Optimization of the extraction process of high levels of chlorogenic acid and ginsenosides from short-term hydroponic-cultured ginseng and evaluation of the extract for the prevention of atopic dermatitis.

Background: Short-term hydroponic-cultured ginseng (sHCG), which is 1-year-old ginseng seedlings cultivated for 4 weeks in a hydroponic system, is a functional food item with several biological effects. However, the optimal extraction conditions for sHCG, and the bioactivity of its extracts, have not been evaluated. Methods: Chlorogenic acid (CGA) and ginsenoside contents were evaluated in sHCG, white ginseng (WG), and red ginseng (RG) using high-performance liquid chromatography. Response surface methodology (RSM) was used to optimize the extraction conditions (temperature and ethanol concentration) to maximize the yield of dry matter, CGA, and four ginsenosides (Re, Rg1, Rb1, and Rd) from sHCG. The optimal extraction conditions were applied to pilot-scale production of sHCG extracts. The expression levels of tumor necrosis factor (TNF)-α/interferon (IFN)-γ-induced thymic and activation-regulated chemokines (TARC/CCL17) were measured after treatment with sHCG, WG, and RG extracts, and the effects of their bioactive compounds (CGA and four ginsenosides) on human skin keratinocytes (HaCaTs) were evaluated. Results: CGA and four ginsenosides, which are bioactive compounds of sHCG, significantly inhibited TNF-α/IFN-γ-induced TARC/CCL17 expression. The optimal sHCG extraction conditions predicted by the RSM models were 80 °C and 60% ethanol (v/v). The sHCG extracts produced at the pilot scale under optimal conditions greatly alleviated TNF-α/IFN-γ-induced TARC/CCL17 production compared with WG and RG extracts. Conclusions: Pesticide-free sHCG extracts, which contain high levels of CGA and the ginsenosides Re, Rg1, Rb1, and Rd as bioactive compounds, may have therapeutic potential for atopic diseases.

Heat-Killed Lactobacillus plantarum KCTC 13314BP Enhances Phagocytic Activity and Immunomodulatory Effects Via Activation of MAPK and STAT3 Pathways.

Identification of novel probiotic strains is of great interest in the field of functional foods. Specific strains of heat-killed bacteria have been reported to exert immunomodulatory effects. Herein, we investigated the immune-stimulatory function of heat-killed Lactobacillus plantarum KCTC 13314BP (LBP). Treatment with LBP significantly increased the production of TNF-α and IL-6 by macrophages. More importantly, LBP was able to enhance the phagocytic activity of macrophages against bacterial particles. Activation of p38, JNK, ERK, NF-κB, and STAT3 was involved in the immunomodulatory function of LBP. LBP treatment significantly increased production of TNF-α by bone marrow-derived macrophages and splenocytes, further confirming the immunostimulatory effect of LBP in primary immune cells. Interestingly, the immunomodulatory effects of LBP were much stronger than those of Lactobacillus rhamnosus GG, a well-known probiotic strain. These results indicate that LBP can be a promising immune-enhancing functional food agent.

5-(3',4'-Dihydroxyphenyl)-γ-valerolactone, a metabolite of procyanidins in cacao, suppresses MDI-induced adipogenesis by regulating cell cycle progression through direct inhibition of CDK2/cyclin O.

Cacao (Theobroma cacao) has a significant polyphenol content and has been reported to elicit anti-obesity effects. Previous studies have focused on the properties of cacao extract and procyanidins, while the potential mechanisms have not been fully elucidated. Here, we investigated the inhibitory effects of procyanidin metabolites on adipogenic cocktail-induced adipogenesis and lipogenesis in 3T3-L1 preadipocytes. It was observed that 5-(3',4'-dihydroxyphenyl)-γ-valerolactone (DHPV), a major procyanidin metabolite, exhibited the greatest inhibitory effects on adipogenesis and lipogenesis. DHPV dose-dependently reduced the expression levels of proteins involved in adipogenesis including peroxisome proliferator-activated receptor γ (PPAR γ) and CCAT/enhancer-binding protein α (C/EBP α), as well as lipogenesis-related factors such as fatty acid synthase and acetyl-CoA carboxylase. These inhibitory effects were primarily due to G1 phase arrest and the suppression of cell proliferation during mitotic clonal expansion, the early stage of adipogenesis. In an extensive kinase array, DHPV directly suppressed activation of the CDK2/cyclin O complex, and inhibited the phosphorylation of C/EBP ß, which is responsible for the induction of PPAR γ and C/EBP α. Taken together, these findings suggest that DHPV is a highly biologically active compound with potential anti-obesity effects and works by inhibiting the intracellular lipid content and cell differentiation.

Gingerenone A Attenuates Monocyte-Endothelial Adhesion via Suppression of I Kappa B Kinase Phosphorylation.

During the early stages of atherosclerosis, monocytes bind and migrate into the endothelial layer, promoting inflammation within the aorta. In order to prevent the development of atherosclerosis, it is critical to inhibit such inflammation. The therapeutic effects of ginger have been investigated in several models of cardiovascular disease. However, although a number of previous studies have focused on specific compounds, the mechanisms of action responsible remain unclear. Here, we investigated five major compounds present in ginger, and observed that gingerenone A exhibited the strongest inhibitory effects against tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS) induced monocyte-endothelial adhesion. Furthermore, gingerenone A significantly suppressed the expression of TNF-α and LPS-induced vascular cell adhesion molecule-1 (VCAM-1) and chemokine (C-C motif) ligand 2 (CCL2), key mediators of the interaction between monocytes, and endothelial cells. Transactivation of nuclear factor-κB (NF-κB), which is a key transcription factor of VCAM-1 and CCL2, was induced by TNF-α and LPS, and inhibited by treatment of gingerenone A. Gingerenone A also inhibited the phosphorylation of NF-κB inhibitor (IκB) α and IκB Kinase. Taken together, these results demonstrate that gingerenone A attenuates TNF-α and LPS-induced monocyte adhesion and the expression of adhesion factors in endothelial cells via the suppression of NF-κB signaling. J. Cell. Biochem. 119: 260-268, 2018. © 2017 Wiley Periodicals, Inc.

Anti-carcinogenic effects of non-polar components containing licochalcone A in roasted licorice root.

BACKGROUND/OBJECTIVE: Licorice has been shown to possess cancer chemopreventive effects. However, glycyrrhizin, a major component in licorice, was found to interfere with steroid metabolism and cause edema and hypertension. The roasting process of licorice modifies the chemical composition and converts glycyrrhizin to glycyrrhetinic acid. The purpose of this study was to examine the anti-carcinogenic effects of the ethanol extract of roasted licorice (EERL) and to identify the active compound in EERL. MATERIALS/METHODS: Ethanol and aqueous extracts of roasted and un-roasted licorice were prepared. The active fraction was separated from the methylene chloride (MC)-soluble fraction of EERL and the structure of the purified compound was determined by nuclear magnetic resonance spectroscopy. The anti-carcinogenic effects of licorice extracts and licochalcone A was evaluated using a MTT assay, Western blot, flow cytometry, and two-stage skin carcinogenesis model. RESULTS: EERL was determined to be more potent and efficacious than the ethanol extract of un-roasted licorice in inhibiting the growth of DU145 and MLL prostate cancer cells, as well as HT-29 colon cancer cells. The aqueous extracts of un-roasted and roasted licorice showed minimal effects on cell growth. EERL potently inhibited growth of MCF-7 and MDA-MB-231 breast, B16-F10 melanoma, and A375 and A2058 skin cancer cells, whereas EERL slightly stimulated the growth of normal IEC-6 intestinal epithelial cells and CCD118SK fibroblasts. The MC-soluble fraction was more efficacious than EERL in inhibiting DU145 cell growth. Licochalcone A was isolated from the MC fraction and identified as the active compound of EERL. Both EERL and licochalcone A induced apoptosis of DU145 cells. EERL potently inhibited chemically-induced skin papilloma formation in mice. CONCLUSIONS: Non-polar compounds in EERL exert potent anti-carcinogenic effects, and that roasted rather than un-roasted licorice should be favored as a cancer preventive agent, whether being used as an additive to food or medicine preparations.

Phloretin suppresses thrombin-mediated leukocyte-platelet-endothelial interactions.

SCOPE: Thrombin playing a pivotal role in coagulation cascade may influence the onset and progression of atherosclerosis as a pro-inflammatory mediator. This study investigated whether phloretin found in apple tree leaves, severed a linkage between thrombosis and atherosclerosis by thrombin. METHODS AND RESULTS: Human endothelial cells were pre-treated with 1-20 µM phloretin and stimulated with 10 U/mL thrombin. Phloretin attenuated adhesion of THP-1 monocytes and platelets to thrombin-inflamed endothelial cells with concurrent inhibition of protease-activated receptor (PAR-1) induction. The thrombin induction of endothelial CD40, endothelial integrin ß3 and P-selectin, and monocytic CD40L was dampened by phloretin. Additionally, phloretin inhibited monocyte secretion of MCP-1, IL-6 and IL-8 responsible for pro-inflammatory activity of thrombin inducing endothelial CD40. The monocyte COX-2 induction and PGE2 secretion due to thrombin were down-regulated by phloretin, deterring endothelial CD40 expression. Thrombin promoted production of PAI-1 and tissue factor in monocytes was attenuated by phloretin through blocking PAR-1 and CD40. Thrombin up-regulated the induction of endothelial connective tissue growth factor independent of PAR-1 activation, which was reversed by phloretin. CONCLUSION: Phloretin disturbed tethering and stable adhesion of monocytes and platelets onto endothelium during increased thrombosis by thrombin. Phloretin would be a potent agent preventing thrombosis and atherosclerosis.

Dietary fat increases solid tumor growth and metastasis of 4T1 murine mammary carcinoma cells and mortality in obesity-resistant BALB/c mice.

INTRODUCTION: High-fat diets (HFDs) are known to cause obesity and are associated with breast cancer progression and metastasis. Because obesity is associated with breast cancer progression, it is important to determine whether dietary fat per se stimulates breast cancer progression in the absence of obesity. This study investigated whether an HFD increases breast cancer growth and metastasis, as well as mortality, in obesity-resistant BALB/c mice. METHODS: The 4-week-old, female BALB/c mice were fed HFD (60% kcal fat) or control diet (CD, 10% kcal fat) for 16 weeks. Subsequently, 4T1 mammary carcinoma cells were injected into the inguinal mammary fat pads of mice fed continuously on their respective diets. Cell-cycle progression, angiogenesis, and immune cells in tumor tissues, proteases and adhesion molecules in the lungs, and serum cytokine levels were analyzed with immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA). In vitro studies were also conducted to evaluate the effects of cytokines on 4T1 cell viability, migration, and adhesion. RESULTS: Spleen and gonadal fat-pad weights, tumor weight, the number and volume of tumor nodules in the lung and liver, and tumor-associated mortality were increased in the HFD group, with only slight increases in energy intake and body weight. HF feeding increased macrophage infiltration into adipose tissues, the number of lipid vacuoles and the expression of cyclin-dependent kinase (CDK)2, cyclin D1, cyclin A, Ki67, CD31, CD45, and CD68 in the tumor tissues, and elevated serum levels of complement fragment 5a (C5a), interleukin (IL)-16, macrophage colony-stimulating factor (M-CSF), soluble intercellular adhesion molecule (sICAM)-1, tissue inhibitors of metalloproteinase (TIMP)-1, leptin, and triggering receptor expressed on myeloid cells (TREM)-1. Protein levels of the urokinase-type plasminogen activator, ICAM-1, and vascular cell adhesion molecule-1 were increased, but plasminogen activator inhibitor-1 levels were decreased in the lungs of the HFD group. In vitro assays using 4T1 cells showed that sICAM-1 increased viability; TREM-1, TIMP-1, M-CSF, and sICAM-1 increased migration; and C5a, sICAM-1, IL-16, M-CSF, TIMP-1, and TREM-1 increased adhesion. CONCLUSIONS: Dietary fat increases mammary tumor growth and metastasis, thereby increasing mortality in obesity-resistant mice.

Dehydroglyasperin C isolated from licorice caused Nrf2-mediated induction of detoxifying enzymes.

Our preliminary experiment demonstrated that a n-hexane/EtOH (9:1, volume) extract of Glycyrrhiza uralensis (licorice) caused a significant induction of NAD(P)H:oxidoquinone reductase (NQO1), one of the well-known phase 2 detoxifying enzymes. We isolated dehydroglyasperin C (DGC) as a potent phase 2 enzyme inducer from licorice. DGC induced NQO1 both in wild-type murine hepatoma Hepa1c1c7 and ARNT-lacking BPRc1 cells, indicating that the compound is a monofunctional inducer. The compound induced not only NQO1 but also some other phase 2 detoxifying/antioxidant enzymes, such as glutathione S-transferase, gamma-glutamylcysteine synthase, glutathione reductase, and heme oxygenase 1. Similar to most monofunctional inducers, DGC caused the accumulation of Nrf2 in the nucleus in dose- and time-dependent manners and thereby activated expression of phase 2 detoxifying enzymes. It also resulted in a dose-dependent increase in the luciferase activity in the reporter assay, in which HepG2-C8 cells transfected with antioxidant response element (ARE)-luciferase construct were used, suggesting that the induction of phase 2 detoxifying and antioxidant enzymes could be achieved through the interaction of Nrf2 with the ARE sequence in the promoter region of their genes.

Influence of conjugated linoleic acid isomers on the metastasis of colon cancer cells in vitro and in vivo.

This study investigated the isomer-specific effects of cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12) conjugated linoleic acid (CLA) on the metastasis of colon cancer cells in vitro and in vivo. Cell migration was examined by a Boyden chamber assay in SW480 cells. MMP-9 activity was monitored by gelatin zymography, and MMP-9 protein and mRNA levels were determined by Western blot and RT-PCR analysis, respectively, in SW480 cells. For the experimental metastasis, BALB/c mice were injected intravenously with CT-26 cells in the tail vein. Mice were fed a diet containing either no CLA or 0.1% c9,t11 or t10,c12 CLA for 4 weeks. In experimental metastasis, the numbers of pulmonary nodules were significantly lower in mice fed CLA isomers than in mice fed a control diet (P<.05). Results from the Boyden chamber assay revealed that c9,t11 CLA significantly inhibited cell migration (P<.05), whereas t10,c12 CLA had no effect on cell migration. The activity of MMP-9 was significantly inhibited by c9,t11 CLA (P<.05) but not by t10,c12 CLA. However, neither MMP-9 protein nor mRNA levels were altered by either of these CLA isomers. We have demonstrated that diets containing 0.1% c9,t11 and t10,c12 CLA were equally effective in inhibiting colon cancer cell metastasis in vivo. However, in vitro, only c9,t11 but not t10,c12 inhibited colon cancer cell migration and MMP-9 activity.

Neuroprotective effects of roasted licorice, not raw form, on neuronal injury in gerbil hippocampus after transient forebrain ischemia.

AIM: To observe neuroprotective effects of raw and roasted licorice against hypoxia and ischemic damage. METHODS: When elucidating the protective effects of raw and roasted licorice, we analyzed the lactate dehydrogenase (LDH) release using PC12 cells after hypoxia in an in vitro study and after transient forebrain ischemia in an in vivo study on Mongolian gerbils. RESULTS: Raw and roasted licorice significantly reduced LDH release from PC12 cells exposed to an hypoxic chamber for 1 h. In the roasted licorice-treated group, the decrease of LDH release was more pronounced compared to that of the raw licorice-treated group. In roasted licorice-treated animals, approximately 66%-71% of CA1 pyramidal cells in the ischemic hippocampus were stained with cresyl violet compared to the control group. However, in the raw licorice-treated animals, no significant neuroprotection against ischemic damage was shown. In addition, ischemic animals in roasted licorice-treated group maintained the Cu, Zn-superoxide dismutase (SOD1) activity and protein levels compared to the control group, while in raw licorice-treated group SOD1 activity and protein levels were reduced significantly. High pressure liquid chromatography analysis showed that non-polar compounds containing glycyrrhizin-degraded products, such as glycyrrhetinic acid (GA) and glycyrrhetinic acid monoglucuronide (GM), were increased in roasted licorice. CONCLUSION: Roasted licorice had neuroprotective effects against ischemic damage by maintaining the SOD1 levels. In addition, the difference in protective ability between raw and roasted licorice may be associated with non-polar compounds, such as GA and GM.

Downstream components of RhoA required for signal pathway of superoxide formation during phagocytosis of serum opsonized zymosans in macrophages.

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47(PHOX). Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47(PHOX) may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.

Inhibition of hydrogen peroxide-induced endothelial apoptosis by 2',4',7-trihydroxyflavanone, a flavonoid form.

Oxidative stress contributes to cellular injury following clinical and experimental ischemia/reperfusion episodes. Oxidative injury can induce cellular and subcellular damage that results in apoptotic cell death. We tested whether 2',4',7-trihydroxyflavanone, a synthetic flavonoid derivative, inhibits hydrogen peroxide (H(2)O(2))-induced toxicity in human umbilical endothelial cells (HUVECs). Cultured HUVECs were incubated for 30 minutes with 0.2 mM H(2)O(2) in the absence and presence of 2',4',7-trihydroxyflavanone at a non-toxic concentration of 50 microM. The effect of 2',4',7-trihydroxyflavanone on apoptosis parameters was compared with that of naringenin and two flavonol derivatives, 2',4',7-trihydroxyflavonol and 2',4',6-trihydroxyflavonol. H(2)O(2) induced cell death within 24 hours over the range of 0.05-1.0 mM, and decreased cell viability by approximately 30% at 0.2 mM. This cytotoxicity was associated with nuclear condensation and DNA fragmentation, indicating induction of apoptosis. 2',4',7-Trihydroxyflavanone, as well as naringenin, was effective as an inhibitor of oxidative stress, protecting cell viability with >/=85% viable cells compared with the control, and no apparent nuclear condensation or DNA fragmentation. In contrast, the flavonol derivatives had no such effect. In addition, immunocytochemical data and Western blot analysis revealed that, unlike flavonol derivatives, the expression of Bcl-2 was markedly up-regulated, and that the expression of Bax and activation of caspase-3 were strongly inhibited by this flavanone derivative, thereby implicating antioxidant activity-related anti-apoptotic mechanisms of 2',4',7-trihydroxyflavanone. These results indicate that the synthetic flavonoid derivative 2',4',7-trihydroxyflavanone is an effective antioxidant, preventing endothelial apoptosis induced by H(2)O(2).