Kye-Taek Lim

Identification of Rhus verniciflua Stokes compounds that exhibit free radical scavenging and anti-apoptotic properties.

Rhus verniciflua Stokes (RVS) is a widely used herbal plant with various biological properties. Our previous study using cultured neuronal cells showed that an ethanol extract of RVS had strong antioxidant properties. In this study, we characterized the antioxidant activity of the RVS ethanol extract and identified the active compounds responsible for this activity. From the RVS ethanol extract, we derived three water-eluted fractions and another three fractions eluted by organic solvents, and determined that the water-eluted fractions are what protect against reactive oxygen species (ROS) generated by iron and enzymes. Water-eluted fraction F(2) was the most efficient antioxidant. Moreover, DNA fragmentation and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining experiments revealed that F(2) also protects against thymocyte apoptosis mediated by hydroxyl radicals. Finally, EI-MS, (1)H-NMR, and (13)C-NMR spectra signals confirmed that the fraction contained flavonoid derivatives, including fustin, quercetin, butein, and sulfuretin. These results suggest that the flavonoid derivatives in F(2) are the compounds in the RVS ethanol extract that act as antioxidants.

Antioxidant activity of a Rhus verniciflua Stokes ethanol extract.

A fractionated ethanol extract derived from Rhus Verniciflua Stokes (RVS) was assessed in both organic and aqueous media for the purpose of characterizing the mechanisms of antioxidant activity. RVS, an indigenous plant to Korea, was initially extracted with ethanol and characterized to contain a 90 KDa-ABTS reactive protein possessing 0.662 ng/mg copper. This characterization suggested that a primary component of RVS was Laccase, an oxidase enzyme complex. RVS exhibited a significant (P < 0.01) concentration-dependent inhibition of linoleic acid oxidation in an emulsion system up to 48 hours of incubation. Free radical scavenging activity of both a stable radical (e.g DPPH) and hydroxyl (e.g. *OH) radical followed a concentration-dependent pattern in different model systems. Using a liposome model with peroxyl radicals generated by AAPH, a significant extension of both the lag phase and a reduction of peak propagation of peroxyl radicals by RVS over a concentration range of 1 to 10 microg/ml was observed. RVS ethanol extract was also found to protect human low-density lipoprotein (LDL) from oxidative modification, mediated by cupric ion at 37 degrees C. Finally, RVS was found to be effective at protecting against plasmid DNA strand breakage induced by peroxyl free radicals in an aqueous medium. Our findings show that the ethanol fraction derived from RVS contained significant antioxidant activity in both polar and non-polar mediums.

Hypolipidemic and Antioxidative Effects of the Plant Glycoprotein (36 kDa) from Rhus verniciflua Stokes Fruit in Triton WR-1339-Induced Hyperlipidemic Mice

We investigated the hypolipidemic and antioxidative effects on male ICR mice of a glycoprotein isolated from Rhus verniciflua Stokes (RVS) fruit. The administration of the RVS glycoprotein (100 mg/kg) for two weeks resulted in a significant decrease in such plasma lipid levels as total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL). The levels of TC, TG and LDL in the hyperlipidemic model were significantly increased, whereas the high-density lipoprotein (HDL) level was considerably decreased. The 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity and the level of thiobarbituric acid-reactive substances (TBARS) were significantly elevated, whereas the production of nitric oxide (NO) was diminished. Moreover, the administration of the RVS glycoprotein prior to inducing hyperlipidemic mice suppressed the increase in the plasma lipid levels (TC, TG and LDL), and decrease in the HDL level in Triton WR-1339-induced hyperlipidemic mice. Furthermore, the RVS glycoprotein significantly inhibited the activity of HMG-CoA reductase and the levels of TBARS in the hyperlipidemic mice. In addition, the activities of detoxicant enzymes [catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)] were gradually augmented after a supplement with the RVS glycoprotein. The results suggest that the RVS glycoprotein would be effective in preventing an increase in the plasma lipid levels and in improving the antioxidant levels. This protein might be useful as a therapeutic agent.

Hepatoprotective and hypolipidaemic effects of glycoprotein isolated from Gardenia jasminoides ellis in mice.

1 The present study was performed to investigate the hepatoprotective and hypolipidaemic effects of a 27 kDa glycoprotein isolated from Gardenia jasminoides Ellis (GJE glycoprotein) in glucose/glucose oxidase (G/GO)‐treated BNL CL.2 cells, as well as in CCl4, Triton WR‐1339 and corn oil‐treated mice. 2 In G/GO‐treated BNL CL.2 cells, the results showed that GJE glycoprotein has an inhibitory effect on G/GO‐induced cytotoxicity and intracellular reactive oxygen species production. In addition, GJE glycoprotein has an anti‐oxidant effect against the lipid peroxidation process in the Fe2+/ascorbic acid system. 3 In CCl4 (1.0 mL/kg)‐treated mice, pretreatment with GJE glycoprotein (80 mg/kg) blocked lactate dehydrogenase release and the formation of thiobarbituric acid‐reactive substances. In addition, in these mice GJE resulted in increased nitric oxide production and the activation of anti‐oxidant enzymes, accompanied by the inhibition of the cytotoxic‐related signals hepatic cytochrome c, nuclear factor‐κB and activator protein‐1. 4 In both Triton WR‐1339 (400 mg/kg) and corn oil (1.0 g/kg)‐treated mice, pretreatment with GJE glycoprotein (80 mg/kg) lowered the levels of plasma lipoproteins (triglyceride, total cholesterol and low‐density lipoprotein). 5 On the basis of these results, we assume that GJE glycoprotein can ameliorate liver function, because it has hepatoprotective and hypolipidaemic activities.

Glycoprotein isolated from Cudrania tricuspidata Bureau inhibits iNO and COX-2 expression through modulation of NF-κB in LPS-stimulated RAW 264.7 cells.

Glycoprotein of Cudrania tricuspidata Bureau (CTB glycoprotein) was isolated from CTB fruits which have been used to heal various disorders of the injury and lung as an herbal agent in Korea since long time ago. The CTB glycoprotein was identified to have a molecular weight of 75kDa and consists of carbohydrate (72.5%) and protein moiety (27.5%). To know inhibitory ability of CTB glycoprotein for inflammation mediated by reactive oxygen radicals, firstly we tested about anti-oxidative activity (DPPH, superoxide anion, and hydroxyl radicals) in cell-free system, and then evaluated changes of inflammation-related signals [intracellular reactive oxygen species (iROS), nitric oxide (NO), nuclear factor-kappa B (NF-κB), COX-2, and iNOS] in the LPS (1μg/ml)-treated RAW 264.7cells. The results in this study showed that CTB glycoprotein (100μg/ml) has a strong scavenging activity against DPPH, superoxide anion, and hydroxyl radicals without any pro-oxidant activity in vitro. In the inflammation-related signals, expression of iROS, NO, NF-κB, COX-2, and iNOS were inhibited by treatment with CTB glycoprotein (50μg/ml) in the presence of LPS (1μg/ml). Taken together, our data obtained from these experiments indicated that CTB glycoprotein suppresses expression of the inflammatory-related proteins (iNOS and COX-2) through regulation of NF-κB. Thus, we speculate that CTB glycoprotein may have therapeutic potential for inflammation-associated disorders.

Protective effect of glycoprotein isolated from Ulmus davidiana Nakai on carbon tetrachloride‐induced mouse liver injury

This study was carried out to evaluate the hepatoprotective activity of glycoprotein isolated from the stems of Ulmus davidiana Nakai (UDN), which has been used as an anti‐inflammatory agent in folk medicine. We evaluated lipid peroxidation in glucose/glucose oxidase (G/GO)‐induced BNL CL.2 cells and measured thiobarbituric acid reactive substances (TBARS), lactate dehydrogenase (LDH), nitric oxide (NO), antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)), activity of cytotoxic‐related signals (hepatic cytochrome c, nuclear factor‐kappa B (NF‐κB) and activator protein‐1 (AP‐1)) and levels of plasma lipids (triglyceride (TG) and total cholesterol (TC)) in carbon tetrachloride (CCl4, 1.0 mL kg−1)‐induced A/J mouse. The results in G/GO‐induced BNL CL.2 cells showed that UDN glycoprotein had a dose‐dependent inhibitory effect on lipid peroxidation. The results in carbon tetrachloride (CCl4, 1.0 mL kg−1)‐induced A/J mouse indicated that treatment with UDN glycoprotein (40 mg kg−1) lowered LDH activity and TBARS formation, and increased NO production and antioxidant enzymes activity, compared with control. Also, our finding from CCl4‐treated mice after pretreatment with UDN glycoprotein demonstrated that the activity of cytotoxic‐related signals decreased but the levels of plasma lipids increased, compared with CCl4 treatment alone. Here, we speculate that UDN glycoprotein has a protective character to CCl4‐induced mouse liver injury.

Plant-originated glycoprotein (36 kDa) suppresses interleukin-4 and -10 in bisphenol A-stimulated primary cultured mouse lymphocytes.

Bisphenol A (BPA) is one of the estrogen mimic environmental hormones and a chemical used for the wrapping foods, toy products for children, biomedical equipment, and machines. It can exert toxic effects, such as occurring allergy-related diseases. This study demonstrates that glycoprotein isolated from Rhus verniciflua Stokes (RVS glycoprotein) has an inhibitory activity of T-helper type 2 (Th2) cytokines [Interleukin (IL)-4 and -10]. First, it was shown that RVS glycoprotein inhibits the proliferation of lymphocytes and scavenges intracellular reactive oxygen species (ROS). Then, the activities of mitogen-activated protein kinase (MAPK), GATA-binding protein-3 (GATA-3), t-box expressed in T-cells (T-bet), and Th2 cell-related cytokine (IL-4 and -10) were evaluated in BPA (50 μM)-stimulated primary cultured mouse lymphocytes, using immunoblot analysis and reverse-transcription polymerase chain reaction (RT-PCR). The results showed that the RVS glycoprotein (50 μg/mL) inhibited the proliferation of lymphocytes, intracellular ROS, and activity of p38 MAPK dose dependently. In the transcriptional factors for the oriented differentiation of T-helper cells, the RVS glycoprotein (50 μg/mL) significantly suppressed the GATA-3, whereas it enhanced T-bet. Also, the RVS glycoprotein (100 μg/mL) significantly attenuated Th2-related cytokines (IL-4 and -10). Taken together, the results obtained from this study suggest that the RVS glycoprotein may help in preventing allergy-related immune dysfunction, such as that produced by BPA.

Allergy-related cytokines (IL-4 and TNF-α) are induced by Di(2-ethylhexyl) phthalate and attenuated by plant-originated glycoprotein (75 kDa) in HMC-1 cells

Phthalate esters as plasticizers have been widespread in the environment and may be associated with development of allergic diseases such as asthma and atopic dermatitis. In this study, we demonstrated that the CTB glycoprotein attenuates allergic reactions caused by di(2‐ethylhexyl) phthalate (DEHP) in human mast cells (HMC‐1). This experiment evaluated degranulation of histamine and β‐hexosaminidase as well as activities of protein kinase C (PKC), stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK), activator protein (AP)‐1 and interleukin (IL)‐4 and tumor necrosis factor (TNF)‐α using immunoblotting and reverse transcription‐polymerase chain reaction (RT‐PCR). Our results revealed that the CTB glycoprotein in the presence of DEHP inhibits degranulation of mast cell, translocation of PKC from cytosol to membrane, and phosphorylation of SAPK/JNK in HMC ‐1 cells. We also found that the CTB glycoprotein (100 μg mL−1) has suppressive effects on transcriptional activation of AP‐1, and on the expression of IL‐4 and TNF‐α in DEHP‐treated HMC‐1 cells. We suggest that the CTB glycoprotein inhibits degranulation of mast cells and expressions of cytokines in HMC‐1 cells.

Plant Glycoprotein Modulates the Expression of Interleukin-1β via Inhibition of MAP Kinase in HMC-1 Cells

Dioscorea batatas Decne (DBD) is used to heal various disorders of the kidney and lungs as an herbal agent in Korea. The purpose of the present study was to determine whether the DBD glycoprotein regulates the inflammatory reaction stimulated by phorbol-12-myristate 13-acetate plus calcium ionophore A23187 (PMACI) in human mast cells (HMC-1). The results indicate that DBD glycoprotein decreased gene expression of interleukin (IL)-1β and cyclooxygenase (COX)-2 in PMACI-stimulated HMC-1 cells through blocking of phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and p38 MAPK and DNA binding activities of nuclear factor (NF)-κB and activator protein (AP)-1. The production of intracellular reactive oxygen species (ROS) and nitric oxide (NO) is gradually reduced by concentration-dependent DBD glycoprotein treatment in PMACI-stimulated HMC-1 cells. Hence, we propose the hypothesis that DBD glycoprotein can serve as a potent anti-inflammatory agent in the treatment of inflammatory allergic diseases through inhibition of inflammation-related signal transduction in mast cell activation.

IgE, COX-2, and IL-4 are Expressed by DEHP through p38 MAPK and Suppressed by Plant Glycoprotein (75 kDa) in ICR Mice

The aim of the present study was to evaluate the inhibitory effect of glycoprotein isolated from Cudrania tricuspidata Bureau (CTB glycoprotein) on di(2-ethylhexyl) phthalate (DEHP)-induced allergic inflammatory response in mice. We evaluated the activity of β-hexosaminidase, expression of cyclooxygenase (COX)-2, p38 mitogen-activated protein kinase (MAPK), and activator protein (AP)-1, and production of immunoglobulin (Ig)E and interleukin (IL)-4 in DEHP-treated RBL-2H3 cells and ICR mice. Our results revealed that the CTB glycoprotein inhibited the activity of β-hexosaminidase and production of IgE and IL-4 in serum from DEHP-treated mice. We also found that the CTB glycoprotein reduced arachidonic acid release, COX-2 expression, and AP-1 transcriptional activation through p38 MAPK phosphorylation in DEHP-treated RBL-2H3 cells. The activation of AP-1 was completely blocked by treatment with p38 MAPK inhibitor (SKF86002). The results from these experiments indicate that CTB glycoprotein effectively protects against the allergic inflammation response, mainly through downregulation of MAPK/AP-1 in the mast cell degranulation stage. In conclusion, we suggest that the CTB glycoprotein may be one component of health supplements for the prevention of allergic inflammation.