We investigated the growth inhibitory activity of several flavonoids, including apigenin, luteolin, kaempherol, quercetin, butein, isoliquiritigenin, naringenin, genistein, and daizein against B16 mouse melanoma 4A5 cells. Isoliquiritigenin and butein, belonging to the chalcone group, markedly suppressed the growth of B16 melanoma cells and induced cell death. The other flavonoids tested showed little growth inhibitory activity and scarcely caused cell death. In cells treated with isoliquiritigenin or butein, condensation of nuclei and fragmentation of nuclear DNA, which are typical phenomena of apoptosis, were observed by Hoechst 33258 staining and by agarose gel electrophoresis of DNA. Flowcytometric analysis showed that isoliquiritigenin and butein increased the proportion of hypodiploid cells in the population of B16 melanoma cells. These results demonstrate that isoliquiritigenin and butein inhibit cell proliferation and induce apoptosis in B16 melanoma cells. Extracellular glucose decreased the proportion of hypodiploid cells that appeared as a result of isoliquiritigenin treatment. p53 was not detected in cells treated with either of these chalcones, however, protein of the Bcl-2 family were detected. The level of expression of Bax in cells treated with either of these chalcones was markedly elevated and the level of Bcl-XL decreased slightly. Isoliquiritigenin did not affect Bcl-2 expression, but butein down-regulated Bcl-2 expression. From these results, it seems that the pathway by which the chalcones induce apoptosis may be independent of p53 and dependent on proteins of the Bcl-2 family. It was supposed that isoliquiritigenin induces apoptosis in B16 cells by a mechanism involving inhibition of glucose transmembrane transport and promotion of Bax expression. On the other hand, it was suggested that butein induces apoptosis via down-regulation of Bcl-2 expression and promotion of Bax expression. This mechanism differs from the isoliquiritigenin induction pathway.

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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 (IC50, 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 IC50 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.

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.

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1 The main objective of this study was to analyse the role and mode of action of the mast cell mediator histamine in leukocyte-endothelium interactions in small venules in vivo. For this purpose, we used a histological approach (combined with intravital microscopy) that allows studies of rapid mediatorinduced venular leukocyte accumulation, re¯ecting leukocyte rolling, in the undisturbed microcirculation of the rat mesentery where rolling is normally absent. 2 We ®rst examined the relative importance of histamine and 5-hydroxytryptamine (5-HT) in acute mast cell-dependent leukocyte recruitment. The mast cell secretagogue compound 48/80 (i.p. for 15 min) induced a marked venular accumulation of polymorphonuclear leukocytes (PMNL) which was almost abolished by combined histamine 1 (H 1 )-and histamine 2 (H 2 )-receptor blockade. In contrast, the 5-HTreceptor antagonist methysergide was inactive in this regard. Moreover, exogenous 5-HT was less active than exogenous histamine in evoking venular PMNL accumulation (histamine response dose-dependent; 5-HT response bell shaped). Prostaglandin D 2 did not cause PMNL accumulation. 3 The venular PMNL response to exogenous histamine peaked between 15 min and 1 h, was still signi®cantly elevated at 2 h, and then returned to prechallenge values after 3 h. At all time points, the histamine-induced PMNL accumulation was nearly abolished by i.v. treatment with the polysaccharide fucoidin (which blocks rolling but not ®rm adhesion per se), suggesting that the PMNL response to histamine was due to rolling rather than ®rm adhesion over the entire 3 h period. At no time point did histamine trigger accumulation of mononuclear leukocytes (MNL). 4 To examine the role of histamine-receptors in the histamine-induced PMNL accumulation (i.e. rolling), the animals were pretreated with diphenhydramine (H 1 -receptor antagonist), cimetidine, or ranitidine (H 2 -receptor antagonists). Diphenhydramine alone inhibited the venular PMNL response to histamine by 52%, while both H 2 -receptor antagonists were completely inactive. However, the combination of cimetidine and diphenhydramine reduced the histamine-induced PMNL rolling by 82%. Furthermore, in contrast to an H 3 -receptor agonist, challenge with either the H 1 -receptor agonist 2-thiazolylethylamine or two di erent H 2 -receptor agonists (impromidine, dimaprit) was su cient to provoke signi®cant venular PMNL accumulation. 5 Treatment with the nitric oxide-synthase inhibitor L-NAME did not a ect the histamine-induced PMNL rolling. On the other hand, 3 h pretreatment with dexamethasone reduced the PMNL response to histamine by 73%, and¯ow cytometric analysis showed that the dexamethasone treatment almost completely inhibited binding of soluble P-selectin to rat isolated PMNLs. 6 We conclude that initial leukocyte recruitment after mast cell activation in the rat mesentery is critically dependent on histamine release. The cellular response to histamine was speci®cally due to PMNL rolling, involved activation of both H 1 -and H 2 -receptors, ...

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1 Although intravital microscopy is the method of choice for observation of in¯ammatory leukocyte rolling and adhesion in small venules in vivo, a problem with this technique is that surgical exposure of suitable tissues per se triggers the rolling mechanism. In this study, we describe an approach to investigate induction of rolling in undisturbed microvessels. For this purpose, intravital microscopic observation of leukocyte rolling and adhesion in the rat mesentery was combined with histological determination of the intravascular concentrations of polymorphonuclear and mononuclear leukocytes (PMNL and MNL). 2 By relating the histologically determined number of intravascular leukocytes to either microvessel volume or to the erythrocyte concentration, the baseline MNL and PMNL content was found to be 3 ± 6 fold higher in venules than in systemic blood. This increase in microvessel leukocyte concentration did not seem to be related to leukocyte-endothelium interactions, because the leukocyte concentration was similarly elevated in arterioles where rolling and adhesion did not take place. 3 Preparation of the rat mesentery for intravital microscopy time-dependently increased the venular PMNL concentration to over 100 fold the systemic PMNL concentration 45 min after exteriorization of the small intestine. The MNLs were much less responsive to the preparative manipulation. By treatment with the polysaccharide fucoidin (inhibits rolling but not ®rm adhesion per se), or by use of intravital microscopy immediately before tissue ®xation, approximately 90% of the accumulated venular PMNLs were found to represent rolling cells. Intraperitoneal injection of 1073 M histamine increased the venular PMNL (but not the MNL) concentration to almost 50 fold the systemic PMNL value. The histamine response did not vary with venular diameter, and the relative contribution of rolling vs ®rmly adherent cells to the PMNL, accumulation was again &90%. Intraperitoneal injection of leukotriene C 4 , but not prostaglandin E 2 , caused a signi®cant increase in venular PMNL concentration. 5 Systemic treatment with the anti-P-selectin monoclonal antibody PB1.3 had no eect on the histamine-induced venular PMNL accumulation (i.e. rolling) in female Wistar or male Sprague-Dawley rats. On the other hand, identical treatment with PB1.3 very eectively inhibited the histamine-induced PMNL response in the mesentery of rabbits. 6 In conclusion, we have shown that a histologically determined increase in leukocyte concentration in rat mesenteric venules may be used as an index of mediator-induced leukocyte rolling if the relative contribution of rolling and ®rm leukocyte adhesion is ®rst determined, for example by the means described in this study. This relatively simple approach may be very useful for studying various aspects of leukocyte rolling when the`spontaneous' rolling triggered by preparation of tissues for intravital microscopy is undesirable.