cell line) suggest that these curcuminoids may not be substrates for Pgp. Treating the cells with non-toxic doses of curcuminoids increased their sensitivity to vinblastine only in the Pgp expressing drug resistant cell line, KB-V1, and curcumin I retained the drug in KB-V1 cells more effectively than curcumin II and III, respectively. Effects of each curcuminoid on rhodamine123, calcein-AM, and bodipy-FL-vinblastine accumulation confirmed these findings. Curcumin I, II and III increased the accumulation of fluorescent substrates in a dose-dependent manner, and at 15 microM, curcumin I was the most effective. The inhibitory effect in a concentration-dependent manner of curcuminoids on verapamil-stimulated ATPase activity and photoaffinity labeling of Pgp with the [(125)I]-iodoarylazidoprazosin offered additional support; curcumin I was the most potent modulator. Taken together, these results indicate that curcumin I is the most effective MDR modulator among curcuminoids, and may be used in combination with conventional chemotherapeutic drugs to reverse MDR in cancer cells.     

姜黄素类化合物体外抗凝血与抗血栓作用研究

目的研究姜黄素类化合物体外抗凝血与抗血栓活性,为探寻姜黄活血化瘀药效物质提供参考。方法采用家兔血浆复钙时间法、凝血酶时间法及体外血栓法、全血血块法,分别对3个天然姜黄素类化合物姜黄素、去甲氧基姜黄素、双去甲氧基姜黄素的体外抗凝血与抗血栓活性进行测定。结果姜黄素、去甲氧基姜黄素、双去甲氧基姜黄素均能延长家兔血浆复钙时间(P<0.01)及凝血酶时间(P<0.01),且均能加快体外血栓(P<0.01)及全血凝块的溶解(P<0.01),其中去甲氧基姜黄素的作用最强。结论姜黄素类化合物具有较好的体外抗凝血与抗血栓作用,空间不对称结构能加强姜黄素类化合物结构母核的抗凝活性。     

Inhibition of multidrug resistance proteins MRP1 and MRP2 by a series of alpha,beta-unsaturated carbonyl compounds

To study the possible interplay between glutathione metabolism of and MRP inhibition by thiol reactive compounds, the interactions of a series of alpha,beta-unsaturated carbonyl compounds with multidrug resistance proteins 1 and 2 (MRP1/ABCC1 and MRP2/ABCC2) were studied. Alpha,beta-unsaturated carbonyl compounds react with glutathione, and therefore either their parent compound or their intracellularly formed glutathione metabolite(s) can modulate MRP-activity. Inhibition was studied in Madin-Darby canine kidney cells stably expressing MRP1 or MRP2, and isolated Sf9-MRP1 or Sf9-MRP2 membrane vesicles. In the latter model system metabolism is not an issue. Of the series tested, three distinct groups could be discriminated based on differences in interplay of glutathione metabolism with MRP1 inhibition. Curcumin inhibited MRP1 transport only in the vesicle model pointing at inhibition by the parent compound. The glutathione conjugates of curcumin also inhibit MRP1 mediated transport, but to a much lesser extent than the parent compound curcumin. In the cellular model system, it was demonstrated that glutathione conjugation of curcumin leads to inactivation of its inhibitory potential. Demethoxycurcumin and bisdemethoxycurcumin inhibited MRP1 in both the vesicle and cellular model pointing at inhibitory potency of at least the parent compound and possibly their metabolites. A second group, including caffeic acid phenethyl ester inhibited MRP1-mediated calcein transport only in the MDCKII-MRP1 cells, and not in the vesicle model indicating that metabolism appeared a prerequisite to generate the active inhibitor. Finally cinnamaldehyde, crotonaldehyde, trans-2-hexanal, citral, and acrolein did not inhibit MRP1. For MRP2, inhibition was much less in both model systems, with the three curcuminoids being the most effective. The results of this study show the importance to study the complex interplay between MRP-inhibitors and their cellular metabolism, the latter affecting the ultimate potential of a compound for cellular MRP-inhibition.     

姜黄素类化合物在不同品系姜黄根茎内积累规律研究

研究姜黄素（Cur）、去甲氧基姜黄素（DMC）、双去甲氧基姜黄素（BDMC）3种主要姜黄素类成分在不同姜黄品系根茎内的动态积累规律,为姜黄规范化生产、适时采收和优质品种选育提供依据。不同姜黄品系姜黄素类化合物在姜黄根茎内的积累规律基本一致,相对含量随生育进程而降低,积累量随生育进程而增加,可将总姜黄素积累量作为姜黄适宜采收期的评价指标;缅甸姜黄素和双去甲氧基姜黄素含量均较高,可作为高姜黄素含量和高双去甲氧基姜黄素含量的优良育种 材料。     

PNIPAM nanoparticles for targeted and enhanced nose-to-brain delivery of curcuminoids: UPLC/ESI-Q-ToF-MS/MS-based pharmacokinetics and pharmacodynamic evaluation in cerebral ischemia model

Stroke is a one of the leading causes of disease and deaths worldwide, which causes irreversible deterioration of the central nervous system. Curcuminoids are reported to have a potential role in the amelioration of cerebral ischemia but they exhibit low serum and tissue levels due to low solubility and poor absorption. Curcumin (CUR), demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC)-loaded PNIPAM nanoparticles (NPs) were prepared by free radical polymerization and characterized for particles size, entrapment efficiency, zeta potential, in vitro release and ex vivo permeation study. Optimized CUR, DMC and BDMC-loaded NPs had the mean size of 92.46?±?2.8, 91.23?±?4.2 and 94.28?±?1.91?nm; zeta potential of ?16.2?±?1.42, ?15.6?±?1.33 and ?16.6?±?1.21 mV; loading capacity of 39.31?±?3.7, 38.91?±?3.6 and 40.61?±?3.6% and entrapment efficiency of 84.63?±?4.2, 84.71?±?3.99 and 85.73?±?4.31%, respectively. Ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectroscopy based bioanalytical method was developed and validated for pharmacokinetics, biodistribution, brain-targeting efficiency and brain drug-targeting potential studies post-intranasal (i.n.) administration which showed enhanced bioavailability of curcuminoids in brain as compared to intravenous administration. Improved neurobehavioural activity (locomotor and grip strength) and reduced cytokines levels (TNF-α and IL-1β) was observed in middle cerebral artery occlusion induced cerebral ischemic rats after i.n. administration of curcuminoids NPs. Finally, the toxicity study was performed which revealed safe nature of developed NPs.     

姜黄素类物的提取、分离及精制

[目的]探索姜黄素类物的提取、分离及精制方法。[方法]姜黄药材经乙醇渗漉得到总姜黄素醇提物,经水洗、石油醚洗之后得到脱脂浸膏。利用干法柱层析分离得到双脱甲氧基姜黄素粗品及姜黄素、脱甲氧基姜黄素混合物。再经湿法柱层析洗脱得到二脱甲氧基姜黄素,通过制备薄层色谱法分离得到姜黄素和一脱甲氧基姜黄素粗品,经丙酮-水重结晶后分别得到姜黄素,一脱甲氧基姜黄素。[结果]测得姜黄素、一脱甲氧基姜黄素和二脱甲氧基姜黄素的熔点分别为182～184℃,168～170℃和226～228℃,三者的高效液相色谱(HPLC)谱图3倍保留时间内峰面积归一化法测得的纯度超过96%。[结论]实验确立的方法可有效分离姜黄素、一脱甲氧基姜黄素和二脱甲氧基姜黄素,分离周期短,节省溶剂。     

基于信息熵赋值法的正交联用Box-Behnken设计-响应面法优化黄丝郁金醋炙工艺研究

目的基于信息熵赋权法的正交联用Box-Behnken设计-响应面法(BBD-RSM)优化黄丝郁金醋炙工艺,优化其醋制炮制工艺。方法以HPLC法测定醋郁金中姜黄素、去甲氧基姜黄素和双去甲氧基姜黄素的量,作为评价指标,采用正交试验考察米醋用量、闷润时间、炒制温度、炒制时间对黄丝郁金醋炙工艺的影响;在正交试验的基础上,进一步采用BBD-RSM考察闷润时间、炒制温度和炒制时间对该炮制工艺的影响。结果正交试验确定的黄丝郁金最佳醋炙工艺为加入10%米醋,拌匀闷润10 min,炒制温度130℃,炒制10 min;BBD-RSM确定最佳炮制工艺为闷润时间12 min,炒制温度150℃,炒制时间8 min。验证实验结果表明该工艺条件重复性良好,具有合理性和可行性。结论此实验方法可行,模型、数据可靠,优化了黄丝郁金醋炙工艺。     

基于UPLC-Triple-TOF-MS和网络药理学快速建立郁金潜在中药质量标志物成分库

目的 根据中药质量标志物（quality marker,Q-Marker）的理念,建立不同基源郁金的潜在Q-Marker库。方法 采用超高效液相色谱-三重四极杆-飞行时间质谱联用（UPLC-Triple-TOF-MS）技术,建立郁金化学成分高分辨质谱数据库,通过网络药理学等方法构建“化学成分-靶点-通路”预测郁金潜在的Q-Marker。结果 郁金药材中共鉴定出46个化学成分,其中共有成分12个。以共有成分为Q-Marker候选物进行网络药理学分析,预测姜黄酮、莪术双环烯酮、莪术二酮、莪术烯醇、莪术醇、二氢姜黄素、去甲氧基姜黄素和莪术呋喃二烯酮可作用于5羟色胺受体（HTR1A、HTR2A、HTR1D、HTR1B）、阿片受体（OPRK1、OPRM1、OPRD1、OPRL1）等靶点,通过调控神经活性配体-受体相互作用、血清素能突触、钙信号通路、cAMP信号通路、逆行内源性大麻素信号等重要通路发挥抗抑郁的作用。结论 通过UPLC-Triple-TOF-MS技术和网络药理学方法,可以快速分析和确定中药郁金的化学成分,建立郁金的潜在Q-Marker库。     

总姜黄素3种单体高纯度同时分离方法

目的研究姜黄色素(curcuminoids,Cur)中姜黄素(curcumin,CurⅠ)、脱甲氧基姜黄素(deme-thoxycurcumin,CurⅡ)和双脱甲氧基姜黄素(bisdemethoxycurcumin,CurⅢ)3种单体的分离、纯化工艺。方法采用以氯仿-甲醇溶剂系统进行梯度洗脱的柱层析法分离Cur中的3种单体,并采用重结晶法对其进行纯化,通过薄层色谱、熔点测定及高效液相色谱法(high performance liquid chromatography,HPLC)对3种姜黄素单体的进行检测并计算纯度。结果可获得3种高纯度的姜黄素单体,HPLC测定结果分别为CurⅠ纯度99.47%、CurⅡ纯度99.64%、CurⅢ纯度98.64%。结论硅胶柱层析法合理、科学,效率高,可为姜黄素单体的进一步研究与开发应用提供实验依据。     

基于传统性效及现代研究的姜黄质量标志物分析

姜黄为我国传统中药,味辛、苦,性温,具有破血行气、通经止痛之功效,其用药历史悠久,最早收载于《新修本草》。对姜黄化学成分及主要药理活性进行总结,并基于传统性效及现代研究两方面对姜黄质量标志物进行预测分析。建议对姜黄的芳姜黄酮、α-姜黄酮、β-姜黄酮、姜黄素、去甲氧基姜黄素、双去甲氧基姜黄素及黄酮类等成分进行定性、定量分析,进一步开展其所含的萜类和甾醇类等成分化学物质组的深入研究,为明确姜黄的质量标志物和姜黄质量评价研究提供科学依据。