The Anthocyanin Delphinidin 3-Rutinoside Stimulates Glucagon-Like Peptide-1 Secretion in Murine GLUTag Cell Line via the Ca2+/Calmodulin-Dependent Kinase II Pathway.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from enteroendocrine L-cells. Although several nutrients induce GLP-1 secretion, there is little evidence to suggest that non-nutritive compounds directly increase GLP-1 secretion. Here, we hypothesized that anthocyanins induce GLP-1 secretion and thereby significantly contribute to the prevention and treatment of diabetes. Delphinidin 3-rutinoside (D3R) was shown to increase GLP-1 secretion in GLUTag L cells. The results suggested that three hydroxyl or two methoxyl moieties on the aromatic ring are essential for the stimulation of GLP-1 secretion. Notably, the rutinose moiety was shown to be a potent enhancer of GLP-1 secretion, but only in conjunction with three hydroxyl moieties on the aromatic ring (D3R). Receptor antagonist studies revealed that D3R-stimulates GLP-1 secretion involving inositol 1,4,5-trisphosphate receptor-mediated intracellular Ca2+ mobilization. Treatment of GLUTag cells with a Ca2+/calmodulin-dependent kinaseII (CaMKII) inhibitor (KN-93) abolished D3R-stimulated GLP-1 secretion. In addition, treatment of GLUTag cells with D3R resulted in activation of CaMKII. Pre-treatment of cells with a G protein-coupled receptor (GPR) 40/120 antagonist (GW1100) also significantly decreased D3R-stimulated GLP-1 secretion. These observations suggest that D3R stimulates GLP-1 secretion in GLUTag cells, and that stimulation of GLP-1 secretion by D3R is mediated via Ca2+-CaMKII pathway, which may possibly be mediated by GPR40/120. These findings provide a possible molecular mechanism of GLP-1 secretion in intestinal L-cells mediated by foods or drugs and demonstrate a novel biological function of anthocyanins in regards to GLP-1 secretion.

Flavonoids in foods: a review.

The flavonoids in edible plants and foods are surveyed. The food materials rich in flavonoids, such as grape, tea, and cocoa receive particular attention. Also, the ingestion of flavonoids, the absorption and the bioavailability in vivo are discussed. Flavonoids have antioxidant and other living-body modulating activities. When expressing these functionalities, interactions between flavonoids and proteins are important. The interactions with various enzymes and receptors are described. Moreover, as an example of a processed food containing flavonoids, fermented red vinegar that we have investigated is highlighted. The structural determination, antioxidant activity and the generation mechanism of the new acylated polyphenols in red vinegar are described.

New acylated anthocyanins from purple yam and their antioxidant activity.

Purple yam (Dioscorea alata L.), which is widely distributed in tropical and subtropical regions, is characterized by its color and viscosity. Previous studies have shown that purple yams contain a variety of acylated anthocyanins that exhibit higher levels of antioxidant activity than the corresponding nonacylated compounds. In this study, the pigments found in purple yams from the Philippines (D. alata) were isolated and evaluated in terms of antioxidant activity. Four new acylated anthocyanins, alanins (1-4) were isolated from the MeOH extracts of purple yam, which were subsequently determined to be cyanidin (1, 2, and 4) and peonidin (3) type compounds, along with four known anthocyanins (5-8). The structures of 1-4 were determined by spectroscopic methods, including NMR and MS analyses. The antioxidant activities of anthocyanins 1-8 were investigated using oxygen radical absorbing capacity and ferric reducing antioxidant power assays.

Anti-inflammatory activity and molecular mechanism of delphinidin 3-sambubioside, a Hibiscus anthocyanin.

Delphinidin 3-sambubioside (Dp3-Sam), a Hibiscus anthocyanin, was isolated from the dried calices of Hibiscus sabdariffa L, which has been used for folk beverages and herbal medicine although the molecular mechanisms are poorly defined. Based on the properties of Dp3-Sam and the information of inflammatory processes, we investigated the anti-inflammatory activity and molecular mechanisms in both cell and animal models in the present study. In the cell model, Dp3-Sam and Delphinidin (Dp) reduced the levels of inflammatory mediators including iNOS, NO, IL-6, MCP-1, and TNF-α induced by LPS. Cellular signaling analysis revealed that Dp3-Sam and Dp downregulated NF-κB pathway and MEK1/2-ERK1/2 signaling. In animal model, Dp3-Sam and Dp reduced the production of IL-6, MCP-1 and TNF-α and attenuated mouse paw edema induced by LPS. Our in vitro and in vivo data demonstrated that Hibiscus Dp3-Sam possessed potential anti-inflammatory properties.

Comparison of the inhibitory effects of delphinidin and its glycosides on cell transformation.

Although anthocyanins are major forms distributed in many plant foods and promising as chemopreventive source, many molecular data are obtained from anthocyanidins, showing their low bioavailability. This study aims to clarify the inhibitory effects of delphinidin glycosides on cell transformation comparing them to those of delphinidin. Screening data revealed that delphinidin 3-sambubioside could directly bind to MAPK/ERK kinase 1. Affinity assay data confirmed that delphinidin 3-sambubioside had higher binding affinity to MAPK/ERK kinase 1 than ERK1/2 and B-Raf. Colony assay data further demonstrated that delphinidin 3-sambubioside inhibited 12-O- tetradecanoylphorbol-13-acetate-induced phosphorylation of MAPK/ERK kinase 1 and sequentially suppressed cell transformation. All of these effects caused by delphinidin 3-sambubioside were weaker than those by its aglycon, delphinidin. Our data suggested that the weaker anti- transformation activity of delphinidin glycosides compared to that of their aglycon is due to lower binding affinity to the target molecule MAPK/ERK kinase 1.

Abscisic acid metabolism and anthocyanin synthesis in grape skin are affected by light emitting diode (LED) irradiation at night.

The effects of blue and red light irradiation at night on abscisic acid (ABA) metabolism and anthocyanin synthesis were examined in grape berries. The expressions of VlMYBA1-2, VlMYBA2, UDP-glucose-flavonoid 3-O-glucosyltransferase (VvUFGT), 9-cis-epoxycarotenoid dioxygenase (VvNCED1), and ABA 8'-hydroxylase (VvCYP707A1) were also investigated. Endogenous ABA, its metabolite phaseic acid (PA), and the expressions of VvNCED1 and VvCYP707A1 were highest in red light-emitting diode (LED)-treated skin. In contrast, anthocyanin concentrations were highest in blue LED-treated skin, followed by red LED treatment. However, the expressions of VlMYBA1-2, VlMYBA2, and VvUFGT did not necessarily coincide with anthocyanin concentrations. The quality of coloring may depend on the amount of malvidin-based anthocyanin, which increased toward harvest in blue and red LED-treated skin, unlike in untreated controls. An increase in sugars was also observed in blue and red LED-treated skin. These results suggest that blue LED irradiation at night may be effective in increasing anthocyanin and sugar concentrations in grape berries. However, there is evidence that another factor may influence anthocyanin concentrations in grape berry skin significantly more than endogenous ABA: ABA concentrations were highest in red LED-treated skin, which had lower anthocyanin concentrations than blue LED-treated skin.

Transepithelial transport of 6-O-caffeoylsophorose across Caco-2 cell monolayers.

The aim of this study was to clarify the transport behaviour and mechanism of caffeic acid analogue bearing a sugar-moiety, 6-O-caffeoylsophorose (CS), in Caco-2 cells. The absorption of CS was investigated by its transport across Caco-2 cell monolayers using a high-performance liquid chromatography-time-of-flight-mass spectrometry (LC-TOF-MS). The permeation of CS was concentration-dependent and reached the plateau at >6 mM. The apparent permeability (P(app)) of CS in the apical-to-basolateral direction was 5.4×10(-7) cm/s, while in the reversed direction the P(app) value was significantly reduced (1.9×10(-7) cm/s). CS transport was competitively inhibited by phloretin, an inhibitor of monocarboxylic acid transporter (MCT). Benzoic acid, an MCT substrate, also reduced CS transport. A less significant change of CS transport was observed across Caco-2 cell monolayers pretreated with quercetin, a suppressor of tight-junction. These findings strongly indicate that CS, a caffeic acid analogue bearing sophorose moiety, can be transported across Caco-2 cell monolayers via the MCT pathway.

Functional new acylated sophoroses and deglucosylated anthocyanins in a fermented red vinegar.

The new acylated polyphenols were isolated from a red-colored vinegar produced via fermentation with purple-fleshed sweet potato storage roots, and identified mainly by MS and NMR. The three acylated sophoroses were determined as 6-O-(E)-caffeoyl-(2-O-(6-O-acyl)-beta-d-glucopyranosyl)-d-glucopyranoses, where acyl was (E)-caffeoyl, p-hydroxybenzoyl, and (E)-feruloyl, respectively. The four acylated anthocyanins were also determined as cyanidin 3-O-(6-O-(E)-caffeoyl-(2-O-(6-O-(E)-feruloyl)-beta-d-glucopyranosyl)-beta-d-glucopyranoside), in addition to peonidin 3-O-(6-O-(E)-caffeoyl-(2-O-(6-O-acyl)-beta-d-glucopyranosyl)-beta-d-glucopyranosides), where acyl was (E)-caffeoyl, p-hydroxybenzoyl, and (E)-feruloyl, respectively. The diacylated sophoroses showed higher antioxidant capacity than that of monoacylated analogue 6-caffeoylsophorose, so the multiacylation established to enhance their antioxidant capacity. Similarly, 5-deglucosylated anthocyanins also gave somewhat stronger antioxidation than corresponding sweet potato anthocyanins. In rat intestinal alpha-glucosidase inhibition study, the diacylated sophoroses preferably inhibited maltase rather than sucrase with an IC(50) value of <300 microM, indicating a potential role as antidiabetic phytochemicals. These acylated polyphenols in a red vinegar were expected to play important functional roles for health.

Determination of acylated anthocyanin in human urine after ingesting a purple-fleshed sweet potato beverage with various contents of anthocyanin by LC-ESI-MS/MS.

Eighty-seven healthy volunteers ingested a purple-fleshed sweet potato beverage with various contents of anthocyanin (beverage A; 22.1 mg/250 ml, B; 107.8, C; 84.9). An acylated anthocyanin, peonidin 3-caffeoylsophoroside-5-glucoside, was detected in the urine 2 h after ingestion. The concentrations were 15.1+/-2.2 microg/l of urine (mean+/-SEM), 46.6+/-5.3, and 53.3+/-2.2 for beverages A, B, and C respectively.

Gastrointestinal uptake of nasunin, acylated anthocyanin in eggplant.

We previously showed that nasunin, acylated anthocyanins in eggplant peel, comprises two isomers, cis-nasunin and trans-nasunin. In this study, gastrointestinal absorption of cis- and trans-nasunins was studied in rats. Orally administered nasunins were quickly absorbed in their original acylated forms and maximally appeared in blood plasma after 15 min. When the maximum plasma concentration and area under the plasma concentration curve were normalized by orally administered dose (micromoles per kilogram), there was no significant difference in the uptake efficiency between two isomers and both exhibited a plasma level almost identical to that of delphinidin 3-O-beta-D-glucopyranoside. However, metabolites such as 4'-O-methyl analogues and extended glucuronides which were observed for delphinidin 3-O-beta-D-glucopyranoside and cyanidin 3-O-beta-D-glucopyranoside metabolisms were not detected in urine or blood plasma. Moreover, deacylated and glycolytic products of nasunins such as delphinidin 3-O-beta-D-glucopyranoside or delphinidin (aglycone) were also not detected in blood plasma even after oral administration for 8 h. These results indicated that nasunins were absorbed in their original acylated forms and exhibit a bioavailability almost identical to that of nonacylated anthocyanins.

Delphinidin 3-sambubioside, a Hibiscus anthocyanin, induces apoptosis in human leukemia cells through reactive oxygen species-mediated mitochondrial pathway.

Delphinidin 3-sambubioside (Dp3-Sam), a Hibiscus anthocyanin, was isolated from the dried calices of Hibiscus sabdariffa L. Dp3-Sam could induce a dose-dependent apoptosis in human leukemia cells (HL-60) as characterized by cell morphology, DNA fragmentation, activation of caspase-3, -8, and -9, and inactivation of poly(ADP)ribose polymerase (PARP). Molecular data showed that Dp3-Sam induced Bid truncation, mitochondrial membrane potential (DeltaPsi(m)) loss, and cytochrome c release from mitochondria to cytosol. Moreover, Dp3-Sam caused a time- and dose-dependent elevation of intracellular reactive oxygen species (ROS) level in HL-60 cells, and antioxidants such as N-acetyl-L-cysteine (NAC) and catalase could effectively block Dp3-Sam-induced ROS generation, caspase-3 activity, and DNA fragmentation. These data indicate that Dp3-Sam might induce apoptosis in HL-60 cells through a ROS-mediated mitochondrial dysfunction pathway. These findings enhance our understanding for anticancer function of Hibiscus anthocyanins in herbal medicine.

Molecular Mechanisms Behind the Chemopreventive Effects of Anthocyanidins.

Anthocyanins are polyphenolic ring-based flavonoids, and are widespread in fruits and vegetables of red-blue color. Epidemiological investigations and animal experiments have indicated that anthocyanins may contribute to cancer chemoprevention. The studies on the mechanism have been done recently at molecular level. This review summarizes current molecular bases for anthocyanidins on several key steps involved in cancer chemoprevention: (i) inhibition of anthocyanidins in cell transformation through targeting mitogen-activated protein kinase (MAPK) pathway and activator protein 1 (AP-1) factor; (ii) suppression of anthocyanidins in inflammation and carcinogenesis through targeting nuclear factor kappa B (NF- $\kappa$ B) pathway and cyclooxygenase 2 (COX-2) gene; (iii) apoptotic induction of cancer cells by anthocyanidins through reactive oxygen species (ROS) / c-Jun NH(2)-terminal kinase (JNK)-mediated caspase activation. These data provide a first molecular view of anthocyanidins contributing to cancer chemoprevention.

Caffeoylsophorose, a new natural alpha-glucosidase inhibitor, from red vinegar by fermented purple-fleshed sweet potato.

The suppressive effect on the postprandial blood glucose rise through alpha-glucosidase (AGH) inhibition was investigated in this study in order to clarify an antihyperglycemic function of 6-O-caffeoylsophorose (CS) from diacylated anthocyanin. The administration of CS (100 mg/kg) following maltose (2 g/kg) to Sprague-Dawley rats resulted in the maximal blood glucose level after 30 min being significantly decreased by 11.1% compared to the control. A reduction in the serum insulin secretion was also observed in parallel to the decrease in blood glucose level. No blood glucose change was apparent when sucrose or glucose was ingested, suggesting that the antihyperglycemic effect of CS was achieved by maltase inhibition, rather than by sucrase or glucose transport inhibition. An AGH inhibitory assay demonstrated that the non-competitive maltase inhibition of CS was partly due to acylation by phenolic acid with sugar, the presence of hydroxyl groups in the aromatic ring, and the presence of an unsaturated alkyl chain in the acylated moiety.

Anthocyanidins inhibit activator protein 1 activity and cell transformation: structure-activity relationship and molecular mechanisms.

Anthocyanins are the chemical components that give the intense color to many fruits and vegetables, such as blueberries, red cabbages and purple sweet potatoes. Extensive studies have indicated that anthocyanins have strong antioxidant activities. To investigate the mechanism of anthocyanidins as an anticancer food source, six kinds of anthocyanidins representing the aglycons of most anthocyanins, were used to examine their effects on tumor promotion in mouse JB6 cells, a validated model for screening cancer chemopreventive agents and elucidating the molecular mechanisms. Of the six anthocyanins tested, only those with an ortho-dihydroxyphenyl structure on the B-ring suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation and activator protein-1 transactivation, suggesting that the ortho-dihydroxyphenyl may contribute to the inhibitory action. Delphinidin, but not peonidin, blocked the phosphorylation of protein kinases in the extracellular signal-regulated protein kinase (ERK) pathway at early times and the c-Jun N-terminal kinase (JNK) signaling pathway at later times. p38 kinase was not inhibited by delphinidin. Furthermore, two mitogen-activated protein kinase (MAPK) specific inhibitors (SP600125 for JNK and UO126 for ERK) could specifically block the activation of JNK and ERK and cell transformation. Those results demonstrate that anthocyanidins contribute to the inhibition of tumorigenesis by blocking activation of the MAPK pathway. These findings provide the first molecular basis for the anticarcinogenic action of anthocyanidins.

Potential chemopreventive properties of anthocyanin-rich aqueous extracts from in vitro produced tissue of sweetpotato (Ipomoea batatas L.).

Anthocyanin-rich aqueous extracts from cell suspension cultures of a high anthocyanin-producing sweetpotato PL (purple line) cell line grown under two different media conditions, MM (multiplication medium) and APM (high anthocyanin-producing medium) and from the cell line's donor tissue, field-grown storage root (SR) of sweetpotato, cv. Ayamurasaki, were evaluated for antioxidative (DPPH test), antimutagenic (Salmonella/reversion assay; mutagen, Trp-P-1), and antiproliferative (human promyelocytic leukaemia cells HL-60) activities. Both cell line extracts MM and APM exhibited higher radical scavenging activities (RSA), 3.8- and 1.4-fold, respectively, than the SR extract. The antimutagenic activity of all extracts was found to be dose-dependent. At a dose of 1 mg/plate, the highest activity exhibited APM (73% inhibition of Trp-P-1-induced reverse mutation of Salmonella typhimurium TA98), followed by MM (54% inhibition) and SR (36% inhibition). The MM extract was the strongest inhibitor of the proliferation of human promyelocytic leukemia cells. At a concentration of 1.6 mg/mL medium during 24 h, it suppressed the growth of 47% of HL-60 cells. A significantly lower growth suppression effect displayed APM and SR extracts (21 and 25%, respectively). Total anthocyanin levels and anthocyanin composition in evaluated samples seem to be related to their activities. The MM extract, which exhibited the highest RSA and antiproliferation activities, contained the highest level of anthocyanins. Among them, nonacylated cyanidin 3-sophoroside-5-glucoside dominated. It is speculated that the presence of this anthocyanin contributed toward enhanced activities of MM extract.

Anthocyanidins induce apoptosis in human promyelocytic leukemia cells: structure-activity relationship and mechanisms involved.

Anthocyanidins are the aglycon nucleuses of anthocyanins, which are reddish pigments widely spread in colored fruits and vegetables. To investigate their anti-cancer effect, induction of apoptosis was tested in human promyelocytic leukemia cells (HL-60), which is a valid model for testing antileukemic or general antitumoral compounds. Of six anthocyanidins representing the aglycons of most of anthocyanins, only those with an ortho-dihydroxyphenyl structure on the B-ring induce apoptosis, suggesting that the ortho-dihydroxyphenyl structure of anthocyanidins may contribute to the induction of apoptosis. Delphinidin, the most potent inducer, causes apoptosis in a time- and dose-dependent manner. The efficacious induction of apoptosis was observed at 100 micro M for 6 h. Concomitant with the apoptosis, delphinidin stimulates JNK pathway activation including JNK phosphorylation and c-jun gene expression, and activates caspase-3. Antioxidants including N-acetyl-L-cysteine (NAC) and catalase effectively block delphinidin-induced JNK phosphorylation, caspase-3 activation, and DNA fragmentation. Moreover, anthocyanidins directly cause HL-60 cells to generate intracellular hydrogen peroxide. Thus, anthocyanidins may trigger an apoptotic death program through an oxidative stress-involved JNK signaling pathway. The induction of apoptosis by anthocyanins may be the pivotal mechanism by which its chemopreventive action against cancer is based.

Caffeoylsophorose in a red vinegar produced through fermentation with purple sweetpotato.

Recently, a new red vinegar has been developed via fermentation with the storage root of purple-fleshed sweetpotato, Ipomoea batatas L. cv. Ayamurasaki. The red vinegar had a higher antioxidative activity than white or black vinegars. The red vinegar contained some new components possibly derived from the original purple sweetpotato. A major component was isolated using preparative HPLC, and the chemical structure was determined to be 6-O-(E)-caffeoyl-(2-O-beta-d-glucopyranosyl)-alpha-d-glucopyranose (caffeoylsophorose) by MS and NMR. Because the caffeoylsophorose showed a high antioxidative activity, it plays an important functional role in red vinegar as do anthocyanins and other components. Examination of the mechanism of formation is now in progress.

Anti-hyperglycemic effect of diacylated anthocyanin derived from Ipomoea batatas cultivar Ayamurasaki can be achieved through the alpha-glucosidase inhibitory action.

To clarify a postprandial glucose suppression effect of diacylated anthocyanin with alpha-glucosidase (AGH) inhibitory activity, a single oral administration study of it in male 8-week-old Sprague-Dawley rats was performed. The diacylated anthocyanin used in this study was peonidin 3-O-[2-O-(6-O-E-feruloyl-beta-D-glucopyranosyl)-6-O-E-caffeoyl-beta-D-glucopyranoside]-5-O-beta-D-glucopyranoside isolated from storage roots of the purple sweet potato (Ipomoea batatas cv. Ayamurasaki), which showed a potent maltase inhibitory activity with an IC(50) value of 200 microM preferable to sucrase inhibition. When the diacylated anthocyanin (100 mg/kg) was administered following maltose (2 g/kg), a maximal blood glucose level (BGL) at 30 min was significantly decreased by 16.5% (P < 0.01) compared to vehicle. A minimum 10 mg/kg dose of the anthocyanin was necessary for the suppression of glycemic rise, and the ED(20) (69 mg/kg) was estimated to be approximately 30-fold lower than that of the therapeutic drug acarbose (ED(20) = 2.2 mg/kg). A reduction of serum insulin secretion was also observed corresponding to the decrease in BGL. No significant change in BGL was observed when sucrose or glucose was ingested, suggesting that the anti-hyperglycemic effect of the anthocyanin was achieved by maltase inhibition, not by sucrase or glucose transport inhibition at the intestinal membrane.

Direct absorption of acylated anthocyanin in purple-fleshed sweet potato into rats.

Absorption of acylated anthocyanins in purple-fleshed sweet potato (Ipomoea batatas cv. Ayamurasaki) in rats was studied to obtain evidence that the acylated anthocyanins themselves could exert a physiological function in vivo. Peonidin 3-caffeoylsophoroside-5-glucoside (Pn 3-Caf*sop-5-glc) in purple-fleshed sweet potato was directly absorbed into rat and present as an intact acylated form in plasma. After oral administration of the purple-fleshed sweet potato anthocyanin (PSA) concentrate containing 38.9 micromol of Pn 3-Caf*sop-5-glc/kg of body weight, Pn 3-Caf*sop-5-glc was detected in the plasma, and the C(max) value and t(max) were estimated as 50.0 +/- 6.8 nmol/Lof plasma and 30 min, respectively. Furthermore, the plasma antioxidant capacity was significantly elevated from 58.0 +/- 12.0 to 89.2 +/- 6.8 micromol of Trolox equivalent/L of plasma 30 min after the administration of the PSA concentrate.

Anthocyanin compositions in sweetpotato (Ipomoea batatas L.) leaves.

The anthocyanin composition of three varieties, Simon No. 1, Kyushu No. 119, and Elegant Summer, in sweetpotato (Ipomoea batatas L.) leaves was examined for promoting new uses. Fifteen anthocyanin compounds were identified and measured. HPLC clearly showed quantitative differences, but not qualitative ones. The anthocyanins were acylated cyanidin and peonidin type. The result suggests that the major anthocyanin composition of sweetpotato leaves is cyanidin type.