Antidiabetic Effect of Fermented Mesembryanthemum crystallinum L. in db/db Mice Involves Regulation of PI3K-Akt Pathway.

Type 2 diabetes (T2D) is a serious health issue with increasing incidences worldwide. However, current medications have limitations due to side effects such as decreased appetite, stomach pain, diarrhea, and extreme tiredness. Here, we report the effect of fermented ice plant (FMC) in the T2M mouse model of db/db mice. FMC showed a greater inhibition of lipid accumulation compared to unfermented ice plant extract. Two-week oral administration with FMC inhibited body weight gain, lowered fasting blood glucose, and improved glucose tolerance. Serum parameters related to T2D including insulin, glycosylated hemoglobin, adiponectin, and cholesterols were improved as well. Histological analysis confirmed the protective effect of FMC on pancreas and liver destruction. FMC treatment significantly increased the expression and phosphorylation of IRS-1, PI3K, and AKT. Additionally, AMP-activated protein kinase phosphorylation and nuclear factor erythroid 2-related factor 2 were also increased in the liver tissues of db/db mice treated with FMC. Overall, our results indicate the anti-diabetic effect of FMC; therefore, we suggest that FMC may be useful as a therapeutic agent for T2D.

Perilla frutescens Leaf Extract Attenuates Vascular Dementia-Associated Memory Deficits, Neuronal Damages, and Microglial Activation.

Vascular dementia (VaD) is characterized by a time-dependent memory deficit and essentially combined with evidence of neuroinflammation. Thus, polyphenol-rich natural plants, which possess anti-inflammatory properties, have received much scientific attention. This study investigated whether Perilla frutescens leaf extract (PFL) exerts therapeutic efficacy against VaD. Sprague Dawley rats were divided into five groups: SO, sham-operated and vehicle treatment; OP, operated and vehicle treatment; PFL-L, operated and low-dose (30 mg/kg) PFL treatment; PFL-M, operated and medium-dose (60 mg/kg) PFL treatment; and PFL-H, operated and high-dose (90 mg/kg) PFL treatment. Two-vessel occlusion and hypovolemia (2VO/H) were employed as a surgical model of VaD, and PFL was given orally perioperatively for 23 days. The rats underwent the Y-maze, Barnes maze, and passive avoidance tests and their brains were subjected to histologic studies. The OP group showed VaD-associated memory deficits, hippocampal neuronal death, and microglial activation; however, the PFL-treated groups showed significant attenuations in all of the above parameters. Using lipopolysaccharide (LPS)-stimulated BV-2 cells, a murine microglial cell line, we measured PFL-mediated changes on the production of nitric oxide (NO), TNF-α, and IL-6, and the activities of their upstream MAP kinases (MAPKs)/NFκB/inducible NO synthase (iNOS). The LPS-induced upregulations of NO, TNF-α, and IL-6 production and MAPKs/NFκB/iNOS activities were globally and significantly reversed by 12-h pretreatment of PFL. This suggests that PFL can counteract VaD-associated structural and functional deterioration through the attenuation of neuroinflammation.

Immune-Enhancing Effect of Sargassum horneri on Cyclophosphamide-Induced Immunosuppression in BALB/c Mice and Primary Cultured Splenocytes.

Sargassum horneri (SH) is a seaweed that has several features that benefit health. In this study, we investigated the immune-enhancing effect of SH, focusing on the role of spleen-mediated immune functions. Chromatographic analysis of SH identified six types of monosaccharide contents, including mannose, rhamnose glucose, galactose xylose and fucose. SH increased cell proliferation of primary cultured naïve splenocytes treated with or without cyclophosphamide (CPA), an immunosuppression agent. SH also reversed the CPA-induced decrease in Th1 cytokines. In vivo investigation revealed that SH administration can increase the tissue weight of major immune organs, such as the spleen and thymus. A similar effect was observed in CPA-injected immunosuppressed BALB/c mice. SH treatment increased the weight of the spleen and thymus, blood immune cell count and Th1 cytokine expression. Additionally, the YAC-1-targeting activities of natural killer cells, which are important in innate immunity, were upregulated upon SH treatment. Overall, our study demonstrates the immune-enhancing effect of SH, suggesting its potential as a medicinal or therapeutic agent for pathologic conditions involving immunosuppression.

Verbascoside-Rich Abeliophyllum distichum Nakai Leaf Extracts Prevent LPS-Induced Preterm Birth Through Inhibiting the Expression of Proinflammatory Cytokines from Macrophages and the Cell Death of Trophoblasts Induced by TNF-α.

Background: Preterm birth is a known leading cause of neonatal mortality and morbidity. The underlying causes of pregnancy-associated complications are numerous, but infection and inflammation are the essential high-risk factors. However, there are no safe and effective preventive drugs that can be applied to pregnant women. Objective: The objectives of the study were to investigate a natural product, Abeliophyllum distichum leaf (ADL) extract, to examine the possibility of preventing preterm birth caused by inflammation. Methods: We used a mouse preterm birth model by intraperitoneally injecting lipopolysaccharides (LPS). ELISA, Western blot, real-time PCR and immunofluorescence staining analyses were performed to confirm the anti-inflammatory efficacy and related mechanisms of the ADL extracts. Cytotoxicity and cell death were measured using Cell Counting Kit-8 (CCK-8) analysis and flow cytometer. Results: A daily administration of ADL extract significantly reduced preterm birth, fetal loss, and fetal growth restriction after an intraperitoneal injection of LPS in mice. The ADL extract prevented the LPS-induced expression of TNF-α in maternal serum and amniotic fluid and attenuated the LPS-induced upregulation of placental proinflammatory genes, including IL-1ß, IL-6, IL-12p40, and TNF-α and the chemokine gene CXCL-1, CCL-2, CCL3, and CCL-4. LPS-treated THP-1 cell-conditioned medium accelerated trophoblast cell death, and TNF-α played an essential role in this effect. The ADL extract reduced LPS-treated THP-1 cell-conditioned medium-induced trophoblast cell death by inhibiting MAPKs and the NF-κB pathway in macrophages. ADL extract prevented exogenous TNF-α-induced increased trophoblast cell death and decreased cell viability. Conclusions: We have demonstrated that the inhibition of LPS-induced inflammation by ADL extract can prevent preterm birth, fetal loss, and fetal growth restriction.

Epigallocatechin-3-Gallate Regulates Anti-Inflammatory Action Through 67-kDa Laminin Receptor-Mediated Tollip Signaling Induction in Lipopolysaccharide-Stimulated Human Intestinal Epithelial Cells.

BACKGROUND/AIMS: Inflammatory bowel disease (IBD) is a condition that involves chronic inflammation in all or part of the digestive tract. Often painful and debilitating, IBD can lead to life-threatening complications and increase the risk for colon cancer. In this study, we investigated the epigallocatechin-3-gallate (EGCG) mediated anti-inflammation response in lipopolysaccharide (LPS)-stimulated human colorectal cells through the negative regulator of Toll-like receptor (TLR) signaling. METHODS: human intestinal epithelial cells (HT-29) were used in all experiments. Cell cytotoxicity and nitric oxide (NO) were evaluated by WST-1 and the Griess reagent. Western blot analysis and ELISA were used to determine inflammatory mediators and 67-kDa laminin receptor (67LR)-mediated Tollip signaling pathways. RESULTS: Treatment of EGCG and LPS did not affect the cytotoxicity in HT-29 cells. LPS treatment dose-dependently increased the pro-inflammatory cytokine, such as interleukin (IL)-8, whereas EGCG significantly reduced the LPS-stimulated IL-8 production. Additionally, EGCG treatment markedly increased the Toll-interacting protein (Tollip) expression, which negatively regulates the TLR signaling in a dose and time-dependent manner. In particular, in the result from an RNA interference-mediated assay, our finding showed that silencing of Tollip resulted in abrogation of the inhibitory action of EGCG on LPS-induced production of pro-inflammatory mediators (inducible nitric oxide synthase-mediated NO/COX2, and IL-8) and activation of MAPKs and NF-κB signaling pathways. Interestingly, we also found that Tollip expression induced by EGCG could be modulated through 67LR expressed on the surface of HT-29 cells. CONCLUSIONS: Our novel finding indicates that 67LR and Tollip signaling activated by EGCG treatment is essential for inhibition of inflammation in human intestinal epithelial cells.

Neuroprotective effect of Capsicum annuum var. abbreviatum against hydrogen peroxide-induced oxidative stress in HT22 hippocampus cells.

Phenolic compounds isolated from pepper (Capsicum annum) have been demonstrated to have neuroprotective effects, whereas the physiological properties of Capsicum annuum var. abbreviatum (CAA) have not been studied. Thus, we investigate the chemical composition and neuroprotective activity of CAA extract (CAAE) in HT22 hippocampus cells against H2O2-induced neurotoxicity. CAAE treatment resulted in a significant protection of H2O2-exposed HT22, this protection ultimately occurred through an inhibition of MDA and ROS levels and an induction of SOD activity. Furthermore, CAAE treatment reduced H202-induced apoptosis though decreasing the expression of pro-apoptotic factors (Bax, cytochrome c, and cleaved caspases-3) while increasing the expression of the anti-apoptotic factors (Bcl-2), as well as the accumulation of nucleus-Nrf2-mediated HO-1 signaling. Interestingly, CAAE has a high concentration of unique phenolic compositions (chlrogenic acid, tangeretin, etc.) than other capsicum annum extracts. Altogether, these findings suggest that CAAE can be a useful natural resource for alleviating neurodegenerative diseases.

Gamma-irradiated ß-glucan induces immunomodulation and anticancer activity through MAPK and NF-κB pathways.

BACKGROUND: This study was designed to evaluate the antitumor activity of low-molecular-weight ß-glucan (LMBG) produced by gamma irradiation (50 kGy), using in vivo and in vitro models. RESULTS: The results indicate that treatment with LMBG increased the proliferation of murine peritoneal macrophages, and their production of tumor necrosis factor α and nitric oxide, to a greater extent than treatment with high-molecular-weight ß-glucan (HMBG). The activation of peritoneal macrophages by LMBG was mediated by both mitogen-activated protein kinases and nuclear factor-κB signaling. Interestingly, when administered prophylactically, LMBG significantly inhibited tumor growth and lung metastasis in mice injected with B16BL6 melanoma cells compared with the HMBG-treated group. In comparison with HMBG treatment, LMBG treatment also elevated cell proliferation, cytokine (interferon-γ and interleukin-2) production, and CD8(+) T cell populations in splenocytes from tumor-bearing mice. CONCLUSION: These data indicate that LMBG is important in eliciting antitumor activity through a non-specific immune response and may play a major role as a value-added product in the medical industry.

Quercetin negatively regulates TLR4 signaling induced by lipopolysaccharide through Tollip expression.

Polyphenolic compounds have been regarded as one of the most promising dietary agents for the prevention and treatment of inflammation-related chronic diseases; however, the anti-inflammatory activities of flavonoids, such as quercetin, are not completely characterized, and many features remain to be elucidated. In this study, we showed the molecular basis for the downregulation of TLR4 signal transduction by quercetin. Quercetin markedly elevated the expression of the Toll-interacting protein, a negative regulator of TLR signaling. Lipopolysaccharide-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II) and production of pro-inflammatory cytokines (tumor necrosis factor-α, IL-1ß, IL-6, and IL-12p70) were inhibited by quercetin, and this action was prevented by Toll-interacting protein silencing. In addition, quercetin-treated macrophages inhibited lipopolysaccharide-induced activation of mitogen-activated protein kinases, such as extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase, and the translocation of nuclear factor-κB and p65 through Toll-interacting protein. Treatment with quercetin resulted in a significant decrease in prostaglandin E2 and cyclooxygenase-2 levels as well as inducible nitric oxide synthase-mediated nitric oxide production induced by lipopolysaccharide. Taken together, these findings represent new insights into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and effective therapeutic intervention for the treatment of inflammatory disease.

Procyanidin dimer B2-mediated IRAK-M induction negatively regulates TLR4 signaling in macrophages.

Polyphenolic compounds have been found to possess a wide range of physiological activities that may contribute to their beneficial effects against inflammation-related diseases; however, the molecular mechanisms underlying this anti-inflammatory activity are not completely characterized, and many features remain to be elucidated. In this study, we investigated the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by procyanidin dimer B2 (Pro B2) in macrophages. Pro B2 markedly elevated the expression of the interleukin (IL)-1 receptor-associated kinase (IRAK)-M protein, a negative regulator of TLR signaling. Lipopolysaccharide (LPS)-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II) and production of pro-inflammatory cytokines (tumor necrosis factor-α, IL-1ß, IL-6, and IL-12p70) were inhibited by Pro B2, and this action was prevented by IRAK-M silencing. In addition, Pro B2-treated macrophages inhibited LPS-induced activation of mitogen-activated protein kinases such as extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase and the translocation of nuclear factor κB and p65 through IRAK-M. We also found that Pro B2-treated macrophages inactivated naïve T cells by inhibiting LPS-induced interferon-γ and IL-2 secretion through IRAK-M. These novel findings provide new insights into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and the immune-pharmacological role of Pro B2 in the immune response against the development and progression of many chronic diseases.

Rubus fruticosus leaf extract inhibits vascular dementia-induced memory impairment and neuronal loss by attenuating neuroinflammation.

Vascular dementia (VaD) is characterized by progressive memory impairment, which is associated with microglia-mediated neuroinflammation. Polyphenol-rich natural plants, which possess anti-inflammatory activities, have attracted scientific interest worldwide. This study investigated whether Rubus fruticosus leaf extract (RFLE) can attenuate VaD. Sprague-Dawley rats were separated into five groups: SO, sham-operated and treated with vehicle; OP, operated and treated with vehicle; RFLE-L, operated and treated with low dose (30 mg/kg) of RFLE; RFLE-M, operated and treated with medium dose (60 mg/kg) of RFLE; and RFLE-H, operated and treated with high dose (90 mg/kg) of RFLE. Bilateral common carotid artery and hypotension were used as a modeling procedure, and the RFLE were intraorally administered for 5 days (preoperative 2 and postoperative 3 days). The rats then underwent memory tests including the novel object recognition, Y-maze, Barnes maze, and passive avoidance tests, and neuronal viability and neuroinflammation were quantified in their hippocampi. The results showed that the OP group exhibited VaD-associated memory deficits, neuronal death, and microglial activation in hippocampi, while the RFLE-treated groups showed significant attenuation in all above parameters. Next, using BV-2 microglial cells challenged with lipopolysaccharide (LPS), we evaluated the effects of RFLE in dynamics of proinflammatory mediators and the upstream signaling pathway. RFLE pretreatment significantly inhibited the LPS-induced release of nitric oxide, TNF-α, and IL-6 and upregulation of the MAPKs/NF-κB/iNOS pathway. Collectively, we suggest that RFLE can attenuate the histologic alterations and memory deficits accompanied by VaD, and these roles are, partly due to the attenuation of microglial activation.

Anti-Atopic Activities of Sargassum horneri Hot Water Extracts in 2,4-Dinitrochlorobezene-Induced Mouse Models.

Atopic dermatitis (AD) is a chronic inflammation associated with skin hypersensitivity caused by environmental factors. The objent of this study was to assess the hot water extracts of Sargassum horneri (SHHWE) on AD. AD was induced by spreading 2,4-dinitrochlorobenzene (DNCB) on the BALB/c mice. The efficacy of SHHWE was tested by observing the immunoglobulin E (IgE), cytokine, skin clinical severity score and cytokine secretions in concanavalin A (Con A)-stimulated splenocytes. The levels of interleukine (IL)-4, IL-5 and IgE, the pro-inflammatory cytokines that are closely related, were notably suppressed in a does-dependent manner by SHHWE, whereas the level of interferon γ (IFN-γ), the atopy-related Th1 cytokine inhibiting the production of Th2 cytokines, was increased. Therefore, these results show that SHHWE has a potent anti-inhibitory effect on AD and is highly valuable for cosmetic development.

Green tea polyphenol epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor on lipopolysaccharide-stimulated dendritic cells.

Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1ß, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases.

Inhibitory effect of ethanolic extract of Abeliophyllum distichum leaf on 3T3-L1 adipocyte differentiation.

BACKGROUND/OBJECTIVES: Abeliophyllum distichum is a plant endemic to Korea, containing several beneficial natural compounds. This study investigated the effect of A. distichum leaf extract (ALE) on adipocyte differentiation. MATERIALS/METHODS: The cytotoxic effect of ALE was analyzed using cell viability assay. 3T3-L1 preadipocytes were differentiated using induction media in the presence or absence of ALE. Lipid accumulation was confirmed using Oil Red O staining. The mRNA expression of adipogenic markers was measured using RT-PCR, and the protein expressions of mitogen-activated protein kinase (MAPK) and peroxisome proliferator-activated receptor gamma (PPARγ) were measured using western blot. Cell proliferation was measured by calculating the incorporation of Bromodeoxyuridine (BrdU) into DNA. RESULTS: ALE reduced lipid accumulation in differentiated adipocytes, as indicated by Oil Red O staining and triglyceride assays. Treatment with ALE decreased the gene expression of adipogenic markers such as Pparγ, CCAAT/enhancer binding protein alpha (C/ebpα), lipoprotein lipase, adipocyte protein-2, acetyl-CoA carboxylase, and fatty acid synthase. Also, the protein expression of PPARγ was reduced by ALE. Treating the cells with ALE at different time points revealed that the inhibitory effect of ALE on adipogenesis is higher in the early period treatment than in the terminal period. Furthermore, ALE inhibited adipocyte differentiation by reducing the early phase of adipogenesis and mitotic clonal expansion. This was indicated by the lower number of cells in the Synthesis phase of the cell cycle (labeled using BrdU assay) and a decrease in the expression of early adipogenic transcription factors such as C/ebpß and C/ebpδ. ALE suppressed the phosphorylation of MAPK, confirming that the effect of ALE was through the suppression of early phase of adipogenesis. CONCLUSIONS: Altogether, the results of the present study revealed that ALE inhibits lipid accumulation and may be a potential agent for managing obesity.

Abeliophyllum distichum Ameliorates High-Fat Diet-Induced Obesity in C57BL/6J Mice by Upregulating the AMPK Pathway.

The use of natural compounds as anti-obesity agents has been gaining attention over the past few years. Abeliophyllum distichum Nakai is endemic to Korea. In the present study, an A. distichum leaf extract (AE) was analyzed for its anti-obesity effects in mice fed a high-fat diet. Seven-week-old male C57BL/6J mice were divided into five groups, namely, normal diet (ND), high-fat diet (HD), HD + Garcinia (GE300), HD + AE low dose (AE100), and HD + AE high dose (AE300). After 8 weeks of the experimental period, treatment with AE reduced body weight and ameliorated high-fat diet-induced changes in serum lipid levels. Histological analysis revealed that treatment with AE decreased lipid accumulation in the liver and brown adipose tissue. Also, AE reduced the adipocyte size in epididymal fat. The reduction in adipose tissue mass in the AE-treated groups was clearly visible in micro-computed tomography images. The expression levels of lipogenic genes, such as PPARγ, C/EBPα, ACC, and FAS, were significantly reduced in the AE300 group. The levels of p-AMPK and p-ACC were increased in the AE300 group compared to the HD group, indicating that the anti-obesity effect of AE was mediated through the AMPK pathway.

Effect of gamma irradiation on spleen cell function and cytotoxicity of doxorubicin.

The present study was attempted to evaluate the effects of gamma-irradiated doxorubicin (IRD) on spleen cell proliferation, cytokines release (IFN-gamma and IL-2) and lung metastasis in mice. Gamma irradiation induced degradation of doxorubicin molecule and cytotoxicity on melanoma (B16BL6) and myoblast (H9c2) cell lines were determined by high performance liquid chromatography (HPLC) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazole) assay, respectively. Non-irradiated doxorubicin (NIRD) was used as a control. The mice injected with NIRD (2mg/kg body weight for 5 days, 24h interval) showed a considerable decrease (P<0.05) in the body, spleen weight, proliferation and cytokine release (IL-2 and IFN-gamma) as compared to control. However, a non-significant variation was observed in IRD treated mice compared with normal. Tumor bearing mice treated with NIRD and IRD (2mg/kg body weight, five doses at 48 h interval) showed diverse results on spleen cell cytokine release, proliferation and metastasis. HPLC results revealed the formation of several trace level degradation (P<0.05) products of IRD. IRD displayed a non-significant variation of cytotoxicity on B16BL6 cells, and low percentage (P<0.01) of cardiotoxicity on H9c2 cells as compared to NIRD. Altogether, this present study emphasis that gamma irradiation altered the property of doxorubicin.

Effect of gamma irradiated hyaluronic acid on acetaminophen induced acute hepatotoxicity.

The hepatoprotective efficacy of irradiated hyaluronic acid (HA) on acetaminophen (APAP) induced acute hepatotoxicity was investigated. BALB/c mice (4-6 weeks of age) were pretreated with unirradiated HA (UIHA), 5 and 50 kGy gamma irradiated HA (GIHA) for 14 days and were dosed APAP (500 mg/kg b.wt). After 9h of APAP dosing animals were euthanized. The degree of acute hepatotoxicity was measured by aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The expression of interferon-gamma (IFN-gamma) in serum and alpha-and mu-class of gluthathione-S-transferase (GSTs), CYP 2E1 class of cytochrome monooxygenase and glutathione (GSH) in liver were quantified. Histological evaluation was done by Hematoxiylin and Eiosin staining, Periodic acid schiffs staining, Manson trichrome staining and histological scorings were done. The degree of acute hepatotoxicity was markedly lower in UIHA and 5 kGy than in 50 kGy GIHA pretreated group and there was negligible difference between 5 and 50 kGy GIHA pretreated group. The expression of interferon-gamma (IFN-gamma) was significantly (P<0.05) suppressed in 5 and 50 kGy GIHA pretreated group. Histological scorings showed a significant protection of liver in UIHA and 5 kGy GIHA pretreated mice. Expression of alpha class GSTs was significantly increased in 5 and 50 kGy GIHA pretreated group. To conclude suppression of IFN-gamma and increase in alpha-class GSTs expression may exert a protective role in acute hepatotoxicity of APAP and 5 kGy GIHA showed comparable protective effect to that of UIHA.

Immune-enhancement effects of tuna cooking drip and its enzymatic hydrolysate in Balb/c mice.

Tuna cooking drip (TCD) is a protein rich by-product of canned tuna processing that is typically discarded. In this study, the immune-enhancing activities of TCD and its enzymatic hydrolysate (EH-TCD) were investigated by intraperitoneally administering Balb/c male mice with TCD and EH-TCD for 2 weeks. This administration resulted in an increase in the weight of the spleen and thymus (200-800 mg/kg) and enhanced the proliferation rates of splenocytes (200-800 mg/kg). TCD and EH-TCD significantly increased the production of immunostimulatory cytokines (interleukin-10 and interleukin-2). In addition, TCD and EH-TCD increased serum IgG1 and IgG2a levels in a concentration-dependent manner. Particularly, EH-TCD had a greater immune-enhancing effect than TCD. These results suggest that TCD and EH-TCD exert immune-enhancing effects through an IgG antibody response and T cell activation, and EH-TCD can be used as an immunostimulatory agent.

Gamma irradiation of aloe-emodin induced structural modification and apoptosis through a ROS- and caspase-dependent mitochondrial pathway in stomach tumor cells.

PURPOSE: The changes in molecular structure and the physiological properties of a gamma-irradiated aloe-emodin were examined. MATERIALS AND METHODS: Aloe-emodin was gamma-irradiated at doses ranging from 0 to 150 kGy, and the molecular structure was then analyzed using high-performance liquid chromatography (HPLC). AGS cells were cultured in RPMI medium and treated gamma irradiated aloe-emodin. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis efficiency was investigated by cell cycle arrest, cell morphology, and signaling pathway. The structure of new radiolytic peak was identified by the hydrogen-nuclear magnetic resonance (1H NMR). RESULTS: HPLC results showed that gamma irradiation induced new radiolytic peaks that were distinguishable from the aloe-emodin standard, and the area of new peaks was increased as the radiation dose increased. Gamma-irradiated aloe-emodin treatment significantly increased the cytotoxicity in AGS tumor cells. We also found that 150 kGy aloe-emodin increased the expression of Bax, cytosolic cytochrome c, PARP cleavage, and the activation of caspases-8, -9, -3, Bid, and Bcl-2. Treatment of 150 kGy aloe-emodin induced ROS production, DNA fragmentation, alterations of cell morphology, and the migration in AGS cells. Gamma-irradiated aloe-emodin induced an increase of sub-G1 phase and depolarization of mitochondrial membrane potential in AGS cells. We also confirmed that fractionated AEF1 (new radiolytic peak) induce the cell death, migration, an increase of sub-G1 phase and cytochrome c in a ROS-dependent manner. CONCLUSIONS: The radiolysis product (AEF1) of aloe-emodin transformed by gamma-irradiation strongly induced apoptotic cell death in AGS cells, indicating AEF1 is a potential candidate drug for use in anti-cancer drug.

Gamma irradiation enhanced Tollip-mediated anti-inflammatory action through structural modification of quercetin in lipopolysaccharide-stimulated macrophages.

The changes in molecular structure and anti-inflammatory action of a gamma-irradiated quercetin were examined. Quercetin was gamma-irradiated at doses of 0, 15, 30, 50, 100 and 150kGy, which induced new radiolytic peaks (the highest radiolytic peak at a dose of 30kGy). Treatment of intact- and gamma-irradiated quercetin did not induce a significant cellular toxicity of macrophages at concentrations ranging from 12.5 to 50µM. Treatment of LPS-stimulated macrophages with gamma-irradiated quercetin (30kGy) showed a higher inhibitory action than intact-quercetin groups in the excessive expression of inducible nitric oxide synthases-mediated nitric oxide, prostaglandin E2, pro-inflammatory cytokines level, such as tumor necrosis factor-α, interleukin-6 and interleukin-1ß, reactive oxygen species, as well as cell surface molecules (CD80, CD86, and MHC class I/II). The inhibition of LPS-stimulated pro-inflammatory mediators was mediated through a suppression of mitogen-activated protein kinases and nuclear factor-κB pathways. In addition, gamma-irradiated quercetin (30kGy) markedly elevated the expression of the Toll-interacting protein compared to intact-quercetin. The inhibitory action of intact- and gamma-irradiated quercetin on the production of IL-6 and TNF-α was not observed in the down-regulation of Tollip. Therefore, these findings represent new insights into the understanding of the changes in molecular structure and the physiological properties of natural products through the application of radiation technology.

Gamma-Irradiated Luteolin Inhibits 3-Isobutyl-1-Methylxanthine-Induced Melanogenesis Through the Regulation of CREB/MITF, PI3K/Akt, and ERK Pathways in B16BL6 Melanoma Cells.

Luteolin was gamma irradiated at doses of 0, 15, 30, 50, 70, and 100 kGy. We observed that the luteolin peak decreased simultaneously with the appearance of new radiolytic peaks, using high-performance liquid chromatography (HPLC). The highest new radiolytic peak (GLM) of radiolytic product in gamma-irradiated luteolin was observed at a dose of 70 kGy, and the GLM was identified by nuclear magnetic resonance and high-performance-liquid-chromatography-quadrupole-time-of-flight (HPLC-Q-TOF) mass spectrometry. We examined whether 70 kGy gamma-irradiated luteolin has more effective anti-melanogenic effects than intact luteolin. Seventy kilograys of gamma-irradiated luteolin inhibited melanin synthesis and intracellular tyrosinase activity without cytotoxicity, whereas the intact luteolin-treated group did not show anti-melanogenic activity in 3-isobutyl-1-methylxanthine-stimulated B16BL6 melanoma cells. The expression of melanogenic enzymes, such as tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2, was decreased by 70 kGy gamma-irradiated luteolin treatment, owing to the suppression of microphthalamia-associated transcription factor and 3',5'-cyclic adenosine monophosphate (cAMP) response element binding protein. In addition, gamma-irradiated luteolin decreased the phosphorylation of phosphoinositide 3-kinase (PI3K)/Akt and extracellular regulated kinase (ERK). The anti-melanogenic effects of 70 kGy gamma-irradiated luteolin were attenuated by the treatment of two specific inhibitors (PD98059 and LY294002), and these results indicate that the anti-melanogenic effects were mediated by ERK and PI3K signaling pathways. Therefore, our findings suggest that gamma-irradiated luteolin can be a potential cosmeceutical agent for skin whitening.