Magnetic nanoparticles immobilized thrombin ligand fishing to screen thrombin inhibitors in natural products.

In this study, thrombin was immobilized with magnetic particles modified by glutaraldehyde. The changes in secondary structures of immobilized enzyme revealed an increment in conformational rigidity and stability, which can be reflected in temperature and pH stability as well as the tolerance of organic reagents. The optimal reutilization times of magnetic particle immobilized thrombin were 7 times, and the half-life of enzyme activity preserved at room temperature was 5 days, which was 2.5 times higher than that of free enzyme. Ligusticum chuanxiong and Anemarrhenae Rhizoma with high enzyme inhibitory activity were selected for primary screening, and six potential inhibitors of thrombin were identified by HPLC/MS. The results showed that three compounds in Anemarrhenae Rhizoma had better predictive thrombin inhibitory activity. Through the in vitro thrombin activity inhibition experiment, it was also verified that mangiferin and neo-mangiferin had an ideal thrombin activity inhibition effect, which was consistent with the results of molecular docking.

The correlation of molecule weight of chitosan oligomers with the corresponding viscosity and antibacterial activity.

In order to explore the correlation between the viscosity of chitosan oligomers-acetic solution and its viscosity average molecular weight (Mv), and determine the Mv range with a strong bactericidal effect. A series of chitosan oligomers were obtained by degraded chitosan (728.5 kDa) with dilute acid and chitosan oligomer (101.5 kDa) was characterized by FT-IR, XRD, H NMR and C NMR. The bactericidal effect of chitosan oligomers with different Mv on E. coli, S. aureus and C. albicans was measured by plate counting method. And the bactericidal rate was taken as the evaluation indicator, the optimum conditions were determined by single-factor experiments. The result showed that the molecular structure of chitosan oligomers and original chitosan (728.5 kDa) were similar. The viscosity of the chitosan oligomers in acetic acid solution was positively correlated with the Mv, and the chitosan oligomers with the Mv of 52.5-145.0 kDa had a strong bactericidal performance. In addition, the bactericidal rate of chitosan oligomers on experimental strains was more than 90% when the concentration of 0.5 g/L (bacteria) and 1.0 g/L (fungi), pH6.0, incubation time of 30 min. Thus, chitosan oligomers had a potential application value when the Mv was in the range of 52.5-145.0 kDa.

Synergistic antimicrobial system based on nisin and α-hydroxy organic acids.

Synergistic antimicrobial is a promising way to overcome microbial contamination in food and drugs. In the study, the synergistic effect between nisin and α-hydroxy organic acids on E. coli and S. aureus was investigated. The experimental results showed that the combined antibacterial ability of nisin-citric acid system was the most prominent. The FCI index also indicated that the combination of nisin and citric acid had synergistic effects on E. coli. When nisin was combined with citric acid, the inhibition rates of E. coli and S. aureus were increased to 4.43 and 1.49 times, respectively. Nisin-citric acid complex system could effectively slow down the proliferation of S. aureus and E. coli at lower concentrations, and can quickly destroy the cell membrane after 4 h of action. Therefore, the combination of nisin and citric acid is expected to be a potential solution for food and drug preservation.

Synergistic antimicrobial effect of nisin-octanoic acid nanoemulsions against E. coli and S. aureus.

Food safety is a major public health concern all over the world. Therefore, the prevention of food contamination is becoming extremely crucial. In this study, an antimicrobial nanoemulsion composed of water-soluble nisin and fat-soluble octanoic acid was successfully prepared. The results showed that the average particle size and the polymer dispersity index of the nisin-octanoic acid (NOA) nanoemulsion were around 52.21 nm and 0.253, respectively. The NOA nanoemulsion required less amounts of nisin and octanoic acid to achieve the effective antimicrobial effect against Escherichia coli and Staphylococcus aureus. In addition, the growth curves of E. coli and S. aureus were determined. The OD600 of NOA nanoemulsion was significantly lower than free nisin after being incubated for 24 h (p < 0.001), indicating that the antimicrobial effect of NOA nanoemulsion was outstanding. Meanwhile, the synergistic antimicrobial property of NOA nanoemulsion against E. coli and S. aureus was significantly better than free nisin under nonacid conditions (p < 0.05). Overall, the results of this study may provide guidance for the further application of nisin in more forms.

Separation and Purification of Bamboo Leaf Flavones by Polyvinylpolypyrrolidone Adsorption.

In view of the adsorption performance of polyvinylpolypyrrolidone (PVPP) to flavones, the adsorption and purification of bamboo leaf flavones (BLFs) by PVPP were studied. The flavones solution was adsorbed by PVPP column chromatography, and then establish a relatively effective method for elution and purification of flavones from bamboo leaf. The optimal separation conditions of column chromatography were determined as the following: the feed concentration of 10 mg/mL, the ratio of diameter to height of 1:1.9, eluents of deionized water (21 mL) and 70% ethanol (800 mL) with a flow rate of 0.33 mL/min. The purity of flavones obtained from ethanol eluents (80-480 mL) was 96.2%. This showed that the PVPP had an ideal adsorption and purification effect on BLFs.

Immobilization of lipase on silica nanoparticles by adsorption followed by glutaraldehyde cross-linking.

In this study, Candida antarctica lipase B was immobilized on silica (SiO2) nanoparticles by physical adsorption, and then cross-linked with glutaraldehyde (GA) to prepare cross-linked immobilized lipase (CLIL). During the condition of 1.28 mg/mL lipase concentration, 25 â„ƒ temperature, 2 h adsorption time, 0.01% GA (V/V) 7.5 mL and 2 h cross-linking time, the highest recovery activity of CLIL reached 87.82 ± 0.07% (22.55 ± 0.025 U/mg). Scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) confirmed that lipase was immobilized on the surface of SiO2 nanoparticles. The changes in secondary structures of CLIL indicated that cross-linking changed the secondary structure of lipase protein, which made the structure of CLIL more stable. Compared with the free lipase, the thermal stability and storage stability of CLIL was significantly improved, and the t1/2 at 60 °C was extended. Studies had shown that it was a feasible method to obtain CLIL by cross-linking after adsorbing lipase on SiO2 nanoparticles.

Degree and distribution of substitution of hydroxypropyl-ß-cyclodextrin in enantioselective liquid-liquid extraction and countercurrent chromatographic enantioseparation.

Nine types of hydroxypropyl-ß-cyclodextrin (HP-ß-CD) with different degrees and distributions of substitution were synthesised, and nine racemates were selected to investigate the effect of different degrees and distributions of substitution of HP-ß-CD on the enantioseparation factor. 1H NMR and GC/MS were used to characterise the synthesised HP-ß-CD. The degree and distribution of substitution had a significant influence on enantioselective liquid-liquid extraction and enantioseparation by countercurrent chromatography. For most of the tested racemates, increasing both the degree of substitution and distribution of substitution at the C-2 position for HP-ß-CD would lead to an increasing enantioseparation factor; the optimal enantioseparation factor of 2-phenylbutyric acid, tropic acid, 2,3-diphenylpropionic acid, 2-(4-hydroxylphenyl) propanoic acid, and naproxen was increased to 1.77, 1.53, 1.67, 1.61, and 1.75, respectively. The enantioseparation of racemic naproxen, 2-(4-hydroxylphenyl) propanoic acid, and 2,3-diphenylpropionic acid by countercurrent chromatography was optimised using HP-ß-CD with a degree of substitution of 16.5, and peak resolution was significantly improved to 1.03, 1.35, and 1.01, respectively.

Enantioseparation of N-methyl duloxetine, duloxetine, and fluoxetine by countercurrent chromatography using anionic ß-cyclodextrin as chiral selector.

Two anionic ß-cyclodextrins as chiral selectors were successfully applied in the enantioseparation of N-methyl duloxetine, duloxetine, and fluoxetine by countercurrent chromatography. Sulfobutyl ether-ß-cyclodextrin and carboxymethyl-ß-cyclodextrin showed opposite enantioselectivity for both duloxetine and N-methyl duloxetine enantiomers. Two biphasic solvent systems, n-hexane: 0.1 mol/L phosphate buffer pH 7.6 with 50 mmol/L of sulfobutyl ether-ß-cyclodextrin (1:1, v/v) and n-hexane: 0.1 mol/L phosphate buffer pH 7.2 with 50 mmol/L of carboxymethyl-ß-cyclodextrin (1:1, v/v), were selected for N-methyl duloxetine. Enantioseparation of duloxetine was achieved by recycling countercurrent chromatography using a solvent system composed of n-butyl acetate: 0.1 mol/L phosphate buffer pH 7.2 with 20 mmol/L of sulfobutyl ether-ß-cyclodextrin or carboxymethyl-ß-cyclodextrin (1:1, v/v). A solvent system composed of n-hexane: n-butyl acetate: 0.1 mol/L phosphate buffer pH 7.6 containing 20 mmol/L of sulfobutyl ether-ß-cyclodextrin (6:4:10, v/v) was selected for enantioseparation of fluoxetine.

Hypoglycemic activity and mechanisms of myricetin.

Myricetin has been reported to have a wide variety of beneficial physiological functions. The present study was designed to investigate the mechanism of high purity myricetin, as a hypoglycemic functional component on high fat diet (HFD) fed streptozotocin (STZ) induced diabetic rats. Four-week antihyperglycemic effects of myricetin were assayed. The results showed that continuous administration of myricetin (50 and 200 mg/kg body weight) in HFD/STZ induced diabetic rats dose-dependently reduced the body serum glucose and insulin. Furthermore, administrations of myricetin significantly increased the expression of insulin receptor (InsR) and glucose transporter 4 (GLUT4) gene and increased the expression of glucose-6-phosphatase (G-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK) gene. Moreover, myricetin protected pancreatic tissue from HFD fed STZ induced apoptosis through regulation of Bcl-2 associated X (Bax) gene and B-cell lymphoma-2 (Bcl-2) gene. The experimental results show that myricetin has significant health benefits and can be explored as a potentially promising dietary supplement for auxiliary hypoglycemic.

[In vitro screening of natural compounds with antitumor activity by trypsin].

In order to investigate the feasibility of in vitro screening the antitumor activity of natural compounds by trypsin, porcine trypsin was used to for screening test, which is marked by inhibition of enzyme activity. Four compounds, namely daidzin, genistin, matrine and oxymatrine, were selected as test subjects. The natural antitumor drug camptothecin was used as the control. The inhibitory effect was detected by two experimental methods: direct detection of trypsin activity inhibition and hydrolysis of bovine serum albumin by trypsin. The results showed the inhibitory effects of the four natural compounds on trypsin, and the inhibition rates of the four natural compounds were significantly different. The enzyme activity assay showed that the inhibitory effect of matrine was better than that of oxymatrine, indicating that trypsin had a good screening resolution. The inhibitory effect was significantly increased with the increased ratio of sample to trypsin, suggesting the structure-activity correlation and dose-effect correlation of the screening methods. Altogether, the experimental method of screening antitumor activity of natural compounds by trypsin has good application values. Since porcine trypsin is similar to human trypsin in terms of molecular structure and performance, it is more applicable for screening of antitumor efficacy of natural pharmacodynamic compounds.

Immobilization of lipase by dialdehyde cellulose crosslinked magnetic nanoparticles.

Cellulose microcrystalline (MCC) was widely used in pharmaceutical and chemical industries because of its low degree of polymerization and large specific surface area. As its modified form, dialdehyde cellulose (DAC) was used for cross-linking and immobilizing Rhizopus lipase together with magnetic nanoparticles (MNPs) due to its active aldehyde groups. In this study, in order to maintain the original enzyme activity as much as possible and improve the stability of lipase, the Rhizopus lipase was successfully immobilized on the magnetic dialdehyde cellulose nanoparticles (MDC). Specifically, the immobilization conditions including dosage of DAC, concentration of enzyme, immobilization time and temperature together with pH value of the reaction medium were optimized. Maximum immobilization yield (60.03 ± 0.49%) and recovery activity (88.88 ± 0.61%) can be obtained under the optimal process conditions. The changes in secondary structures of immobilized enzyme revealed the increment in conformational rigidity, which can be reflected in temperature and pH stability as well as tolerance of organic reagents. Additionally, the recovery activity of immobilized enzyme still reached 50.60 ± 0.59% after 30 d of storage and 52.10 ± 0.57% retained after 6 cycles. These results indicated the ideal application prospect of MDC in immobilized enzymes.

Enantioseparation of ondansetron by countercurrent chromatography using sulfobutyl ether-ß-cyclodextrin as chiral selector.

Ondansetron, a highly selective 5-hydroxytryptamine 3 receptor antagonist, was successfully enantioseparated by recycling countercurrent chromatography using sulfobutyl ether-ß-cyclodextrin as chiral selector. Important factors for the enantioseparation were optimized, including different organic solvent, type of substituted ß-cyclodextrin, pH of aqueous phase, concentration of chiral selector, and separation temperature. A biphasic solvent system composed of n-hexane: n-butyl acetate: 0.1 mol/L phosphate buffer solution pH 9.2 with 50 mmol/L of sulfobutyl ether-ß-cyclodextrin (2.5:7.5:10, v/v/v) was selected. Under optimized separation conditions, 5 mg of ondansetron was enantioseparated using recycling countercurrent chromatography, yielding 1.2 and 1.5 mg of ondansetron enantiomers with 97.5 and 95.8% purity and the recovery reached 48-60%.

Catalytic esterification performance of protease in micro-aqueous system.

OBJECTIVE: To evaluate the catalytic esterification performance of proteases in micro-aqueous systems and to study the suitable conditions for maintaining protease activity. RESULTS: It was found that the protease showed better enzyme catalytic activity in the micro-aqueous phase containing 4% boric acid-borax buffer than that of the pure organic phase. The protease activity was easily activated by 0.20 M boric acid-borax buffer, and the enzyme activity was still high for a long time in alkaline environment (pH 8.40-9.60) and under the temperature of 40-55 °C. Experiments using protease and Candida lipase to synthesize sucrose-6-ethyl ester showed that protease had better esterification activity than Candida lipase in the micro-aqueous phase.

Study on the relationship between crosslinking degree and properties of TPP crosslinked chitosan nanoparticles.

In order to reveal the relationship between the degree of crosslinking and the properties of chitosan nanoparticles, a potassium polyvinyl sulfate (PVSK) titration method was established for the determination of free amino group in chitosan, which showed that there was a window effect in the crosslinking degree and particle size of three kinds of molecular weight chitosan cross-linked by different content of tripolyphosphate (TPP), and the nanoparticles with moderate crosslinking degree were smaller. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) showed the moderate degree of cross-linking nanoparticles had strong antibacterial properties. IR analysis showed that the interaction between chitosan nanoparticles with different crosslinking degree and TPP were different. X-ray diffraction analysis showed that the cross-linked chitosan nanoparticles were amorphous. Above all, the crosslinking degree of TPP cross-linked chitosan nanoparticles was related to the particle size and antibacterial properties.

Preparation and characterization of crosslinked porous starch hemostatic.

In order to develop starch hemostatic materials with excellent hemostatic properties, the preparation of crosslinked porous starch (SPS) with sodium trimetaphosphate (STMP) as cross-linking agent was studied in this paper. When the solid-liquid ratio of porous starch (PS) was 30%, the mass ratio of cross-linking agent to starch was 0.04-1, and the SPS was crosslinked at pH 10.0, 55 °C for 50-60 min, the water absorption ratio and swelling ratio of SPS reach up to 160.5% and 239.1%, respectively. The characterization by infrared spectra, scanning electron microscopy and X-Ray diffraction spectra confirmed that the structure of SPS is similar to that of PS. The degradation experiment in vitro indicated that the degradation effect of PS was better than that of SPS. The whole blood coagulation kinetics experiment showed that SPS could promote the formation of blood clot, and the adsorption experiment of red blood cells in vitro showed that SPS could adsorb red blood cells. The average hemostasis time of SPS in tail amputation 1 cm and liver laceration were 181.03 s and 179.30 s.

A Comparison of the Resolution of Selective (+/-)-Glycidyl Butyrate by Using Free and Nano-SiO2 Immobilized Porcine Pancreatic Lipase.

To explore the hydrolyzed properties of nano-SiO2 immobilized porcine pancreatic lipase, the selective hydrolysis of immobilized lipase for glycidyl butyrate was compared with the free enzyme. The hydrolysis selectivity of the immobilized biocatalyst was evaluated and compared with the free enzyme using the enantiomeric excess (ee) of resolving racemic glycidyl butyrate as the indicator. The enantiomeric excess of the immobilized biocatalyst could be increased by 4.5%-10.0% which compared with the free enzyme under every single technological condition. The ee was improved from 84.7% for free enzyme to 91.6% for the immobilized enzyme with 61.2% conversion. Compared with free enzyme, the conversion rate of the immobilized enzyme was increased slightly, but the % enantiomeric excess of the immobilized enzyme was increased greatly.

Study the effect of trypsin enzyme activity on the screening of applying frontal affinity chromatography.

Frontal affinity chromatography (FAC) combined with enzyme has been widely used for drug screening. In this paper, the effect of target enzyme activity on screening of bioactive compounds was studied through applying FAC. Trypsin with different degree of inactivation were prepared as target enzyme by thermal denaturation. Their primary structure was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and use Fourier transform infrared (FTIR) and ultraviolet-visible (UV-vis) spectroscopy to detect group structure. Ultimately, it was found that the main structure of enzyme with decreased activity remained unchanged. The oxymatrine and matrine which can interact with trypsin were selected to study their binding to trypsin with different activities in FAC. The results showed that oxymatrine and matrine had a significant difference in the breakthrough volume among seven kinds of columns prepared by trypsins with different activities, at the different concentration. It indicated that trypsins with different activities in FAC could combine with oxymatrine and matrine. The binding constant (Kd) variation between oxymatrine, matrine and trypsin with different activities are 5.520 ±â€¯0.038 and 3.577 ±â€¯0.071, within error range, which indicated that the activity of target enzyme with primary unchanged structure has no effect on screening of bioactive components by FAC.

Preparation of immobilized lipase on magnetic nanoparticles dialdehyde starch.

Dialdehyde starch (DAS) is a kind of modified starch which contains many active aldehyde groups and has good biocompatibility. In this study, magnetic dialdehyde starch nanoparticles were successfully used to immobilize lipase. The lipase was immobilized onto magnetic nanoparticles by using DAS instead of glutaraldehyde as a crosslinker. The parameters like DAS dosage, enzyme concentration and immobilization time were optimized. Enzymatic properties studies exhibited that after DAS cross-linking, the storage stability of the immobilized enzyme reached 82.5%, and the recycling rate reached 53.6%, whereas in case of glutaraldehyde cross linker, it was 79.4% and 46.8%, the former also exhibited better stability and durability. Compared with the free enzyme, the immobilized enzyme indicated higher acid-base tolerance and thermal stability, and had good enzymatic properties. Magnetic dialdehyde starch nanoparticles may have application prospects as an excellent enzyme carrier, which provides a reference for the preparation of other immobilized enzymes with excellent performance.

Enzymatic acylation of flavone isolated from extractive of bamboo leaves with oleic acid and antioxidant activity of acylated product.

This present study aims to establish a relatively cheap and effective method for preparation of ester of flavone isolated from bamboo-leaf extracts. According to this method, the solubility of bamboo leaf extract in a lipid medium could be improved, which would expand the utilization of bamboo leaf extract. Acylation of flavone with oleic acid was efficient in a system containing dried tert-amyl-alcohol and molecular sieves. The bioconversion yield ranged from 24 to 63% in different conditions. the type of fatty acids, the amount of the lipase, water content and temperature of the system were investigated in detail to influence on the performance of the acylation reaction. High purity of ester (>95%) was obtained by a two-step solvent purification procedure and chromatographic separation. Followed by the 1H and 13C nuclear magnetic resonance spectroscopy analysis, single product of 6''-O-oleate isoorientin ester was detected. Introducing an acyl group into isoorientin significantly improved its lipophilicity yet slightly reduced their antioxidant activity, while its antioxidant activity still significantly better than VE.

Preparation and properties of gum arabic cross-link binding nisin microparticles.

Nisin is a small cationic peptide composed of 34 amino acid residues, it exhibits a wide spectrum antimicrobial activity against Gram-positive bacteria, but nisin has the disadvantages of poor stability and short duration of antibacterial activity. In order to improve the stability of nisin during food processing and storage period, the gum arabic (GA) is used to get GA-nisin microparticles. The analysis of zeta potential, SEM and FT-IR were used to characterize the combination of GA and nisin, test the antibacterial activity on Staphylococcus aureus and Escherichia coli, the bactericidal effect of GA-nisin microparticles decreased within 24 h compare to free nisin, however, GA-nisin microparticles after 121 °C treatment for 10 min, the activity loss rate is less than free nisin and has better sustained antibacterial effect at 25 °C. The results shown GA-nisin microparticles have better high-temperature stability and sustained bactericidal ability than the free nisin.