Jisun Lee

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.

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.

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]

The immunostimulating activity of quercetin 3-O-xyloside in murine macrophages via activation of the ASK1/MAPK/NF-κB signaling pathway

Quercetin is a natural plant flavonoid that has been reported to possess a wide range of beneficial health effects, including anti-cancer and anti-inflammatory activities. Glycosylation of natural flavonoids with various sugar moieties can affect their physical, chemical, and biological properties. In this study, quercetin 3-O-xyloside (Quer-xyl) was enzymatically synthesized, and the immunomodulatory activities of quercetin and Quer-xyl were evaluated and compared. The results showed that Quer-xyl more effectively induced the secretion of TNF-α and IL-6 than quercetin by 2.5 and 1.5-fold, respectively. Quer-xyl dose-dependently induced the inducible nitric oxide synthase (iNOS) expression and increased the production of nitric oxide (NO) 1.3-fold more than quercetin. Quer-xyl also increased the phosphorylation of ASK1 and MAPKs (JNK and p38). Treatment with NQDI-1 (an inhibitor of ASK1) significantly attenuated the Quer-xyl-induced up-regulation of TNF-α secretion. The activation and subsequent nuclear translocation of NF-κB were substantially enhanced upon treatment with Quer-xyl (2.5-20 μM), while NQDI-1 treatment blocked the nuclear translocation of NF-κB. These results demonstrated that Quer-xyl can enhance the early innate immunity more effectively than quercetin by activating macrophages to secrete TNF-α and IL-6 through up-regulation of the redox-dependent ASK1/MAPK/NF-κB signaling pathway, suggesting for the first time that Quer-xyl may represent a new immunostimulator.

Neuroprotective effects of phytosterols and flavonoids from Cirsium setidens and Aster scaber in human brain neuroblastoma SK-N-SH cells.

AIMS We investigated the neuroprotective effects and action mechanism of three major compounds [daucosterol (Dau), pectolinarin (Pec), and astragalin (Ast)] isolated from edible plants against H2O2-induced cell death of human brain neuroblastoma SK-N-SH cells. MAIN METHODS Cytotoxicity was determined by MTT and lactate dehydrogenase (LDH) assays. Apoptotic cell death was monitored by annexin V-FITC/PI double staining and by TUNEL assay. The formation of reactive oxygen species (ROS), expression of antioxidant enzymes and phosphorylation of mitogen-activated protein kinase (MAPK) were determined by 2,7-dichlorofluorescein diacetate (DCF-DA) assay, RT-PCR, and western blotting, respectively. KEY FINDINGS The ethyl acetate fractions from Cirsium setidens (CSEA) and Aster scaber (ASEA) showed neuroprotective effects in SK-N-SH cells. The phytochemicals were isolated from CSEA and ASEA and identified by spectral analyses, as β-sitosterol, Dau, Pec, Ast, or isoquercitrin. Pretreatment with Dau, Pec, or Ast showed protective effects against H2O2-induced cell death and inhibited ROS generation by oxidative stress. HO-1 mRNA and protein levels were increased by the presence of H2O2 and were further elevated by pretreatment with Dau and Ast. Dau pretreatment resulted in further increases of H2O2-induced enhancement in levels of CAT and SOD2. Pretreatment with Dau, Pec, and Ast inhibited phosphorylation of MAPK, such as extracellular protein regulated protein kinase, p38, and c-Jun N-terminal kinase by H2O2. SIGNIFICANCE Dau exerts its neuroprotective effects by down regulation of MAPK pathways and upregulation of the HO-1, CAT and SOD2 antioxidant genes and is associated with reduced oxidative stress in SK-N-SH cells.

Corn silk maysin induces apoptotic cell death in PC-3 prostate cancer cells via mitochondria-dependent pathway

AIMS Despite recent advances in prostate cancer diagnostics and therapeutics, the overall survival rate still remains low. This study was aimed to assess potential anti-cancer activity of maysin, a major flavonoid of corn silk (CS, Zea mays L.), in androgen-independent human prostate cancer cells (PC-3). MAIN METHODS Maysin was isolated from CS of Kwangpyeongok, a Korean hybrid corn, via methanol extraction and preparative C18 reverse phase column chromatography. Maysin cytotoxicity was determined by either monitoring cell viability in various cancer cell lines by MTT assay or morphological changes. Apoptotic cell death was assessed by annexin V-FITC/PI double staining, depolarization of mitochondrial membrane potential (MMP), expression levels of Bcl-2 and pro-caspase-3 and by terminal transferase mediated dUTP-fluorescein nick end labeling (TUNEL) staining. Underlying mechanism in maysin-induced apoptosis of PC-3 cells was explored by evaluating its effects on Akt and ERK pathway. KEY FINDINGS Maysin dose-dependently reduced the PC-3 cell viability, with an 87% reduction at 200 μg/ml. Maysin treatment significantly induced apoptotic cell death, DNA fragmentation, depolarization of MMP, and reduction in Bcl-2 and pro-caspase-3 expression levels. Maysin also significantly attenuated phosphorylation of Akt and ERK. A combined treatment with maysin and other known anti-cancer agents, including 5-FU, etoposide, cisplatin, or camptothecin, synergistically enhanced PC-3 cell death. SIGNIFICANCE These results suggested for the first time that maysin inhibits the PC-3 cancer cell growth via stimulation of mitochondria-dependent apoptotic cell death and may have a strong therapeutic potential for the treatment of either chemo-resistant or androgen-independent human prostate cancer.

7,8-Dihydroxyflavone inhibits adipocyte differentiation via antioxidant activity and induces apoptosis in 3T3-L1 preadipocyte cells

AIMS Anti-obesity effects of a natural plant flavonoid 7,8-dihydroxyflavone (7,8-DHF) were evaluated using 3T3-L1 preadipocyte cells. MAIN METHODS The cell viability was determined using MTT assay. Effects of 7,8-DHF on intracellular lipid droplets and intracellular reactive oxygen species (ROS) were measured using a 2,7-dichlorofluorescein diacetate (DCF-DA) assay and Oil Red O staining method, respectively. Apoptotic cell death was monitored by annexin V-FITC/PI double staining and by a TUNEL assay. Antioxidant enzyme mRNA levels and protein expression of adipogenic transcription factors were determined by real-time PCR and Western blotting, respectively. KEY FINDINGS Whereas the cell viability of 3T3-L1 preadipocytes was not affected by lower concentrations of 7,8-DHF (<20 μM), higher concentrations of 7,8-DHF (>20 μM) induced apoptotic cell death. 7,8-DHF (<20 μM) significantly reduced the intracellular lipid droplets and the expression of major adipogenic transcription factors, such as CCAAT/enhancer-binding protein-α (C/EBP-α), C/EBP-β, and peroxisome proliferator activated receptor-γ (PPAR-γ). 7,8-DHF treatment also dose-dependently reduced the intracellular ROS level, attenuated MAPK pathway activation, and increased the expression of antioxidant enzymes, such as Mn-superoxide dismutase (Mn-SOD), catalase (CAT), and heme oxygenase-1 (HO-1). SIGNIFICANCE The results of this study indicated that 7,8-DHF inhibits the adipogenesis of 3T3-L1 preadipocyte cells by down-regulating the expression of adipogenic transcription factors, reduces lipid accumulation, and attenuates ROS accumulation by inducing antioxidant enzymes in differentiated 3T3-L1 cells, suggesting for the first time that 7,8-DHF has an anti-obesity effect in vitro via its anti-oxidant activity.

Ginseng marc-derived low-molecular weight oligosaccharide inhibits the growth of skin melanoma cells via activation of RAW264.7 cells.

Panax ginseng C.A. Meyer has been traditionally consumed to prevent or treat various medical disorders due to its diverse health benefits. Polysaccharides isolated from Panax ginseng have been known to possess various pharmacological activities, including immune modulating, anti-diabetic, and anti-obesity properties. Despite the increasing number of reports on the bioactivities of ginseng polysaccharides, little is known regarding the medicinal potential of ginseng-derived oligosaccharides. In this study, we prepared a lower-molecular weight oligosaccharide (GOS, MW. 2.2kDa) from ginseng polysaccharides (MW. 11-605kDa) by enzymatic degradation and evaluated for its immunostimulating activities in RAW 264.7 murine macrophage cells. GOS was shown to be a glucan type oligosaccharide mainly containing glucose residues (97.48 in molar %). Treatment with GOS (100-500μg/ml) dose-dependently enhanced the production of TNF-α, IL-6, and NO in RAW 264.7 cells. Western blot analysis indicated that GOS dose-dependently induced the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38, and nuclear factor κB (NFκB), which are upstream signalling molecules for cytokine production. While GOS was not cytotoxic to the RAW 264.7 macrophage cells at the concentration tested (up to 1000μg/ml), when B16F10 melanoma cells were co-cultured with the GOS-activated macrophages, the cell viability of melanoma cells was dose-dependently decreased through the induction of apoptotic cell death. Taken together, these results suggested that ginseng marc-derived GOS has anti-cancer activity in vitro against melanoma cells by potentiating macrophage function.

Immunostimulating activity of polyhydric alcohol isolated from Taxus cuspidata

A polyhydric alcohol (PAL) was isolated from Taxus cuspidata and its immunostimulatory activities were assessed. The primary monosaccharide composition of the PAL was determined to be glucose, where HPAEC analysis showed no significant amount of any other sugars. However, glycerol and xylitol were identified as the main sugar alcohols. Fourier-transform infrared (FT-IR) analysis indicated that the purified PAL is a complex glycitol, which structurally contains significant amount of hydroxyl groups. MALDI-TOF mass spectroscopy also demonstrated that PAL is a complex glycitol built in hexose polymerization. Enzyme linked immunosorbent assay showed that the PAL stimulates the release of the proinflammatory cytokines TNF-α and IL-6 in a dose-dependent manner. Furthermore, treatment of RAW 264.7 cells with PAL for 24h remarkably increased the phosphorylation levels of ERK, p38 and JNK in a dose-dependent manner, whereas the total protein levels of ERK (t-ERK), p38 (t-p38) and JNK (t-JNK) remained unchanged. These results clearly demonstrate that PAL stimulates the immune response in RAW 264.7 cells through the activation of MAPKs (ERK, p38 and JNK) signaling pathway. To the best of our knowledge, this is the first study to demonstrate the primary structure and immune-stimulating activities of PAL from the fruit of T. cuspidata.

Corn silk maysin ameliorates obesity in vitro and in vivo via suppression of lipogenesis, differentiation, and function of adipocytes

Present study was aimed to investigate the potential anti-obesity effects of maysin, a major flavonoid of corn silk, in vitro and in vivo using 3T3-L1 preadipocyte cells and C57BL/6 mice. Maysin decreased the levels of intracellular lipid droplets and triglycerides (TG), and down-regulated the protein expression levels of C/EBP-β, C/EBP-α, PPAR-γ, and aP2 in 3T3-L1 preadipocyte cells, suggesting that maysin inhibits lipid accumulation and adipocyte differentiation. In addition, maysin was shown to induce the apoptotic cell death in 3T3-L1 preadipocyte cells via activation of caspase cascades and mitochondrial dysfunction, which may ultimately lead to reduction of adipose tissue mass. Furthermore, oral administration of maysin (25mg/kg body weight) decreased weight gain and epididymal fat weight in high-fat diet (HFD)-fed C57BL/6 mice. Administration of maysin also reduced serum levels of TG, total-cholesterol, LDL-cholesterol, and glucose. Taken collectively, these results suggest for the first time that the purified maysin exerts an anti-obesity effect in vitro and in vivo. These observations may support the applicability of maysin as a potent functional ingredient in health-beneficial foods or as a therapeutic agent to prevent or treat obesity.