Glycosylation Enhances the Physicochemical Properties of Caffeic Acid Phenethyl Ester.

In this study, we synthesized a glycosylated derivative of caffeic acid phenethyl ester (CAPE) using the amylosucrase from Deinococcus geothermalis with sucrose as a substrate and examined its solubility, chemical stability, and anti-inflammatory activity. Nuclear magnetic resonance spectroscopy showed that the resulting glycosylated CAPE (G-CAPE) was the new compound caffeic acid phenethyl ester-4-O-α-D-glucopyranoside. G-CAPE was 770 times more soluble than CAPE and highly stable in Dulbecco's modified Eagle's medium and buffered solutions, as estimated by its half-life. The glycosylation of CAPE did not significantly affect its anti-inflammatory activity, which was assessed by examining lipopolysaccharide-induced nitric oxide production and using a nuclear factor erythroid 2-related factor 2 reporter assay. Furthermore, a cellular uptake experiment using high-performance liquid chromatography analysis of the cell-free extracts of RAW 264.7 cells demonstrated that G-CAPE was gradually converted to CAPE within the cells. These results demonstrate that the glycosylation of CAPE increases its bioavailability by helping to protect this vital molecule from chemical or enzymatic oxidation, indicating that G-CAPE is a promising candidate for prodrug therapy.

The Effect of Bisphasic Calcium Phosphate Block Bone Graft Materials with Polysaccharides on Bone Regeneration.

In this study, bisphasic calcium phosphate (BCP) and two types of polysaccharide, carboxymethyl cellulose (CMC) and hyaluronic acid (HyA), were used to fabricate composite block bone grafts, and their physical and biological features and performances were compared and evaluated in vitro and in vivo. Specimens of the following were prepared as 6 mm diameter, 2 mm thick discs; BPC mixed with CMC (the BCP/CMC group), BCP mixed with crosslinked CMC (the BCP/c-CMC group) and BCP mixed with HyA (the BCP/HyA group) and a control group (specimens were prepared using particle type BCP). A scanning electron microscope study, a compressive strength analysis, and a cytotoxicity assessment were conducted. Graft materials were implanted in each of four circular defects of 6 mm diameter in calvarial bone in seven rabbits. Animals were sacrificed after four weeks for micro-CT and histomorphometric analyses, and the findings obtained were used to calculate new bone volumes (mm³) and area percentages (%). It was found that these two values were significantly higher in the BCP/c-CMC group than in the other three groups (p < 0.05). Within the limitations of this study, BCP composite block bone graft material incorporating crosslinked CMC has potential utility when bone augmentation is needed.

Synthesis and biological evaluation of a novel baicalein glycoside as an anti-inflammatory agent.

Baicalein-6-α-glucoside (BG), a glycosylated derivative of baicalein, was synthesized by using sucrose and the amylosucrase of Deinococcus geothermalis and tested for its solubility, chemical stability, and anti-inflammatory activity. BG was 26.3 times more soluble than baicalein and highly stable in buffered solutions and Dulbecco׳s modified Eagle medium containing 10% fetal bovine serum. BG treatment decreased the production of nitric oxide in RAW 264.7 cells treated with lipopolysaccharide (LPS). Luciferase reporter assays, western blots, reverse transcription-polymerase chain reaction, and flow cytometric analyses indicated that BG activated nuclear factor erythroid 2-related factor 2 (Nrf2), an antioxidant transcription factor that confers protection from various inflammatory diseases, induced Nrf2-dependent gene expression, and suppressed the production of reactive oxygen species elicited by LPS more effectively than baicalein. Cellular uptake of BG assessed by confocal microscopy and HPLC analysis of the cell-free extracts of RAW 264.7 cells demonstrated that BG was gradually converted to baicalein inside the cells. These results explain that glycosylation increased the bioavailability of baicalein by helping to protect this vital molecule from chemical or enzymatic oxidation. Therefore, BG, a glycosylated derivative of baicalein, can be an alternative to baicalein as a therapeutic drug.

Probing the critical residues for intramolecular fructosyl transfer reaction of a levan fructotransferase.

Levan fructotransferase (LFTase) preferentially catalyzes the transfructosylation reaction in addition to levan hydrolysis, whereas other levan-degrading enzymes hydrolyze levan into a levan-oligosaccharide and fructose. Based on sequence comparisons and enzymatic properties, the fructosyl transfer activity of LFTase is proposed to have evolved from levanase. In order to probe the residues that are critical to the intramolecular fructosyl transfer reaction of the Microbacterium sp. AL-210 LFTase, an error-prone PCR mutagenesis process was carried out, and the mutants that led to a shift in activity from transfructosylation towards hydrolysis of levan were screened by the DNS method. After two rounds of mutagenesis, TLC and HPLC analyses of the reaction products by the selected mutants revealed two major products; one is a di-D-fructose- 2,6':6,2'-dianhydride (DFAIV) and the other is a levanbiose. The newly detected levanbiose corresponds to the reaction product from LFTase lacking transferring activity. Two mutants (2-F8 and 2-G9) showed a high yield of levanbiose (38-40%) compared with the wild-type enzyme, and thus behaved as levanases. Sequence analysis of the individual mutants responsible for the enhanced hydrolytic activity indicated that Asn-85 was highly involved in the transfructosylation activity of LFTase.