Sword Bean (Canavalia gladiata) Pod Exerts Anti-Allergic and Anti-Inflammatory Effects through Modulation of Th1/Th2 Cell Differentiation.

Allergy is an immunoglobulin E (IgE)-mediated process, and its incidence and prevalence have increased worldwide in recent years. Therapeutic agents for allergic diseases are continuously being developed, but side effects follow when used for a long-term use. Therefore, treatments based on natural products that are safe for the body are urgently required. Sword bean (Canavalia gladiata) pod (SBP) has been traditionally used to treat inflammatory diseases, but there is still no scientific basis for its anti-allergic effect. Accordingly, this study investigates the anti-allergic effect and its mechanism of SBP in vitro and in vivo. SBP reduced the nitric oxide production and decreased mRNA and protein expression of inflammatory mediates (inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)), and inhibited the phosphorylation of nuclear factor kappa B (NF-κB), a major signaling molecule in the inflammatory response. Additionally, SBP extract treatment inhibited phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling activity to further inhibit degranulation and allergy mediator generation and control the balance of Th1/Th2 cells, which can induce an allergic reaction when disrupted. Furthermore, the SBP extract exhibited anti-allergic effects in anti-dinitrophenyl IgE-induced RBL-2H3 cells and ovalbumin-treated mice. These findings have potential clinical implications for the treatment as well as prevention of allergic diseases.

Immature sword bean pods (Canavalia gladiata) inhibit adipogenesis in C3H10T1/2 cells and mice with high-fat diet-induced obesity.

BACKGROUND: Sword bean (SB; Canavalia gladiata) is a perennial vine used as a food and medicinal plant in Asia. SB is rich in nutrients, such as flavonoids and urease, and has various functions, including beneficial effects on dysentery, nausea, and hemorrhoids, as well as anti-inflammatory and antioxidant activity. Various plant parts are used; however, little is known about the physiological effects of SB pods (SBP). In this study, the anti-obesity effects of SBP extract were evaluated. METHODS: To investigate the anti-obesity effects of SBP extract, we confirmed the SBP extract downregulated lipogenesis-related genes and upregulated genes involved in lipolysis and brown adipocyte markers in differentiated C3H10T1/2 adipocytes in vitro. Next, we use a high-fat diet (HFD)-induced obesity mouse model to determine the anti-obesity effects of SBP extract. RESULTS: Treatment with SBP extract significantly reduced adipocytes. The extract decreased the HFD-induced increases in body weight and plasma triglyceride levels in mice after 8 weeks. mRNA and protein levels of the adipogenesis and lipogenesis-related factors CCAAT/enhancer binding protein-ß, CCAAT/enhancer binding protein-α, peroxisome proliferator-activated receptor-γ (PPARγ), and their target genes Ap2, SREBP-1c, FAS, and SCD-1 were reduced by SBP extract. In contrast, AMP-activated protein kinase and sirtuin1, involved in the thermogenic catabolism of fat, were activated by SBP extract in adipocytes and white adipose tissue, increasing the expression of peroxisome proliferator-activated receptor gamma coactivator-1α, peroxisome proliferator-activated receptor-α (PPARα), and uncoupling protein 1 and activating thermogenic activity. CONCLUSION: SBP extract exerts an anti-obesity effect by inhibiting lipogenesis-related factors and activating fat-catabolizing factors; it is, therefore, a promising functional food and natural anti-obesity agent.

Optimization of pectinase-assisted extraction condition of mulberry (Morus alba L.) fruit using response surface methodology and its effect on anthocyanin synthesis pathway-related metabolites.

Mulberry (Morus alba L.) fruit (MF) is a rich source of functional compounds, such as anthocyanin. However, during solvent extraction, these compounds are not fully dispersed into the substrate, leading to incomplete extraction. Moreover, raw MF rapidly ripens and deteriorates after harvesting; hence, innovative methods to process MF are needed. Here, a pectinase-assisted extraction method is developed to liberate polyphenols and anthocyanins from cell wall matrices in MF. We optimized the procedure to maximize water solubility index (WSI), total phenolic (TP) content, and total anthocyanin (TA) content using a central composite design to perform a response surface methodology (RSM) analysis. The optimal conditions predicted by the RSM were a 1:5 w/v material/water ratio with 3.5% pectinase (v/w) and 1.5% citric acid (w/w) for 113 min at 50°C. Under these conditions, the WSI, TP, and TA were significantly higher compared with those in the untreated control. The results well matched (within 5% differences) with the predicted RSM values. Furthermore, metabolite analysis revealed that the levels of cyanidin-3-O-glucoside, delphinidin hexoside, and quercetin were higher in pectinase-assisted MF extraction compared with the untreated control. This work demonstrated that pectinase-assisted extraction using citric acid could be an efficient technique to enhance the value of MF and its potential applications in the food industry. PRACTICAL APPLICATION: A pectinase-assisted extraction method was optimized to enhance the WSI, TP, and TA yields from MF extracts. The optimal conditions were predicted to be 1:5 w/v material/water ratio, 3.5% pectinase (v/w), and 1.5% CA (w/w) with a 113 min reaction time at 50°C. Under these conditions, WSI, TP, and TA were significantly increased compared with the untreated control. These results suggested the potential of mulberry plants for use in the food industry via the development of a simple, efficient process to extract functional compounds from MF.

Improvement of Fatigue Symptoms and Endurance Capacity by the Combined Administration of Cervus elaphus L., Angelica gigas Nakai, and Astragalus membranaceus Bunge.

Fatigue is a common phenomenon usually observed in healthy, as well as in nonhealthy, individuals that affects their performance and quality of life. Efficient supplementation to relieve fatigue is of significant importance. This study was designed to investigate the efficacy of three prescreened natural resources (Cervus elaphus L. [CEL], Angelica gigas Nakai [AGN], and Astragalus membranaceus Bunge [AMB]) against fatigue symptoms induced by heavy exercise. Effects on muscle fatigue and endurance capacity during exercise were investigated in C2C12 myoblasts and exercised mice. A combination of CEL, AGN, and AMB (CEL:AGN:AMB, 1:2:1) treatment in myoblasts reduced intracellular reactive oxygen species levels induced by hydrogen peroxide by ∼20 times (P < .001). The optimal mixture extract combination was determined as CEL:AGN:AMB, 1:2:1 (CAA), which was recombined by applying the extraction yield of individual substance for in vivo study. Compared to the exercise control (EC) group, the serum lactate dehydrogenase level decreased by ∼40% due to CAA administration. The proliferator-activated receptor gamma coactivator 1-alpha protein expression increased significantly (P < .05) after CAA administration compared to that observed in the normal control group. In parallel, CAA treatment significantly (P < .05) enhanced the maximum running time compared to the EC group. Overall, combinatorial administration exhibited greater efficacy compared to each individual treatment, indicating that CAA could be used as an efficient ergogenic and antifatigue supplement.

Fermentation of Chestnut (Catanea crenata Sieb) Inner Shell Enhances Anti-Obesity Effects in 3T3-L1 and C3H10T1/2 Adipocytes.

Chestnut inner shell (CIS) is rich in phenols and flavonoids such as gallic acid and ellagic acid, which are known to exhibit effective antioxidant and anti-obesity properties. Fermentation using lactic acid bacteria can enhance the physiological activity by increasing the contents of such functional ingredients. In this study, we evaluated the anti-obesity effects of a CIS extract subjected to a fermentation process (fermented CIS [FCIS]). Treatment with CIS and FCIS extracts (125, 250, and 500 µg/mL) increased cell viability and did not induce apoptosis, indicating no toxicity. The extract suppressed the gene expression of adipogenic factors, peroxisome proliferation-activated receptor gamma, CCAAT/enhancer binding protein (C/EBP) alpha, and C/EBP beta (by 7.75% and 67.59%, 21.41% and 66.27% in 500 µg/mL, respectively), and consequently suppressed the expression of downstream lipogenic factors such as fatty acid synthase, stearoyl CoA desaturase-1, citrate synthase, and ATP citrate lyase. The expression of factors involved in fat catabolism and ß-oxidation increased in a dose-dependent manner, thereby preventing fat accumulation. This observation was consistent with the significant decrease in the staining intensity for lipid droplets, which indicated that lipid accumulation was decreased by 15.46% and 29.44% in 3T3L-1 and 27.01% and 46.68% in C3H10T1/2. Together, these results demonstrate the higher anti-obesity effects of FCIS extract than that of CIS extract, indicating the potential applicability of FCIS as an effective natural raw material to curb obesity.

Mustard Leaf Extract Suppresses Psychological Stress in Chronic Restraint Stress-Subjected Mice by Regulation of Stress Hormone, Neurotransmitters, and Apoptosis.

Mustard leaf (Brassica juncea var. crispifolia L. H. Bailey) has been reported to have psychological properties such as anti-depressant activities. However, studies on chronic stress and depression caused by restraint have not been conducted. Therefore, this study aimed to evaluate the effects of a mustard leaf (ML) extract on chronic restraint stress (CRS) in mice. Male mice were subjected to a CRS protocol for a period of four weeks to induce stress. The results showed that the ML extract (100 and 500 mg/kg/perorally administered for four weeks) significantly decreased corticosterone levels and increased neurotransmitters levels in stressed mice. Apoptosis by CRS exposure was induced by Bcl-2 and Bax expression regulation and was suppressed by reducing caspase-3 and poly (ADP-ribose) polymerase expression after treatment with the ML extract. Our results confirmed that apoptosis was regulated by increased expression of brain-derived neurotrophic factor (BDNF). Additionally, cytokine levels were regulated by the ML extract. In conclusion, our results showed that the ML extract relieved stress effects by regulating hormones and neurotransmitters in CRS mice, BDNF expression, and apoptosis in the brain. Thus, it can be suggested that the studied ML extract is an agonist that can help relieve stress and depression.

Anti-Inflammatory Effect of Immature Sword Bean Pod (Canavalia gladiata) in Lipopolysaccharide-Induced RAW264.7 Cells.

Sword bean has been known as a traditional medicinal plant to treat cancer, sinus infection, and suppurative disease. It also possesses hypertension-relieving, antioxidation, and antibacterial effects. However, studies on the efficacy of sword bean are limited to mature beans. Few studies have focused on immature sword bean pod (ISBP). Therefore, this study aimed to investigate the anti-inflammatory effect of ISBP in RAW264.7 cells stimulated with lipopolysaccharide (LPS). After LPS-induced RAW264.7 cells were treated with ISBP at concentrations (0.5, 1, 2, and 5 mg/mL), levels of nitrite oxide (NO) and prostaglandin E2 (PGE2) production, protein, and mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), inflammatory cytokine secretion level, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity were determined. Under inflammatory conditions induced by LPS, ISBP reduced levels of inflammatory mediators NO and PGE2 by 60% and 23%, respectively. It also decreased protein and mRNA expression levels of iNOS and COX-2 known to synthesize inflammatory mediators. Inflammatory cytokines, interleukin (IL)-6, and IL-1ß, levels were decreased, while interferon gamma level was increased by ISBP based on enzyme-linked immunosorbent assay (ELISA) and real time-polymerase chain reaction results. Finally, ISBP showed the ability to inhibit NF-κB activity. In conclusion, ISBP can alleviate inflammation by controlling inflammation-related substances, and may have efficacy as a healthful functional food and natural anti-inflammatory drug.

Effect of Achyranthes japonica Nakai extract on immunity and anti-inflammation in dogs.

Achyranthes japonica Nakai (A. japonica) is a medicinal herb found widely distributed throughout Korea. The biological activities of A. japonica are well-documented and include anti-fungal, anti-inflammatory, and immunity enhancement. The objective of the present study was to investigate the immune-related activities of A. japonica extract in dogs. The extract was acquired by ethanol extraction and purified by filtration. To examine the effect of A. japonica extract on immune cell viability, human lymphocytes, such as Jurkat T-cells and Ramos B-cells, were exposed to the extract. After treatment with the extract, the number of Ramos B-cells was increased, whereas Jurkat T-cells remained unaffected. Griess assay revealed decreased nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated mouse macrophage Raw 264.7 cells after exposure to A. japonica extract. To evaluate the in-vivo effect in dogs, feed containing A. japonica extract was provided to 8 dogs for 2 months. Blood samples were collected before, during, and after consumption of the feed. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples and the number of T-cells and B-cells were assessed using flow cytometry with anti-dog fluorescein isothiocyanate (FITC)-conjugated CD3 and anti-dog phycoerythrin (PE)-conjugated CD21 antibodies, respectively. We observed a significant increase in the average number of B-cells in the PBMCs during ingestion of the feed containing A. japonica. In addition, enzyme-linked immunosorbent assay (ELISA) revealed a decrease in the levels of tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine, in 3 out of 8 dogs and increased levels of interleukin-10 (IL-10), an anti-inflammatory cytokine, in 4 out of 8 dogs. Taken together, we believe that these changes indicate that A. japonica extract is beneficial in improving the immunity of dogs by stimulating B-cells and inducing production of anti-inflammatory responses.

Achyranthes japonica Nakai (A. japonica) est une herbe médicinale retrouvée largement distribuée à travers la Corée. Les activités biologiques d'A. japonica sont bien documentées et inclus des effets antifongique, anti-inflammatoire et de stimulation de l'immunité. L'objectif de la présente étude était d'examiner les activités reliées à l'immunité d'un extrait d'A. japonica chez des chiens. L'extrait fut obtenu par extraction à l'éthanol et purification par filtration. Pour examiner l'effet de l'extrait d'A. japonica sur la viabilité de cellules immunitaires, des lymphocytes humains, tels que les cellules T Jurkat et les cellules B Ramos, furent exposés à l'extrait. Après traitement avec l'extrait, le nombre de cellules B Ramos était augmenté, alors que celui des cellules T Jurkat était inchangé. L'épreuve de Griess a révélé une diminution de production d'oxyde nitreux (NO) chez les macrophages de souris Raw 264,7 stimulés par le lipopolysaccharide (LPS) à la suite de l'exposition à l'extrait d'A. japonica. Afin d'évaluer les effets in vivo chez les chiens, de la nourriture contenant l'extrait d'A. japonica fut donnée à huit chiens pour une période de 2 mois. Des échantillons sanguins furent prélevés avant, durant et après consommation de l'aliment. Des mononucléaires du sang périphérique (PBMCs) furent isolés des échantillons sanguins et le nombre de cellules T et de cellules B fut évalué en utilisant la cytométrie de flux et des anticorps anti-CD3 de chien conjugués à l'isothiocyanate de fluorescéine (FITC) et des anticorps anti-CD21 de chien conjugués à la phycoérythrine (PE), respectivement. Nous avons observé une augmentation significative du nombre moyen de cellules B dans le PBMCs durant l'ingestion de la nourriture contenant A. japonica. De plus, une épreuve immuno-enzymatique (ELISA) a révélé une diminution des niveaux du facteur alpha nécrosant des tumeurs (TNF-α), une cytokine pro-inflammatoire, chez trois des huit chiens et des niveaux augmentés d'interleukine-10 (IL-10), une cytokine anti-inflammatoire, chez quatre des huit chiens. Pris globalement, nous croyons que ces changements indiquent qu'un extrait d'A japonica est bénéfique pour améliorer l'immunité chez les chiens en stimulant les cellules B et en induisant la production de réponses anti-inflammatoires.(Traduit par Docteur Serge Messier).

Platycodon grandiflorum Extract Reduces High-Fat Diet-Induced Obesity Through Regulation of Adipogenesis and Lipogenesis Pathways in Mice.

Obesity is caused by an energy imbalance between food intake and energy expenditure, and has detrimental effects on human health. Platycodon (Platycodon grandiflorum) widely grows in Korea, Japan, and China. It has long been used for food and as a medicinal product. However, the mechanism of the improvement of obesity by platycodon was still not clear. Therefore, we investigated the detailed mechanisms of the antiobesity activity of platycodon extracts. Twenty mice (C57BL/6J) were placed into five groups. The test group received 1 g/kg platycodon extracts. The positive control group received 10 mg/kg orlistat, while the negative control and normal control groups received phosphate-buffered saline. The extracts were given orally daily for 8 weeks. The in vivo treatment of platycodon extracts reduced body weight gain by 7.5%, improved plasma lipid profiles. In the groups given platycodon extracts, leptin was significantly decreased whereas adiponectin was increased. Furthermore, platycodon extracts downregulated lipogenic gene (e.g., lipoprotein lipase, acetyl-CoA carboxylase, and fatty acid synthase) expression and increased lipolysis genes (e.g., carnitine palmitoyltransferase 1α, hormone-sensitive lipase, and uncoupling proteins 2) in liver and white adipose tissue. In addition, platycodon extracts inhibited the expression of key adipogenic transcriptional factors. In conclusion, we have demonstrated that platycodon extracts ameliorate high-fat diet-induced obesity and its related metabolic disease by regulating multiple pathways. Dietary supplementation of platycodon extracts as a functional food and medicinal ingredients may be suitable for prevention and treatment of obesity.

Effects of Low Temperature-Aged Garlic on Exercise Performance and Fatigue in Mice.

We developed low temperature-aged garlic (LTAG) to remove its unique and spicy flavor and evaluated the anti-fatigue properties of LTAG against exercise-induced fatigue in mice. In the results, the treadmill running time to exhaustion in the mice fed LTAG was prolonged compared with the control. There was significant difference in blood parameters of glucose, lactate, lactate dehydrogenase (LDH), and free fatty acid (FFA) concentration between the LTAG-fed mice and the control. In addition, LTAG effectively increased the content of glycogen and creatine kinase and the activity of antioxidant enzymes in the muscle. The mechanism underlying the anti-fatigue activity of LTAG is hypothesized to involve increase in postexercise tissue glycogen accumulation to improve the aerobic and anaerobic exercise capacity. LTAG may have an ergogenic effect on endurance exercise while decreasing the levels of FFA, LDH, and lactate, which are associated with the anti-fatigue effect. Thus, LTAG has potential as a pharmacological anti-fatigue agent.

Anti-inflammatory effect of aerial bulblets of Dioscorea japonica Thunb extract through inhibition of NF-κB and MAPK signalling pathway in RAW 264.7.

BACKGROUND: Yam (Dioscorea japonica Thunb) is a well-known health food in Korea and is widely distributed in the temperate and tropical regions. Although various medical effects of yam have been demonstrated, there is little current knowledge on the efficacy of Youngyeoja (YYJ; the aerial bulblets of the yam plant), their physiological effects, and their mechanism of action. METHODS: To investigate the anti-inflammatory effects of YYJ, we examined the level of inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells treated with YYJ extract. Nitric oxide (NO) and prostaglandin E2 (PGE2) levels were determined using enzyme-linked immunosorbent assays. Expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were evaluated using real-time polymerase chain reaction and western blotting. In addition, activation of nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) was detected using western blotting. RESULTS: Treatment of macrophages with LPS markedly induced the production of NO and PGE2. YYJ treatment inhibited the induction of inflammatory mediators and the expression of iNOS and COX-2. More importantly, LPS-induced phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) was suppressed by treatment with YYJ, suggesting YYJ inhibited NF-κB activation. Furthermore, YYJ inhibited the LPS-induced phosphorylation of MAPKs. CONCLUSION: YYJ was shown to have a potent anti-inflammatory effect in LPS-stimulated RAW 264.7 cells, which may be attributed to its inhibitory effect on NF-κB and MAPK activation, consequently blocking the production of inflammatory factors. Therefore, these results suggest that the YYJ extracts could be used as anti-inflammatory agents.

Low temperature-aged garlic extract suppresses psychological stress by modulation of stress hormones and oxidative stress response in brain.

BACKGROUND: Garlic is a folk medicine known for its multiple physiological activities, but the neuro-modulatory effect of garlic against psychological stress has rarely been explored. The current study was conducted to determine the potential antipsychological stress effect of low temperature-aged garlic (LTAG). METHODS: After acute restraint stress exposure, mice were administered with raw garlic (RG, 500 mg/kg, p.o.) or LTAG (500 mg/kg, p.o.). We investigated corticosterone, cortisol, and monoamines levels, and the mRNA expression of genes relevant to oxidative stress. RESULTS: RG and LTAG treatment significantly decreased stress-related hormones such as corticotropin-releasing factor, adrenocorticotropic hormone, corticosterone, and cortisol. Moreover, RG and LTAG administration significantly restored acute restraint stress-induced changes in concentrations of brain neurotransmitters (serotonin, norepinephrine, dopamine, and epinephrine). In addition, RG and LTAG improved the antioxidant defense system by causing an increase in mRNA expression of superoxide dismutase, catalase, and glutathione peroxidase in the brain. CONCLUSION: This study suggests an antipsychological stress and neuroprotective effect of RG and LTAG under stress conditions.

Oxyresveratrol Increases Energy Expenditure through Foxo3a-Mediated Ucp1 Induction in High-Fat-Diet-Induced Obese Mice.

The phytochemical oxyresveratrol has been shown to exert diverse biological activities including prevention of obesity. However, the exact reason underlying the anti-obese effects of oxyresveratrol is not fully understood. Here, we investigated the effects and mechanism of oxyresveratrol in adipocytes and high-fat diet (HFD)-fed obese mice. Oxyresveratrol suppressed lipid accumulation and expression of adipocyte markers during the adipocyte differentiation of 3T3-L1 and C3H10T1/2 cells. Administration of oxyresveratrol in HFD-fed obese mice prevented body-weight gains, lowered adipose tissue weights, improved lipid profiles, and increased glucose tolerance. The anti-obese effects were linked to increases in energy expenditure and higher rectal temperatures without affecting food intake, fecal lipid content, and physical activity. The increased energy expenditure by oxyresveratrol was concordant with the induction of thermogenic genes including Ucp1, and the reduction of white adipocyte selective genes in adipose tissue. Furthermore, Foxo3a was identified as an oxyresveratrol-induced gene and it mimicked the effects of oxyresveratrol for induction of thermogenic genes and suppression of white adipocyte selective genes, suggesting the role of Foxo3a in oxyresveratrol-mediated anti-obese effects. Taken together, these data show that oxyresveratrol increases energy expenditure through the induction of thermogenic genes in adipose tissue and further implicates oxyresveratrol as an ingredient and Foxo3a as a molecular target for the development of functional foods in obesity and metabolic diseases.

Rosmarinic Acid, a Component of Rosemary Tea, Induced the Cell Cycle Arrest and Apoptosis through Modulation of HDAC2 Expression in Prostate Cancer Cell Lines.

Rosmarinic acid (RA), a main phenolic compound contained in rosemary which is used as tea, oil, medicine and so on, has been known to present anti-inflammatory, anti-oxidant and anti-cancer effects. Histone deacetylases (HDACs) are enzymes that play important roles in gene expression by removing the acetyl group from histone. The aberrant expression of HDAC in human tumors is related with the onset of human cancer. Especially, HDAC2, which belongs to HDAC class I composed of HDAC 1, 2, 3 and 8, has been reported to be highly expressed in prostate cancer (PCa) where it downregulates the expression of p53, resulting in an inhibition of apoptosis. The purpose of this study is to investigate the effect of RA in comparison with suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor used as an anti-cancer agent, on survival and apoptosis of PCa cell lines, PC-3 and DU145, and the expression of HDAC. RA decreased the cell proliferation in cell viability assay, and inhibited the colony formation and tumor spheroid formation. Additionally, RA induced early- and late-stage apoptosis of PC-3 and DU145 cells in Annexin V assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, respectively. In western blot analysis, RA inhibited the expression of HDAC2, as SAHA did. Proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E1 were downregulated by RA, whereas p21 was upregulated. In addition, RA modulated the protein expression of intrinsic mitochondrial apoptotic pathway-related genes, such as Bax, Bcl-2, caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1) (cleaved) via the upregulation of p53 derived from HDAC2 downregulation, leading to the increased apoptosis of PC-3 and DU145 cells. Taken together, treatment of RA to PCa cell lines inhibits the cell survival and induces cell apoptosis, and it can be used as a novel therapeutic agent toward PCa.

Effects of Zanthoxylum piperitum ethanol extract on osteoarthritis inflammation and pain.

Degenerative arthritis, also known as osteoarthritis (OA), is the most common type of arthritis, which is caused by degenerative damage of the cartilage, which primarily protects the joints, leading to inflammation and pain. The objective of this study was to investigate the in vivo and in vitro effects of treatment with ZPE-LR (90% EtOH extract of Zanthoxylum piperitum) on pain severity and inflammation. When using an in vivo OA model MIA (monosodiumidoacetate-induced arthritis) rats, ZPE-LR (100 mg/kg) oral-administratio significantly inhibited MIA-induced change in loaded weight ratio on the left foot, and articular cartilage thickness. To confirm the positive effects on pain relief, acetic acid, heat and formalin-induced pain were remarkably decreased by 50 and 100 mg/kg ZPE-LR oral-administration. Pain related KCNJ6 mRNA expression as well as K + current was increased after ZPE-LR treatment in BV-2 cells. To confirm the positive effects on inflammation, TPA (12-O-tetradecanoylphorbol-13-acetate) induced inflammation measured by mouse ear thickness and biopsy punch weight and TPA-induced iNOS, COX-2 mRNA and protein expression were remarkably suppressed by 50 and 100 mg/kg ZPE-LR oral-administration. In addition, TPA-induced iNOS, COX-2 mRNA level and protein expression were reduced. Acetic acid, heat and formalin-induced pain were remarkably decreased by 50 and 100 mg/kg ZPE-LR oral-administration. We examined in vitro ZPE-LR effects in LPS-induced RAW 264.7 cells. LPS-induced p65 translocation to the nucleus was prohibited by ZPE-LR 100 µg/ml oral administration. Moreover, ROS generation by LPS was significantly inhibited by ZPE-LR 50 and 100 µg/ml treatment. To investigate new ZPE-LR activating mechanisms, the gene fishing method (not a typical term, should probably use PCR based genetic screening) was used. LPS-induced HPRT1 (hypoxanthine phosphoribosyltransferase 1) was decreased by ZPE-LR. However, RPL8 (Ribosomal protein L8) which showed no change in mRNA expression due to LPS, did show increased mRNA levels after ZPE-LR treatment. Our data elucidate mechanisms underlying ZPE-LR and suggest ZPE-LR may be a potential therapeutic agent to modulate osteoarthritis inflammation and pain.

Phytochemical-induced reactive oxygen species and endoplasmic reticulum stress-mediated apoptosis and differentiation in malignant melanoma cells.

BACKGROUND: Phytochemicals are derived from plants, vegetables and daily products and exert chemopreventive effects. Malignant melanoma is highly metastatic, and melanoma patients can develop chemotherapeutic resistance against conventional melanoma therapies. METHODS: In the present study, we investigated the anti-cancer effect of the phytochemicals kaempferol (Kaem), genistein (Gen), and 3'3-diindolylmethane (DIM) on melanoma cell viability. We also evaluated the altered expression of cell cycle-related genes. We verified the production of intracellular reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress at both the protein and cellular level using a western blot, TUNEL assay, and Dihydrodichlorofluorescein diacetate (DCF-DA) assay. RESULTS: Treatment of A375SM melanoma cells with phytochemicals resulted in inhibition of cell growth. Treatment with phytochemicals increased the gene expression of p21 and decreased the gene expression of cyclin E and/or cyclin B. The three phytochemicals activated the ROS-p38-p53 apoptotic pathway by increasing the level of phosphorylated p38 MAPK and p53, and they activated the ER stress-mediated apoptotic pathway by increasing the level of phosphorylated eIF2α and C/EBP homologous protein (CHOP). Both the ROS-p38-p53 and ER stress-mediated pathway induced the mitochondrial apoptotic pathway by attenuating Bcl-2 expression and upregulating BAX. Detection of morphological changes demonstrated that Kaem and Gen can induce differentiation in A375SM cell line. CONCLUSION: These results indicate that phytochemicals are potentially useful in treatments for melanoma due to their ability to inhibit melanoma cell growth and division via the ROS and ER stress pathway.

Aster yomena extract ameliorates pro-inflammatory immune response by suppressing NF-κB activation in RAW 264.7 cells.

BACKGROUND: Aster yomena, an edible vegetable, is a perennial herb found in Korea, China, Japan, and Siberia. It is used as folk medicine to treat cough, bronchial asthma, and insect bites. A. yomena was recently shown to have antioxidant and anti-asthmatic activities. Studies have not yet evaluated the anti-inflammatory effects of the various solvent fractions of A. yomena. We investigated the anti-inflammatory activity of various solvent fractions (hexane, dichloromethane, ethyl acetate, and butanol) from ethanol extract of A. yomena in activated macrophages. METHODS: Anti-inflammatory effects of A. yomena were investigated to determine the inhibitory effects of A. yomena against inflammation using RAW 264.7 mouse macrophages. To measure the effects of A. yomena on inflammatory mediators and cytokines, we used the following methods: cell viability assay, flow cytometry assay, ELISA assay, real-time PCR and western blotting. RESULTS: The dichloromethane fraction exhibited marked anti-inflammatory activities by inhibiting nitric oxide (NO) and prostaglandin E2 production and mRNA expression of inducible isoforms of NO synthase, cyclooxygenase-2, and cytokines (tumor necrosis factor α, interleukin (IL)-6, IL-1ß) in response to lipopolysaccharide (LPS) stimulation. Moreover, dichloromethane fraction from A. yomena significantly inhibited the transactivation of nuclear factor (NF)-κB and the nuclear translocation of the NF-κB p50 and p65 subunits. CONCLUSION: These results suggest that A. yomena may have anti-inflammatory activity in vitro, suggesting this herb could be a source of natural anti-inflammatory agents.

Inhibitory effects of 3,3'-diindolylmethane on epithelial-mesenchymal transition induced by endocrine disrupting chemicals in cellular and xenograft mouse models of breast cancer.

As a phytoestrogen, 3,3'-diindolylmethane (DIM) plays a chemopreventive role by inhibiting cancer progression. In this study, we examined the effects of 17ß-estradiol (E2), two endocrine disrupting chemicals (EDCs), triclosan (TCS) and bisphenol A (BPA), and DIM on epithelial-mesenchymal transition (EMT) and metastatic behaviors of estrogen receptor (ER)-positive MCF-7 breast cancer cells. An in vitro assay revealed that E2 (10-9 M), TCS (10-5-10-7 M), and BPA (10-5-10-7 M) induced MCF-7 cell proliferation compared to a control through the ER pathway. In addition, E2, TCS, and BPA changed the cell morphology from the epithelial to the mesenchymal phenotype and increased the migration and invasion capacity of MCF-7 cells via ER; however, co-treatment with DIM (20 µM) effectively suppressed E2, TCS, and BPA-induced cell proliferation, EMT, migration, and invasion of MCF-7 cells. Western blot assay revealed that DIM regulated the protein expression of EMT- and metastasis-related genes toward the inhibition of these processes. Moreover, E2, TCS, and BPA increased the protein expression of CXCR4, which is a receptor of chemokine CXCL12 that is positively involved in breast cancer metastasis via an ER-dependent pathway. Conversely, DIM and a CXCR4 antagonist (AMD3100) decreased CXCR4 protein expression, which led to inhibition of the EMT process, indicating that DIM may suppress E2, TCS or BPA-induced EMT, migration, and invasion of MCF-7 breast cancer cells by suppressing CXCR4 protein expression. These in vitro effects of E2, TCS, BPA, and DIM were also identified in a xenografted mouse model transplanted with MCF-7 breast cancer cells. Taken together, DIM is a potent chemopreventive compound for preventing metastatic behaviors of breast cancer cells induced by EDCs with cancer-related toxicity.

Cholesterol-lowering effect of Aralia elata (Miq.) Seem via the activation of SREBP-2 and the LDL receptor.

BACKGROUND: Hyperlipidemia causes arteriosclerosis, a risk factor for coronary heart disease. Prevention of hyperlipidemia by improving dietary habits has recently attracted attention. In this regard, we investigated whether Aralia elata (Miq.) Seem (AE) extract inhibits hepatic cholesterol accumulation and modulate the cellular signaling pathway. METHODS: To determine AE's cholesterol regulating mechanism, we measured cholesterol level, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and cholesterol regulating-related gene expression in HepG2 cells and in high-fat diet (HFD)-induced mice using ELISA and RT-PCR assay. RESULTS: The AE extract reduced cholesterol levels and HMG-CoA reductase activity in hepatocellular carcinoma HepG2 cells. In addition, it also reduced the plasma cholesterol concentrations in HFD-induced mice. Furthermore, the AE extract increased the gene expression of the LDL-receptor (LDL-R); sterol-regulatory-element binding protein-2 (SREBP-2); ATP-binding cassette, sub-family A, member 1 (ABCA1); and scavenger receptor class B member 1 (SR-B1) in a dose-dependent manner. However, the AE extract did not affect the gene expression of acetyl-coenzyme A acetyltransferase (ACAT) in either the HepG2 cells or mice. CONCLUSION: We demonstrated that the AE extract activated genes related to cholesterol metabolism, such as SREBP-2 and LDL-R, which resulted in hypocholesterolemic activities.

Kaempferol, a phytoestrogen, suppressed triclosan-induced epithelial-mesenchymal transition and metastatic-related behaviors of MCF-7 breast cancer cells.

As a phytoestrogen, kaempferol is known to play a chemopreventive role inhibiting carcinogenesis and cancer progression. In this study, the influences of triclosan, an anti-bacterial agent recently known for an endocrine disrupting chemical (EDC), and kaempferol on breast cancer progression were examined by measuring their effects on epithelial-mesenchymal transition (EMT) and metastatic-related behaviors of MCF-7 breast cancer cells. Morphological changes of MCF-7 cells were observed, and a wound-healing assay was performed after the treatment of triclosan and kaempferol. The effects of triclosan and kaempferol on protein expression of EMT-related markers such as E-cadherin, N-cadherin, Snail, and Slug and metastasis-related markers such as cathepsin B, D, MMP-2 and -9 were investigated by Western blot assay. In microscopic observations, triclosan (10-6M) or E2 (10-9M) induced transition to mesenchymal phenotype of MCF-7 cells compared with the control. Co-treatment of ICI 182,780 (10-8M), an ER antagonist, or kaempferol (25µM) with E2 or triclosan restored the cellular morphology to an epithelial phenotype. In a wound-healing scratch and a transwell migration assay, triclosan enhanced migration and invasion of MCF-7 cells, but co-treatment of kaempferol or ICI 182,780 reduced the migration and invasion ability of MCF-7 cells to the control level. In addition, kaempferol effectively suppressed E2 or triclosan-induced protein expressions of EMT and metastasis promoting markers. Taken together, triclosan may be a distinct xenoestrogenic EDC to promote EMT, migration, and invasion of MCF-7 breast cancer cells through ER. On the other hand, kaempferol can be an alternative chemopreventive agent to effectively suppress the metastatic behavior of breast cancer induced by an endogenous estrogen as well as exogenous xenoestrogenic compounds including triclosan.