Antioxidative functions of natto,a kind of fermented soybeans: effect on LDL oxidation and lipid metabolism in cholesterol-fed rats

Natto water-soluble fractions, low-molecular-weight viscous substance, and soybean water extract, which had an inhibitory effect on the oxidation of low-density lipoproteins (LDL) in vitro, were fed to rats for 3 weeks. These fractions had no influence on the growth of rats, which were fed a basal diet containing 1% cholesterol, but lowered plasma triglyceride and total cholesterol. Inhibition of copper-oxidation of plasma and LDL ex vivo, and a reduction in lipid peroxidation in liver and aorta in vivo, were also observed. The antioxidant enzymes were not induced in rats fed natto fraction diets. These results demonstrate that ingestion of the natto fractions led to inhibition of LDL oxidation, and that the fractions perform direct antioxidant action in the body. It is suggested that natto fractions might help to prevent arteriosclerosis, as they appear to reduce lipid peroxidation and improve lipid metabolism.     

Inhibitory effect of hot-water extract from dried fruit of Crataegus pinnatifida on low-density lipoprotein (LDL) oxidation in cell and cell-free systems

The dried fruit of Crataegus pinnatifida, a local soft drink material and medical herb, was found to possess potential against oxidative stress. In the preliminary study, the antioxidant potential of a hot-water extract obtained from the dried fruit of C. pinnatifida (CF-H) was evaluated in terms of its capacity of quenching 1,1-diphenyl-2-picrylhydrazyl free radicals (EC(50) = 0.118 mg/mL). After content analysis, it was found that CF-H is mainly composed of polyphenols including flavonoids (6.9%), procyanidins (2.2%), (+)-catechin (0.5%), and (-)-epicatechin (0.2%). The antioxidative bioactivity of CF-H had been assess previously using the models of CuSO(4) as cell-free system and sodium nitroprusside (SNP) plus macrophage RAW 264.7 cells as cell system to induce human low-density lipoprotein oxidation. CF-H was found to inhibit relative electrophoretic mobility and thiobarbituric acid reactive substances at the concentration of 0.5-1.0 mg/mL in the cell-free system and at 0.01-0.10 mg/mL in the cell system. Furthermore, it was found that CF-H decreased the SNP-induced cell lipid peroxidation and reduced glutathione depletion.     

Inhibitory effect of blueberry polyphenolic compounds on oleic acid-induced hepatic steatosis in vitro

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide and is closely associated with metabolic syndromes, such as obesity, diabetes, and insulin resistance. Nonalcoholic fatty liver (NAFL), also called simple steatosis, is the initial phase of NAFLD, which is accompanied the characteristic pathological overaccumulation of lipids without inflammation. To prevent NAFLD from reaching the NAFL stage through dietary therapy, in the present work, wild Chinese blueberries (Vacciniun spp.) were selected for their well-known benefits in inhibiting metabolic syndrome. After being purified from wild Chinese blueberries, polyphenol-rich extracts were subsequently separated into three fractions, namely, anthocyanin-rich fraction, phenolic acid-rich fraction, and ethyl acetate extract. The inhibition of oleic acid (OA)-induced triglyceride (TG) deposition in HepG 2 cells was referred to as the potential activity of preventing NAFL. Biochemical indicators, such as cytotoxicity, TG level, levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and intracellular reactive oxygen species, were used to evaluate the analogous pathological stage of NAFLD. The results show that OA ≤ 1.0 mM exhibits a dose-dependent induction of TG accumulation, and no inflammation was observed based on the changes in ALT and AST levels. Therefore, 1.0 mM OA was used to simulate an in vitro fatty liver. Blueberry polyphenol-rich extract efficiently inhibited OA-induced TG accumulation in HepG2 cells, and the phenolic acid-rich fraction performed efficiently. Seven phenolic acids were subsequently identified using a high-performance liquid chromatography assay, and the main types were caffeic, chlorogenic, ferulic, p-coumaric, and cinnamic acids. These phenolic acid standards also displayed good efficiency in inhibiting TG accumulation in HepG2 cells. These results imply that wild Chinese blueberries have a potential preventive effect on NAFLD in its early stage, and phenolic acids are the most efficient component.     

Inhibitory effect of (-)-epigallocatechin 3-gallate, a polyphenol of green tea, on neutrophil chemotaxis in vitro and in vivo

The effect of (-)-epigallocatechin 3-gallate (EGCG), a major polyphenol of green tea, on neutrophil migration has been studied using multiwell-type Boyden chambers in vitro and a fluorescein isothiocyanate-labeled ovalbumin (FITC-OVA)-induced rat allergic inflammation model in vivo. EGCG inhibited rat neutrophil chemotaxis toward cytokine-induced neutrophil chemoattractant-1 (CINC-1) in a concentration-dependent manner. In addition, CINC-1-induced neutrophil chemotaxis was suppressed by the pretreatment of rat neutrophils with EGCG at the concentration over 15 microg/mL. EGCG caused concentration-dependent suppression of the transient increase in CINC-1-induced intracellular free calcium level in both rat neutrophils and rat CXC chemokine receptor 2 (CXCR2)-transfected HEK 293 cells. EGCG inhibited CINC-1 production by IL-1beta-stimulated rat fibroblasts (NRK-49F cells) and lipopolysaccharide-stimulated rat macrophages at the concentration over 50 microg/mL, a comparatively high concentration. Oral administration of EGCG (1.0 mg or 1.5 mg/rat) at 1 h before the challenge with FITC-OVA suppressed neutrophil infiltration into the air pouch (inflammatory site) in the air-pouch type FITC-OVA-induced allergic inflammation in rats. Chemokine levels in the pouch fluids, however, were not influenced by EGCG administration. The results suggest that EGCG suppressed neutrophil infiltration by a direct action on neutrophils, but not by indirect actions, including the suppression of chemokine production at the inflammatory site.     

Antioxidative activity of sulfur-containing compounds in Allium species for human low-density lipoprotein (LDL) oxidation in vitro

Sulfur-containing compounds contributing to health promotion in Allium species are produced via enzymic and thermochemical reactions. Sulfur-containing amino acids and volatile organosulfur compounds were prepared for an antioxidative assay. The inhibitory activity of S-alk(en)yl-l-cysteines and their sulfoxides, volatile alk(en)yl disulfides and trisulfides, and vinyldithiins in Allium species against lipid hydroperoxide (LOOH) formation in human low-density lipoprotein (LDL) was examined. It was elucidated that the alk(en)yl substituents (methyl, propyl, and allyl) and the number of sulfur atoms in the compounds were important for the antioxidative activity. 3,4-Dihydro-3-vinyl-1,2-dithiin, which is produced by a thermochemical reaction of allyl 2-propenethiosulfinate, exhibited the highest antioxidative activity of human LDL among sulfur-containing compounds.     

Inhibitory effect of a quercetin metabolite, quercetin 3-O-beta-D-glucuronide, on lipid peroxidation in liposomal membranes.

To study the antioxidant activity of quercetin 3-O-beta-D-glucuronide (Q3GA), which is one of the quercetin metabolites in the blood after intake of quercetin-rich food, the inhibitory effect of Q3GA on lipid peroxidation was estimated using phosphatidylcholine large unilamellar vesicles (PC LUV) as a biomembrane model. Iron ion, an aqueous peroxyl radical generator, a peroxynitrite generator, or lipoxygenase was used as the inducer of lipid peroxidation. In all cases, Q3GA inhibited lipid peroxidation significantly, although its inhibitory effect was lower than that of quercetin aglycon. The ultrafiltration of PC LUV containing Q3GA revealed that Q3GA has low but significant affinity with the membranes of phospholipid bilayers. It is therefore likely that Q3GA acts as an efficient antioxidant in membranous lipid peroxidation through its localization in the phospholipid bilayer. This conjugated quercetin metabolite seems to retain the ability to protect cellular and subcellular membranes from peroxidative attack by reactive oxygen species and peroxidative enzymes.     

Study on furundu, a traditional Sudanese fermented roselle (Hibiscus sabdariffa L.) seed: effect on in vitro protein digestibility, chemical composition, and functional properties of the total proteins

Furundu, a meat substitute, is traditionally prepared by cooking the karkade (Hibiscus sabdariffa L.) seed and then fermenting it for 9 days. Physicochemical and functional properties of raw and cooked seed and of furundu ferments were analyzed. Furundu preparation resulted in significant changes in karkade seed major nutrients. Total polyphenols and phytic acid were also reduced. The increase in total acidity and fat acidity coupled with a decrease in pH indicates microbial hydrolysis of the major nutrients; proteins, carbohydrates, and fats. In vitro digestibility of the seed proteins reached the maximum value (82.7%) at the sixth day of fermentation, but thereafter it significantly decreased. The effect of furundu preparation on N solubility profiles and functional properties, such as emulsification and foaming properties and other related parameters, is investigated in water and in 1 M NaCl extracts from defatted flour samples. The results show that cooking followed by fermentation affects proteins solubility in water and 1 M NaCl. The foaming capacity (FC) from the flour of raw seed decreased as a result of cooking. Fermentation for 9 days significantly increased the FC of the cooked seed, restoring the inherent value. Foam from fermented samples collapsed more rapidly during a period of 120 min as compared to the foam from raw and cooked karkade seeds; stability in 1 M NaCl was lower as compared to those in water. In water, the emulsion stability (ES) from the fermented samples was significantly higher than that of the raw seed flour. Addition of 1 M NaCl significantly decreased the ES of the fermented samples.     

Positional effect on protein and oil content and composition of soybeans

Soybean (Glycine max [L.] Merr.) protein and oil qualities, with respect to monogastric nutrition, have been linked to the relative abundance of specific protein subunits and fatty acids, respectively. An analysis of field-grown soybean seeds by near-infrared spectroscopy revealed significant differences in their protein and oil contents as a function of nodal position. Seed proteins from the plant apex were high in protein and low in oil content, while those from the basal region exhibited an opposite pattern of accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total seed proteins revealed that the beta-subunit of beta-conglycinin content was 4-fold higher in seeds from the apical nodes than in seeds from basal nodes. The glycinin A3 polypeptide content gradually increased in successively lower nodes from the top of the plant. Its accumulation was drastically reduced when nitrogen was applied at specific growth stages. Exogenous nitrogen did not alter the pattern of beta-subunit accumulation, but accrual of the acidic and basic polypeptides of glycinin was diminished. The remaining seed storage protein components were not influenced by nodal position or nitrogen application. Gas chromatographic analysis of fatty acids indicated that only oleic (18:0) and linoleic (18:2) acids showed variability in accumulation at different nodes. Neither the abundance nor the distribution of the fatty acids was altered by nitrogen application.     

Novel fibrinolytic enzyme in fermented shrimp paste, a traditional asian fermented seasoning

A novel fibrinolytic enzyme was purified from fermented shrimp paste, a popular seasoning used in Asian countries. The enzyme is a monomer with an apparent molecular weight of 18 kDa, and it is composed primarily of beta-sheet and random coils. The N-terminal amino acid sequence was determined to be DPYEEPGPCENLQVA. It is a neutral protease with an optimal activity from pH 3 to 7. No inhibition was observed with PMSF, Pepstatin A, E64, and 1,10-phenanthroline, but the enzyme was slightly inhibited by EDTA and Cu(2+). It was relatively specific to fibrin or fibrinogen as a protein substrate, yet it hydrolyzed none of the plasma proteins in the studies. In vitro, the enzyme was resistant to pepsin and trypsin digestion. It also had an anticoagulant activity measured with activated partial thrombin time and prothrombin time tests. The novel fibrinolytic enzyme derived from traditional Asian foods is useful for thrombolytic therapy. In addition, this enzyme has a significant potential for food fortification and nutraceutical applications, such that its use could effectively prevent cardiovascular diseases.     

Juice and phenolic fractions of the berry Aristotelia chilensis inhibit LDL oxidation in vitro and protect human endothelial cells against oxidative stress

Oxidative modification of low-density lipoprotein (LDL) particles is a key event in the development of atherosclerosis. Oxidized LDL induces oxidative stress and modifies gene expression in endothelial cells. Berries constitute a rich dietary source of phenolic antioxidants. We found that the endemic Chilean berry Aristotelia chilensis (ach) has higher phenol content and scores better for total radical-trapping potential and total antioxidant reactivity in in vitro antioxidant capacity tests, when compared to different commercial berries. The juice of ach is also effective in inhibiting copper-induced LDL oxidation. In human endothelial cell cultures, the addition of ach juice significantly protects from hydrogen peroxide-induced intracellular oxidative stress and is dose-dependent. The aqueous, anthocyanin-rich fraction of ach juice accounts for most of ach's antioxidant properties. These results show that ach is a rich source of phenolics with high antioxidant capacity and suggest that it may have antiatherogenic properties.     

Supplementation of plasma with olive oil phenols and extracts: influence on LDL oxidation

Phenols present in olive oil may contribute to the health effects of the Mediterranean lifestyle. Olive oil antioxidants increase the resistance of low-density lipoproteins (LDL) against oxidation in vitro, but human intervention studies have failed to demonstrate similar consistent effects. To better mimic the in vivo situation, plasma was incubated with either individual olive oil phenols or olive oil extracts with different phenolic compositions, and LDL was subsequently isolated and challenged for its resistance to oxidation. The results show that the ortho-dihydroxy phenols (hydroxytyrosol and oleuropein-aglycone) are more efficient than their mono-hydroxy counterparts (tyrosol and ligstroside-aglycone) in increasing the resistance of LDL to oxidation. However, the concentration of antioxidants required to inhibit LDL oxidation when added to whole plasma was substantially higher as compared to previous data where antioxidants are directly added to isolated LDL. In conclusion, this study supports the hypothesis that extra virgin olive oil phenols protect LDL in plasma against oxidation. The explanation that in vitro studies show protective effects in contrast to the lack of effect in the majority of human studies may be that the dose of the phenols and thus their plasma concentration in humans was too low to influence ex vivo LDL oxidizability. Further studies are required to gain a better understanding of the potential health benefits that extra virgin olive oil may provide.     

Inhibitory effects of oolong tea polyphenols on pancreatic lipase in vitro

Fifty-four polyphenols isolated from tea leaves were evaluated for their inhibitory activities against pancreatic lipase, the key enzyme of lipid absorption in the gut. (-)-Epigallocatechin 3-O-gallate (EGCG), which is one of major polyphenols in green tea, showed lipase inhibition with an IC50 of 0.349 microM. Moreover, flavan-3-ol digallate esters, such as (-)-epigallocatechin-3,5-digallate, showed higher activities of inhibition on lipase with an IC50 of 0.098 microM. On the other hand, nonesterified flavan-3-ols, such as (+)-catechin, (-)-epicatechin, (+)-gallocatechin, and (-)-epigallocatechin, showed zero and/or the lowest activities against pancreatic lipase (IC50 > 20 microM). These data suggested that the presence of galloyl moieties within the structure was required for enhancement of pancreatic lipase inhibition. It is well-known that flavan-3-ols are polymerized by polyphenol oxidase and/or heating in a manufacturing process of oolong tea. Oolonghomobisflavans A and B and oolongtheanin 3'-O-gallate, which are typical in oolong tea leaves, showed strong inhibitory activities with IC50 values of 0.048, 0.108, and 0.068 microM, respectively, even higher than that of EGCG. The oolong tea polymerized polyphenols (OTPP) were prepared for the assay from oolong tea extract, from which the preparation effectively subtracted the zero and/or less-active monomeric flavan-3-ols by preparative high-performance liquid chromatography. The weight-average molecular weight (Mw) and number-average molecular-weight (Mn) values of OTPP were 2017 and 903, respectively, by using gel permeation choromatography. OTPP showed a 5-fold stronger inhibition against pancreatic lipase (IC50 = 0.28 microg/mL) by comparison with that of the tannase-treated OTPP (IC50 = 1.38 microg/mL). These data suggested that the presence of galloyl moieties within their chemical structures and/or the polymerization of flavan-3-ols were required for enhancement of pancreatic lipase inhibition.     

Inhibitory effect of caffeic acid phenethyl ester on angiogenesis, tumor invasion, and metastasis

Caffeic acid phenethyl ester (CAPE) derived from honeybee propolis has been used as a folk medicine and has several proven biological activities. The present study investigated the effect of CAPE on angiogenesis, tumor invasion, and metastasis. A cytotoxicity assay of CAPE in CT26 colon adenocarcinoma cells showed a dose-dependent decrease in cell viability but no significant influence on the growth of human umbilical vein epithelial cells (HUVEC). A low concentration of CAPE (1.5 microg/mL) inhibited 52.7% of capillary-like tube formation in HUVEC culture on Matrigel. CAPE (6 microg/mL)-treated CT26 cells showed not only inhibited cell invasion by 47.8% but also decreased expression of matrix metalloproteinase (MMP)-2 and -9. Vascular endothelial growth factor (VEGF) production from CT26 cells was also inhibited by treatment with CAPE (6 microg/mL). Intraperitoneal injection of CAPE (10 mg/kg/day) in BALB/c mice reduced the pulmonary metastatic capacity of CT26 cells accompanied with a decreased plasma VEGF level. CAPE treatment also prolonged the survival of mice implanted with CT26 cells. These results indicate that CAPE has potential as an antimetastatic agent.     

Lactoferrin in infant formulas: effect on oxidation

Lactoferrin is an iron transport protein present in human milk at an average concentration of 1.4 mg/mL. Commercially modified infant formulas based on cow's milk contain much lower amounts of lactoferrin (0.1 mg/mL lactoferrin) and soy based formulas have none. In addition to its role in iron transport, lactoferrin has bacteriostatic and bactericidal activities. Infant formulas are supplemented with relatively large amounts of iron (up to 12 mg/L). The effect of various concentrations of added lactoferrin and supplemental iron on lipid oxidation was tested in two different infant formulas. The extent of oxidation in the formulas as a function of time was determined by formation of hydroperoxides, production of hexanal, and fluorescence. On the basis of all three of these determinations, lactoferrin acted as an antioxidant in the absence and presence of different concentrations of supplemented iron. Lactoferrin inhibited oxidation in a concentration-dependent manner even at concentrations beyond its capacity to bind iron at its two high affinity binding sites. Lactoferrin can be used, therefore, as a dual purpose additive in infant formulas and similar food products for its antioxidant and its antimicrobial properties.     

Antiallergic effect of milk fermented with lactic acid bacteria in a murine animal model

The objective of this study was to assess the antiallergic effect of fermented milk prepared, respectively, with Streptococcus thermophilus MC, Lactobacillus acidophilus B, Lactobacillus bulgaricus Lb, L. bulgaricus 448, and Bifidobacterium longum B6. Female BALB/c mice fed fermented milk were immunized intraperitoneally with ovalbumin (OVA)/complete Freund's adjuvant (CFA) to evaluate the immune response by observing the secretion of cytokines IL-2, IL-4, and IFN-gamma and serum antibody IgE. The results showed that supplementation with lactic acid bacteria fermented milk did not significantly change the IL-2 spontaneous and OVA-stimulated secretions of splenocytes. However, both spontaneous and OVA-stimulated secretions of splenocytes from mice fed lactic acid bacteria fermented milk showed significantly (P < 0.05) lower levels of IL-4 (Th2 cytokine) than those from OVA/CFA-immunized mice fed non-fermented milk (OVA/CFA-milk group). The spontaneous secretion of IFN-gamma (Th1 cytokine) by splenocytes from mice fed L. bulgaricus 448 or L. bulgaricus Lb fermented milk significantly increased as compared to that from the OVA/CFA-milk group. The results showed that the ratios of IFN-gamma to IL-4 of both spontaneous and OVA-stimulated secretions in splenocytes from mice fed lactic acid bacteria fermented milk increased significantly as compared to that of PBS- or OVA/CFA-milk groups. The serum levels of OVA-specific IgE in fermented milk fed groups, especially the group fed S. thermophilus MC fermented milk, were significantly lower than those in the OVA/CFA-milk group through a 6 week feeding experiment. The results showed that milk fermented with lactic acid bacteria demonstrated in vivo antiallergic effects on OVA/CFA-immunized mice via increasing the secretion ratio of IFN-gamma/IL-4 (Th1/Th2) by splenocytes and decreasing the serum level of OVA-specific IgE.     

Enzymatic O-methylation of flavanols changes lag time, propagation rate, and total oxidation during in vitro model triacylglycerol-rich lipoprotein oxidation

3'-O-Methyl derivatives of flavan-3-ols, (+)-catechin (C), (-)-epicatechin (EC), and (-)-catechin gallate (CG) were prepared enzymatically. Hexanal (EC and CG family, 5 mmol/L) and conjugated diene (C and EC family, 0.25-10 mmol/L) formation following CuSO4-mediated triacylglycerol-rich lipoprotein oxidation was measured. All EC and CG compounds significantly reduced hexanal formation (p < 0.02). O-Methylation improved the ability of CG (more polar) while reducing the ability of EC (less polar) to limit hexanal formation. 3'-O-methyl EC was 18% (p < 0.001) and 4'-O-methyl 65% (p < 0.001) less able than EC to suppress hexanal formation. At >1 micromol/L all EC and C compounds significantly increased lag time. Parent compounds were more effective (> 4-fold increase) than metabolites (1.5-fold increase). Parent compounds did not influence propagation rate (DeltaOD/min). At >1 mmol/L O-methylated EC and C reduced propagation by 20-40% (p < 0.01). Notably, at 0.25 mmol/L O-methylated EC and C increased propagation rates 22% (p < 0.01) despite prolonging lag time.     

Inhibitory effect of red koji extracts on mushroom tyrosinase

Red koji has been recognized as a cholesterol-lowering diet supplement because of it contains fungi metabolites, monacolins, which reduce cholesterol synthesis by inhibiting HMG-CoA reductase. In this study, water extracts of red koji were loaded onto a C(18) cartridge, and the acetonitrile eluate was collected as test fraction. Red koji water extracts and its C(18) cartridge acetonitrile eluent had total phenols concentrations of 5.57 and 1.89 mg/g of red koji and condensed tannins concentrations of 2.71 and 1.20 mg/g of red koji, respectively. Both exhibited an antioxidant activity and an inhibitory activity to mushroom tyrosinase. The higher antioxidant activity of the red koji acetonitrile eluent was due to the existence of a high percentage of condensed tannins. The results from the kinetic study for inhibition of mushroom tyrosinase by red koji extracts showed that the compounds in the extracts competitively inhibited the oxidation of tyrosine catalyzed by mushroom tyrosinase with an ID(50) of 5.57 mg/mL.     

Inhibitory effect of protein hydrolysates on calcium carbonate crystallization

Protein hydrolysates, prepared by enzymatic digestion of soybean protein and egg white albumin using several proteases, inhibited the crystal growth of calcium carbonate. Each hydrolysate showed different inhibitory activities, suggesting the key role of peptide structures in the inhibition. The deamidation of protein hydrolysates by glutaminase increased not only the inhibitory activity toward the crystal growth of calcium carbonate but also the resistance of the hydrolysates against peptic digestion. Furthermore, the addition of sodium chloride, citric acid, or lactose into the reaction mixture enhanced the inhibitory activity. The protein hydrolysates inhibited both nucleation and crystal growth of calcium carbonate and also affected the crystal morphology.     

Hypotriglyceridemic effect of Anka (a fermented rice product of monascus sp.) in rats

Experimental rats with hypertriglyceridemia were prepared by feeding a high-fructose diet. Dried Anka powder (2%), a rice product fermented with Monascus sp., was mixed with basic high-fructose (30%) or basal-diet feed. Serum and liver lipids were measured after 6 months. The concentrations of serum triglycerides, total cholesterol, VLDL-C, and LDL-C had significantly decreased, whereas that of HDL-C had slightly increased in 30% fructose-Anka-fed rats as compared with the 30% fructose-fed rats, but hepatic lipase activity had increased in the Anka-fed groups. The ratio of lipoprotein lipase/hepatic lipase was not significantly different between 30% fructose-Anka-fed rats and 30% fructose-fed rats. The dietary intake and weight of these two groups were approximately the same. Similar results were obtained in noninduced hypertriglyceridemic rats. The concentrations of triglycerides and cholesterol did not significantly differ in the liver. Interestingly, Anka can suppress serum triglycerides in rats with induced hypertriglyceridemia. The antioxidant enzyme SOD activity was also measured in serum, and no significant change was observed. On the basis of these findings, we suggest that Anka may be used to suppress hypertriglyceridemia and hyperlipidemia in rats and possibly in man.