Woo-Sik Jeong

Pharmacodynamics of dietary phytochemical indoles I3C and DIM: Induction of Nrf2-mediated phase II drug metabolizing and antioxidant genes and synergism with isothiocyanates

The antioxidant response element (ARE) is a critical regulatory element for the expression of many phase II drug metabolizing enzymes (DME), phase III transporters and antioxidant enzymes, mediated by the transcription factor Nrf2. The aim of this study was to examine the potential activation and synergism of Nrf2‐ARE‐mediated transcriptional activity between four common phytochemicals present in cruciferous vegetables; the indoles: indole‐3‐carbinol (I3C), 3,3′‐diindolylmethane (DIM); and the isothiocyanates (ITCs): phenethyl isothiocyanate (PEITC) and sulforaphane (SFN). The cytotoxicity of the compounds was determined in a human liver hepatoma cell line (HepG2‐C8). The combination index was calculated to assess the synergistic effects on the induction of ARE‐mediated gene expressions. Quantitative real‐time polymerase chain reaction (qPCR) was employed to measure the mRNA expressions of Nrf2 and Nrf2‐mediated genes. I3C and DIM showed less cytotoxicity than SFN and PEITC. Compared with I3C, DIM was found to be a stronger inducer of ARE. Synergism was observed after combined treatments of 6.25 µm I3C + 1 µm SFN, 6.25 µm I3C + 1 µm PEITC and 6.25 µm DIM + 1 µm PEITC, while an additive effect was observed for 6.25 µm DIM + 1 µm SFN. Induction of endogenous Nrf2, phase II genes (GSTm2, UGT1A1 and NQO1) and antioxidant genes (HO‐1 and SOD1) was also observed. In summary, the indole I3C or DIM alone could induce or syngergistically induce in combination with the ITCs SFN or PEITC, Nrf2‐ARE‐mediated gene expression, which could potentially enhance cancer chemopreventive activity. Copyright

6-shogaol-rich extract from ginger up-regulates the antioxidant defense systems in cells and mice.

The rhizome of ginger (Zingiber officinale Roscoe) is known to have several bioactive compounds including gingerols and shogaols which possess beneficial health properties such as anti-inflammatory and chemopreventive effects. Based on recent observations that 6-shogaol may have more potent bioactivity than 6-gingerol, we obtained a 6-shogaol-rich extract from ginger and examined its effects on the nuclear factor E2-related factor2 (Nrf2)/antioxidant response element (ARE) pathway in vitro and in vivo. 6-Shogaol-rich extract was produced by extracting ginger powder with 95% ethanol at 80 °C after drying at 80 °C (GEE8080). GEE8080 contained over 6-fold more 6-shogaol compared to the room temperature extract (GEE80RT). In HepG2 cells, GEE8080 displayed much stronger inductions of ARE-reporter gene activity and Nrf2 expression than GEE80RT. GEE8080 stimulated phosphorylations of mitogen-activated protein kinases (MAPKs) such as ERK, JNK, and p38. Moreover, the GEE8080-induced expressions of Nrf2 and HO-1 were attenuated by treatments of SB202190 (a p38 specific inhibitor) and LY294002 (an Akt specific inhibitor). In a mouse model, the GEE8080 decreased the diethylnitrosamine (DEN)-mediated elevations of serum aspartate transaminase and alanine transaminase as well as the DEN-induced hepatic lipid peroxidation. Inductions of Nrf2 and HO-1 by GEE8080 were also confirmed in the mice. In addition, the administration of GEE8080 to the mice also restored the DEN-reduced activity and protein expression of hepatic antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase. In conclusion, GEE8080, a 6-shogaol-rich ginger extract, may enhance antioxidant defense mechanism through the induction of Nrf2 and HO-1 regulated by p38 MAPK and PI3k/Akt pathway in vitro and in vivo.

Antioxidant and Hepatoprotective Effects of the Red Ginseng Essential Oil in H2O2-Treated HepG2 Cells and CCl4-Treated Mice

The aim of this study was to evaluate the antioxidant mechanisms of red ginseng essential oil (REO) in cells as well as in an animal model. REO was prepared by a supercritical CO2 extraction of waste-products generated after hot water extraction of red ginseng. In HepG2 cells, REO diminished the H2O2-mediated oxidative stress and also restored both the activity and expression of antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase. Administration of REO inhibited the phosphorylation of upstream mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38. In mice, the CCl4-mediated elevation of serum aspartate transaminase and alanine transaminase as well as the induction of hepatic lipid peroxidation were decreased by REO administration. REO treatments also resulted in up-regulation of the antioxidant enzyme expression in the liver. Moreover, increased phosphorylations of MAPKs were inhibited after REO administration. Overall, REO seems to protect the liver from oxidative stress through the activation and induction of antioxidant enzymes via inhibition of MAPKs pathways.

Anti-Inflammatory Effect of Procyanidins from Wild Grape (Vitis amurensis) Seeds in LPS-Induced RAW 264.7 Cells

In the present study, the anti-inflammatory effect and underlying mechanisms of wild grape seeds procyanidins (WGP) were examined using lipopolysaccharide- (LPS-) stimulated RAW 264.7 cells. We used nitric oxide (NO) and prostaglandin E2 (PGE2) and reactive oxygen species (ROS) assays to examine inhibitory effect of WGP and further investigated the mechanisms of WGP suppressed LPS-mediated genes and upstream expression by Western blot and confocal microscopy analysis. Our data indicate that WGP significantly reduced NO, PGE2, and ROS production and also inhibited the expression of proinflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expressions. Consistently, WGP significantly reduced LPS-stimulated expression of proinflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin- (IL-) 1β. Moreover, WGP prevented nuclear translocation of nuclear factor-κB (NFκB) p65 subunit by reducing inhibitory κB-α (IκBα) and NFκB phosphorylation. Furthermore, we found that WGP inhibited LPS-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK). Taken together, our results demonstrated that WGP exerts potent anti-inflammatory activity through the inhibition of iNOS and COX-2 by regulating NFκB and p38 MAPK pathway.

Procyanidins from Wild Grape (Vitis amurensis) Seeds Regulate ARE-Mediated Enzyme Expression via Nrf2 Coupled with p38 and PI3K/Akt Pathway in HepG2 Cells

Procyanidins, polymers of flavan-3-ol units, have been reported to exhibit many beneficial health effects such as antioxidant and anti-carcinogenic effects. In this study, we investigated the cancer chemopreventive properties of procyanidins from wild grape (Vitis amurensis) seeds in particular their roles in inducing phase II detoxifying/antioxidant enzymes as well as in modulating the upstream kinases. Ethanolic extract of V. amurensis seeds was fractionated with a series of organic solvents and finally separated into six fractions, F1–F6. Chemical properties of the procyanidins were analyzed by vanillin assay, BuOH-HCl test, and depolymerization with phloroglucinol followed by LC/MS analysis. The F5 had the highest procyanidin content among all the fractions and strongly induced the reporter activity of antioxidant response element as well as the protein expression of nuclear factor E2-related factor (Nrf2) in HepG2 human hepatocarcinoma cells. The procyanidin-rich F5 also strongly induced the expression of the phase II detoxifying and antioxidant enzymes such as NAD(P)H:quinone oxidoreductase1 and hemeoxygenase1. Phosphorylations of the upstream kinases such as MAPKs and PI3K/Akt were significantly increased by treatment with procyanidin fraction. In addition, the procyanidin-mediated Nrf2 expression was partly attenuated by PI3K inhibitor LY294002, and almost completely by p38 inhibitor SB202190, but neither by JNK inhibitor SP600125 nor by MEK1/2 inhibitor U0126. Taken together, the procyanidins from wild grape seeds could be used as a potential natural chemopreventive agent through Nrf2/ARE-mediated phase II detoxifying/antioxidant enzymes induction via p38 and PI3K/Akt pathway.

Red ginseng marc oil inhibits iNOS and COX-2 via NFκB and p38 pathways in LPS-stimulated RAW 264.7 macrophages.

In this study, we investigated the anti-inflammatory effects of red ginseng marc oil (RMO) in the RAW 264.7 macrophage cell line. RMO was prepared by a supercritical CO2 extraction of waste product generated after hot water extraction of red ginseng. RMO significantly inhibited the production of oxidative stress molecules such as nitric oxide and reactive oxygen species in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Levels of inflammatory targets including prostaglandin E2, tumor necrosis factor-α, interleukin (IL)-1β and IL-6 were also reduced after the treatment with RMO. In addition, RMO diminished the expressions of inducible nitric oxide synthase and cyclooxygenase 2 at both mRNA and protein levels. Blockade of nuclear translocation of the p65 subunit of nuclear factor κB (NFκB) was also observed after the treatment of RMO. Furthermore, RMO decreased the phosphorylations of p38 mitogen-activated protein kinase (MAPK) and its upstream kinases including MAPK kinases 3/6 (MKK3/6) and TAK 1 (TGF-β activated kinase 1). Gas chromatographic analysis on RMO revealed that RMO contained about 10% phytosterols including sitosterol, stigmasterol and campesterol which may contribute to the anti-inflammatory properties of RMO. Taken together, these results suggest that the anti-inflammatory effect of RMO in LPS-induced RAW 264.7 macrophages could be associated with the inhibition of NFκB transcriptional activity, possibly via blocking the p38 MAPK pathway.

Protective effects of the key compounds isolated from Corni fructus against β-amyloid-induced neurotoxicity in PC12 cells.

β-Amyloid (Aβ) peptide is the major component of senile plaques and is considered to have a causal role in the development and progression of Alzheimer’s disease (AD). There is compelling evidence supporting the notion that Aβ-induced cytotoxicity is mediated though the generation of ROS. In the present study, we investigated the neuroprotective effects of ursolic acid (UA), p-coumaric acid (p-CA), and gallic acid (GA) isolated from Corni fructus (CF) against Aβ(25–35)-induced toxicity in PC12 cell. Exposure of PC12 cells to 50 μM Aβ(25–35) increased cellular oxidative stress, the number of apoptotic cells and caspase-3 activity and finally caused significant cell death. However, UA, p-CA, and GA not only suppressed the generation of ROS but also attenuated DNA fragmentation and eventually attenuated Aβ-induced apoptosis in a dose-dependent manner. In protecting cells against Aβ neurotoxicity, UA and GA possessed stronger ability against ROS generation than p-CA, while p-CA showed the strongest anti-apoptotic activity. Particularly, p-CA protected cells at the concentration range from 0.5 up to 125 μM without any adverse effect. Taken together, these effects of UA, p-CA, and GA may be partly associated with the neuroprotective effect of CF. Furthermore, our findings might raise a possibility of therapeutic applications of CF for preventing and/or treating neurodegenerative diseases.

p-Coumaric Acid and Ursolic Acid from Corni fructus Attenuated β-Amyloid25–35-Induced Toxicity through Regulation of the NF-κB Signaling Pathway in PC12 Cells

Neuroinflammatory responses induced by amyloid-beta peptide (Aβ) are important causes in the pathogenesis of Alzheimers disease (AD). Blockade of Aβ has emerged as a possible therapeutic approach to control the onset of AD. This study investigated the neuroprotective effects and molecular mechanisms of p-coumaric acid (p-CA) and ursolic acid (UA) from Corni fructus against Aβ(25-35)-induced toxicity in PC12 cells. p-CA and UA significantly inhibited the expression of iNOS and COX-2 in Aβ(25-35)-injured PC12 cells. Blockade of nuclear translocation of the p65 subunit of nuclear factor κB (NF-κB) and phosphorylation of IκB-α was also observed after p-CA and UA treatment. For the upstream kinases, UA exclusively reduced ERK1/2, p-38, and JNK phosphorylation, but p-CA suppressed ERK1/2 and JNK phosphorylation. Both compounds comprehensively inhibited NF-κB activity, but possibly with different upstream pathways. The results provide new insight into the pharmacological modes of p-CA and UA and their potential therapeutic application to AD.

Volatile pollutants emitted from selected liquid household products.

Background, aim, and scopeTo identify household products that may be potential sources of indoor air pollution, the chemical composition emitted from the products should be surveyed. Although this kind of survey has been conducted by certain research groups in Western Europe and the USA, there is still limited information in scientific literature. Moreover, chemical components and their proportions of household products are suspected to be different with different manufacturers. Consequently, the current study evaluated the emission composition for 42 liquid household products sold in Korea, focusing on five product classes (deodorizers, household cleaners, color removers, pesticides, and polishes).Materials and methodsThe present study included two phase experiments. First, the chemical components and their proportions in household products were determined using a gas chromatograph and mass spectrometer system. For the 19 target compounds screened by the first phase of the experiment and other selection criteria, the second phase was done to identify their proportions in the purged-gas phase.ResultsThe number of chemicals in the household products surveyed ranged from 9 to 113. Eight (product class of pesticides) to 17 (product class of cleaning products) compounds were detected in the purged-gas phase of each product class. Several compounds were identified in more than one product class. Six chemicals (acetone, ethanol, limonene, perchloroethylene (PCE), phenol, and 1-propanol) were identified in all five product classes. There were 13 analytes occurring with a frequency of more than 10% in the household products: limonene (76.2%), ethanol (71.4%), PCE (66.7%), phenol (40.5%), 1-propanol (35.7%), decane (33%), acetone (28.6%), toluene (19.0%), 2-butoxy ethanol (16.7%), o-xylene (16.7%), chlorobenzene (14.3%), ethylbenzene (11.9%), and hexane (11.9%). All of the 42 household products analyzed were found to contain one or more of the 19 compounds.DiscussionThe chemical composition varied broadly along with the product classes or product categories, and it was different from that reported in other studies abroad, although certain target chemicals were identified in both studies. This finding supports an assertion that chemical components emitted from household products may be different in different products and with different manufacturers. The chlorinated pollutants identified in the present study have not been reported to be components of cleaning products in papers published since the early 1990s. Limonene was identified as having the highest occurrence in the household products in the present study, although it was not detected in any of 67 household products sold in the U.S.ConclusionsThe emission composition of selected household products was successfully examined by purge-and-trap analysis. Along with other exposure information such as use pattern of household products and the indoor climate, this composition data can be used to estimate personal exposure levels of building occupants. This exposure data can be employed to link environmental exposure to health risk. It is noteworthy that many liquid household products sold in Korea emitted several toxic aromatic and chlorinated organic compounds. Moreover, the current finding suggests that product types and manufacturers should be considered, when evaluating building occupants’ exposure to chemical components emitted from household products.Recommendations and perspectivesThe current findings can provide valuable information for the semiquantitative estimation of the population inhalation exposure to these compounds in indoor environments and for the selection of safer household products. However, although the chemical composition is known, the emissions of household products might include compounds formed during the use of the product or compounds not identified as ingredients by this study. Accordingly, further studies are required, and testing must be done to determine the actual composition being emitted. Similar to eco-labeling of shampoos, shower gels, and foam baths proposed by a previous study, eco-labeling of other household products is suggested.

Optimization of Extraction Conditions for the 6-Shogaol-rich Extract from Ginger (Zingiber officinale Roscoe)

6-Shogaol, a dehydrated form of 6-gingerol, is a minor component in ginger (Zingiber officinale Roscoe) and has recently been reported to have more potent bioactivity than 6-gingerol. Based on the thermal instability of gingerols (their dehydration to corresponding shogaols at high temperature), we aimed to develop an optimal process to maximize the 6-shogaol content during ginger extraction by modulating temperature and pH. Fresh gingers were dried under various conditions: freeze-, room temperature (RT)- or convection oven-drying at 60 or 80°C, and extracted by 95% ethanol at RT, 60 or 80°C. The content of 6-shogaol was augmented by increasing both drying and extraction temperatures. The highest production of 6-shogaol was achieved at 80°C extraction after drying at the same temperature and the content of 6-shogaol was about 7-fold compared to the lowest producing process by freezing and extraction at RT. Adjustment of pH (pH 1, 4, 7 and 10) for the 6-shogaol-richest extract (dried and extracted both at 80°C) also affected the chemical composition of ginger and the yield of 6-shogaol was maximized at the most acidic condition of pH 1. Taken together, the current study shows for the first time that a maximized production of 6-shogaol can be achieved during practical drying and extraction process of ginger by increasing both drying and extracting temperatures. Adjustment of pH to extraction solvent with strong acid also helps increase the production of 6-shogaol. Our data could be usefully employed in the fields of food processing as well as nutraceutical industry.