Cover Picture: Mol. Nutr. Food Res. 11'2010

Regular issues provide a wide range of research and review articles covering all aspects of Molecular Nutrition & Food Research. Selected topics of issue 11 are: Flavan‐3‐ol C‐glycosides – preparation and model experiments mimicking their human intestinal transit Resveratrol confers resistance against taxol via induction of cell cycle arrest in human cancer cell lines Kaempferol induced apoptosis via endoplasmic reticulum stress and mitochondria‐dependent pathway in human osteosarcoma U‐2 OS cells Diosgenin present in fenugreek improves glucose metabolism by promoting adipocyte differentiation and inhibiting inflammation in adipose tissues Plasma phospholipids n‐3 polyunsaturated fatty acid is associated with metabolic syndrome     

Chinese Yam Extracts Containing β‐Sitosterol and Ethyl Linoleate Protect against Atherosclerosis in Apolipoprotein E‐Deficient Mice and Inhibit Muscular Expression of VCAM‐1 In Vitro

Atherosclerosis is a chronic inflammatory disease, which is associated with increased expression of adhesion molecules and monocyte recruitment into the arterial wall. This study evaluated whether hexane extracts from the edible part (DB‐H1) or bark region (DB‐H2) of Dioscorea. batatas Decne have anti‐atherosclerotic properties in vivo and in vitro experiments. We also identified bioactive components in the hexane extracts. Thirty‐six apolipoprotein E (ApoE^?/?) mice and 12 control (C57BL/6J) mice were given a Western‐type diet for 11 or 21 wk. To examine the effects of yam extracts on lesion development, ApoE^?/? mice were orally administered DB‐H1 or DB‐H2 for the duration of the study (200 mg/kg b.w./day, 3 times per wk). Both DB‐H1 and DB‐H2 significantly reduced the total atherosclerotic lesion area in the aortic root. In addition, plasma concentrations of total cholesterol, oxidized‐low‐density lipoprotein, and c‐reactive protein were decreased by administration of DB‐H1 and DB‐H2. Consistent with the in vivo observations, DB‐H1 and DB‐H2 inhibited tumor necrosis factor (TNF)‐α–induced vascular cell adhesion molecule‐1 expression and adhesion of THP‐1 monocytes to TNF‐α–activated vascular smooth muscle cells. It was also found that treatment with DB‐H1 or DB‐H2 resulted in the inhibition nitric oxide (NO) and reactive oxygen species production and iNOS expression in macrophages. Thus, DB‐H1 and DB‐H2 seem to influence atherosclerosis by affecting the production of inflammatory mediators in vivo. Our results suggest that yam extracts have the potential to be used in the prevention of atherosclerosis.     

Anti‐inflammatory and cytotoxic effects of ginseng extract bioconverted by Leuconostoc mesenteroides KCCM 12010P isolated from kimchi

A bioconversion technique using microorganisms has been applied to ginseng to increase content of bioactive ginsenoside and biofunctionality such as anticancer, anti‐obesity and antioxidant activities. The objective of this study was to screen lactic acid bacteria for bioconversion of ginsenosides and to evaluate anti‐inflammatory and cytotoxic effects of bioconverted ginseng extract. Strains isolated from kimchi were screened for their β‐glucosidase activities using esculin agar. Selected strain was identified based on 16S rRNA sequencing and carbohydrate fermentation. During ginseng fermentation, viable cell number and pH were determined. Bioconverted ginsenosides were analysed by HPLC. Anti‐inflammatory effects were evaluated using RAW 264.7 cells, and cytotoxic effects were determined by MTT assay. Among 166 isolates screened, Leuconostoc mesenteroides was selected for ginseng bioconversion, as it showed a higher β‐glucosidase activity and viable cell number than any of the other tested strains. After fermentation for 2 days, viable cell number was 8.8 log CFU mL^?1 and final pH was 4.8. Ginsenoside Rb₂ was bioconverted into ginsenoside Rg₃ (Rb₂ → Rd → Rg₃) by L. mesenteroides. The nitric oxide contents of 2‐day‐fermented extract decreased by as much as 25%, compared to a non‐fermented extract. The cell viabilities of HepG2, HT‐29, HeLa and LoVo treated with fermented ginseng extract also decreased by 49.7%, 20.2%, 21.0% and 8.7%, respectively, compared to those of control cells treated with non‐fermented extract. Ginseng extract bioconverted by L. mesenteroides showed anti‐inflammatory and anticancer effects. Therefore, bioconverted ginseng extract might have applications in the pharmaceutical and/or functional food industry.     

Anti‐inflammatory and anticancer activities of water‐soluble peptide extracts of buffalo and cow milk Cheddar cheeses

This article aimed to assess the anti‐inflammatory and anticancer potential of water‐soluble peptide (WSP) extracts from buffalo and cow milk Cheddar cheeses. Anti‐inflammatory activity was evaluated on the basis of nitric oxide (NO) production in lipopolysaccharide‐stimulated macrophage (RAW‐264.7) cells. A cell viability assay, cell cycle arrest and apoptosis were performed to explore anticancer activity in a colon cancer model (HT‐29). The WSP extracts of both Cheddar cheeses effectively inhibited NO production in activated macrophages. Maximum growth inhibition was observed in the HT‐29 cells at concentrations of 400 and 500 μg/mL. A significant increase in cell population at G₀/G₁ phase of the cell cycle was observed. Moreover, the WSP extracts also induced extensive apoptosis in colon cancer cells.     

Quercetin‐induced apoptotic cascade in cancer cells: Antioxidant versus estrogen receptor α‐dependent mechanisms

The flavonol quercetin, especially abundant in apple, wine, and onions, is reported to have anti‐proliferative effects in many cancer cell lines. Antioxidant or pro‐oxidant activities and kinase inhibition have been proposed as molecular mechanisms for these effects. In addition, an estrogenic activity has been observed but, at the present, it is poorly understood whether this latter activity plays a role in the quercetin‐induced anti‐proliferative effects. Here, we studied the molecular mechanisms of quercetin committed to the generation of an apoptotic cascade in cancer cells devoid or containing transfected estrogen receptor α (ERα; i.e., human cervix epitheloid carcinoma HeLa cells). Although none of tested quercetin concentrations increase reactive oxygen species (ROS) generation in HeLa cells, quercetin stimulation prevents the H₂O₂‐induced ROS production both in the presence and in the absence of ERα. However, this flavonoid induces the activation of p38/MAPK, leading to the pro‐apoptotic caspase‐3 activation and to the poly(ADP‐ribose) polymerase cleavage only in the presence of ERα. Notably, no down‐regulation of survival kinases (i.e., AKT and ERK) was reported. Taken together, these findings suggest that quercetin results in HeLa cell death through an ERα‐dependent mechanism involving caspase‐ and p38 kinase activation. These findings indicate new potential chemopreventive actions of flavonoids on cancer growth.     

Resveratrol inhibits migration and invasion of human breast‐cancer cells

Metastasis is the primary cause of death from breast cancer. Cell migration and invasion play important roles in neoplastic metastasis. The insulin‐like growth factor (IGF‐1) stimulates cell migration through activation of PI‐3K/Akt signaling pathway. IGF‐1 induces the tumorigenicity of many types of cancer cells and is critical for metastatic cell spread in estrogen receptor (ER)‐negative breast‐cancer cells. Matrix metalloproteinase‐2 (MMP‐2) is a key enzyme in the degradation of extracellular matrices and its expression has been dysregulated in breast cancer invasion and metastasis. Resveratrol exhibited potential anticarcinogenic activities in several studies. However, the inhibitory effects of resveratrol on the expression of MMP‐2, migration and invasion of breast‐cancer cell have not been demonstrated yet. In the present study, we investigated the anti‐invasive mechanism of resveratrol in human breast cancer MDA‐MB 435cells. Here, we showed that IGF‐1 is a potent stimulant of the migration of ER‐negative human breast‐cancer cells. Resveratrol could inhibit IGF‐1‐mediated cell migration of MDA‐MB 435 in vitro. The inhibitory effect of resveratrol was mediated in part through the suppression of the activation of PI‐3K/Akt signaling pathway. Furthermore, IGF‐1‐mediated expression of MMP‐2 was significantly inhibited by resveratrol in concomitance with alteration of cell invasion.     

RAPA: a novel in vitro method to evaluate anti‐bacterial skin cleansing products

Development of efficacious anti‐bacterial skin cleansing products has been limited by the availability of a pre‐clinical (in vitro) method to predict clinical efficacy adequately. We report a simple and rapid method, designated as rapid agar plate assay (RAPA), that uses the bacteriological agar surface as a surrogate substrate for skin and combines elements of two widely used in vivo (clinical) methods (Agar Patch and Cup Scrub). To simulate the washing of the human hand or forearm skin with the test product, trypticase soy agar plates were directly washed with the test product and rinsed under running tap water. After air‐drying the washed plates, test bacteria (Staphylococcus aureus or Escherichia coli) were applied and the plates were incubated at 37°C for 18–24 h. Using S. aureus as the test organism, anti‐bacterial bar soap containing triclocarbanilide showed a strong linear relationship (R² = 0.97) between bacterial dose and their per cent reduction. A similar dose‐response relationship (R² = 0.96) was observed for anti‐bacterial liquid hand soap against E. coli. RAPA was able to distinguish between anti‐bacterial products based on the nature and level of actives in them. In limited comparative tests, results obtained by RAPA were comparable with the results obtained by clinical agar patch and clinical cup scrub methods. In conclusion, RAPA provides a simple, rugged and reproducible in vitro method for testing the relative efficacy of anti‐bacterial skin cleansing products with a likelihood of comparable clinical efficacy. Further testing is warranted to improve the clinical predictability of this method.     

Anticancer activity of bovine α‐lactalbumin treated with microbial transglutaminase

This study investigated the effects of bovine α‐lactalbumin (α‐La) treated with microbial transglutaminase on human cancer cells, cell cultures and growth rate assays. The anticancer activity for 10 mg/mL of bovine α‐lactalbumin (α‐La) was measured as ～90% in a human colorectal cancer cell line HCT 116. For the human bone cancer cell line SJSA‐1, α‐La hydrolysis resulted in higher cytotoxicity compared to untreated tumour cells. The formation of polymers of α‐La was suppressed by the addition of ethylenediaminetetraacetic acid, indicating that polymers of α‐La are promoted by metal ions such as Ca²⁺. The effect of α‐La on the morphology of SJSA‐1 cells was manifested as morphological changes compatible with apoptosis. Bovine milk α‐La with and without microbial transglutaminase is considered a valuable food ingredient and a nutraceutical for human health.     

Salvianolic acid B inhibits low‐density lipoprotein oxidation and neointimal hyperplasia in endothelium‐denuded hypercholesterolaemic rabbits

BACKGROUND: Atherosclerosis and restenosis are inflammatory responses involving free radicals and lipid peroxidation and may be prevented/cured by antioxidant‐mediated lipid peroxidation inhibition. Salvianolic acid (Sal B), a water‐soluble antioxidant obtained from a Chinese medicinal herb, is believed to have multiple preventive and therapeutic effects against human vascular diseases. In this study the in vitro and in vivo inhibitory effects of Sal B on oxidative stress were determined. RESULTS: In human aortic endothelial cells (HAECs), Sal B reduced oxidative stress, inhibited low‐density lipoprotein (LDL) oxidation and reduced oxidised LDL‐induced cytotoxicity. Sal B inhibited Cu²⁺‐induced LDL oxidation in vitro (with a potency 16.3 times that of probucol) and attenuated HAEC‐mediated LDL oxidation as well as reactive oxygen species (ROS) production. In cholesterol‐fed New Zealand White rabbits (with probucol as positive control), Sal B intake reduced Cu²⁺‐induced LDL oxidation, lipid deposition in the thoracic aorta, intimal thickness of the aortic arch and thoracic aorta and neointimal formation in the abdominal aorta. CONCLUSION: The data obtained in this study suggest that Sal B protects HAECs from oxidative injury‐mediated cell death via inhibition of ROS production. The antioxidant activity of Sal B may help explain its efficacy in the treatment of vascular diseases. Copyright © 2010 Society of Chemical Industry     

Anti‐inflammatory properties of high pressure‐assisted extracts of Grifola frondosa in lipopolysaccharide‐activated RAW 264.7 macrophages

The aim of this study was to determine the in vitro anti‐inflammatory properties of the shake extract (SE) and the high pressure‐assisted extract (PE) of the mycelia of Grifola frondosa in a lipopolysaccharide‐stimulated RAW 264.7 macrophage model. The content of total polysaccharides and β‐glucans of PE at 600 MPa (PE‐600) was 41.2 and 6.2 mg g^?1 dry weight, respectively, which were significantly higher than SE extracts. The results showed that treatment with 500 μg mL^?1 of PE by 600 MPa (PE‐600) did not reduce RAW 264.7 cell viability but did significantly inhibit the production of LPS‐induced NO, PGE₂ and intracellular ROS. The PE‐600 inhibited the activation of NF‐kB and then reduced the production of LPS‐induced TNF‐α, IL‐6 and IL‐1β in a dose‐dependent manner. Thus, the PE could be used as an alternative extraction method for improving the extraction efficacy of G. frondosa and serve as an alternative source of anti‐inflammatory agents.     

Anti‐proliferative activity of bovine blood hydrolysates towards cancer cells in culture

Four proteins, α/β globulin, serum albumin, γ‐globulin and fibrinogen, were isolated from bovine blood and hydrolysed using papain. Hydrolysates were assessed for non‐cellular and cellular antioxidant activity. The anti‐proliferative activity of hydrolysed fractions was assessed in a number of cancer cell lines including U937 lymphoma cells, MCF‐7 breast cancer cells, HepG2 hepatocytes and Caco‐2 epithelial colorectal adenocarcinoma cells. Anti‐inflammatory activity of the hydrolysates was also assessed. Hydrolysates generated from γ‐globulin or fibrinogen had significant antioxidant activity in non‐cellular assays. Hydrolysates were also found to be highly toxic to different cancer cell lines, in particular U937 lymphoma cells when assessed using the MTT assay. The fibrinogen hydrolysate was the most toxic sample and toxicity appeared to correlate with its non‐cellular antioxidant activity. None of the hydrolysates had significant anti‐inflammatory activity. The high cytotoxicity of the γ‐globulin and the fibrinogen hydrolysates towards cancer cells may indicate a potential use as anti‐proliferative agents.     

S‐Allyl cysteine,S‐ethyl cysteine and S‐propyl cysteine alleviate oxidative stress‐induced damage within PC12 cells

BACKGROUND: The PC12 cell line is a suitable model for the investigation of neurodegenerative diseases. In this study, PC12 cells were used to examine in vitro antioxidative and antiapoptotic protection by S‐allyl cysteine (SAC), S‐ethyl cysteine (SEC) and S‐propyl cysteine (SPC). PC12 cells were treated with these agents at 5 and 10 µmol L^?1 before exposure to hydrogen peroxide (H₂O₂). RESULTS: H₂O₂ treatment significantly decreased mitochondrial membrane potential (MMP) and cell viability and increased lactate dehydrogenase (LDH) release and DNA fragmentation (P < 0.05). The pre‐treatments with SAC, SEC and SPC significantly and concentration‐dependently elevated cell viability and MMP and lowered LDH release and DNA fragmentation (P < 0.05). H₂O₂ treatment also significantly increased levels of malondialdehyde (MDA), reactive oxygen species (ROS) and oxidised glutathione (GSSG) and decreased glutathione (GSH) content (P < 0.05). The pre‐treatments with SAC, SEC and SPC significantly decreased subsequent H₂O₂‐induced formation of MDA, ROS and GSSG (P < 0.05) and also alleviated H₂O₂‐induced GSH depletion (P < 0.05). Finally, H₂O₂ treatment significantly decreased Na⁺‐K⁺‐ATPase activity and elevated caspase‐3 activity (P < 0.05). The pre‐treatments with SAC, SEC and SPC significantly attenuated H₂O₂‐induced Na⁺‐K⁺‐ATPase activity reduction and caspase‐3 activity elevation (P < 0.05). CONCLUSION: The results obtained support that the three cysteine‐containing compounds studied are potent neuroprotective agents. Copyright © 2008 Society of Chemical Industry     

Cranberry extract and quercetin modulate the expression of cyclooxygenase‐2 (COX‐2) and IκBα in human colon cancer cells

BACKGROUND: Cranberry (Vaccinium marcocarpon) fruit and quercetin, a major flavonoid found in cranberries, are likely contributors to chemoprevention, and their anti‐inflammatory activities may play a potential role in colon cancer prevention. The aim of this study was to examine the effect of cranberry extract and quercetin on basal expression of cyclooxygenase‐2 (COX‐2) and IκBα as well as the effect on phorbol 12‐myristate 13‐acetate (PMA)‐induced COX‐2 expression in colon cancer cells. RESULTS: HT‐29 human colon adenocarcinoma cells were treated with various concentrations of cranberry extract or quercetin and/or PMA, and the protein expression of COX‐2 and IκBα was determined. The results indicated that cranberry extract and quercetin decreased COX‐2 expression and suppressed degradation of IκBα in unstimulated cells. In PMA‐stimulated cells, cranberry extract was also able to decrease COX‐2 expression and suppress degradation of IκBα. CONCLUSION: The results suggest that a possible mechanism involved in the anti‐cancer activity of cranberry and quercetin is partly mediated through its anti‐inflammatory action. These findings indicate that cranberry and quercetin may reduce the risk of colon cancer possibly by suppressing inflammatory responses. Copyright © 2008 Society of Chemical Industry     

Sesamin attenuates intercellular cell adhesion molecule‐1 expression in vitro in TNF‐α‐treated human aortic endothelial cells and in vivo in apolipoprotein‐E‐deficient mice

Sesame lignans have antioxidative and anti‐inflammatory properties. We focused on the effects of the lignans sesamin and sesamol on the expression of endothelial‐leukocyte adhesion molecules in tumor necrosis factor‐α (TNF‐α)‐treated human aortic endothelial cells (HAECs). When HAECs were pretreated with sesamin (10 or 100 μM), the TNF‐α‐induced expression of intercellular cell adhesion molecule‐1 (ICAM‐1) was significantly reduced (35 or 70% decrease, respectively) by Western blotting. Sesamol was less effective at inhibiting ICAM‐1 expression (30% decrease at 100 μM). Sesamin and sesamol reduced the marked TNF‐α‐induced increase in human antigen R (HuR) translocation and the interaction between HuR and the 3'UTR of ICAM‐1 mRNA. Both significantly reduced the binding of monocytes to TNF‐α‐stimulated HAECs. Sesamin significantly attenuated TNF‐α‐induced ICAM‐1 expression and cell adhesion by downregulation of extracellular signal‐regulated kinase 1/2 and p38. Furthermore, in vivo, sesamin attenuated intimal thickening and ICAM‐1 expression seen in aortas of apolipoprotein‐E‐deficient mice. Taken together, these data suggest that sesamin inhibits TNF‐α‐induced extracellular signal‐regulated kinase/p38 phosphorylation, nuclear translocation of NF‐κB p65, cytoplasmic translocalization of HuR and thereby suppresses ICAM‐1 expression, resulting in reduced adhesion of leukocytes. These results also suggest that sesamin may prevent the development of atherosclerosis and inflammatory responses.     

Apoptotic Activity of Lactobacillus plantarum DGK‐17‐Fermented Soybean Seed Extract in Human Colon Cancer Cells via ROS–JNK Signaling Pathway

Fermented food has been always possesses upper hand compared to normal food due to its antibacterial, antioxidant, and anticancer properties. Soybeans, which have high nutritional value, are widely consumed in Korea. In this study, soybean seed powder fermented with Lactobacillus plantarum DGK‐17, which was previously isolated from kimchi, showed anticancer potential. Fermented soybean extract (FSE) resulted in morphological changes, reduction of cancer cell colony formation and apoptotic cell death of HCT‐116 colon cancer cells in a dose‐dependent manner, and IC₅₀ value of 111 μg. FSE treatment caused reduction of cell growth in a dose‐dependent manner via release of lactate dehydrogenase. FSE treatment induced HCT‐116 apoptotic cell death as confirmed by the presence of fragmented nuclei, oxidative burst, and reduced mitochondrial membrane potential (ΔΨ_m). Further, FSE treatment sensitized cells to ER stress via IRE1‐α induction. FSE treatment also resulted in JNK activation, subsequently causing activation of Bax and downregulation of BCl2. Weakened mitochondrial membrane potential (ΔΨ_m) also caused release of Cyto C, further activating caspase‐mediated cell death. Therefore, this study reveals the apoptotic role of DGK‐17‐fermented soybean seed extract in human colon cancer HCT‐116 cells.     

Characterization of cosmetic cream with Mesembryanthemum crystallinum plant extract: influence of formulation composition on physical stability and anti‐oxidant activity

Cosmetic or pharmaceutical composition containing superoxide dismutase (SOD) was usually used in topical administration, particularly, in fighting against skin ageing and in the protection of the skin against radiation exposure. Mesembryanthemum crystallinum is a halophyte plant widely used in the traditional medicine, characterized by the presence of anti‐oxidants enzymes in responses to abiotic stresses. In the present study, we prepared a formulation with M. crystallinum extract characterized by naturally occurring SOD and catalase in association with other anti‐oxidants molecules. The SOD activity was measured by 3‐(4,5‐dimethyldiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide/riboflavin method, catalase by colorimetric method and the total anti‐radical activity was measured by 1,1‐diphenyl‐2‐picrylhydrazyl radical (DPPH) method. Formulations contain a significant SOD activity (8.33 U mg^?1), a catalase activity (0.5 × 10⁷ UC) and an anti‐radical activity (30% of DPPH inhibition). The formulation storage (15 days at 4°C) showed a marked loss of total anti‐oxidant capacity. The addition of the M. crystallinum extract induced also a reduction in formulation viscosity and pH.     

Marie Ménard apples with high polyphenol content and a low‐fat diet reduce 1,2‐dimethylhydrazine‐induced colon carcinogenesis in rats: Effects on inflammation and apoptosis

Inflammation may increase cancer risk, therefore, we studied whether polyphenol‐rich Marie Ménard (MM) apples with reported anti‐inflammatory activity prevent 1,2‐dimethylhydrazine (DMH)‐induced colon carcinogenesis in rats and, likewise whether high‐fat (HF) diet promoting carcinogenesis, may affect inflammation. DMH‐induced rats were fed for 15 weeks with: an HF diet (23% corn oil w/w); an HF diet containing 7.6% w/w lyophilized MM (apple diet (AD)); a low‐fat (LF) diet and an HF diet containing piroxicam (PXC) (0.01% w/w) as control. Mucin depleted foci (MDF), precancerous lesions in the colon, were dramatically reduced in the AD, LF, and PXC groups compared with the HF. Peritoneal macrophage activation, an index of systemic inflammation, was significantly decreased in the AD, LF, and PXC groups. TNF‐α, iNOS, IL‐1β, IL‐6 m‐RNA expression in the colon, as well as CD68 cells and plasmatic PGE2 were lower in the AD, but not in the LF group. Apoptosis in the MDF of both the AD and LF‐fed rats was significantly higher than in HF rats. In conclusion, AD has a strong chemopreventive effect, reducing inflammation, and increasing apoptosis, while the chemopreventive effect of the LF diet seems mediated mainly by increased apoptosis in MDF.     

Red mold dioscorea‐induced G2/M arrest and apoptosis in human oral cancer cells

BACKGROUND: Monascus‐fermented products are among the most commonly used traditional food supplements. Dioscorea is known to exhibit anticancer properties. In this study the effects of the ethanol extract of red mold dioscorea (RMDE) on cell proliferation, cell cycle and apoptosis in human oral cancer cells were investigated. RESULTS: RMDE exercised growth inhibition on squamous cell carcinoma‐25 (SCC‐25) cells. RMDE‐mediated G2/M phase arrest was associated with the down‐regulation of NF‐κB, resulting in the inhibition of cyclin B1 and CDK1 expression; this may be the mechanism by which RMDE inhibits cancer cells. Furthermore, the proapoptotic activity of RMDE was revealed by the Annexin V‐FITC/PI double‐staining assay. In addition, the proapoptotic effect of RMDE was evident by the inhibition of Bax expression in the mitochondria, resulting in the activation of caspase‐9 and caspase‐3 and subsequent triggering of the mitochondrial apoptotic pathway. RMDE also enhanced caspase‐8 activity, indicating the involvement of the death receptor pathway in RMDE‐mediated SCC‐25 cell apoptosis. CONCLUSION: RMDE treatment inhibited the growth of SCC‐25 cells by arresting cell cycle at the G2/M phase and induced apoptosis in a time‐ and dose‐dependent manner. Therefore RMDE may be a good candidate for development as a dietary supplement against oral cancer. Copyright © 2010 Society of Chemical Industry