Yong Il Park

Enzymatic synthesis of novel phloretin glucosides.

ABSTRACT A UDP-glycosyltransferase from Bacillus licheniformis was exploited for the glycosylation of phloretin. The in vitro glycosylation reaction confirmed the production of five phloretin glucosides, including three novel glucosides. Consequently, we demonstrated the application of the same glycosyltransferase for the efficient whole-cell biocatalysis of phloretin in engineered Escherichia coli.

Purification, characterization and immunomodulating activity of a pectic polysaccharide isolated from Korean mulberry fruit Oddi (Morus alba L.).

A water-soluble polysaccharide (JS-MP-1) was isolated and purified from the Korean mulberry fruits Oddi (Morus alba L.) by crushing the fresh fruits then performing ethanol precipitation and DEAE-cellulose ion exchange chromatography. The neutral monosaccharide composition of the purified JS-MP-1 was determined to be composed mainly of galactose (37.6%, in mole percent), arabinose (36.3%), and rhamnose (18.4%), while other major sugars such as glucose, xylose, mannose, and fucose were present as minor components. HPLC analysis revealed that JS-MP-1 contains both galacturonic acid (GalA) and glucuronic acid (GlcA) at approximately 4:1 in mole percent. Monosaccharide composition, Fourier-transform infrared (FTIR) analysis, biochemical analysis, and elemental analysis suggested that JS-MP-1 is an acidic heteropolysaccharide, most likely a rhamnoarabinogalacturonan type plant pectic polysaccharide, with an apparent molecular mass of 1600 kDa containing no, or if any, negligible level of sulfate esters and proteins. Enzyme-Linked Immunosorbent Assay and RT-PCR analysis demonstrated that JS-MP-1 significantly stimulates murine RAW264.7 macrophage cells to release chemokines (RANTES and MIP-1α) and proinflammatory cytokines (TNF-α and IL-6) and to induce the expression of iNOS and COX-2, which are responsible for the production of NO and prostaglandin PGE2, respectively. These results suggest that the mulberry fruit-derived polysaccharide JS-MP-1 can act as a potent immunomodulator, and these observations may support the applicability of this polysaccharide as an immunotherapeutic adjuvant or the water extracts of the mulberry fruit as a beneficial health food.

CHARACTERIZATION OF A RENEWABLE EXTRACELLULAR POLYSACCHARIDE FROM DEFATTED MICROALGAE DUNALIELLA TERTIOLECTA

Extracellular polysaccharide (EPS) was isolated from defatted micro-algae Dunaliela tertiolecta and defined as linear (1→4)-α-D-glucan based on monosaccharide composition, enzymatic and spectroscopic analyses. Optimization and characterization of acidic and enzymatic hydrolyses of EPS have been performed for its potential use as a renewable biorefinery material. The hydrolytic methods were improved to assess the effect of substrate specificity, reaction time, pH, ionic strength and temperature on efficiency of glucose production. EPS was effectively converted into glucose within one-step enzymatic or acidic hydrolysis under optimized conditions. Over 90% recovery of glucose was achieved for both hydrolytic approaches. High potential production of EPS and high yield conversion of this substrate to glucose may allow further exploration of microalga D. tertiolecta as a potential biomass producer for biotechnological and industrial exploitation of bioethanol.

Glucosylation of Isoflavonoids in Engineered Escherichia coli

A glycosyltransferase, YjiC, from Bacillus licheniformis has been used for the modification of the commercially available isoflavonoids genistein, daidzein, biochanin A and formononetin. The in vitro glycosylation reaction, using UDP-α-D-glucose as a donor for the glucose moiety and aforementioned four acceptor molecules, showed the prominent glycosylation at 4′ and 7 hydroxyl groups, but not at the 5th hydroxyl group of the A-ring, resulting in the production of genistein 4′-O-β-D-glucoside, genistein 7-O-β-D-glucoside (genistin), genistein 4′,7-O-β-D-diglucoside, biochanin A-7-O-β-D-glucoside (sissotrin), daidzein 4′-O-β-D-glucoside, daidzein 7-O-β-D-glucoside (daidzin), daidzein 4′, 7-O-β-D-diglucoside, and formononetin 7-O-β-D-glucoside (ononin). The structures of all the products were elucidated using high performance liquid chromatography-photo diode array and high resolution quadrupole time-of-flight electrospray ionization mass spectrometry (HR QTOFESI/MS) analysis, and were compared with commercially available standard compounds. Significantly higher bioconversion rates of all four isoflavonoids was observed in both in vitro as well as in vivo bioconversion reactions. The in vivo fermentation of the isoflavonoids by applying engineered E. coli BL21(DE3)/ΔpgiΔzwfΔushA overexpressing phosphoglucomutase (pgm) and glucose 1-phosphate uridyltransferase (galU), along with YjiC, found more than 60% average conversion of 200 μM of supplemented isoflavonoids, without any additional UDP-α-D-glucose added in fermentation medium, which could be very beneficial to large scale industrial production of isoflavonoid glucosides.

Enzymatic Biosynthesis of Novel Resveratrol Glucoside and Glycoside Derivatives.

ABSTRACT A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d- and l-sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α-d-glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3-O-β-d-glucoside, resveratrol 4′-O-β-d-glucoside, resveratrol 3,5-O-β-d-diglucoside, and resveratrol 3,5,4′-O-β-d-triglucoside. The conversion rates and numbers of products formed were found to vary with the other NDP sugar donors. Resveratrol 3-O-β-d-2-deoxyglucoside and resveratrol 3,5-O-β-d-di-2-deoxyglucoside were found to be produced using TDP-2-deoxyglucose as a donor; however, the monoglycosides resveratrol 4′-O-β-d-galactoside, resveratrol 4′-O-β-d-viosaminoside, resveratrol 3-O-β-l-rhamnoside, and resveratrol 3-O-β-l-fucoside were produced from the respective sugar donors. Altogether, 10 diverse glycoside derivatives of the medically important resveratrol were generated, demonstrating the capacity of YjiC to produce structurally diverse resveratrol glycosides.

Neuroprotective effects of corn silk maysin via inhibition of H2O2-induced apoptotic cell death in SK-N-MC cells

AIMS Neuroprotective effects of maysin, which is a flavone glycoside that was isolated from the corn silk (CS, Zea mays L.) of a Korean hybrid corn Kwangpyeongok, against oxidative stress (H2O2)-induced apoptotic cell death of human neuroblastoma SK-N-MC cells were investigated. MAIN METHODS Maysin cytotoxicity was determined by measuring cell viability using MTT and lactate dehydrogenase (LDH) assays. Intracellular reactive oxygen species (ROS) were measured using a 2,7-dichlorofluorescein diacetate (DCF-DA) assay. Apoptotic cell death was monitored by annexin V-FITC/PI double staining and by a TUNEL assay. Antioxidant enzyme mRNA levels were determined by real-time PCR. The cleavage of poly (ADP-ribose) polymerase (PARP) was measured by western blotting. KEY FINDINGS Maysin pretreatment reduced the cytotoxic effect of H2O2 on SK-N-MC cells, as shown by the increase in cell viability and by reduced LDH release. Maysin pretreatment also dose-dependently reduced the intracellular ROS level and inhibited PARP cleavage. In addition, DNA damage and H2O2-induced apoptotic cell death were significantly attenuated by maysin pretreatment. Moreover, maysin pretreatment (5-50 μg/ml) for 2h significantly and dose-dependently increased the mRNA levels of antioxidant enzymes (CAT, GPx-1, SOD-1, SOD-2 and HO-1) in H2O2 (200 μM)-insulted cells. SIGNIFICANCE These results suggest that CS maysin has neuroprotective effects against oxidative stress (H2O2)-induced apoptotic death of human brain SK-N-MC cells through its antioxidative action. This report is the first regarding neuroprotective health benefits of corn silk maysin by its anti-apoptotic action and by triggering the expression of intracellular antioxidant enzyme systems in SK-N-MC cells.

Cell Wall Polysaccharides of Marine Algae

Marine algae are interesting as a plentiful source of many bioactive compounds, including polysaccharides, which represent various structurally different polymers of high diversity in monosaccharide composition, absolute and anomeric configuration, glycosidic linkages, molecular mass, and the presence and distribution of various functional groups. These polysaccharides are used by algae as cell wall structural components or food reserve. Algal polysaccharides are highly indicative for main algal taxa. Sulfated galactans of periodical unit/linkage sequence (agars, carrageenans) are typical for red algae, alginates, and fucoidans for brown algae; sulfated glucuronoxylorhamnans (ulvans) and other sulphated glycans for green algae. Algal cell wall polysaccharides are extracted from the raw material and further purified by preparative chromatography and/or chemical treatment. Many algal polysaccharides are assigned as phycocolloids due to their good solubility in water and their ability to create colloid systems, including gels and films. They also demonstrate various biological activities (immunomodulation, antitumor, anticoagulant, antiviral, and many other activities), which are prerequisites of pharmaceutical and medicinal applications. Finally, initially inactive or weakly active natural algal polysaccharides can be partially degraded or structurally modified to be fitted to various medicinal applications.

Inhibitory effect of phloretin and biochanin A on IgE-mediated allergic responses in rat basophilic leukemia RBL-2H3 cells.

AIMS Anti-allergic effects and action mechanism of phloretin (Phl) and biochanin A (BioA) on the IgE-antigen complex-mediated allergic responses in rat basophilic leukemia RBL-2H3 cells were investigated. MAIN METHODS Cell viability, formation of reactive oxygen species (ROS), DPPH radical-scavenging activity, β-hexosaminidase release, production of interleukin (IL)-4, IL-13, and tumor necrosis factor-alpha (TNF-α) and phosphorylation of Akt and mitogen-activated protein kinase (MAPK) were determined by MTT assay, 2,7-dichlorofluorescein diacetate (DCF-DA) assay, DPPH radical-scavenging assay, reverse transcriptase polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and western blot analysis, respectively. KEY FINDINGS Ph1 and BioA dose-dependently inhibited the formation of ROS and the release of β-hexosaminidase from the RBL-2H3 cells and also showed DPPH radical-scavenging activity. Ph1 and BioA suppressed the antigen-induced phosphorylation of the downstream signaling intermediates, including MAPK and Akt, which are critical for the production of pro-inflammatory cytokines, and also significantly attenuated the production of IgE-mediated pro-inflammatory cytokines, such as IL-4, IL-13, and TNF-α. SIGNIFICANCE Phloretin and biochanin A attenuate the degranulation and allergic cytokine production through inhibition of intracellular ROS production and the phosphorylation of Akt and the MAPKs, such as ERK1/2, p38, and JNK. The results of this study suggested that these two plant flavonoids may have potent anti-allergic activity in vitro.

Structural analysis and anti-obesity effect of a pectic polysaccharide isolated from Korean mulberry fruit Oddi (Morus alba L.)

A water-soluble polysaccharide JS-MP-1 was isolated from Korean mulberry fruits Oddi (Morus alba L.). Sugar linkage analysis and NMR data confirmed that it is a rhamnogalacturonan type I (RG I) polymer carrying arabinan and arabinogalactan (AG II) side chains. JS-MP-1 reduced dose-dependently the viability of 3T3-L1 pre-adipocyte cells, significantly stimulated the cleavage of caspases 9 and 3 and poly (ADP-ribose) polymerase (PARP) and decreased the ratio of Bcl-2 to Bax expression level that led to mitochondrial dysfunction and apoptosis in pre-adipocyte cells. The apoptotic death was mediated by stimulation of MAPKs (ERK and p38) signalling pathway. These results suggest that JS-MP-1 is able to reduce the number of fat cells and the mass of adipose tissue via inhibition of pre-adipocyte proliferation and thus JS-MP-1 itself or a crude aqueous Oddi extract containing this polysaccharide can be used as functional ingredient of health-beneficial food supplements for the treatment or prevention of obesity disorders.

Immunostimulating activity of maysin isolated from corn silk in murine RAW 264.7 macrophages

Corn silk (CS) has long been consumed as a traditional herb in Korea. Maysin is a major flavonoid of CS. The effects of maysin on macrophage activation were evaluated, using the murine macrophage RAW 264.7 cells. Maysin was isolated from CS by methanol extraction, and preparative C18 reverse phase column chromatography. Maysin was nontoxic up to 100 μg/ml, and dose-dependently increased TNF-α secretion and iNOS production by 11.2- and 4.2-fold, respectively, compared to untreated control. The activation and subsequent nuclear translocation of NF-κB was substantially enhanced upon treatment with maysin (1-100 μg/ml). Maysin also stimulated the phosphorylation of Akt and MAPKs (ERK, JNK). These results indicated that maysin activates macrophages to secrete TNF-α and induce iNOS expression, via the activation of the Akt, NF-κB and MAPKs signaling pathways. These results suggest for the first time that maysin can be a new immunomodulator, enhancing the early innate immunity. [BMB Reports 2014; 47(7): 382-387]