Antibiofilm Formation Activity of Lupinifolin Against Methicillin-Resistant Staphylococcus aureus.

<b>Background and Objective:</b> Biofilm formation activity of Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is one of the crucial factors rendering this pathogenic bacterium difficult to be eradicated. It has been reported that lupinifolin, is a major phytochemical agent isolated from <i>Derris reticulata</i> Craib. stem possesses antibacterial activity against MRSA. This study aimed to investigate the effects of lupinifolin and its combinations with some antibacterial drugs, including ampicillin, cloxacillin or vancomycin, on the biofilm formation activity of MRSA. <b>Materials and Methods:</b> The crystal violet biofilm formation assay was performed to evaluate the biofilm formation activity. <b>Results:</b> Lupinifolin produced a significant inhibitory activity against MRSA biofilm formation with the median inhibitory concentration (IC₅₀) of 7.96±3.05 µg mL¹ (n = 6) at 24 hrs incubation. Lupinifolin at the concentrations of sub-MICs (1, 2, 4 and 8 µg mL¹) combined with the antibacterial drugs at their sub-MICs also exhibited substantial antibiofilm formation activities. The maximal antibiofilm activity was found with the combination of lupinifolin (8 µg mL¹) and vancomycin (1 µg mL¹) by the percentage inhibition of 102.39±0.89 (n = 8). The antibiofilm formation activities of the combinations between lupinifolin and the antibacterial drugs at various concentrations tested were also significantly higher than those of lupinifolin alone. <b>Conclusion:</b> These results indicated that lupinifolin can potentially be developed as an antibacterial enhancer for the management of biofilm-associated bacterial infections caused by MRSA, in which the current pharmacological treatment is still limited.

Synergistic Activity of Lupinifolin in Combinations with Antibiotics Against Staphylococcus aureus.

<b>Background and Objective:</b> Antibacterial resistance is one of the top global public health problems. The use of natural substances, which can enhance the antibacterial activity of currently used medications, is a promising alternative to oppose antibacterial resistance. The pharmacological activities of lupinifolin, a prenylated flavanone isolated from stems of <i>Derris reticulata</i> Craib., against growth and biofilm formation of <i>Streptococcus mutans</i> and <i>Staphylococcus aureus</i> have been previously documented. Nonetheless, interactions between lupinifolin and other antibacterial agents have not been determined. This study aimed to investigate the effects of lupinifolin in combinations with some antibacterial agents, specifically ampicillin, cloxacillin or vancomycin, against <i>S. mutans</i>, Methicillin-Sensitive <i>S. aureus</i> (MSSA) and Methicillin-Resistant <i>S. aureus</i> (MRSA). <b>Materials and Methods:</b> The checkerboard assay was performed to determine the antibacterial activity of lupinifolin plus the testing antibacterial agents. The Fractional Inhibitory Concentration Index (FICI) was calculated to indicate the interaction between lupinifolin and the antibacterial agent tested. <b>Results:</b> Lupinifolin exerted the synergistic activity when using in combination with ampicillin or cloxacillin against MSSA with the FICIs of <u><</u>0.5. The potential synergistic effect was also observed with lupinifolin plus ampicillin or cloxacillin against MRSA. However, the combination of lupinifolin plus vancomycin resulted in no interaction against MRSA. The combined effects of lupinifolin and ampicillin or cloxacillin against <i>S. mutans</i> were somewhat ambiguous with the borderline values of FICI of 0.5156 and 0.5625, respectively. <b>Conclusion:</b> Lupinifolin potentially plays a role as an antibacterial intensifier against some pathogenic gram-positive bacteria, particularly MSSA and MRSA. Nonetheless, further experiments are required to explain the precise mechanism of synergy.

CaV1.2 and CaV1.3 voltage-gated L-type Ca2+ channels in rat white fat adipocytes.

L-type channel antagonists are of therapeutic benefit in the treatment of hyperlipidaemia and insulin resistance. Our aim was to identify L-type voltage-gated Ca2+ channels in white fat adipocytes, and determine if they affect intracellular Ca2+, lipolysis and lipogenesis. We used a multidisciplinary approach of molecular biology, confocal microscopy, Ca2+ imaging and metabolic assays to explore this problem using adipocytes isolated from adult rat epididymal fat pads. CaV1.2, CaV1.3 and CaV1.1 alpha1, beta and alpha2delta subunits were detected at the gene expression level. The CaV1.2 and CaV1.3 alpha1 subunits were identified in the plasma membrane at the protein level. Confocal microscopy with fluorescent antibodies labelled CaV1.2 in the plasma membrane. Ca2+ imaging revealed that the intracellular Ca2+ concentration, [Ca2 +]i was reversibly decreased by removal of extracellular Ca2+, an effect mimicked by verapamil, nifedipine and Co2+, all blockers of L-type channels, whereas the Ca2+ channel agonist BAY-K8644 increased [Ca2+]i. The finding that the magnitude of these effects correlated with basal [Ca2+]i suggests that adipocyte [Ca2+]i is controlled by L-type Ca2+ channels that are constitutively active at the adipocyte depolarized membrane potential. Pharmacological manipulation of L-type channel activity modulated both basal and catecholamine-stimulated lipolysis but not insulin-induced glucose uptake or lipogenesis. We conclude that white adipocytes have constitutively active L-type Ca2+ channels which explains their sensitivity of lipolysis to Ca2+ channel modulators. Our data suggest CaV1.2 as a potential novel therapeutic target in the treatment of obesity.

Anticariogenic Activities of Derris reticulata Ethanolic Stem Extract Against Streptococcus mutans.

BACKGROUND AND OBJECTIVE: Streptococcus mutans is a dominant causative pathogen of dental caries, which is a major oral health problem affecting million people worldwide. Derris reticulata is a medicinal plant possessing antimicrobial activity against several Gram-positive pathogenic bacteria. None the less, its effects on growth and cariogenic properties of S. mutans has not been clearly established. This study aimed to investigate the antibacterial and anti cariogenic activities of the D. reticulata ethanolic stem extract. MATERIALS AND METHODS: The TLC analysis was performed to authenticate the D. reticulata sample. Minimum inhibition concentration and minimum bactericidal concentration were determined by using broth dilution and drop plate methods, respectively. Sucrose dependent and sucrose independent-adherences, biofilm formation and glycolytic pH drop assays were performed to evaluate the anticariogenic activity. RESULTS: The ethanolic stem extract of D. reticulata possessed the antibacterial activity against S. mutans with the MIC and MBC of 0.875±0.250 and 1.750±0.500 mg mL-1, respectively. The extract at the lower concentrations of sub-MIC also had significant inhibitory actions against the cariogenic properties of S. mutans, including surface adherence, biofilm formation and glycolytic acid production. CONCLUSION: The D. reticulata stem extract had a substantial anticariogenic activities and thus potentially be developed as an oral health care product for dental caries prevention in the near future.

Etiology of the membrane potential of rat white fat adipocytes.

The plasma membrane potential (Vm) is key to many physiological processes; however, its ionic etiology in white fat adipocytes is poorly characterized. To address this question, we employed the perforated patch current clamp and cell-attached patch clamp methods in isolated primary white fat adipocytes and their cellular model 3T3-L1. The resting Vm of primary and 3T3-L1 adipocytes were -32.1 ± 1.2 mV (n = 95) and -28.8 ± 1.2 mV (n = 87), respectively. Vm was independent of cell size and fat content. Elevation of extracellular K(+) to 50 mM by equimolar substitution of bath Na(+) did not affect Vm, whereas substitution of bath Na(+) with the membrane-impermeant cation N-methyl-D-glucamine(+)-hyperpolarized Vm by 16 mV, data indicative of a nonselective cation permeability. Substitution of 133 mM extracellular Cl(-) with gluconate-depolarized Vm by 25 mV, whereas Cl(-) substitution with I(-) caused a -9 mV hyperpolarization. Isoprenaline (10 µM), but not insulin (100 nM), significantly depolarized Vm. Single-channel ion activity was voltage independent; currents were indicative for Cl(-) with an inward slope conductance of 16 ± 1.3 pS (n = 11) and a reversal potential close to the Cl(-) equilibrium potential, -29 ± 1.6 mV. Although the reduction of extracellular Cl(-) elevated the intracellular Ca(2+) of adipocytes, this was not as large as that produced by elevation of extracellular K(+). In conclusion, the Vm of white fat adipocytes is well described by the Goldman-Hodgkin-Katz equation with a predominant permeability to Cl(-), where its biophysical and single-channel properties suggest a volume-sensitive anion channel identity. Consequently, changes in serum Cl(-) homeostasis or the adipocyte's permeability to this anion via drugs will affect its Vm, intracellular Ca(2+), and ultimately its function and its role in metabolic control.

Anti-implantation effects of indomethacin and celecoxib in rats.

Pregnant Wistar rats were used to investigate the anti-implantation effect of cyclooxygenase (COX) inhibitors, indomethacin (nonselective COX-1/COX-2 inhibitor) and celecoxib (specific COX-2 inhibitor). Indomethacin at doses of 2.5 and 5 mg/kg/day and celecoxib at doses of 40, 80, and 160 mg/kg/day were orally administered once daily to each group (n = 8) on Days 3-5 of pregnancy (Day 1 = sperm detection). Indomethacin and celecoxib at anti-implantation dosages were further investigated for the effects on changes in endometrial vascular permeability in pregnant rats and uterine decidualization in pseudopregnant rats. The results demonstrated that indomethacin at a dose of 5 mg/kg/day as well as celecoxib at doses of 80 and 160 mg/kg/day could significantly reduce the proportion of pregnant rats. At the anti-implantation dosages, they exhibited no significant effect on proportion of rats with blue dye sites in the endometrial vascular permeability study, but they could significantly reduce the uterine decidualization. From these findings, the anti-implantation effect of the two COX inhibitors may principally be from decidualization defects, and COX inhibitors should, therefore, be used with caution in childbearing age women. On the other hand, specific COX-2 inhibitors with their good gastric safety profile may have a potential role in nonhormonal postcoital contraception.