Biochemical characterization of a novel glucose-tolerant GH3 ß-glucosidase (Bgl1973) from Leifsonia sp. ZF2019.

Beta-glucosidase (Bgl) is an enzyme with considerable food, beverage, and biofuel processing potential. However, as many Bgls are inhibited by their reaction end product glucose, their industrial applications are greatly limited. In this study, a novel Bgl gene (Bgl1973) was cloned from Leifsonia sp. ZF2019 and heterologously expressed in E. coli. Sequence analysis and structure modeling revealed that Bgl1973 was 748 aa, giving it a molecular weight of 78 kDa, and it showed high similarity with the glycoside hydrolase 3 (GH3) family Bgls with which its active site residues were conserved. By using pNPGlc (p-nitrophenyl-ß-D-glucopyranoside) as substrate, the optimum temperature and pH of Bgl1973 were shown to be 50 °C and 7.0, respectively. Bgl1973 was insensitive to most metal ions (12.5 mM), 1% urea, and even 0.1% Tween-80. This enzyme maintained 60% of its original activity in the presence of 20% NaCl, demonstrating its excellent salt tolerance. Furthermore, it still had 83% residual activity in 1 M of glucose, displaying its outstanding glucose tolerance. The Km, Vmax, and kcat of Bgl1973 were 0.22 mM, 44.44 µmol/min mg, and 57.78 s-1, respectively. Bgl1973 had a high specific activity for pNPGlc (19.10 ± 0.59 U/mg) and salicin (20.43 ± 0.92 U/mg). Furthermore, molecular docking indicated that the glucose binding location and the narrow and deep active channel geometry might contribute to the glucose tolerance of Bgl1973. Our results lay a foundation for the studying of this glucose-tolerant ß-glucosidase and its applications in many industrial settings. KEY POINTS: • A novel ß-glucosidase from GH3 was obtained from Leifsonia sp. ZF2019. • Bgl1973 demonstrated excellent glucose tolerance. • The glucose tolerance of Bgl1973 was explained using molecular docking analysis.

Microemulsion Delivery System Improves Cellular Uptake of Genipin and Its Protective Effect against Aß1-42-Induced PC12 Cell Cytotoxicity.

Genipin has attracted much attention for its hepatoprotective, anti-inflammatory, and neuroprotection activities. However, poor water solubility and active chemical properties limit its application in food and pharmaceutical industries. This article aimed to develop a lipid-based microemulsion delivery system to improve the stability and bioavailability of genipin. The excipients for a genipin microemulsion (GME) preparation were screened and a pseudo-ternary phase diagram was established. The droplet size (DS), zeta potential (ZP), polydispersity index (PDI), physical and simulated gastrointestinal digestion stability, and in vitro drug release properties were characterized. Finally, the effect of the microemulsion on its cellular uptake by Caco-2 cells and the protective effect on PC12 cells were investigated. The prepared GME had a transparent appearance with a DS of 16.17 ± 0.27 nm, ZP of -8.11 ± 0.77 mV, and PDI of 0.183 ± 0.013. It exhibited good temperature, pH, ionic strength, and simulated gastrointestinal digestion stability. The in vitro release and cellular uptake data showed that the GME had a lower release rate and better bioavailability compared with that of free genipin. Interestingly, the GME showed a significantly better protective effect against amyloid-ß (Aß1-42)-induced PC12 cell cytotoxicity than that of the unencapsulated genipin. These findings suggest that the lipid-based microemulsion delivery system could serve as a promising approach to improve the application of genipin.

Physicochemical and rheological characterization of pectin-rich polysaccharides from Gardenia jasminoides J. Ellis flower.

Gardenia (Gardenia jasminoides J. Ellis) is regarded as an edible medicine plant in China. Here, gardenia flower polysaccharide fraction (GFPF) was extracted by water at 90°C and its chemical composition, rheological properties, and antioxidant activities of GFPF were investigated. The GFPF extraction yield was 18.04 ± 1.81% (W/W) and mainly comprised neutral sugars (46.83 ± 3.14%), uronic acid (35.21 ± 0.17%), protein (1.63 ± 0.34%), and total phenol (9.49 ± 0.08 mgGAE/g). Galacturonic acid (41.05 ± 0.59%) was the main monosaccharide, and galactose, glucose, arabinose, rhamnose, xylose, mannose, and glucuronic acid were also detected in GFPF. Its degree of esterification was 32.76 ± 1.52%. FT-IR spectra analysis showed a similar absorption pattern between GFPF and pectin from apple. The results suggested that GFPF was low methoxy pectin. Thermogravimetric analysis and zeta potential analysis indicated that the pectin was stable under high temperature and alkaline condition. Steady rheology showed that the GFPF dispersion was a shear thinned pseudoplastic fluid with high apparent viscosities at concentration above 2%. The degree of pseudoplasticity of the solutions increased with the concentrations increased and the temperatures decreased. DPPH and ABTS free radical scavenging assay indicated that GFPF had relatively high antioxidant activity. The results showed that gardenia flower was rich in pectin polysaccharides with low methoxy pectin. It had high apparent viscosities at concentration above 2% and had good antioxidant activity. The data suggested that GFPF can be a new resource of low methoxy pectin with potential application as thicker or gelling agents in food industry.

Evaluation of reactive oxygen species scavenging activities and DNA damage prevention effect of Pleioblastus kongosanensis f. aureostriatus leaf extract by chemiluminescence assay.

Reactive oxygen species scavenging effect of Pleioblastus kongosanensis f. aureostriatus leaf extract against O2(-), OH and H2O2 were investigated by chemiluminescence methods in vitro. Bamboo grass leaves were extracted with 70% ethanol solution and sequentially partitioned with solvents in an order of increasing polarity. Among fractions of different polarity, BuOH and EtOAc fractions showed powerful scavenging activities than others, and showed better scavenging ability on OH than that of O2(-)and H2O2, with IC50 of 0.55 µg/mL and 0.60 µg/mL, respectively. Both OH-induced DNA damage model by chemiluminescence assay and plasmid pUC18 double-strand break model by agarose gel electrophoresis showed that BuOH and EtOAc fractions had remarkable concentration-dependent prevention effect on the OH-induced damage of DNA attribute to their good scavenging effects on ROS. Results from the compositional analysis of different fractions indicate that the flavonoids in the Pleioblastus kongosanensis f. aureostriatus leaf may be responsible for its ROS scavenging activity and DNA damage prevention ability.

Altitudinal variation of antioxidant components and capability in Indocalamus latifolius (Keng) McClure leaf.

Indocalamus latifolius (Keng) McClure leaf is a popular food material in East Asia due to its antioxidant and anticorrosive activities. To utilize it more effectively, we investigated the discrepancy of antioxidant activities and active compound content in Indocalamus latifolius leaf along with the altitude change. Total flavonoids, phenolics, titerpenoids and eight characteristic active constituents, i.e, orientin, isoorientin, vitexin, homovitexin, p-coumaric acid, chlorogenic acid, caffeic acid, and ferulic acid, were determined by UV-spectrophotometer and synchronous RP-HPLC, respectively. Antioxidant activity was measured using DPPH and FRAP methods. Our data showed that the content of TP and TF, DPPH radical scavenging ability and ferric reduction power of Indocalamus latifolius leaf changed as altitude altered, with the trends of decreasing gradually when lower than 700 m and then increasing to 1,000 m. Chlorogenic acid and orientin were the main characteristic compounds in Indocalamus latifolius leaf and were also affected by altitude. Our result indicated that higher altitude with an adverse environment is conducive to secondary metabolite accumulation for Indocalamus latifolius. It would provide a theoretical basis to regulate the leaf collection conditions in the industrial use of Indocalamus latifolius leaf.

Seasonal variations of the antioxidant composition in ground bamboo Sasa argenteastriatus leaves.

Sasa argenteastriatus, with abundant active compounds and high antioxidant activity in leaves, is a new leafy bamboo grove suitable for exploitation. To utilize it more effectively and scientifically, we investigate the seasonal variations of antioxidant composition in its leaves and antioxidant activity. The leaves of Sasa argenteastriatus were collected on the 5th day of each month in three same-sized sample plots from May 2009 to May 2011. The total flavonoids (TF): phenolics (TP) and triterpenoid (TT) of bamboo leaves were extracted and the contents analyzed by UV-spectrophotometer. Our data showed that all exhibited variations with the changing seasons, with the highest levels appearing in November to March. Antioxidant activity was measured using DPPH and FRAP methods. The highest antioxidant activity appeared in December with the lowest in May. Correlation analyses demonstrated that TP and TF exhibited high correlation with bamboo antioxidant activity. Eight bamboo characteristic compounds (orientin, isoorientin, vitexin, homovitexin and p-coumaric acid, chlorogenic acid, caffeic acid, ferulic acid) were determined by RP-HPLC synchronously. We found that chlorogenic acid, isoorientin and vitexin are the main compounds in Sasa argenteastriatus leaves and the content of isovitexin and chlorogenic acid showed a similar seasonal variation with the TF, TP and TT. Our results suggested that the optimum season for harvesting Sasa argenteastriatus leaves is between autumn and winter.