Anti-Inflammatory Effects and Macrophage Activation Induced by Bioavailable Cinnamon Polyphenols in Mice.

SCOPE: Cinnamon is a commonly used spice and herb that is rich in polyphenols. Due to the limited bioavailability of oral polyphenols, it remains unclear to which extent they can reach cells and exert a biological effect. This study aims to investigate the impact of bioavailable cinnamon polyphenols on lipopolysaccharide (LPS)-stimulated macrophages. METHODS AND RESULTS: A polyphenol fraction is prepared from cinnamon (Cinnamomi ramulus) (CRPF) by boiling cinnamon in water and adsorbing the extract onto a hydrophobic resin. Mice are orally administered CRPF for 7 days and then subjected to three independent experiments: endotoxemia, serum collection, and macrophage isolation. Upon intraperitoneal lipopolysaccharide challenge, CRPF decreases serum levels of inflammatory cytokines, involving suppression of liver and spleen macrophages. When normal macrophages are cultured in serum obtained from CRPF-treated mice, they exhibit an anti-inflammatory phenotype. However, macrophages from CRPF-treated mice show an increased production of inflammatory cytokines when cultured in fetal bovine serum and stimulated with LPS. CONCLUSION: The study provides evidence for the presence of bioavailable cinnamon polyphenols with anti-inflammatory properties and macrophage activation. These findings suggest that cinnamon polyphenols have the potential to modulate macrophage function, which could have implications for reducing inflammation and improving immune function.

Butein-Enriched Fractions of Butea monosperma (Lam.) Taub. Flower Decrease Weight Gains and Increase Energy Expenditure in High-Fat Diet-Induced Obese Mice.

Butea monosperma (Lam.) Taub. has been applied to treat inflammatory, metabolic, and infectious diseases. However, the antiobesity effects of B. monosperma (Lam.) Taub. flower (BMF) and the underlying mechanisms have not been determined. In this study, we analyzed the various extraction procedures, investigated the antiobesity effects, and identified the main chemical constituents of BMF. The BMF was subjected to acid hydrolysis in 5% H2SO4 in methanol at 50°C for 48 h and partitioned with ethyl acetate. The acid-hydrolyzed BMF ethyl acetate extracts (BMFE) strongly induced the expression of uncoupling protein 1 (Ucp1) and other thermogenic genes in C3H10T1/2 adipocytes. Daily oral administration of 70 mg/kg BMFE (BMFE70) to mice with diet-induced obesity resulted in less body weight gain, increased glucose tolerance, higher rectal temperature, and increased oxygen consumption. Qualitative and quantitative analyses along with treatments in Akt1 knockout mouse embryonic fibroblasts indicate that butein is a major active ingredient of BMFE, which stimulates Ucp1 gene expression. These data show the effects of butein-containing B. monosperma flower extract on thermogenesis and energy expenditure, further suggesting the potential role of BMFE as a functional ingredient in obesity and related metabolic diseases.

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.

Understanding the functional role of genistein in the bone differentiation in mouse osteoblastic cell line MC3T3-E1 by RNA-seq analysis.

Genistein, a phyto-estrogen, can potentially replace endogenous estrogens in postmenopausal women, but the underlying molecular mechanisms remain incompletely understood. To obtain insight into the effect of genistein on bone differentiation, RNA sequencing (RNA-seq) analysis was used to detect differentially expressed genes (DEGs) in genistein-treated vs. untreated MC3T3-E1 mouse osteoblastic cells. Osteoblastic cell differentiation was monitored by measuring osteoblast differentiation factors (ALP production, bone mineralization, and expression of osteoblast differentiation markers). From RNA-seq analysis, a total of 132 DEGs (including 52 up-regulated and 80 down-regulated genes) were identified in genistein-treated cells (FDR q-value < 0.05 and fold change > 1.5). KEGG pathway and Gene Ontology (GO) enrichment analyses were performed to estimate the biological functions of DEGs and demonstrated that these DEGs were highly enriched in functions related to chemotactic cytokines. The functional relevance of DEGs to genistein-induced osteoblastic cell differentiation was further evaluated by siRNA-mediated knockdown in MC3T3-E1 cells. These siRNA knockdown experiments (of the DEGs validated by real-time qPCR) demonstrated that two up-regulated genes (Ereg and Efcab2) enhance osteoblastic cell differentiation, while three down-regulated genes (Hrc, Gli, and Ifitm5) suppress the differentiation. These results imply their major functional roles in bone differentiation regulated by genistein.

Sulfuretin has therapeutic activity against acquired lymphedema by reducing adipogenesis.

Acquired lymphedema is a pathological condition associated with lymphatic dysfunction caused by surgical treatments for cancer. Although global estimates of the prevalence of acquired lymphedema have been rising, there are currently no effective therapeutics available. Since adipose tissue accumulation is a clinical hallmark of lymphedema, we hypothesized that regulation of adipogenesis in lymphedematous tissue could be used as a therapeutic intervention against lymphedema. Toward this, we investigated the possibility of anti-adipogenic 30% ethanol Rhus verniciflua Stokes (RVS) extract as a potential lymphedema treatment. Oral administration of RVS extract ameliorated volumetric symptoms of lymphedema in a mouse model. RVS administration also reduced adipose tissue accumulation in lymphedematous tissue and downregulated expression of adipocyte markers, including Pparγ and Fabp4. Sulfuretin was identified as a major bioactive compound in the 30% ethanol RVS extract in liquid chromatography-mass spectrometry analysis. Similar to the activities of RVS, sulfuretin inhibited adipocyte differentiation in 3T3-L1 preadipocytes. Moreover, treatment with sulfuretin on lymphedema-induced mice reduced lymphedema volume, decreased the expression of adipogenic markers, but induced the expression of markers associated with lymphangiogenesis. Taken together, our data raise the possibility that sulfuretin might be used in therapeutic interventions against acquired lymphedema.

Antioxidant Properties of Aqueous Extract of Roasted Hulled Barley in Bulk Oil or Oil-in-Water Emulsion Matrix.

UNLABELLED: Antioxidant properties of the aqueous extracts of hulled barley (Hordeum vulgare L.) that had been roasted at 210 °C for 20 min were determined in bulk oil and oil-in-water (O/W) emulsions. Bulk oils were heated at 60, 100, and 180 °C, and O/W emulsions were oxidized under riboflavin photosensitization. The content of phenolic compounds was analyzed by high-performance liquid chromatography, and in vitro antioxidant assays were also conducted. The major phenolics contained in the aqueous extract of roasted hulled barley (AERB) were p-coumaric, ferulic, protocatechuic, chlorogenic, 4-hydroxybenzoic, and vanillic acids. Depending on the concentration and oxidation temperature, AERB had antioxidant or prooxidant properties in bulk oil. At 60 °C, AERB at a concentration of 0.5% acted as a prooxidant, whereas at 1.0% it acted as an antioxidant. At 100 °C, AERB acted as an antioxidant irrespective of concentration. In 180 °C conditions, 0.5% AERB acted as a prooxidant, whereas other concentrations of AERB acted as antioxidants. In the case of riboflavin photosensitized O/W emulsions, AERB showed antioxidant properties irrespective of concentration. Antioxidant abilities of AERB are affected by the food matrix, including bulk oil and O/W emulsions, and concentrations of AERB, even though diverse phenolic compounds may display high antioxidant properties in in vitro assays. PRACTICAL APPLICATION: Roasted barley has been widely used as a tea ingredient in East Asian countries such as Korea, China, and Japan. The highly antioxidative properties of the aqueous extracts of roasted barley have been confirmed in bulk oil and O/W emulsions as well as in vitro assays because of the presence of phenolic compounds. The results of this study can contribute to the development of antioxidant-rich beverages using roasted barley by aiding in the selection of proper food matrix-containing extracts of high phenolic compounds, as well as by expanding consumers' choices for healthy beverages.

Black rice (Oryza sativa L.) extracts induce osteoblast differentiation and protect against bone loss in ovariectomized rats.

Osteoporosis, an age associated skeletal disease, exhibits increased adipogenesis at the expense of osteogenesis from common osteoporotic bone marrow cells. In this study, black rice (Oryza sativa L.) extracts (BRE) were identified as osteogenic inducers. BRE stimulated the alkaline phosphatase (ALP) activity in both C3H10T1/2 and primary bone marrow cells. Similarly, BRE increased mRNA expression of ALP and osterix. Oral administration of BRE in OVX rats prevented decreases in bone density and strength. By contrast, BRE inhibited adipocyte differentiation of mesenchymal C3H10T1/2 cells and prevented increases in body weight and fat mass in high fat diet fed obese mice, further suggesting the dual effects of BRE on anti-adipogenesis and pro-osteogenesis. UPLC analysis identified cyanidin-3-O-glucoside and peonidin-3-O-glucoside as main anti-adipogenic effectors but not for pro-osteogenic induction. In mechanism studies, BRE selectively stimulated Wnt-driven luciferase activities. BRE treatment also induced Wnt-specific target genes such as Axin2, WISP2, and Cyclin D1. Taken together, these data suggest that BRE is a potentially useful ingredient to protect against age related osteoporosis and diet induced obesity.

Pinus densiflora Sieb. et Zucc. alleviates lipogenesis and oxidative stress during oleic acid-induced steatosis in HepG2 cells.

Excess accumulation of lipids and oxidative stress in the liver contribute to nonalcoholic fatty liver disease (NAFLD). We hypothesized that Pinus densiflora Sieb. et Zucc. (PSZ) can protect against NAFLD by regulating lipid accumulation and oxidative stress in the liver. To investigate the effect of PSZ upon NAFLD, we used an established cellular model: HepG2 cells treated with oleic acid. Then, the extent of hepatic steatosis and oxidative stress was assessed and levels of inflammatory markers measured. Oleic acid-treated HepG2 cells, compared with controls, had greater lipid accumulation. PSZ decreased lipid accumulation by 63% in oleic acid-treated HepG2 cells. Additionally, PSZ decreased the target gene expression of lipogenesis such as sterol regulatory element binding protein-1c, fatty acid synthase, stearoyl-CoA desaturase-1, diacylglycerol O-acyltransferase-1, and acetyl-CoA carboxylase-1 by 1.75, 6.0, 2.32, 1.93 and 1.81 fold, respectively. In addition, Oleic acid-treated HepG2 cells elicited extensive accumulation of tumor necrosis factor-α (TNFα) by 4.53 fold, whereas PSZ-treated cells decreased the expression of TNFα mRNA by 1.76 fold. PSZ significantly inhibited oxidative stress induced by reactive oxygen species. These results suggest that PSZ has effects on steatosis in vitro and further studies are needed in vivo to verify the current observations.

Reciprocal regulation of adipocyte and osteoblast differentiation of mesenchymal stem cells by Eupatorium japonicum prevents bone loss and adiposity increase in osteoporotic rats.

Pathological increases in adipogenic potential with decreases in osteogenic differentiation occur in osteoporotic bone marrow cells. Previous studies have shown that bioactive materials isolated from natural products can reciprocally regulate adipogenic and osteogenic fates of bone marrow cells. In this study, we showed that Eupatorium japonicum stem extracts (EJE) suppressed lipid accumulation and inhibited the expression of adipocyte markers in multipotent C3H10T1/2 and primary bone marrow cells. Conversely, EJE stimulated alkaline phosphatase activity and induced the expression of osteoblast markers in C3H10T1/2 and primary bone marrow cells. Daily oral administration of 50 mg/kg of EJE for 6 weeks to ovariectomized rats prevented body weight increase and bone mineral density decrease. Finally, activity-guided fractionation led to the identification of coumaric acid and coumaric acid methyl ester as bioactive anti-adipogenic and pro-osteogenic components in EJE. Taken together, our data indicate a promising possibility of E. japonicum as a functional food and as a therapeutic intervention for preventing osteoporosis and bone fractures.

Prunus yedoensis bark inhibits lipopolysaccharide-induced inflammatory cytokine synthesis by IκBα degradation and MAPK activation in macrophages.

The bark of Prunus yedoensis is used in antitussive medicines and in oral herbal formulations for inflammatory skin disorders. In the present study, we explored whether P. yedoensis bark extract (PYE) and its solvent partitioned fractions could modulate lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α and interleukin (IL)-6 in vivo and in vitro. In addition, we examined the effect of PYE extract and its fractions on LPS-induced NF-κB and mitogen-activated protein kinase (MAPK) signaling in mouse peritoneal macrophages. Oral treatment of PYE decreased serum levels of TNF-α and IL-6 in LPS injected mice. PYE inhibited LPS-induced TNF-α and IL-6 in macrophages at the transcriptional level and also suppressed LPS-induced IκBα degradation and MAPK activation in vitro. Among the fractions, the chloroform fraction, which contains genistein, naringenin, sakuranetin, prunetin, and amygdalin, showed inhibitory effects at much lower concentrations than the water and ethyl acetate fractions. Taken together, our results indicate that PYE was able to inhibit LPS-induced expression of TNF-α and IL-6, the latter of which was more prominent. The effects of PYE on inflammatory cytokine synthesis may involve modulation of NF-κB and MAPK activation.

ATF-2/CREB/IRF-3-targeted anti-inflammatory activity of Korean red ginseng water extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Korean Red Ginseng (KRG) is one of the representative traditional herbal medicines prepared from Panax ginseng Meyer (Araliaceae) in Korea. It has been reported that KRG exhibits a lot of different biological actions such as anti-aging, anti-fatigue, anti-stress, anti-atherosclerosis, anti-diabetic, anti-cancer, and anti-inflammatory activities. Although systematic studies have investigated how KRG is able to ameliorate various inflammatory diseases, its molecular inhibitory mechanisms had not been carried out prior to this study. MATERIALS AND METHODS: In order to investigate these mechanisms, we evaluated the effects of a water extract of Korean Red Ginseng (KRG-WE) on the in vitro inflammatory responses of activated RAW264.7 cells, and on in vivo gastritis and peritonitis models by analyzing the activation events of inflammation-inducing transcription factors and their upstream kinases. RESULTS: KRG-WE reduced the production of nitric oxide (NO), protected cells against NO-induced apoptosis, suppressed mRNA levels of inducible NO synthase (iNOS), cyclooxygenase (COX)-2, and interferon (IFN)-ß, ameliorated EtOH/HCl-induced gastritis, and downregulated peritoneal exudate-derived NO production from lipopolysaccharide (LPS)-injected mice. The inhibition of these inflammatory responses by KRG-WE was regulated through the suppression of p38, c-Jun N-terminal kinase (JNK), and TANK-binding kinase 1 (TBK1) and by subsequent inhibition of activating transcription factor (ATF)-2, cAMP response element-binding protein (CREB), and IRF-3 activation. Of ginsensides included in this extract, interestingly, G-Rc showed the highest inhibitory potency on IRF-3-mediated luciferase activity. CONCLUSION: These results strongly suggest that the anti-inflammatory activities of KRG-WE could be due to its inhibition of the p38/JNK/TBK1 activation pathway.

Dichloromethane extracts of Sophora japonica L. stimulate osteoblast differentiation in mesenchymal stem cells.

Sophora japonica L. fruit prevents bone loss by inhibiting osteoclast activity. We hypothesized that S japonica L. extracts could promote osteoblast differentiation. To test this hypothesis, we investigated the effect of S japonica L. on osteoblast differentiation and identified the bioactive compound(s) from S japonica L. The mature fruit of S japonica L. was partitioned with ethanol, hexane, dichloromethane (DCM), ethyl acetate, and butanol, and their effects were tested on osteoblast differentiation of C3H10T1/2 cells. DCM fractionated extracts were identified as the most osteogenic fractions. DCM fractionated extracts dose-dependently stimulated alkaline phosphatase activity and matrix mineralization. The DCM fractions also induced expression of osteoblast markers such as alkaline phosphatase, osterix, and osteocalcin in C3H10T1/2 and primary bone marrow cells. Genistein was found abundantly in the DCM fractions. Furthermore, the genistein and DCM fractions similarly modulated the expression of estrogen target genes and were both active in transfection assays that measured estrogen agonistic activity. Finally, pharmacological inhibition by treatment with an estrogen receptor antagonist or specific inhibition of gene expression by small interference RNAs targeted to estrogen receptor-ß abolished the effects of the DCM extracts, further supporting the idea that the genistein in the DCM extracts mediated the pro-osteogenic effects. Taken together, we identified genistein as the key phytoestrogen responsible for the effects of S japonica L. on osteoblast differentiation.

Butein is a novel anti-adipogenic compound.

Rhus verniciflua Stokes (RVS) has been used as a traditional herbal medicine for its various biological activities including anti-adipogenic effects. Activity-guided separation led to the identification of the anti-adipogenic functions of butein. Butein, a novel anti-adipogenic compound, robustly suppressed lipid accumulation and inhibited expression of adipogenic markers. Molecular studies showed that activated transforming growth factor-ß (TGF-ß) and suppressed signal transducer and activator of transcription 3 (STAT3) signaling pathways were mediated by butein. Analysis of the temporal expression profiles suggests that TGF-ß signaling precedes the STAT3 in the butein-mediated anti-adipogenic cascade. Small interfering RNA-mediated silencing of STAT3 or SMAD2/3 blunted the inhibitory effects of butein on adipogenesis indicating that an interaction between two signaling pathways is required for the action of butein. Upon butein treatments, stimulation of TGF-ß signaling was still preserved in STAT3 silenced cells, whereas regulation of STAT3 signaling by butein was significantly impaired in SMAD2/3 silenced cells, further showing that TGF-ß acts upstream of STAT3 in the butein-mediated anti-adipogenesis. Taken together, the present study shows that butein, a novel anti-adipogenic compound from RVS, inhibits adipocyte differentiation through the TGF-ß pathway followed by STAT3 and peroxisome proliferator-activated receptor γ signaling, further implicating potential roles of butein in TGF-ß- and STAT3-dysregulated diseases.

Polyphenols differentially inhibit degranulation of distinct subsets of vesicles in mast cells by specific interaction with granule-type-dependent SNARE complexes.

Anti-allergic effects of dietary polyphenols were extensively studied in numerous allergic disease models, but the molecular mechanisms of anti-allergic effects by polyphenols remain poorly understood. In the present study, we show that the release of granular cargo molecules, contained in distinct subsets of granules of mast cells, is specifically mediated by two sets of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, and that various polyphenols differentially inhibit the formation of those SNARE complexes. Expression analysis of RBL-2H3 cells for 11 SNARE genes and a lipid mixing assay of 24 possible combinations of reconstituted SNAREs indicated that the only two active SNARE complexes involved in mast cell degranulation are Syn (syntaxin) 4/SNAP (23 kDa synaptosome-associated protein)-23/VAMP (vesicle-associated membrane protein) 2 and Syn4/SNAP-23/VAMP8. Various polyphenols selectively or commonly interfered with ternary complex formation of these two SNARE complexes, thereby stopping membrane fusion between granules and plasma membrane. This led to the differential effect of polyphenols on degranulation of three distinct subsets of granules. These results suggest the possibility that formation of a variety of SNARE complexes in numerous cell types is controlled by polyphenols which, in turn, might regulate corresponding membrane trafficking.

Genistein mediates the anti-adipogenic actions of Sophora japonica L. extracts.

Previous studies showed that feeding diets containing the mature fruits of Sophora japonica L. prevented body weight gain and reduced fat mass in high-fat diet-induced obese mice. This observation has led to the hypothesis that extracts from S. japonica L. may inhibit adipocyte differentiation of preadipocytes. To elucidate the possible mechanisms for the anti-obesity action of S. japonica L., its effects on adipocyte differentiation were investigated in C3H10T1/2 mesenchymal stem cells and 3T3-L1 preadipocyte cells. The mature fruit of S. japonica L. was partitioned with ethanol, hexane, dichloromethane, ethyl acetate (EtOAc), and butanol to identify the active fractions. The EtOAc fraction extracts inhibited morphological differentiation and lipid accumulation in the C3H10T1/2 and 3T3-L1 preadipocytes. Molecular studies indicated that the EtOAc fraction extracts also reduced the expression of peroxisome proliferator-activated receptor γ and other adipocyte markers. Furthermore, among the fractions, the EtOAc fraction extracts had the highest total phenolic contents, suggesting that the polyphenols in the EtOAc fractions mediated the anti-adipogenic effects. Finally, high-performance liquid chromatography identified genistein, a known anti-adipogenic compound, as the probable mediator of the anti-adipogenic effects of the EtOAc fractions. This work validates the beneficial roles of S. japonica L. in controlling body weight and obesity-related metabolic diseases.

Selenium attenuates A beta production and A beta-induced neuronal death.

The objective of the present study was to examine the role of selenium in the metabolism of A beta and in A beta-induced neuronal death. Selenium treatment significantly reduced A beta 40, A beta 42, and sAPP beta production by reducing A beta producing beta-secretase and gamma-secretase activities. The lipid peroxidation product 4-Hydroxynonenal (HNE)-induced transcription of beta-secretase (BACE1) was blocked by selenium. Finally, our data show that selenium protects against HNE and A beta-mediated toxicity in primary cultured neurons. The present study suggests that selenium may be able to salvage the neuronal degeneration of Alzheimer's disease, thereby limiting beta-amyloid production and neuronal death.

Diets containing Sophora japonica L. prevent weight gain in high-fat diet-induced obese mice.

Obesity, a worldwide epidemic, is associated with metabolic diseases such as insulin resistance, dyslipidemia, hypertension, and heart disease. Many strategies, including natural alternative antiobesity agents, have been widely used to prevent obesity. Polyphenolic compounds and flavonoids from natural products are shown to inhibit adipogenesis. Because mature fruits of Sophora japonica L. were previously shown to contain antiadipogenic compounds, we hypothesized that diets with mature fruits of S japonica L. would prevent body weight gain in high-fat diet-induced obesity. Four-week-old mice were fed either a control high-fat diet, or high-fat diet containing 1% or 5% of S japonica L. for 4 weeks. The administration of S japonica L. fed in combination with a 30% high-fat diet significantly decreased body weight gain. S japonica L. also reduced serum and hepatic triglyceride, serum total, and high-density lipoprotein cholesterol. Consistent with the effects of lowering glucose level and fat mass, S japonica L. caused a decrease in the number of large adipocytes and a concomitant increase in the number of small adipocytes, which may explain at least in part the antiobesity effects of S japonica L. Together, these data provide evidence for roles of S japonica L. in the control of body weight and obesity-related metabolic diseases.