Cyanidin and cyanidin-3-glucoside derived from Vigna unguiculata act as noncompetitive inhibitors of pancreatic lipase.

The consumption of legumes positively correlated with the reduction of body weight. In the present study, we identified and evaluated pancreatic lipase inhibitors from Vigna unguiculata and unraveled their mode of inhibition. The highly sensitive fluorometric method was adopted to access the pancreatic lipase activity and the ethanolic extract of Vigna unguiculata showed the maximum inhibition (IC50 of 15.2 µg/ml). Cyanidin and cyanidin-3-glucoside are the major anthocyanins observed in Vigna unguiculata. The IC50 value of cyanidin was 28.29 µM which was 6.5-fold higher than the cyanidin-3-glucoside (188.28 µM). We determined an apparent Ki of 27.28 µM for cyanidin and cyanidin-3-glucoside (88.97 µM) with noncompetitive inhibition. Collectively, these results suggest that the glycosylation of the anthocyanidins significantly reduces lipase inhibition. The noncompetitive inhibition of pancreatic lipase by Vigna unguiculata anthocyanins may exert significant pharmacological activities toward obesity complications by calorie restriction. PRACTICAL APPLICATIONS: The results of this study emphasize the importance of legumes in our diet to combat obesity-related complications. Consumption of legumes minimizes fat absorption by inhibiting the action of the fat-digesting enzyme.

Tofu and fish oil independently modulate serum lipid profiles in rats: Analyses of 10 class lipoprotein profiles and the global hepatic transcriptome.

Soy protein and fish oil are food components that decrease the risk of cardiovascular disease. Previous studies demonstrated that these food components reduced serum cholesterol levels and suppressed hepatic lipogenesis. However, the underlying mechanisms of action of these food components remain unclear. Ten classes of serum lipoprotein profiles showed that dietary tofu, a soybean curd, suppressed cholesterol absorption, while fish oil reduced most of the lipoprotein classes in rats. Tofu and fish oil both halved the level of the lipoprotein class LAC1 (LDL-anti-protease complex), a 15-nm LDL-anti-protease complex, which is speculated to be a cause of atherosclerosis. Moreover, a global transcriptome analysis revealed that tofu inhibited the mRNA expression of genes involved in hepatic lipogenesis, while fish oil stimulated that of genes related to fatty acid degradation. Therefore, tofu and fish oil independently regulate lipid metabolism. The decrease observed in LAC1 may have been due to reduced cholesterol absorption in the tofu diet group and the interference of lipogenesis via the activation of polyunsaturated fatty acid detoxification in the fish oil group.

Mode of pancreatic lipase inhibition activity in vitro by some flavonoids and non-flavonoid polyphenols.

Numerous reports have shown plant metabolites as potential inhibitors of pancreatic lipase (PL). The most notable group is plant polyphenols. However, a limited number of reports diagnosed their mode of inhibition delineating conflicting results. To elucidate the kinetic mode of PL inhibition, some selected flavonoid and non-flavonoid polyphenol standards were first screened for their lipase inhibition potency by their half maximal inhibitory concentration (IC50) followed by inhibition kinetic analysis. Of the phenolics tested, only gallic acid (GA) and galloyl moiety containing epicatechin, viz., epigallocatechin (EGC) and epigallocatechin gallate (EGCG) showed, comparative to others, higher PL inhibitions (IC50, 387.2, 237.3, and 391.2µM respectively). Analysis of enzyme inhibition modalities at various substrate concentrations revealed a dose-dependent inhibition of reaction velocity. Inhibitory rates decreased by the order of EGCG>EGC>GA (Ki, 13.29>35.0>44.61µM respectively). The results, when verified by visual inspection of Lineweaver-Burk as well as Dixon plots, showed inhibitions of PL by GA, EGC, and EGCG that were best fit to competitive inhibitions. A role of the galloyl moiety in enzyme-inhibitor binding has been evident from their structural resemblance. Depicting it further, ethyl gallate (EG), showed a similar competitive inhibition, therefore, indicating a galloyl moiety driven competitive inhibition of PL.

Tea polyphenols as novel and potent inhibitory substances against renin activity.

Renin inhibitory activities of three tea products were investigated for the first time in this work. Water extracts from fermented oolong and black tea showed strong renin inhibitory activities. By the means of ultrafiltration, gradient high performance liquid chromatography and spectroscopic analysis, four active compounds were separated from aqueous black tea extract and identified as theasinensin B, theasinensin C, strictinin, and a hexose sulfate with a galloyl moiety, which had IC50 values of 19.33, 40.21, 311.09, and 50.16 µM against renin activity, respectively. Further detection indicated that the potent inhibitor theasinensin B was present only in black tea, and that monomeric catechins did not contribute significantly to the renin inhibitory activities of tea products. These results revealed novel and potent tea-derived renin inhibitors and suggested another potential pathway for tea consumption to control hypertension.

Inhibitory effect of catechin-related compounds on renin activity.

Renin is a crucial enzyme in the renin-angiotensin system, and the inhibition of its activity is considered as a useful approach to the treatment of hypertension. The inhibitory effect of catechin-related compounds on renin was investigated in this work. It was found that epigallocatechin gallate (EGCg) possessed the strongest activity with an IC50 value of 44.53 µM and acted in an uncompetitive manner. Gallated catechins exerted higher inhibition than the ungallated forms, and gallic acid exhibited an inhibitory potency close to that of epicatechin gallate (ECg). Results indicated that the galloyl moiety and ortho-trihydroxy phenyl structures might be favorable for the renin-inhibitory activity of these compounds.

Phenolic antioxidants in some Vigna species of legumes and their distinct inhibitory effects on α-glucosidase and pancreatic lipase activities.

Phenolic extracts of 4 Vigna species of legumes (mung bean, moth bean, and black and red varieties of adzuki beans) were evaluated for phenolic contents, antioxidant activities, and inhibitory properties against α-glucosidase and pancreatic lipase. Results showed that adzuki bean varieties contain higher phenolic indexes than mung bean and moth beans. Adzuki bean (black) variety was found to be the most active 2,2'-diphenyl-1-picrylhydrazyl and superoxide anion scavenger. However, the hydrogen peroxide scavenging and metal chelating abilities were significantly higher in adzuki bean (red) variety. Mung bean exhibited least antioxidant activities in all the methods tested. Phenolic extracts from these legumes also showed distinct variations in the inhibition of enzymes associated with hyperglycemia and hyperlipidemia. Inhibitory activities of all the extracts against lipase were found to be more potent than α-glucosidase. Although, α-glucosidase inhibitory activity was superior in the black variety of adzuki bean (IC(50,) 26.28 mg/mL), both adzuki bean varieties (black and red) along with moth bean showed strong inhibitory activities on lipase with no significant difference in their IC(50) values (7.32 to 9.85 mg/mL). These results suggest that Vigna species of legumes are potential source of antioxidant phenolics and also great sources of strong natural inhibitors for α-glucosidase and lipase activities. This information may help for effective utilization of these legumes as functional food ingredients for promoting health. Practical Application: Vigna species of legumes are good sources of phenolic antioxidants and strong natural inhibitors of enzymes associated with diabetes and obesity. Therefore, utilization of these legumes in the development of functional foods with increased therapeutic value would be a significant step toward health promotion and wellness.

Simple, selective, and rapid quantification of 1-deoxynojirimycin in mulberry leaf products by high-performance anion-exchange chromatography with pulsed amperometric detection.

1-Deoxynojirimycin (DNJ) occurs in mulberry and other plants and is a highly potent glycosidase inhibitor reported to suppress blood glucose levels, thus preventing diabetes. Derivatization is required for quantification of DNJ upon use of spectral detection methods. Because of this difficulty, the DNJ contents of mulberry-based food products are rarely stated, even if DNJ is their active component. A simple, selective, and rapid method of high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) to quantify DNJ in mulberry-based food products was developed. Stability testing of DNJ under heat treatment was also performed. A water extract of mulberry tea sample was subjected to HPAEC-PAD in a CarboPac MA1 column with a sodium hydroxide gradient. DNJ was clearly separated at a retention time of 7.26 min without interference and was selectively detected in the water extract. The detection limit was 5 ng. Heat stability studies suggested that DNJ was heat stable. HPAEC-PAD was not subject to interference, was highly selective for DNJ, and was superior to other high-performance liquid chromatography (HPLC) techniques in terms of sample preparation, resolution, and sensitivity. The method allowed simple, selective, and rapid analysis of DNJ in food matrices and might be useful for development of mulberry-based food products. Heat treatment could be an option for sterilizing mulberry-based products.

Purification and identification of 1-deoxynojirimycin (DNJ) in okara fermented by Bacillus subtilis B2 from Chinese traditional food (Meitaoza).

This study was to purify an alpha-glucosidase inhibitor from okara (soy pulp) fermented by Bacillus subtilis B2 and to identify its chemical structure. Membrane dialysis, active charcoal, CM-Sepharose chromatography, and preparative thin-layer chromatography (TLC) were used in the purification, while positive mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectrometry were used in the identification. The MS and NMR data showed that the purified alpha-glucosidase inhibitor was 1-deoxynojirimycin (DNJ) with a molecular weight of 163 Da. This is the first time that DNJ was isolated from foods fermented with Bacillus species. Okara fermentation with B. subtilis B2 might be used to produce a food-derived DNJ product as a functional food for diabetic patients.

Continuous orally administered coffee enhanced the antigen-specific Th1 response and reduced allergic development in a TCR-transgenic mice model.

Coffee is a globally consumed beverage. Although recent studies have suggested that coffee reduced the risk of lifestyle-related diseases, there are few studies regarding allergic response. This study investigates the effects of orally administered coffee (91 ml/kg/d) on allergic responses using a T cell receptor (TCR)-transgenic DO11.10 mouse allergic model. Splenocytes from coffee-administered naïve mice increased antigen (Ag)-specific interleukin (IL)-12p40 secretion. When Ag sensitization and coffee administration were concurrently performed, the splenocytes from coffee-administered mice showed a decrease of IL-2 and an increase of IL-12p40 secretion. The Ag-specific cutaneous response and serum IgE level were reduced in coffee-administered mice, although, after establishing the allergy, coffee administration did not suppress the allergic reaction. These results suggest that coffee could induce a Th1-type response of the immune system and prevent an allergy developing. Further studies on the optimum dose, cultivar differences, and roasted degree need to be undertaken.

Effects of sources of carbon and nitrogen on production of α-glucosidase inhibitor by a newly isolated strain of Bacillus subtilis B2.

This study examined production of α-glucosidase inhibitors by Bacillus subtilis B2 in Luria-Bertani (LB) fermentation with okara, soy powder, starch or pectin as additional source of carbon and nitrogen. All the fermentation broths of B. subtilis B2 exhibited gradual increase in α-glucosidase inhibitory activity during the fermentation process with or without supplemented source of carbon or nitrogen. Addition of okara into the LB medium greatly enhanced the strength (nearly twice as much of that without okara supplement) of α-glucosidase inhibitory activity of fermentation broth. The α-glucosidase inhibitory activity of B. subtilis B2 fermentation broth was positively correlated (p<0.05) with the bacterial populations grown in LB medium containing okara. Glucose and sucrose were not detected in LB medium during the entire fermentation process and were both reduced drastically in media containing okara, soy powder, starch or pectin after 6days of fermentation. The fermented LB medium containing okara by B. subtilis B2 possessed very strong α-glucosidase inhibitory activity and contained little glucose and sucrose, suggesting that fermentation of B. subtilis B2 in LB added with okara might be considered as a strategy for preparing functional foods for diabetic patients.

Orally administered bisphenol A disturbed antigen specific immunoresponses in the naïve condition.

Bisphenol A [2,2-bis(4-hydoxyphenyl)propane; BPA] is an endocrine disrupter widely used in polycarbonate plastics and epoxy resins. We investigated the effects of orally administered BPA on antigen-specific responses of the naïve immune system.BPA was orally administered to T cell receptor transgenic mice, and the antigen-specific responses of immune cells were investigated. Administered BPA moderately reduced interleukin (IL)-2, 4, and interferon (IFN)-gamma secretion and increases in IgA and IgG2a production.Additionally, it was found that orally administered BPA increased antigen-specific IFN-gamma production of T cells and modified whole antigen presenting cells (APCs) to suppress antigen-specific cytokine production from T cells. These findings suggest that BPA can augment the Th1-type responses of naïve immune systems, though the bioavailability of orally administered BPA was low in our experiments.

Effect of dietary fat on skin reactivity against histamine, Th1 and Th2 cytokine levels and some serum parameters in mice.

Changes in diet may be associated with the increase in allergic disease; change to high-calorie and high-fat diets may be a factor. In this study our objective was to determine skin reactivity of histamine and serum cytokine concentrations in mice fed diets containing different amounts of fat. Histamine reactivity was performed on mice back skin and serum cytokine concentrations were measured by ELISA in mice injected with anti-CD3 antibody. We measured serum interferon-gamma as a Th1-type cytokine and interleukin-4 as a Th2-type cytokine. Mice fed a high fat diet displayed enhanced skin reactivity of histamine and higher IL-4 levels in serum. These data suggest that a high fat diet may play a role in enhancing allergic reactions.

Comparative effects of non-gelatinized corn and rice starches on the life-span of ICR mice.

We compared the effects of non-gelatinized rice and corn starches on the life-span of ICR mice. Six groups of male ICR mice consisting of 30 animals each were maintained on purified experimental diets containing either corn or rice starch and different amounts of soybean oil (6, 12 or 24%) throughout their life-time. Plots of the survival rates of the mice indicate that rice compared to corn starch conferred a longer life-span to ICR mice, although a significant difference due to the starch type was only observed in the mice fed on the 24% fat diet (p=0.012). A divergent effect of rice and corn starches on the survival rate was apparent when observations were combined with respect to the starch type regardless of the dietary fat level (p=0.005). In addition, two-way ANOVA data indicate that the mean survival time was longer for the mice given rice starch (593-645 days) than for those fed corn starch (538-580 days) (p=0.011). However, no significant difference in these parameters due to dietary fat levels was observed. The results of our study indicate that starch type is one of the determinants of longevity in mice.

Effects of isothiocyanates on tumor necrosis factor-alpha production by J774A.1 (BALB/c macrophage) cells.

The effects of isothiocyanates (ITCs) on tumor necrosis factor-alpha (TNF-alpha) production by the J774A.1 mouse macrophage cell line stimulated with lipopolysaccharide (LPS) were examined. Some individual ITCs examined showed a priming effect, which was expressed as pre-activation, but also inhibited the production of TNF-alpha with concomitant stimulation by LPS, as a triggering stimulant of TNF production. These results suggest that ITCs exert opposing activities that can enhance or inhibit the host defense system.

Analysis of leukocyte rolling and migration--using inhibitors in the undisturbed microcirculation of the rat mesentery--on inflammatory stimulation.

AIM: Our aim was to develop a method of migration analysis using the undisturbed microcirculation of rat mesentery, and using the new method, analyze leukocyte migration in casein-induced inflammation. METHOD: Sprague Dawley (SD) rats were injected with tumor necrosis factor (TNF) alpha, interleukin (IL)-1alpha, or casein intraperitoneally. Following this, the rats were sacrificed and the mesentery tissue removed was fixed and stained with Giemsa. The leukocytes were counted as a rolling index in the venules and as a migration index in the perivascular area. RESULTS: There was no relation between the diameter of venules and leukocyte migration. The time change curves of leukocyte activity in casein inflammation show about a 1 h difference between rolling and migration. From inhibitor experiments of casein-induced migration at 2 h, it has been suggested that selectin-related rolling is necessary. Platelet-activating factor (PAF) also appears partially involved. CONCLUSION: The improved undisturbed microcirculation method is helpful not only for rolling analysis but also in analysis of leukocyte migration. Casein inflammation analyzed using this method revealed that rolling is necessary and also suggested that partial involvement of PAF is necessary for pathogenesis of leukocyte extravasations.

Determination of 1-deoxynojirimycin in mulberry leaves using hydrophilic interaction chromatography with evaporative light scattering detection.

A simple and rapid method for determining 1-deoxynojirimycin (DNJ), a potent glucosidase inihibitor present in mulberry leaves (Morus alba and Morus bombysis), by high-performance liquid chromatography coupled to an evaporative light scattering detector (ELSD) has been developed. DNJ was separated from an extract of mulberry leaves on a TSKgel Amide-80 column, which is a representative column for hydrophilic interaction chromatography. During postcolumn detection, DNJ was detected by ELSD and concurrently identified by mass spectrometry. The detection limit was 100 ng. This method is sufficiently sensitive for determining DNJ in mulberry leaves and other related products.

Simple and rapid determination of 1-deoxynojirimycin in mulberry leaves.

A simple and rapid method for determining 1-deoxynojirimycin (DNJ), a potent glucosidase inhibitor present in mulberry leaves (Morus alba and Morus bombysis), by high performance liquid chromatography coupled to an evaporative light scattering detector (ELSD) has been developed. DNJ was separated from extract of mulberry leaves on TSK gel Amide-80 column, which is a representative column for hydrophilic interaction chromatography. During post column detection, DNJ was detected by ELSD and concurrently identified by mass spectrometry. The detection limit was 100 ng. This method is sufficiently sensitive for determining DNJ in mulberry leaves and other related products.

Inhibitory effects of several flavonoids on E-selectin expression on human umbilical vein endothelial cells stimulated by tumor necrosis factor-alpha.

The present study investigates the suppressive effect of flavonoids on TNF-alpha-stimulated E-selectin expression on HUVECs by carrying out a comparative examination of the 37 flavonoids. Several flavonoids: fisetin, luteolin and apigenin (subclass of flavone), kaempferol and quercetin (flavonols), eriodictyol (flavanones), genistein (isoflavones) and butein (chalcone) exhibit the inhibitory effects. Considerations to the structure of flavonoids, the C2-C3 double bond of C-ring and 4-oxo functional group are essential for their inhibition activities. These results help to explain the pharmacological efficacy of flavonoids as anti-inflammatory compounds.

Inhibitory effect of some flavonoids on tumor necrosis factor-alpha production in lipopolysaccharide-stimulated mouse macrophage cell line J774.1.

Certain naturally occurring flavonoids affect immunoregulatory activities in vitro and in vivo against cytokine production. Since tumor necrosis factor (TNF)-alpha is one of the major inflammatory cytokines, the effects of various dietary flavonoids on TNF-alpha production in lipopolysaccharide (LPS)-stimulated J774.1 cells were evaluated in vitro. Flavones, flavonols, and chalcone are the most potent inhibitors of production of TNF-alpha. Flavanone, naringenin, anthocyanidin, pelargodinin, and cyanidin exhibit moderate inhibitory activity. In contrast, genistein isoflavone displays weak inhibition, while eriodictyol flavanone is inactive. It is clear that the double bond between carbons 2 and 3 and the ketone group at position 4 of flavonoids are necessary for potent inhibitory effect. The difference in inhibitory action appears to depend on the categorized subclass of flavonoids.

An improved method for measuring vascular permeability in rat and mouse skin.

INTRODUCTION: We have improved a rodent vascular permeability measurement method employing fluorescent dye-labeled bovine serum albumin. METHODS: The incubation duration for direct fluorescent detection of skin injected with an inflammatory agent was decided based on regression curve parameters with the correlation coefficient obtained from the least squares method. RESULTS: A suitable incubation time was determined to be 2-6 h. The recovery of FITC-BSA from the skin sample was very good, and the correlation coefficient of the linear regression curve was .99. The linear relation between the previous dye extraction method using brilliant blue 6B and the new and improved fluorescence method was very high. In mice, histamine-induced serum exudation in the back skin increased from 0.31 to 1.25 microg/site in a dose-dependent manner and reached a plateau at 1.25-2.5 microg/site. The serum exudation caused by histamine increased to 10 microg/site and almost reached a plateau at 10-40 microg/site in rats. The time required for the measurement of fluorescence intensity was very short because a microplate reader was used as the measurement apparatus. CONCLUSION: The improved method is easy to use and sensitive and does not necessitate extraction of dye from the skin.