Blood glucose-lowering activity of protocatechuic acid is mediated by inhibiting a-glucosidase

α-Glucosidase inhibitors are effective in controlling postprandial hyperglycemia,which play crucial roles in the management of type 2 diabetes.Protocatechuic acid(PCA)is one of phenolic acids existing not only in various plant foods but also as a major microbial metabolite of dietary anthocyanins in the large colon.The present study investigated the inhibitory mechanism of PCA on a-glucosidase in vitro and examined its effect on postprandial blood glucose levels in vivo.Results from in vitro experiments demonstrated that PCA was a mix-type inhibitor of a-glucosidase.Driven by hydrogen bonds and van der Waals interactions,PCA reversibly bound withα-glucosidase to form a stable a-glucosidase-PCA complex in a spontaneous manner.The computational simulation found that PCA could insert into the active cavity of a-glucosidase and establish hydrogen bonds with catalytic amino acid residues.PCA binding aroused the steric hindrance for substrates to enter active sites and caused the structural changes of interacted catalytic amino acid residues.PCA also exhibited postprandial hypoglycemic capacity in diabetic mice.This study may provide the theoretical basis for the application of PCA as an active ingredient of functional foods in dietary management of diabetes.

Kinetic Studies on the Irreversible Inhibition of Restriction Endonuclease Pst I by Site-Specific Inhibitors

The irreversible modifying effects onPst I of several inhibitors have been studied with the irreversible inhibition kinetic theory of single substrate reaction provided by Tsou, C. L. Pyridoxal phosphate (PLP), p-chloromercuribenzoic acid (PCMB), diisopropyl fluorophosphate (DEP), 2,3-diacetyl (DAC) and N-ethyl-5-phenylisoxazoliun-3′-sulfonate (woodward's reagent K, WRK) modify the lysine, cysine, serteine, arginine and carboxyl groups of the protein molecule respectively. These five inhibitors have been found to inhibit both the prime activity and star activity ofPst I. Used with the irreversible inhibition theory, the apparent inhibition rate constant,A and the microcosmic inhibition rate constants,k +0 andk′ +0 of every inhibitor were calculated. We also found that their inhibition effects belong to the noncompetitive irreversible inhibition. Results show that among the groups to be modified, some have nothing to do with the combination with the substrate, and some may have, but any of them isn't the only factor involved in the specific binding. Despite all this, they may take part in the catalysis of enzyme or have important effects on maintaining the active structure of enzyme molecules. Furthermore, serine and arginine residues are related to the alteration ofPst I conformation and then influence the ability ofPst I recognizing and incising DNA specifically.

Kinetic and binding equilibrium studies of dihydroflavonol 4-reductase from Vitis vinifera and its unusually strong substrate inhibition

Dihydroflavonol 4-reductase (DFR), a member of the short-chain dehydrogenase family, catalyzes the last common step in the biosynthesis of flavan-3-ols and condensed tannins. Initial rates of DFR were measured by monitoring the 340-nm absorbance decrease resulting from the joint consumption of dihydroquercetin (DHQ) and NADPH, as a function of pH, temperature and ionic strength. At pH 6.5 and 30o C, substrate inhibition was observed above 30 μM DHQ. At lower/non-inhibitory DHQ concentrations, NADP+ behaves as a competitive inhibitor with respect to NADPH and as a mixed inhibitor with respect to DHQ, which supports a sequential ordered mechanism, with NADPH binding first and NADP+ released last. Binding-equilib-rium data obtained by means of the chromatographic method of Hummel and Dreyer at pH 7.5 and by isothermal calorimetric titration at pH 6.5 led to the conclusion that ligands of the apoenzyme included NADPH, NADP+ and DHQ. The mechanism which best accounts for substrate inhibition at pH 6.5 in the absence of product involves the formation of a binary non-productive E.DHQ complex. Thus, a productive ternary complex cannot be formed when DHQ binds first. This mechanism of inhibition may prevent the accumulation of unstable leucoanthocyanidins within cells.

Structural characterizations andα-glucosidase inhibitory activities of four Lepidium meyenii polysaccharides with different molecular weights

Four polysaccharides(MCPa,MCPb,MCPc,MCPd)were obtained from Lepidium meyenii Walp.Their structures were characterized by chemical and instrumental methods including total sugar,uronic acid and protein content determi-nation,UV,IR and NMR spectroscopy,as well as monosaccharide composition determination and methylation analy-ses.Four polysaccharides were a group of glucans with different molecular weights ranging from 3.12 to 14.4 kDa,and shared a similar backbone chain consisting of(1→4)-glucose linkages with branches attached to C-3 and C-6.Furthermore,bioactivity assay showed that MCPs had concentration-dependent inhibitory activity onα-glucosidase.MCPb(Mw=10.1 kDa)and MCPc(Mw=5.62 kDa)with moderate molecular weights exhibited higher inhibitory activ-ity compared with MCPa and MCPd.

Enzymatic activities and kinetic properties of β-glucosidase from selected white rot fungi

Beta-glucosidase is among the suite of enzymes produced by white rot fungi (WRF) to biodegrade plant biomass. This study investigated the enzymatic activities and kinetic properties of β-glucosidase from seventeen WRF comprised of the following species from various geographical locations: Pleurotus ostreatus, Auricularia auricular, Polyporus squamosus, Trametes versicolor, Lentinula edodes, and Grifola frondosa. All the WRF studied showed β-glucosidase activities. Significant variations in protein and carbohydrate contents were also recorded. Beta-glucosidase activities after 30 min of incubation ranged from 6.4 μg (T. versicolor) to 225 μg (G. frondosa). The calculated kinetic constant (Km) ranged from 0.47 μM (A. auricular-1120) to 719 μM (L. edodes-7). The Vmax depending on the kinetic transformation model ranged from 0.21 μg·min-1 (T. versicolor) to 9.70 μg·min-1 (G. frondosa-28). Beta-glucosidase activities also exhibited pH optima between 3.5 and 5.0 while temperature optima were between 60°C and 70°C with some media exhibiting a secondary temperature peak at 90°C attributable to the presence of thermostable isoenzyme. WRF if appropriately screened and purified can be harnessed to potentially improve the bio-conversion of cellulose to glucose and also facilitate efficient plant biomass biodegradation and production of useful plant bio-products.

New flavanone-monoterpene hybrids as α-glucosidase inhibitors from the root bark of Morus alba

Objective The Morus alba root bark is a well-known Chinese herbal medicine called Sang-Bai-Pi and has often been used to relieve the hyperglycemic symptom of diabetes patients.The current work aims to further explore its bioactive constituents with α-glucosidase inhibitory activity for the potential treatment of diabetes.Methods A combination of different separating techniques including routine column chromatograph and HPLC especially on chiral columns were applied for the isolation of target molecules,while comprehensive spectroscopic experiments comprising MS,NMR,ECD,etc.were carried out to complete the structural assignment.The anti-hyperglycemic property of the isolates was evaluated by an in vitro α-glucosidase inhibitory bioassay.Results Two pairs of new flavanone-monoterpene hybrid enantiomers were isolated and identified,and an interesting phenomenon of mutual transformation between these cometabolites were detected,which resulted in their regio-isomerization and enantiomerization.The bioassay results revealed remarkable α-glucosidase inhibitory activity for these fascinating molecules.Conclusions The Morus alba root bark is a rich source of bioactive flavonoid derivatives and deserves further investigations to develop new potential chemotherapies for diabetes control and treatment.

A short review on natural gas hydrate, kinetic hydrate inhibitors and inhibitor synergists

Gas hydrate-caused pipeline plugging is an industrial nuisance for petroleum flow assurance that calls for technological innovations.Traditional thermodynamic inhibitors such as glycols and inorganic salts suffer from high dosing,environmental unfriendliness,corrosiveness,and economical burden.The development and use of kinetic hydrate inhibitors(KHIs),mostly polymeric compounds,with their inhibiting effects on hydrate nucleation and growth are considered an effective and economically viable chemical treatment for hydrate prevention.However,the actual performance of a KHI candidate is dependent on various factors including its chemical structure,molecular weight,spatial configuration,effective concentration,pressure and temperature,evaluation methods,use of other additives,etc.This review provides a short but systematic overview of the fundamentals of natural gas hydrates,the prevailing categories of polymeric kinetic hydrate inhibitors with proposed inhibition mechanisms,and the various synergists studied for boosting the KHI performance.Further research endeavors are in need to unveil the KHI working modes under different conditions.The conjunctive use of KHIs and synergists may facilitate the commercial application of effective KHIs to tackle the hydrate plugging problem in the oil and gas flow assurance practices.

Chitosan as green kinetic inhibitors for gas hydrate formation

The kinetic inhibiting effect of a number of chitosans on hydrate formation was investigated using methane and methane/ethane gas mixtures.The results indicated that chitosan was a good kinetic inhibitor.The induction time of gas hydrate formation evidently increased with the degree of deacetylation （DD）,however,when DD was higher than 80%,the effect of DD on the induction time was negligible.Moreover,it was found that the molecular weight （MW） of chitosan and the addition of polyethylene oxide （PEO） had little effect on the induction time.The optimal concentration of chitosan was found to be 0.6 wt%.Finally,the mechanisms of the kinetic inhibitor on the hydrate formation were discussed.

Microbial Degradation of Quinoline: Kinetics Study With Burkholderia picekttii

Objective To investigate the kinetics of quinoline biodegradation by Burkholderia pickttii, a Gram negative rod-shaped aerobe, isolated in our laboratory. Methods HPLC (Hewlett-Packard model 5050 with an UV detector) was used for the analysis of quinoline concentration. GC/MS method was used to identify the intermediate metabolites of quinoline degradation. Results The biodegradation of quinoline was inhibited by quinoline at a high concentration, and the degradation process could be described by the Haldane model. The kinetic parameters based on Haldane substrate inhibition were evaluated. The values were v = 0.44 h-1,Ks=166.7 mg/L, Ki= 650 mg/L, respectively. The quinoline concentration to avoid substrate inhibition was inferred theoretically and determined to be 329 mg/L. Conclusion The biodegradation of quinoline conforms to the Haldane inhibition model and the main intermediate metabolite of quinoline biodegradation is 2-hydroxy-quinoline.

Effect of Mg on Microstructure and Growth Kinetics of Zn-22.3Al-1.1Si Coating

The effects of Mg on the microstructure and growth kinetics of the hot-dip Zn-22.3 Al-1.1 Si-x Mg(x = 0, 0.2, 0.4, and 0.6) coatings were investigated in detail. Scanning electron microscopy with energy dispersive spectroscopy and X-ray diffraction studies revealed the presence of η-Zn, α-Al, Zn/Al eutectoid,(Si), Al/Si eutectic, and Zn/Al/Mg_2 Zn11 ternary eutectic on the top surface of these Mg-containing coatings. Especially, a small amount of MgZn2 phase appears in top surface of Zn-22.3 Al-1.1 Si-0.6 Mg coating. Five phases are found in the alloy layers, i e, Fe_2 Al_5, FeAl_3, τ_(5C), τ_(5H), and τ_1. The addition of 0.2% Mg can delay the emergence of FeAl_3 phase. When the Mg content is more than 0.2%, the outer layers of coating change from τ_(5C) to τ_(5H) phase. The growth of the inhibition layer is diffusion controlled for various Mg content baths. Mg improves the corrosion resistance of the Mg-containing coatings, and the Zn-22.3 Al-1.1 Si-0.6 Mg coating possesses the highest protective properties.

Kinetics of Asymmetric Reduction of Phenylglyoxylic Acid to R-(-)-Mandelic Acid by Saccharomyces Cerevisiae FD11b

The kinetics of asymmetric production of R-（-）-mandelic acid （R-MA） from phenylglyoxylic acid （PGA） catalyzed by Saccharomyces cerevisiae sp. strain FD11b was studied by fed-batch cultures. The concentrations of glucose and PGA were controlled respectively with a dual feeding system. When the electron donor glucose was supplied at the rate of 0.0833mmol·gdw^-1·h^-1, the specific production rate （qp） and the enantiomeric excess of R-MA reached the maximum 0.353mmol·gdw^-1·h^-1 and 97.1%, respectively. The apparent reduction activity of yeast FD 11 b was obviously affected by both substrate PGA and product MA. The qp value reached the maximum 0.36-0.38mmol·gdw^-1·h^-1 when the PGA concentration was controlled between 25 and 35mmol·L^-1. The obvious substrate inhibition of bioconversion was observed at the PGA concentrations higher than 40mmol·L^-1. The accumulation of product MA also caused a severe feed-back inhibition for its production when the product concentration was above 60mmol·L^-1. The kinetic model with the inhibition effect of both substrate and product was simulated by a computer-based least-square arithrnatic. The established kinetic model was in good agreement with the experimental data.

Kinetics of cometabolic degradation of 2-chlorophenol and phenol by Pseudomonas putida

In order to address the complex cometabolic degradation of toxic compounds, batch experiments on the biodegradation of 2-chlorophenol （2-CP） and phenol by Pseudomonas putida were carried out. The experimental results show that 2-CP has an inhibitory effect on cell growth and phenol degradation, which demonstrates that the interaction between substrates affects cell growth and substrate degradation. A kinetic model of cell growth and substrate transformation was also developed. The square of the correlation coefficient from the experiment was 0.97, indicating that this model properly simulates the cometabolic degradation of 2-CP and phenol.

Investigation on the effect of oxalic acid, succinic acid and aspartic acid on the gas hydrate formation kinetics

Gas hydrate reserves are potential source of clean energy having low molecular weight hydrocarbons trapped in water cages.In this work,we report how organic compounds of different chain lengths and hydrophilicities when used in small concentration may modify hydrate growth and either act as hydrate inhibitors or promoters.Hydrate promoters foster the hydrate growth kinetics and are used in novel applications such as methane storage as solidified natural gas,desalination of sea water and gas separation.On the other hand,gas hydrate inhibitors are used in oil and gas pipelines to alter the rate at which gas hydrate nucleates and grows.Inhibitors such as methanol and ethanol which form strong hydrogen bond with water have been traditionally used as hydrate inhibitors.However,due to relatively high volatility a significant portion of these inhibitors ends up in gas stream and brings further complexity to the safe transportation of natural gas.In this study,organic additives such as oxalic acid,succinic acid and L-aspartic acid(all three)having––COOH group(s)with aspartic acid having an additional––NH2 group,are investigated for gas hydrate promotion/inhibition behavior.These compounds are polar in nature and thus have significant solubility in liquid water;the presence of weak acidic and water loving(carboxylic/amine groups)moieties makes these organic acids an excellent candidate for further study.This study would pave ways to identify a novel(read better)promoter/inhibitor for gas hydrate formation.Suitable thermodynamic conditions were generated in a stirred tank reactor coupled with cooling system;comparison of gas hydrate formation kinetics with and without additives were carried out to identify the effect of these acids on the formation and growth of hydrates.The possible mechanisms by which these additives inhibit or promote the hydrate growth are also discussed.

Investigation of the Performance and Kinetics of Anaerobic Digestion at 45°C

The objective of this study was to evaluate the performance of anaerobic digestion (AD) within the intermediate zone, specifically at 45°C. Single-stage batch anaerobic digestion system was developed in the lab and performance was monitored for more than 2 years. The AD system was able to achieve high biogas production with about 62% - 67% methane content. The digester exhibited high acetate accumulation, but sufficient buffering capacity was observed as the pH, alkalinity and volatile fatty acids-to-alkalinity ratio were within recommended values. The system achieved 36.5% reduction of total solids (TS) and 47.8% reduction of volatile solids (VS), which exceeded the required VS destruction efficiency for Class A biosolids. The pathogen counts were less than 1000 MPN/g total solids in the effluent, which also satisfied Class A biosolids requirements. The accumulation of acetate was presumably due to the high temperature which contributed to high hydrolysis rate. Consequently, it produced large amount of toxic salts that combined with the acetate, making them not readily available to be consumed by methanogens. The slower degradation of acetate was observed by the kinetic parameters. Accumulation of acetate contributed to 52% to 71% reduction in acetate degradation process, but was not completely inhibitory. The methanogens existing in the system were mostly thermo-tolerant acetate-utilizing methanogens, and specifically from Methanosarcinaceae species.

Antidiabetic effect of Chrysophyllum albidum is mediated by enzyme inhibition and enhancement of glucose uptake via 3T3-L1 adipocytes and C2C12 myotubes

Objective:To investigate the in vivo and in vitro antidiabetic potential of Chrysophyllum albidum.Methods:The effects of oral treatment with hydro-ethanolic extract(125,250 and 500 mg/kg)of the stem bark of Chrysophyllum albidum and glibenclamide for 21 d on glucose level,serum enzyme markers for liver function,lipid profile,total protein,serum urea,serum creatinine,and body weight were evaluated in experimental diabetic rats administered with 45 mg/kg of streptozotocin.In vitro assays including glucose uptake in C2 C12 cells and 3 T3-L1 adipose tissues,α-glucosidase andα-amylase inhibition were employed to evaluate the possible mechanism of hypoglycemic action of the extract.DPPH and nitric oxide radical antioxidant activity of the extract was also measured.Results:The increased levels of blood glucose,triglycerides,lowdensity lipoprotein,total cholesterol,serum aspartate,and alanine transaminases,creatinine,and urea in the diabetic animals were reduced significantly(P<0.01)after treatment with Chrysophyllum albidum extract.The decreased total protein and high-density lipoprotein concentrations were normalized after treatment.In addition,the extract significantly(P<0.01)increased the transport of glucose in 3 T3-L1 cells and C2 C12 myotubes and exhibited considerable potential to inhibitα-amylase andα-glucosidase.It also demonstrated potent antioxidant action by scavenging considerably DPPH and nitric oxide radicals.Conclusions:Chrysophyllum albidum stem bark extract exhibits considerable antidiabetic effect by stimulating glucose uptake and utilization in C2 C12 myotubes and 3 T3-L1 adipocytes as well as inhibiting the activities ofα-amylase andα-glucosidase.

Studies on Enzyme Kinetics by Microchip and Related Techniques

Both conventional and microchip-based capillary electrophoresis(CE) technologies have been used for the analysis of enzymes. Practical procedures of using CE to determine the Km and Vmax values of an enzyme have been developed. By studying the inhibition to the enzyme, it is possible to select a suitable drug candidate. When compared with the conventional CE method, single lane microchip-based method can improve the speed for the assay three times. By using multiple lane-based microchip, the speed can be further increased.

Inhibition of Aluminium Corrosion Using <i>Carica papaya</i>Leaves Extract in Sulphuric Acid

Inhibition of aluminium corrosion using C. papaya leaves extract in 1.0 M H2SO4 was investigated by using gravimetric analysis at various concentrations and temperatures: 303 K, 313 K and 323 K. Characterization was done by using Scanning Electron Microscope (SEM) and Fourier Transform Infrared (FT-IR) spectroscopy. Results show that, inhibiting ability of the extract was due to its adsorption onto the metal surface through Langmuir adsorption isotherm. Thermodynamic (Gibbs energy, entropy and heats of adsorption) and kinetic parameters (activation energy and entropy of activation) were also determined. All of these agreed to physical adsorption of inhibitor onto the aluminium surface.

Steady-state kinetics of <i>Roystonea regia</i>palm tree peroxidase

Royal palm tree peroxidase (RPTP) has been isolated to homogeneity from leaves of Roystonea regia palm trees. The enzyme purification steps included homogenization, (NH4)SO4 precipitation, extraction of palm leaf colored compounds and consecutive chromatography on Phenyl-Sepharose, TSK-Gel DEAE-5PW and Superdex-200. The novel peroxidase was characterized as having a molecular weight of 48.2 ± 3.0 kDa and an isoelectric point pI 5.4 ± 0.1. The enzyme forms dimers in solution with approximate molecular weight of 92 ± 2 kDa. Here we investigated the steady-state kinetic mechanism of the H2O2-supported oxidation of different organic substrates by RPTP. The results of the analysis of the initial rates vs. H2O2 and reducing substrate concentrations were seen to be consistent with a substrate-inhibited Ping-Pong Bi-Bi reaction mechanism. The phenomenological approach used expresses the peroxidase Ping-Pong mechanism in the form of the Michaelis-Menten equation and affords an interpretation of the effects in terms of the kinetic parameters KmH2O2, KmAH2, kcat, KSIH2O2, KSIAH2 and of the microscopic rate constants k1 and k3 of the shared three-step peroxidase catalytic cycle. Furthermore, the concentration and time-dependences and the mechanism of the suicide inactivation of RPTP by hydrogen peroxide were studied kinetically with guaiacol as co-substrate. The turnover number (r) of H2O2 required to complete the inactivation of the enzyme was 2154 ± 100 and the apparent rate constants of catalysis 185 s–1 and 18 s–1.

Inhibitory Effect of Ferulic Acid on Oxidation of L-DOPA Catalyzed by Mushroom Tyrosinase

The inhibitory effect of ferulic acid on the diphenolase activity of mushroom tyrosinase and the kinetic behavior were studied with L-3,4-dihydroxyphenylalanine （L-DOPA） as substrate. The inhibitor concentration leading to 50% relative activity lost （IC50） was estimated to be 0.15 mmol·L^-1. The inhibition mechanism obtained from Lineweaver-Burk plots shows that ferulic acid is a competitive inhibitor and the inhibition of tyrosinase by ferulic acid is a reversible reaction. The equilibrium constant for ferulic acid binding with the tyrosinase was determined to be 0.25 mmol·L^-1 for diphenolase. Keywords tyrosinase, ferulic acid, kinetics, inhibition, L-DOPA, diphenolase

Inhibitory effects of novel green inhibitors on gas hydrate formation

Natural gas hydrates easily form in pipelines,causing potential safety issues during oil and gas production and transportation.Injecting gas hydrate inhibitors is one of the most effective methods for preventing gas hydrate formation or aggregation.However,some thermodynamic hydrate inhibitors are toxic and harmful to the environment,whereas degradation of kinetic inhibitors is difficult.Therefore,environmentally friendly and easily biodegradable novel green inhibitors have been proposed and investigated.This paper provides a short but systematic review of the inhibitory performance of amino acids,antifreeze proteins,and ionic liquids.For different hydrate formation systems,the influences of the inhibitor type,structure,and concentration on the inhibitory effects are summarized.The mechanism of green inhibitors as kinetic inhibitors is also discussed.The progress described here will facilitate further developments of such green inhibitors for gas hydrate formation.