Optimizing Green Synthesis of Hydrotalcite - Silver Nanoparticles using Syzygium Nervosum based Reducing Agent.

This work aimed to assess the effect of Syzygium nervosum leaf extract as a reducing agent for green synthesis of AgNPs on HT through an optimizing process using response surface methodology (RSM) and the Box-Benken model. The optimal conditions for synthesis of AgNPs on HT include reaction time of 6.15 hours, reaction temperature of 50 oC and the ratio of diluted Syzygium nervosum leaf extract to reduce AgNO3 of 50.37 mL/mg. Under the optimal conditions, the yield of reduction reaction reached 77.54 %, close to the theoretical value of 76.97 %. Besides, the morphology, density, and characteristics of AgNPs on the surface of HT layers have been determined by using Ultraviolet-visible spectroscopy, Field emission scanning electron microscopy, High resolution transmission electron microscopy, selected area diffraction, X-ray diffraction, Dynamic light scattering, Infrared spectroscopy, Fluorescence emission spectroscopy, Brunauer-Emmett-Teller  methods. The spherical AgNPs were synthesized successfully on the surface of HT with the average particle size of 13.0 ± 1.1 nm. Interestingly, HT-Ag hybrid materials can inhibit strongly the growth of E. coli, S. aureus as well as two antibiotic resistance bacterial strains, P. stutzeri B27 and antibiotic resistance E. coli. Overall, the HT-Ag hybrid materials are very promising for application in practice.

A new 8,3'-neolignan from Solanum procumbens Lour.

The phytochemical investigation of the EtOAc-soluble fraction of the aerial parts of Solanum procumbens Lour. has been carried out to obtain seven compounds, including a new 8,3'-neolignan named solacanin A (1). Their chemical structures were elucidated based on the spectroscopic data interpretation. All isolated compounds were tested for their α-glucosidase inhibitory activity. Compounds 1 and 3-6 showed inhibitory activity with IC50 values of 221.5, 18.9, 6.0, 104.1, and 219.7 µM, respectively.[Formula: see text].

Optimizing lime pretreatment of rice straw for biolipid production using oleaginous microorganisms.

This work focuses on the lime pretreatment of rice straw, agricultural waste in northeast Vietnam, for microbial lipid (bio-lipid) production. A response surface methodology approach was performed to optimize parameters for the pretreatment process. These parameters are the concentration of Ca(OH)2, hydrolysis temperature, and maintained time; the effect is considered bio-lipid production yield. The lipid yield was estimated through consolidated bioprocessing of the pretreated rice straw as a substrate and using the fungus strain (Aspergillus oryzae 32) and the yeast strain (Lipomyces starkeyi 22). As a result, the optimal pretreatment conditions for maximum lipid yield were obtained at Ca(OH)2 concentration of 12 g/L and hydrolysis temperature of 110 °C within 60 min. The accumulated lipid in fermentation using these oleaginous microorganisms was 8.5g/100g oven-dry weight of rice straw (10.9g/100g for pretreated rice straw). The biolipid consisted of 42.6% saturated, 21.1% monounsaturated (MUFA), and 35.1% polyunsaturated (PUFA) fatty acids.

Fitting the elementary rate constants of the P-gp transporter network in the hMDR1-MDCK confluent cell monolayer using a particle swarm algorithm.

P-glycoprotein, a human multidrug resistance transporter, has been extensively studied due to its importance to human health and disease. In order to understand transport kinetics via P-gp, confluent cell monolayers overexpressing P-gp are widely used. The purpose of this study is to obtain the mass action elementary rate constants for P-gp's transport and to functionally characterize members of P-gp's network, i.e., other transporters that transport P-gp substrates in hMDR1-MDCKII confluent cell monolayers and are essential to the net substrate flux. Transport of a range of concentrations of amprenavir, loperamide, quinidine and digoxin across the confluent monolayer of cells was measured in both directions, apical to basolateral and basolateral to apical. We developed a global optimization algorithm using the Particle Swarm method that can simultaneously fit all datasets to yield accurate and exhaustive fits of these elementary rate constants. The statistical sensitivity of the fitted values was determined by using 24 identical replicate fits, yielding simple averages and standard deviations for all of the kinetic parameters, including the efflux active P-gp surface density. Digoxin required additional basolateral and apical transporters, while loperamide required just a basolateral tranporter. The data were better fit by assuming bidirectional transporters, rather than active importers, suggesting that they are not MRP or active OATP transporters. The P-gp efflux rate constants for quinidine and digoxin were about 3-fold smaller than reported ATP hydrolysis rate constants from P-gp proteoliposomes. This suggests a roughly 3∶1 stoichiometry between ATP hydrolysis and P-gp transport for these two drugs. The fitted values of the elementary rate constants for these P-gp substrates support the hypotheses that the selective pressures on P-gp are to maintain a broad substrate range and to keep xenobiotics out of the cytosol, but not out of the apical membrane.

The steady-state Michaelis-Menten analysis of P-glycoprotein mediated transport through a confluent cell monolayer cannot predict the correct Michaelis constant Km.

PURPOSE: Typically, the kinetics of membrane transport is analyzed using the steady-state Michaelis-Menten (or Eadie-Hofstee or Hanes) equations. This approach has been successful when the substrate is picked up from the aqueous phase, like a water-soluble enzyme, for which the Michaelis-Menten steady-state analysis was developed. For membrane transporters whose substrate resides in the lipid bilayer of the plasma membrane, like P-glycoprotein (P-gp), there has been no validation of the accuracy of the steady-state analysis because the elementary rate constants for transport were not known. METHODS: Recently, we fitted the mass action elementary kinetic rate constants of P-gp transport of three different drugs through a confluent monolayer of MDCKII-hMDR1 cells. With these elementary rate constants in hand, we use computer simulations to assess the accuracy of the steady-state Michaelis-Menten parameters. This limits the simulation to parameter ranges known to be physiologically relevant. RESULTS: Using over 2,300 different vectors of initial elementary parameters spanning the space bounded by the three drugs, which defines 2,300 "virtual substrates", the concentrations of substrate transported were calculated and fitted to Eadie-Hofstee plots. Acceptable plots were obtained for 1,338 cases. CONCLUSION: The fitted steady-state Vmax values from the analysis correlated to within a factor of 2-3 with the values predicted from the elementary parameters. However, the fitted Km value could be generated by a wide range of underlying "molecular" Km values. This is because of the convolution of the drug passive permeability kinetics into the fitted Km. This implies that Km values measured in simpler systems, e.g., microsomes or proteoliposomes, even if accurate, would not predict the Km values for the confluent monolayer system or, by logical extension, in vivo. Reliable in vitro-in vivo extrapolation seems to require using the elementary rate constants rather than the Michaelis-Menten steady-state parameters.

The elementary mass action rate constants of P-gp transport for a confluent monolayer of MDCKII-hMDR1 cells.

The human multi-drug resistance membrane transporter, P-glycoprotein, or P-gp, has been extensively studied due to its importance to human health and disease. Thus far, the kinetic analysis of P-gp transport has been limited to steady-state Michaelis-Menten approaches or to compartmental models, neither of which can prove molecular mechanisms. Determination of the elementary kinetic rate constants of transport will be essential to understanding how P-gp works. The experimental system we use is a confluent monolayer of MDCKII-hMDR1 cells that overexpress P-gp. It is a physiologically relevant model system, and transport is measured without biochemical manipulations of P-gp. The Michaelis-Menten mass action reaction is used to model P-gp transport. Without imposing the steady-state assumptions, this reaction depends upon several parameters that must be simultaneously fitted. An exhaustive fitting of transport data to find all possible parameter vectors that best fit the data was accomplished with a reasonable computation time using a hierarchical algorithm. For three P-gp substrates (amprenavir, loperamide, and quinidine), we have successfully fitted the elementary rate constants, i.e., drug association to P-gp from the apical membrane inner monolayer, drug dissociation back into the apical membrane inner monolayer, and drug efflux from P-gp into the apical chamber, as well as the density of efflux active P-gp. All three drugs had overlapping ranges for the efflux active P-gp, which was a benchmark for the validity of the fitting process. One novel finding was that the association to P-gp appears to be rate-limited solely by drug lateral diffusion within the inner monolayer of the plasma membrane for all three drugs. This would be expected if P-gp structure were open to the lipids of the apical membrane inner monolayer, as has been suggested by recent structural studies. The fitted kinetic parameters show how P-gp efflux of a wide range of xenobiotics has been maximized.

Exact kinetic analysis of passive transport across a polarized confluent MDCK cell monolayer modeled as a single barrier.

Knowledge of the passive permeability coefficient for new drugs is useful for estimating the fraction absorbed across the gastrointestinal tract. The commonly used approximate formula for the passive permeability coefficient is based on the initial rate of permeation across cell monolayers, requires measurement during the linear phase of permeation, and is not applicable when there is significant back flux of compound or mass balance problem. To develop a rigorous equation that can be used at any time point, i.e., that is valid outside of the linear phase, the mass action equations were integrated for a standard single barrier model of passive permeability. The simple analytical solution found also allows correction for both loss of drug (e.g., due to binding and/or hydrolysis) and sampling volume loss for multiple time point experiments. To test this equation, we measured the passive permeation of three well characterized drugs (amprenavir, quinidine, and loperamide) across confluent monolayers of MDCKII-hMDR1 cells. The potent P-glycoprotein inhibitor GF120918 was used to inhibit P-glycoprotein activity, so only passive permeability was determined. Dramatically different time-dependent behavior was observed for the three compounds, with loperamide showing significant loss of compound, and loperamide and quinidine causing plasma membrane modifications over time. The simple and exact equation for the permeability coefficient developed here works from start of transport to equilibrium, being valid when the commonly used approximate equation may not be. Thus, the exact equation is safer to use in any context, even for single time point estimates in high-throughput permeability assays.