School of Food Science, Washington State University , Pullman, Washington 99164-6376, United States.
J Nat Prod. 2014 May 23;77(5):1102-10. doi: 10.1021/np400781y. Epub 2014 Apr 22.
A fluorometric microplate assay has been developed to determine Escherichia (E.) coli adhesion to uroepithelial cells (UEC). P-fimbriated E. coli were labeled with BacLight Green and preincubated 30 min with human urine or standard. Fluorescent-E. coli were added to UEC in mircoplates at a 400:1 ratio, incubated 1 h, and washed, and the fluorescence intensity was measured. Specific labeling and adherence were confirmed by flow cytometry. A myricetin (1) standard curve (0-30 μg/mL) was developed; the lower limit of detection was 0.1 μg/mL, and half-maximal inhibitory concentration was 0.88 μg/mL (intra- and interassay coefficients of variance were <10% and <15%, respectively). Vaccinium macrocarpon (cranberry) extracts, quercetin (2), and procyanidins B1 (3), B2 (4), and C1 (5) showed similar inhibition. Antiadhesion activity of urine samples from subjects (n = 12) consuming placebo or V. macrocarpon beverage determined using this assay was positively correlated (R(2) = 0.78; p < 0.01) with a radiolabeled-E. coli assay.
已开发出一种荧光微孔板测定法来确定大肠埃希氏菌（Escherichia coli）对尿路上皮细胞（uroepithelial cells，UEC）的黏附。用 BacLight Green 标记带有 P-菌毛的大肠埃希氏菌，并与人尿或标准品预孵育 30 分钟。将荧光标记的大肠埃希氏菌以 400:1 的比例加入微孔板中的 UEC 中，孵育 1 小时，然后进行洗涤，并测量荧光强度。通过流式细胞术确认了特异性标记和黏附。建立了杨梅素（myricetin，1）标准曲线（0-30 μg/mL）；检测下限为 0.1 μg/mL，半最大抑制浓度为 0.88 μg/mL（内和日间变异系数分别<10%和<15%）。蔓越莓（ Vaccinium macrocarpon）提取物、槲皮素（quercetin，2）和原花青素 B1（procyanidins B1，3）、B2（procyanidins B2，4）和 C1（procyanidins C1，5）表现出相似的抑制作用。使用该测定法测定 12 名服用安慰剂或蔓越莓饮料的受试者尿液样本的抗黏附活性与放射性标记的大肠埃希氏菌测定呈正相关（R²=0.78；p<0.01）。
