The synergistic reversal effect of multidrug resistance by quercetin and hyperthermia in doxorubicin-resistant human myelogenous leukemia cells

Purpose: This study aimed to evaluate the multidrug resistance (MDR) reversal activity of quercetin (Que) in combination with hyperthermia (HT) in human myelogenous leukemia cells K562/A.

Methods: The cytotoxicity of Que alone and the effect of Que and HT to doxorubicin (Dox) cytotoxicity were determined using MTT assay in K562 and K562/A cells. K562/A cells was heated with or without Que pretreatment, and the protein and mRNA levels of heat shock protein 70 (HSP70) and P-glycoprotein (P-gp) were determined by flow cytometry (FCM) and RT-PCR, respectively. Intracellular accumulation of Dox, cell cycle and apoptosis were monitored with FCM.

Results: Que alone inhibited cell growth in a dose-dependent manner in K562 and K562/A cells. Either Que or HT alone had a weak reversal effect on Dox resistance, however, combination HT and Que showed a much more significant reversal effect on Dox resistance (reverse fold 9.49). The elevated protein expression and mRNA level of HSP70 and P-gp in response to HT were inhibited by Que. Pretreatment with Que caused the cells to accumulate Dox 8.3-fold higher than in control cells. In addition, Que induced apoptosis and G2/M arrest in a dose-dependent manner, and the combination of Que and HT was found to have a synergistic efeect on apoptosis.

Conclusions: Que pretreatment could significantly inhance the MDR reversal activity of HT in resistant cell line, by sensitizing the cell to reversing MDR activity of HT.     

维药小新塔花和芳香新塔花的HPLC指纹图谱研究

目的 建立小新塔花和芳香新塔花的HPLC指纹图谱分析方法,并同时测定8批小新塔花和22批芳香新塔花中金丝桃苷、槲皮素、胡薄荷酮的量.方法 采用Kromasil C18色谱柱(250 mm×4.6 mm,5 μm),以甲醇-0.08％甲酸水溶液进行梯度洗脱,同时测定了小新塔花和芳香新塔花中金丝桃苷、槲皮素、胡薄荷酮的量,建立了不同产地8批小新塔花和22批芳香新塔花的指纹图谱.结果 根据主成分分析、聚类分析,将30批药材分为2类,建立了2种新塔花的HPLC指纹图谱.不同产地的2种新塔花中金丝桃苷、槲皮素和胡薄荷酮等成分的量存在较大差异;芳香新塔花不含槲皮素,而小新塔花中含有;芳香新塔花中的金丝桃苷和胡薄荷酮量高于小新塔花;小新塔花中金丝桃苷量为49.72～204.33 μg/g,槲皮素量为161.06～213.22μg/g,胡薄荷酮量为36.19～93.29 μg/g;芳香新塔花中金丝桃苷量为174.15～802.24μg/g,胡薄荷酮量为77.43～353.45 μg/g.结论 不同品种的新塔花指纹图谱存在一定的差异,可为新塔花药材的质量控制、品种鉴定及划分提供一定的参考依据.     

槐米中槲皮素的双相动态提取工艺优选

目的: 优选槐米中槲皮素的双相动态提取工艺。 方法: 以槲皮素提取量为评价指标,在单因素试验基础上,通过正交试验考察提取时间、提取温度、提取次数、硫酸体积分数对槲皮素双相动态提取工艺的影响。采用HPLC测定槲皮素含量,色谱条件为Agilent ZORBAX C₁₈色谱柱（4.6 mm×250 mm,5 μm）,流动相甲醇-0.02%H₃PO₄（45∶55）,检测波长254 nm,进样量10 μL。 结果: 槐米中槲皮素的最佳提取工艺为加2%硫酸于85℃提取2次,每次2.5 h;槲皮素提取量达5.22 mg·g^-1,远高于乙醇回流提取法的3.26 mg·g^-1。 结论: 优选的提取工艺稳定可行,采用双相动态工艺提取槲皮素具有提取率高的优势。     

沙棘总黄酮的大鼠肠吸收特性考察

目的:考察沙棘总黄酮在大鼠小肠的吸收特性.方法:运用大鼠在体单向灌流和离体外翻肠囊模型,采用HPLC测定槲皮素、山奈酚、异鼠李素含量,计算沙棘总黄酮在大鼠小肠的吸收参数.结果:沙棘总黄酮中槲皮素、山奈酚、异鼠李素的最佳吸收部位分别为回肠、回肠、十二指肠,吸收速率常数(Ka)分别为3.405×10-2,3.649×10-2,5.671×10-2,沙棘总黄酮质量浓度在50 ～200 mg·L-1时,Ka和表观吸收系数无显著性差异.累积吸收量随药物质量浓度升高呈线性增加.结论:沙棘提取物中3种黄酮类成分均呈现一级动力学特征,吸收机制均为被动扩散,且在小肠有特定吸收部位.     

HPLC测定凤眼草中槲皮素与山柰素的含量

目的:建立以高效液相色谱法测定凤眼草中槲皮素与山柰素含量的方法.方法:Inertsil ODS-3色谱柱(4.6mrn×250 mm,5μm),流动相甲醇-0.4％磷酸溶液(50∶50),检测波长360 nm,流速1.0 mL· min-1.结果:槲皮素在11.1～333μg(r=1)线性关系良好,平均回收率为97.82％,RSD 1.1％;山柰素在2.3～69 μg (r=1)线性关系良好,平均回收率为96.81％,RSD 1.7％.结论:方法简便、快速、重复性好,可用于凤眼草的质量控制.     

HPLC测定复方肝炎颗粒中槲皮素和山奈素的含量

目的： 高效液相法测定复方肝炎颗粒中槲皮素和山奈素的含量。方法： 采用HPLC,样品经80%甲醇超声、酸水解后,采用色谱柱C₁₈ （4.6 mm× 250 mm,5 μm）,甲醇-0.4%磷酸（50:50）为流动相,流速1.0 mL·min^-1,检测波长360 nm,柱温30℃。结果： 槲皮素在0.050 1~0.501 μg（r=0.999 9）和山奈素在0.024 2~0.242 μg（r=0.999 5）均呈良好线性关系;槲皮素和山奈素加样回收率分别为99.69%（RSD 0.22%）,99.30%（RSD 0.82%）（n=9）。结论： 该法操作简便﹑结果准确、重复性好﹑结果准确可靠,可用于复方肝炎颗粒的质量控制要求。     

HPLC程序可变波长法测定绞股蓝中芦丁、人参皂苷Rb1和槲皮素

目的:建立绞股蓝药材中芦丁、人参皂苷Rb1和槲皮素的含量测定方法.方法:TSK ODS-100V(4.6 mm×250mm,5μm)色谱柱;流动相乙腈-0.1％磷酸(30∶70),流速1.0 mL· min-1,柱温30℃,进样量10 μL,检测波长(0 ～ 14 min,257nm;14 ～ 18 min,203 nm;18 ～30 min,257 nm).结果:芦丁、人参皂苷Rb1和槲皮素分别在0.171 2～1.284 0μg、0.828 0～6.210 0 μg,0.204 8～ 1.525 7μg线性关系良好,回归方程分别为Y=732 927.570 1 X+1 523.016 4,r=0.9998、Y=158 991.787 4X +307.545 6,r=0.999 9,Y=745 728.515 6X +2 101.1668(r =0.999 8),平均加样回收率分别为97.33％(RSD 2.02％),98.6t％ (RSD 1.70％),98.38％(RSD 2.59％).结论:所建立的方法准确性高,重复性好,可作为绞股蓝药材的质量控制方法.     

HPLC法测定杠板归中槲皮素和山柰素的含量

目的建立杠板归中槲皮素和山柰素含量的HPLC测定方法。方法色谱柱:Kromasil C18(5μm,250×4.6mm);流动相:甲醇-0.4%磷酸溶液(60:40);检测波长:360nm,柱温:30℃。结果槲皮素和山柰素的回收率分别为99.6%、100.8%;线性范围分别为0.158 9³.178μg、0.006 993.178μg、0.006 99⁰.279 6μg。结论结果准确,重复性好,可用于该产品的质量控制。     

The complex degradation and metabolism of quercetin in rat hepatocyte incubations

Abstract

1.?The current study demonstrated that there is still new information to be obtained on the chemical and biological transformation of the widely studied flavonoid quercetin.

2.?In rat hepatocytes, 35 metabolites of quercetin were observed by using high-resolution mass spectrometry. The metabolites included glucuronides, sulfates, mixed sulfate/glucuronide metabolites and methylated versions of these metabolites.

3.?Several metabolites were formed from chemical degradation products of quercetin which were found to form in Krebs–Henseleit (KH) buffer, degradants of quercetin were also formed in the buffer under the conditions used for hepatocyte incubations.

4.?The degradants and metabolites of quercetin were characterized by using high-resolution MS². It was observed that the glutathione (GSH) conjugates of quercetin formed in large amounts in ammonium bicarbonate solution although the pattern of conjugates formed was different from that observed in hepatocytes suggesting some degree on enzymatic control on GSH conjugate formation in the hepatocyte incubations.

5.?GSH conjugates were not formed when GSH was included in incubations of quercetin in KH buffer alone and only small amounts of quercetin degradation occurred. Instead, GSH was extensively converted into GSSG, thus presumably reducing the levels of oxygen in the incubation thus preventing quercetin degradation.     

HPLC同时测定不同产地荷叶饮片中4种黄酮成分

目的： 采用HPLC同时测定荷叶饮片中4种黄酮类成分的含量,为制定荷叶饮片的质量标准提供实验依据。 方法： 采用Kromasil C₁₈（4.6 mm×200 mm,5 μm）色谱柱,以甲醇（A）-0.2%磷酸水（B）为流动相,梯度洗脱（0~8 min,10%~40%A;8~23 min,40%~70%A;23~30 min,70%~85%A）,流速1 mL·min^-1,柱温30 ℃,二极管阵列检测器,检测波长360 nm。 结果： 槲皮素-3-O-桑布双糖苷、金丝桃苷、异槲皮苷和槲皮素的线性范围依次为6.10~97.60,18.76~300.16,8.48~135.68,9.12~145.92 mg·L^-1,平均回收率（n=6）依次为100.98%（RSD 1.85%）,99.05%（RSD 1.48%）,99.29%（RSD 1.84%）,97.71%（RSD 1.78%）。5个不同产地荷叶饮片中槲皮素-3-O-桑布双糖苷、金丝桃苷、异槲皮苷和槲皮素的含量分别为0.092%~0.161%,0.949%~1.862%,0.067%~0.133%,0.040%~0.062%。 结论： 该方法简便快速,准确可靠,可作为荷叶饮片中4种黄酮成分的含量测定方法。不同产地荷叶饮片中上述4种黄酮成分含量相差较大,建议将黄酮成分纳入荷叶饮片的质量控制标准。