Concentration-dependent doxorubicin efflux (10–30 µM) in the presence or absence of EEAC.

<p>The left panels showed the nonlinear regression analysis of doxorubicin efflux and the right panels demonstrated the Lineweaver-Burk plot analysis of doxorubicin efflux. (<i>a</i>) Wild-type P-gp (1236C-2677G-3435C). (<i>b</i>) Variant-type P-gp (1236T-2677T-3435T). (<i>c</i>) Variant-type P-gp (1236T-2677A-3435T). Data were presented as mean ± SE of at least three experiments, each in triplicate. *p<0.05 as compared to doxorubicin efflux without EEAC (control).</p

Effect of the ethanolic extract of <i>Antrodia cinnamomea</i> (EEAC) on rhodamine123 transport by wild-type and variant-type human P-gp.

<p>Effect of the ethanolic extract of <i>Antrodia cinnamomea</i> (EEAC) on rhodamine123 transport by wild-type and variant-type human P-gp.</p

Effect of EEAC on P-gp mRNA and protein expression level.

<p>(<i>a</i>)(<i>b</i>) Western blot of P-gp expression with C219 monoclonal antibody (2 µg protein/lane). The upper one was the mature fully glycosylated (∼170 kD) and the lower one was the immature P-gp (∼150 kD). The expression of β-actin was used as loading control. Treatment of EEAC 5 µg/mL for 48 and 72 hours demonstrated no effect on P-gp protein expression. (<i>c</i>)(<i>d</i>)(<i>e</i>) Analysis of mRNA levels with real-time quantitative RT-PCR. There were no significant <i>ABCB1</i> mRNA expression differences after treating the P-gp expressed cells with 5 µg/ml EEAC for 24, 48 and 72 hours. (<i>f</i>) Cytotoxicity study of EEAC by MTT assay. The IC₅₀ of EEAC for Flp-In™ 293, <i>ABCB1</i> 1236C-2677G-3435C (wild-type), <i>ABCB1</i> 1236T-2677T-3435T and <i>ABCB1</i> 1236T-2677A-3435T were 21.27, 29.31, 20.60 and 22.74 µg/ml, respectively. There was no significant difference among the IC₅₀. Data were presented as mean ± SE of at least three experiments, each in triplicate.</p

The Functional Influences of Common <i>ABCB1</i> Genetic Variants on the Inhibition of P-glycoprotein by <i>Antrodia cinnamomea</i> Extracts

<div><p><i>Antrodia cinnamomea</i> is a traditional healthy food that has been demonstrated to possess anti-inflammatory, antioxidative, and anticacer effects. The purpose of this study was to evaluate whether the ethanolic extract of <i>A. cinnamomea</i> (EEAC) can affect the efflux function of P-glycoprotein (P-gp) and the effect of <i>ABCB1</i> genetic variants on the interaction between EEAC and P-gp. To investigate the mechanism of this interaction, Flp-In™-293 cells stably transfected with various genotypes of human P-gp were established and the expression of P-gp was confirmed by Western blot. The results of the rhodamine 123 efflux assay demonstrated that EEAC efficiently inhibited wild-type P-gp function at an IC₅₀ concentration of 1.51±0.08 µg/mL through non-competitive inhibition. The IC₅₀ concentrations for variant-type 1236T-2677T-3435T P-gp and variant-type 1236T-2677A-3435T P-gp were 5.56±0.49 µg/mL and 3.33±0.67 µg/mL, respectively. In addition, the inhibition kinetics of EEAC also changed to uncompetitive inhibition in variant-type 1236T-2677A-3435T P-gp. The ATPase assay revealed that EEAC was an ATPase stimulator and was capable of reducing verapamil-induced ATPase levels. These results indicate that EEAC may be a potent P-gp inhibitor and higher dosages may be required in subjects carrying variant-types P-gp. Further studies are required to translate this basic knowledge into clinical applications.</p></div

Evaluation of influences of EEAC on P-glycoprotein efflux function.

<p>(<i>a</i>) Rhodamine 123 efflux assay: EEAC inhibited P-gp mediated efflux of rhodamine 123 in a concentration dependent manner. Verapamil 10 µM was used as a positive control. EEAC significantly inhibited rhodamine 123 efflux in wild-type P-gp, as well as the two variant-types P-gp. *p<0.05 as compared to rhodamine 123 efflux without EEAC (control). (<i>b</i>) Calcein-AM uptake assay: EEAC increased intracellular accumulation of calcein in a dose dependent manner in wild-type P-gp, as well as the two variant-types P-gp. Verapamil 10 µM was used as a positive control. *p<0.05 as compared to calcein-AM uptake without EEAC (control). (<i>c</i>) Doxorubicin (10 µM) efflux assay: EEAC inhibited P-gp mediated efflux of doxorubicin in a concentration dependent manner. EEAC significantly inhibited doxorubicin efflux in wild-type P-gp, as well as the two variant-types P-gp. *p<0.05 as compared to doxorubicin efflux without EEAC (control). (<i>d</i>) The IC₅₀ of the inhibitory effect of EEAC on rhodamine 123 efflux by <i>ABCB1</i> 1236C-2677G-3435C (wild type), <i>ABCB1</i> 1236T-2677T-3435T and <i>ABCB1</i> 1236T-2677A-3435T were 1.51, 5.56 and 3.33 µg/ml, respectively, indicating the inhibitory effect of EEAC on rhodamine 123 efflux by wild-type P-gp was the most potent. Above data were presented as mean ± SE of at least three experiments, each in triplicate. *p<0.05 as compared to rhodamine 123 efflux without EEAC (control).</p

Concentration-dependent rhodamine 123 efflux (10–30 µM) in the presence or absence of EEAC.

<p>The left panels showed the nonlinear regression analysis of rhodamine 123 efflux and the right panels demonstrated the Lineweaver-Burk plot analysis of rhodamine 123 efflux. (<i>a</i>) Wild-type P-gp (1236C-2677G-3435C). (<i>b</i>) Variant-type P-gp (1236T-2677T-3435T). (<i>c</i>) Variant-type P-gp (1236T-2677A-3435T). Data were presented as mean ± SE of at least three experiments, each in triplicate. *p<0.05 as compared to rhodamine 123 efflux without EEAC (control).</p

Effect of EEAC on P-glycoprotein ATPase activity.

<p>(<i>a</i>) Incubation with EEAC (0.01–200 µg/ml) stimulated the P-gp ATPase activity. Data were analyzed in terms of RLUs. (<i>b</i>) Verapamil (25–400 µM) showed its capacity to stimulate P-gp ATPase activity. (<i>c</i>) EEAC (1–100 µg/ml) was tested for its capacity to inhibit 200 µM verapamil-stimulated P-gp ATPase activity. Above results indicated that EEAC stimulated the P-gp ATPase activity and inhibited 200 µM verapamil-stimulated P-gp ATPase activity at concentration 5 µg/ml.</p

Effect of the ethanolic extract of <i>Antrodia cinnamomea</i> (EEAC) on doxorubicin transport by wild-type and variant-type human P-gp.

<p>Effect of the ethanolic extract of <i>Antrodia cinnamomea</i> (EEAC) on doxorubicin transport by wild-type and variant-type human P-gp.</p

Ab Initio Calculations on the Structures and Energetics of Li₄OH, Li₃NaOH, and Li₂Na₂OH Isomers

We performed ab initio electronic structure calculations on the structures and energetics of the mixed hyperalkaliated hydrogen oxides Li4OH, Li3NaOH, and Li2Na2OH. Five equilibrium geometries exist for each complex of Li4OH and Li3NaOH, and seven minima were located for Li2Na2OH. The calculated dissociation energies for the possible dissociation pathways are all endothermic. The global minimum structures of the three complexes have C2v symmetry and contain a hydrogen-bridged, Li−H−Li, three-centered skeleton. We also investigated the charge redistribution within these complexes in their ionic forms. The energetic factors governing the construction of the equilibrium structures and their bonding properties are analyzed

Effect of blood biochemical parameters of SD rats after 4-week treatment with sham + water, BA + water, BA + MgCl₂, and DSW.

<p>Concentration unit: U/L. BA, balloon angioplasty; RDA, recommended dietary allowances; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GGT, γ-glutamyltransferase. Mg²⁺: serum Mg²⁺ concentration. Values are mean ± SEM, n = 6.</p><p>*<i>p</i><0.05 compared to sham + water.</p