coli O157:H7 upon exposure of different concentrations of limonoids Concentration (μg/ml) DMSO IL IBA Ichangin DNAG IOAG 100 23.56 ± 0.71 23.11 ± 0.76 22.97 ± 0.96 23.65 ± 0.95 23.58 ± 1.06 22.96 ± 1.06 50 24.90 ± 1.82 22.97 ± 0.97 23.12 ± 0.92 23.16 ± 0.93 23.27 ± 1.09 23.64 ± 1.08 25 23.62 ± 2.47 23.58 Selleck GDC-973 ± 1.19

23.26 ± 1.23 22.58 ± 1.26 23.68 ± 0.91 23.51 ± 1.26 12.5 23.68 ± 1.84 23.54 ± 1.01 22.69 ± 1.09 23.12 ± 1.08 23.97 ± 1.31 23.69 ± 1.32 6.25 23.91 ± 0.63 23.70 ± 1.09 23.90 ± 1.02 23.55 ± 1.05 23.61 ± 1.05 23.76 ± 1.01 The mean ± SD of three replicates are presented. All the five limonoids inhibit biofilm formation in concentration dependent manner (Figure 2). Biofilm inhibitory activities of limonoids were compared by calculating IC25 values from 3-parameter sigmoid equations (Figure 2). The 3-parameter equation was chosen due to better fit demonstrated for 4 out of 5 limonoids. IC25 values were used for comparison because limonoids demonstrated <50% inhibition of biofilm formation. The R2 values for isolimonic acid,

ichangin, isoobacunoic acid, IOAG and DNAG were 0.99, 0.96, 0.92, 0.88 and 0.99 respectively. PI3K inhibitor drugs Isolimonic acid was the most potent inhibitor of biofilm formation among the tested limonoids with an IC25 of 19.7 μM (Figure 2) followed by ichangin (IC25 = 28.3 μM). IOAG was more potent (IC25= 29.54 μM) than its aglycone isoobacunoic acid (IC25= 57.2 μM). Furthermore, 95% confidence intervals for IC25 values were CHIR-99021 concentration calculated as 8.9-27.1 μM (isolimonic acid), 20.3-38.7 μM (ichangin), 17.9-54.6 μM (IOAG), 43.0-71.5 μM (isoobacunoic acid) and 23.0-66.1 μ M (DNAG). Figure 2 Three parameter models of biofilm formation inhibition by citrus limonoids. Line curves at 50% and 25% represent the IC50 and IC25 values for compounds. Biofilms were grown in 96-well plates and quantified using crystal violet. Percent HSP90 inhibition over solvent control (DMSO) was calculated. To generate 3-parameter models, concentrations were changed to Log10 μM and plotted against percent inhibition. Effect of limonoids on adhesion of EHEC to Caco-2 cells To further understand the effect of limonoids, adherence of EHEC to colon

epithelial Caco-2 cells was studied. Isolimonic acid and ichangin (100 μg/ml) treatment significantly (p<0.05) reduced the number of EHEC cells attached to Caco-2 cells by 0.66 and 0.59 Log10 cfu/ml, respectively (Figure 3A). Isoobacunoic acid, IOAG and DNAG did not affect the number of EHEC cells adhering to Caco-2 cells. To determine, if the observed reduction in adhesion of EHEC was due to reduced cell viability of Caco-2 cells, survival of Caco-2 in presence of 100 μg/ml limonoids at 6 h was assayed by measuring extracellular LDH. Survival of Caco-2 cells in presence of 100 μg/ml limonoids was similar to solvent control (Figure 3B).