Two new secondary metabolites isolated from Avena sativa L. (Oat) seedlings and their effects on osteoblast differentiation.

Seedlings of natural crops are valuable sources of pharmacologically active phytochemicals. In this study, we aimed to identify new active secondary metabolites in Avena sativa L. (oat) seedlings. Two new compounds, avenafuranol (1) and diosgenoside (2), along with eight known compounds (3-10) were isolated from the A. sativa L. seedlings. Their chemical structures were elucidated via 1D and 2D NMR spectroscopy, high-resolution ESIMS, IR spectroscopy, optical rotation analysis, and comparisons with the reported literature. The effect of each isolated compound on alkaline phosphatase (ALP) activity for osteoblast differentiation induced by bone morphogenetic protein-2 (BMP-2) was investigated using the C2C12 immortal mouse myoblast cell line. Compounds 1, 4, 6, 8, and 9 induced dose-dependent increases in ALP expression relative to ALP expression in cells treated with only BMP-2, and no cytotoxicity was observed. These results suggest that A. sativa L. seedlings are a natural source of compounds that may be useful for preventing bone disorders.

Gypenoside UL4-Rich Gynostemma pentaphyllum Extract Exerts a Hepatoprotective Effect on Diet-Induced Nonalcoholic Fatty Liver Disease.

Nonalcoholic steatohepatitis (NASH) arises from nonalcoholic fatty liver disease (NAFLD) as a consequence of oxidative stress. Gynostemma pentaphyllum extract (GPE) is proven to be beneficial for patients suffering from NAFLD. However, the precise mechanism by which GPE confers these benefits remains largely unknown. The purpose of this study was to investigate the underlying mechanism and to determine whether supplementation with the newly discovered GPE gypenoside UL4 mitigates NASH progression. Male c57BL/6 mice were fed a normal chow diet, a methionine choline-deficient (MCD) diet, or an MCD diet supplemented with various doses of UL4-rich GPE for eight weeks. GPE supplementation suppressed oxidative stress induced by the MCD diet by increasing levels of sirtuin 6 and phase 2 anti-oxidant enzymes in mouse liver and HepG2 cells. Additionally, GPE supplementation prevented diet-induced hepatic fat accumulation, hepatocellular injury, inflammation, and fibrosis in mice fed the MCD diet. These results indicate the possible therapeutic potential of dietary supplementation of UL4-rich GPE in preventing the development of fatty liver and its progression to NASH.

Inhibitory effect of esculetin on free-fatty-acid-induced lipid accumulation in human HepG2 cells through activation of AMP-activated protein kinase.

This study aimed to determine the lipid-lowering effect of esculetin (6,7-dihydroxycoumarin), a coumarin derivative, using a cell model of steatosis induced by a mixture of free fatty acids (FFAs). Esculetin dose-dependently inhibited intracellular lipid accumulation by down-regulating the protein expression of lipogenic genes such as sterol regulatory element-binding protein-1c (SREBP1c) and fatty acid synthase (FAS) in FFAs-induced HepG2 cells. Moreover, esculetin significantly elevated the activation of the adenosine monophosphate-activated protein kinase (AMPK) signaling pathways in HepG2 hepatocytes. The anti-lipogenic effects of esculetin mediated by AMPK activation were abolished when FFAs-induced HepG2 cells were treated with a specific inhibitor of AMPK, i.e., compound C. These results suggest that esculetin attenuates hepatic lipid accumulation by inhibiting lipogenesis through the modulation of AMPK signaling pathway on FFAs-induced steatosis in HepG2 cells and may be used for the prevention of nonalcoholic fatty liver disease (NAFLD).

Anti-inflammatory effect of methanol extract from Erigeron Canadensis L. may be involved with upregulation of heme oxygenase-1 expression and suppression of NFκB and MAPKs activation in macrophages.

BACKGROUND/OBJECTIVES: In this study, we determined the anti-inflammatory activities and the underlying molecular mechanisms of the methanol extract from Erigeron Canadensis L. (ECM) in LPS-stimulated RAW264.7 macrophage cells. MATERIALS/METHODS: The potential anti-inflammatory properties of ECM were investigated by using RAW264.7 macrophages. We used western blot assays and real time quantitative polymerase chain reaction to detect protein and mRNA expression, respectively. Luciferase assays were performed to determine the transactivity of transcription factors. RESULTS: ECM significantly inhibited inducible nitric oxide synthase (iNOS)-derived NO and cyclooxygenase-2 (COX-2) derived PGE2 production in LPS-stimulated RAW264.7 macrophages. These inhibitory effects of ECM were accompanied by decreases in LPS-induced nuclear translocations and transactivities of NFκB. Moreover, phosphorylation of mitogen-activated protein kinase (MAPKs) including extracellular signal-related kinase (ERK1/2), p38, and c-jun N-terminal kinase (JNK) was significantly suppressed by ECM in LPS-stimulated RAW264.7 macrophages. Further studies demonstrated that ECM by itself induced heme oxygenase-1 (HO-1) protein expression at the protein levels in dose-dependent manner. However, zinc protoporphyrin (ZnPP), a selective HO-1 inhibitor, abolished the ECM-induced suppression of NO production. CONCLUSIONS: These results suggested that ECM-induced HO-1 expression was partly responsible for the resulting anti-inflammatory effects. These findings suggest that ECM exerts anti-inflammatory actions and help to elucidate the mechanisms underlying the potential therapeutic values of Erigeron Canadensis L.

Black rice extract protected HepG2 cells from oxidative stress-induced cell death via ERK1/2 and Akt activation.

BACKGROUND/OBJECTIVES: The objective of this study was to evaluate the protective effect of black rice extract (BRE) on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. MATERIALS/METHODS: Methanolic extract from black rice was evaluated for the protective effect on TBHP-induced oxidative injury in HepG2 cells. Several biomarkers that modulate cell survival and death including reactive oxygen species (ROS), caspase-3 activity, and related cellular kinases were determined. RESULTS: TBHP induced cell death and apoptosis by a rapid increase in ROS generation and caspase-3 activity. Moreover, TBHP-induced oxidative stress resulted in a transient ERK1/2 activation and a sustained increase of JNK1/2 activation. While, BRE pretreatment protects the cells against oxidative stress by reducing cell death, caspase-3 activity, and ROS generation and also by preventing ERKs deactivation and the prolonged JNKs activation. Moreover, pretreatment of BRE increased the activation of ERKs and Akt which are pro-survival signal proteins. However, this effect was blunted in the presence of ERKs and Akt inhibitors. CONCLUSIONS: These results suggest that activation of ERKs and Akt pathway might be involved in the cytoprotective effect of BRE against oxidative stress. Our findings provide new insights into the cytoprotective effects and its possible mechanism of black rice against oxidative stress.

Protective effects of oligomeric and polymeric procyanidin fractions from defatted grape seeds on tert-butyl hydroperoxide-induced oxidative damage in HepG2 cells.

Oligomeric and polymeric procyanidins have been reported to possess different antioxidant capacities. However, the intracellular antioxidant mechanisms of oligomeric and polymeric procyanidins are still poorly understood. In this study, we evaluated the cytoprotective effects of the oligomeric procyanidin fraction (OPF) and the polymeric procyanidin fraction (PPF) from grape seeds against the oxidative damage induced by tert-butyl hydroperoxide (TBHP) in HepG2 cells. The levels of cellular reactive oxygen species (ROS), cellular lipid peroxidation, glutathione (GSH), and antioxidant enzyme activities were measured as biomarkers of cellular oxidative status. HepG2 cells were treated with different concentrations of procyanidin samples (0-20 µg/ml) for 6h prior to treatment with TBHP for 3h. The incubation of HepG2 cells with TBHP led to an approximately 60% decrease in cell viability. However, pretreatment of the cells with the samples, at 5-20 µg/ml, rescued cell viability in a dose-dependent manner. Cellular generation of ROS, formation of malondialdehyde (MDA), and depletion of GSH were reduced by OPF and PPF. Moreover, TBHP treatment increased the activities of glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD). However, a 6h pretreatment with each of the samples at 20 µg/ml significantly decreased the activities of these enzymes. These results clearly showed that treatment with OPF and PPF protected against oxidative damage by modulating ROS production, GSH levels, MDA generation, and antioxidant enzyme activities in HepG2 cells.

Antioxidant and cytoprotective effects of oligomeric and polymeric procyanidin fractions from defatted grape seed in PC12 cells.

In this study, a crude polyphenol extract (CPE) from defatted grape seeds was evaluated for antioxidant and cytoprotective effects in PC12 cells. The CPE was then fractionated into a catechin and oligomeric procyanidin fraction (F(cat+olig)) and a polymeric procyanidin fraction (F(pol)), and the bioactivities of the fractions were evaluated individually. F(pol) was the richest in total polyphenol, total flavonoid, and polymeric procyanidin but contained less flavan-3-ol than F(cat+olig). Consistent with these results, F(pol) had the highest 2,2-diphenyl-ß-picrylhydrazyl radical scavenging activity and reducing power. Both F(cat+olig) and F(pol) had higher 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic) acid radical scavenging activity than CPE. The resistance of PC12 cells against oxidative stress, after pretreatment with F(pol) (200 µg/mL) for 9 h, was 100.7±2.9% of that in the control. Moreover, pretreating these cells with F(cat+olig) and F(pol) significantly decreased reactive oxygen species generation. These results provide information on a potential new source of antioxidants in the form of a functional food derived from an agricultural by-product.

Nobiletin suppresses adipogenesis by regulating the expression of adipogenic transcription factors and the activation of AMP-activated protein kinase (AMPK).

The objective of this study was to elucidate the effect of nobiletin (5,6,7,8,3',4'-hexamethoxyflavone) on adipogenesis in 3T3-L1 cells. To determine the effect of nobiletin on adipogenesis, preadipocyte differentiation was induced in the presence or absence of nobiletin (10-100 µM) for 4 days. The results revealed that nobiletin markedly inhibited lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity and blocked the expression of adipogenic transcription factors, including peroxisome proliferator-activated receptors (PPARγ) and CCAAT/enhancer binding proteins (C/EBPα). Moreover, nobiletin significantly increased AMP-activated protein kinase (AMPK), a major regulator of cellular energy balance, phosphorylation, and intracellular reactive oxygen species (ROS) generation. This study also investigated the involvement of AMPK in the expression of a major transcription factor, PPARγ. It was found that pretreatment with compound C, a cell permeable inhibitor of AMPK, abolished the inhibitory effects of nobiletin on PPARγ expression. The results suggest that nobiletin exerts antiadipogenic effects through modulation of the PPARγ and AMPK signaling pathway and, therefore, may be a promising antiobesity agent.