Bilberries and their anthocyanins ameliorate experimental colitis.

Bilberries have positive effects in acute and chronic diarrhea. Patients with inflammatory bowel disease (IBD) report on improved symptoms upon ingestion. Bilberries contain approximately 10% of anthocyanins (ACs), which have anti-oxidative, anti-carcinogenic, and anti-inflammatory properties. We investigated whether experimental colitis can be ameliorated by dried bilberries or ACs. Acute and chronic dextrane sodium sulphate (DSS) colitis were induced in Balb/c mice by 2.5% DSS in the drinking water. Mice were fed with dried bilberries or ACs, respectively. Cytokines were determined in supernatants from mesenteric lymph nodes (MLNs) by ELISA and apoptosis was investigated by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assays. Oral administration of bilberries during acute DSS-induced colitis ameliorated disease severity and reduced secretion of IFN-γ and tumor necrosis factor from mesenteric lymph node cells. Dried bilberries also improved chronic DSS-colitis. Ingestion of ACs reduced intestinal inflammation in acute and chronic DSS-colitis with decreased histological scores and cytokine secretion. Both bilberries and ACs prevented inflammation-induced apoptosis in colonic epithelial cells. Taken together, ingestion of dried bilberries had positive effects on various parameters especially in acute DSS-colitis. Oral administration of ACs resulted in an amelioration of acute colitis as well as chronic colitis. These promising results justify a clinical study on their therapeutic effect in inflammatory bowel disease patients.

Preparation and characterization of shellac-coated anthocyanin pectin beads as dietary colonic delivery system.

SCOPE: Anthocyanins are connected with various biological activities. A promising way to enhance the availability of anthocyanins for in situ effects in the lower intestine is colon-specific delivery. METHODS AND RESULTS: Shellac and shellac/hydroxypropyl methylcellulose (HPMC) coated anthocyanin amidated pectin beads as dietary colonic delivery systems were successfully prepared by ionotropic gelation and fluid bed Wurster coating with aqueous shellac solution. Release characteristics, studied in vitro and ex vivo using simulated gastric fluid (SGF), ileostomy fluid and colostomy fluid (CF) revealed a retardation of anthocyanins during simulated passage of stomach and ileum as well as the desired release of pigments in the colon. Coating level was identified as an important parameter. By addition of 5 or 15% of the water-soluble polysaccharide HPMC to the shellac film, resistance in SGF was increased due to the plasticizer properties of the polymer. Incorporation of 15% HPMC (w/w based on shellac) into the shellac film additionally led to increased anthocyanin diffusivity and complete release as well as degradation of the formulation in CF. CONCLUSION: In the used in vitro and ex vivo model system mimicking the human intestinal transit, the potential of shellac and shellac/HPMC coated anthocyanin amidated pectin beads as dietary colon targeting systems was demonstrated.

Flavan-3-ol C-glycosides--preparation and model experiments mimicking their human intestinal transit.

In order to study the human intestinal transit of flavan-3-ol C-glycosides, several C-glycosyl derivatives were prepared by non-enzymatic reaction of (+)-catechin with α-D-glucose, α-D-galactose and α-D-rhamnose, respectively. In contrast to literature data, we propose that the reaction mechanism proceeds in analogy to the rearrangement of flavan-3-ols during epimerization under alkaline conditions. Four of the 12 synthesized flavan-3-ol C-glycosides were incubated under aerobic conditions at 37°C using saliva (2 min) and simulated gastric juice (3 h). To simulate human intestine, the C-glycosides were also incubated under anaerobic conditions at 37°C both in human ileostomy fluid (10 h) and colostomy fluid (24 h), respectively. The flavan-3-ol C-glycosides under study, i.e. (+)-epicatechin 8-C-ß-D-glucopyranoside (1a), (+)-epicatechin 6-C-ß-D-glucopyranoside (1d), (+)-catechin 6-C-ß-D-galactopyranoside (2b), (+)-catechin 6-C-ß-D-rhamnopyranoside (3b) were analyzed in the incubation samples by HPLC-DAD and HPLC-DAD-MS/MS. They were found to be stable in the course of incubation in saliva, simulated gastric juice and ileostomy fluid and underwent degradation in colostomy fluid. While the 6-C-ß-D-glucopyranoside 1d was completely metabolized between 2 and 4 h, decomposition of the 6-C-ß-D-galactopyranoside 2b reached only 16 ± 2% within 4 h of incubation. Linear degradation rates of 1d and 2b in colostomy fluid differed significantly. As microbial metabolism of flavan-3-ols is known not to be influenced by the stereochemistry of the aglycon, varying degradation rates are ascribed to the effect of the sugar moiety. Based on these results we assume that flavan-3-ol C-glycosides pass through the upper gastrointestinal tract (oral cavity, stomach and small intestine) unmodified and are then metabolized by the colonic microflora.

Cytochrome P450 2C19 inhibitory activity of common berry constituents.

The cytochrome P450 enzyme CYP2C19 is involved in the metabolism of many commonly prescribed drugs, including proton pump inhibitors, antiepileptics and antidepressants. CYP2C19 inhibitors from food and food supplements may augment the toxicity of these agents and lead to noncompliance with treatment. The present investigation addresses CYP2C19 inhibition by 18 berry constituents using a chemiluminescent assay. Test compounds displayed inhibitory properties in a concentration-dependent fashion, with IC(50) values ranging from 20.2 microM up to >316 microM. In the order of decreasing effect size, anthocyanidins were followed by anthocyanidin-monoglycosides and procyanidins. Anthocyanidin-diglucosides exhibited weak and biphasic effects. When compared with the CYP2C19 inhibitor fluvoxamine, the flavonoids under study were 50- to 750-fold less potent. It is concluded that the above natural substances are moderate to poor inhibitors of CYP2C19 in vitro.

sPhospholipase A(2) is inhibited by anthocyanidins.

Epidemiological studies suggest that nutritional antioxidants may reduce the incidence of neurodegenerative disorders and age-related cognitive decline. Specifically, protection against oxidative stress and inflammation has served as a rationale for promoting diets rich in vegetables and fruits. The present study addresses secretory phospholipase A(2) (sPLA(2)) as a novel candidate effector of neuroprotection conferred by anthocyanins and anthocyanidins. Using a photometric assay, 15 compounds were screened for their ability to inhibit PLA(2). Of these, cyanidin, malvidin, peonidin, petunidin, and delphinidin achieved K(i) values

Berry anthocyanins and their aglycons inhibit monoamine oxidases A and B.

Monoamine oxidases (MAO) are mitochondrial enzymes that catalyze the oxidation of monoamines in multiple tissues, including the brain. Elevated MAO activity has long been implicated in the etiology of depression, anxiety, and neurodegenerative disease, fuelling the search for inhibitors in the prevention and treatment of these disorders. We hypothesized that emerging neuroprotective effects of anthocyanins from berry fruits may be explained by an affinity of these polyphenols for MAO isoforms A or B. Using a luminometric MAO assay, 25 anthocyanidins, anthocyanidin-3-glycosides, anthocyanidin-3,5-diglucosides, proanthocyanidins, and phenolic metabolites were examined. For MAO A and B, IC(50) values in the low micromolar range were reached by anthocyanidins and anthocyanidin-3-glycosides, as opposed to values in the low millimolar range for phenolic acids. Kinetic analyses, performed with cyanidin and cyanidin-3-glucoside, indicated a competitive interaction of cyanidin with MAO A plus a mixed competitive and non-competitive mode of interaction of cyanidin with MAO B and of cyanidin-3-glucoside with both enzyme isoforms. Thus anthocyanins and their aglycons achieve MAO inhibition in vitro that is compatible with central nervous functionalities. For extrapolation of the present findings to in vivo effects, future studies will need to address in more detail the bioavailability of these dietary constituents.

Anthocyanins and anthocyanidins are poor inhibitors of CYP2D6.

The cytochrome P450 CYP2D6 isoform is involved in the metabolism of about 50% of all psychoactive drugs, including neuroleptic agents, selective serotonin reuptake inhibitors, selective norepinephrine reuptake inhibitors and tricyclic antidepressants. Therefore, inhibition of cytochrome P450 activity by foodstuffs has implications for drug safety. The present study addresses inhibitory effects of polyphenolic anthocyanins and their aglycons that are found in many dietary fruits and vegetables. Using a chemiluminescent assay, we obtained IC(50) values ranging from 55 microM to > 800 microM for 17 individual compounds. According to earlier data on furanocoumarins from grapefruit extract, CYP2D6 inhibition is achieved in the range of 190-900 nM. As the tested anthocyanins and anthocyanidins were shown to be about 1,000-fold less potent, they are unlikely to interfere with drug metabolism by CYP2D6. Further studies are warranted to examine the effects of the above flavonoids on other CYP isoforms for more detailed toxicity profiles.

An examination of anthocyanins' and anthocyanidins' affinity for cannabinoid receptors.

A growing body of evidence suggests that anthocyanins and anthocyanidins may possess analgesic properties in addition to neuroprotective and anti-inflammatory activities. These functionalities suggest a role for the cannabinoid receptor (CB) in mediating biological effects. Competitive radioligand binding assays identified cyanidin (K(i) = 16.2 microM) and delphinidin (K(i) = 21.3 microM) as ligands with moderate affinity to human CB1. For CB2, similar affinities were achieved by cyanidin (K(i) = 33.5 microM), delphinidin (K(i) = 34.3 microM), and peonidin (K(i) = 46.4 microM). Inhibition constants >50 microM were obtained for pelargonidin, malvidin, cyanidin-3,5-di-O-glucoside, cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, and cyanidin-3-O-rutinoside for both CB subtypes.

Anthocyanins as lipoxygenase inhibitors.

Health benefits associated with diets rich in anthocyanins are ascribed to multilevel biological activities including antioxidative and anti-inflammatory effects. The present study addresses lipoxygenase inhibition as a mechanism by which anthocyanins may exert health promoting effects. The inhibitory potential of delphinidin (Dp), cyanidin (Cy), peonidin (Pn), and malvidin (Mv) glycosides, i.e., 3-O-glucosides, 3-O-galactosides, and 3-O-arabinosides as well as their aglycons was analyzed by using soybean lipoxygenase-1 and human neutrophil granulocyte 5-lipoxygenase. The determined IC(50) values comprised a wide range, i.e., from the sub-microM level until practically no effect of inhibition (Mv and its glycosides). With IC(50) values of 0.43 and 0.49 microM Dp 3-O-glucoside (Dp3glc) and Dp 3-O-galactoside (Dp3gal) were found to be the most effective soybean lipoxygenase-1 inhibitors; their strong inhibitory potential was also reflected by the IC(50) values determined for these anthocyanins in the 5-lipoxygenase inhibition exhibiting 2.15 and 6.9 microM, respectively. As to the mechanism of inhibition, experiments carried out with lipoxygenase-1 revealed the uncompetitive type. Considering the powerful inhibitory properties of Dp glycosides in relation to their currently known availability in human metabolism, in vivo prevention of inflammatory diseases by these anthocyanins could be envisaged.

Anthocyanins and their metabolites are weak inhibitors of cytochrome P450 3A4.

The cytochrome P450 enzyme cytochrome P450 3A4 (CYP3A4) controls the metabolism of about 60% of all drugs, and its inhibition may dramatically affect drug safety. Modulation of cytochrome P450 activity has been observed by constituents of fruit extracts including several flavonoids. The present investigation addresses CYP3A4 inhibition by anthocyanins, their aglycons, proanthocyanidins, and phenolic metabolites using a chemiluminescent assay. Test compounds inhibited CYP3A4 activity in a concentration-dependent manner featuring IC(50) values from 12.2 up to 7,842 microM. In the order of decreasing effect size, anthocyanidins were followed by anthocyanins, proanthocyanidins, and phenolic acids. When compared to earlier data on furanocoumarins from grapefruit extract, the inhibitory activity of tested anthocyanins, and anthocyanidins was shown to be about 10,000-fold weaker, and was negligible for phenolic acids (>100 000-fold weaker). Future studies are invited to address effects of the above flavonoids on other CYP isoforms for more detailed toxicity profiles.

Model experiments mimicking the human intestinal transit and metabolism of D-galacturonic acid and amidated pectin.

In order to study the human intestinal transit and metabolism of D-galacturonic acid and amidated pectin a number of model experiments were carried out. Both substrates were incubated under aerobic conditions at 37 degrees C using saliva (2 min) and simulated gastric juice (4 h). Under anaerobic conditions the substrates were incubated at 37 degrees C using human ileostomy and colostomy fluids, each obtained from three different donors, for 10 and for 24 h, respectively. D-Galacturonic acid, SCFA (acetic acid, propionic acid, and butyric acid), as well as methanol were analyzed photometrically after carbazole reaction, GC-flame ionization detection (GC-FID), and headspace solid-phase microextraction GC/MS (HS-SPME-GC/MS), respectively. D-Galacturonic acid and amidated pectin were found to be stable during incubations with saliva and simulated gastric juice, whereas both substrates underwent degradation in the course of human ileostomy and colostomy fluid incubations. D-Galacturonic acid was practically completely decomposed within 10 h and SCFA, with acetic acid as the major representative, were formed up to 98% of the incubated substrate in colostomy effluent. The amidated pectin was only degraded in part, revealing stable amounts of 22-35% and 3-17% in ileostomy (after 10 h) and colostomy fluid (after 24 h), respectively. SCFA were generated up to 59% of the applied amidated pectin. In parallel, 19-60% and 52-67% of the available methyl ester groups were cleaved in the course of incubations with ileostomy and colostomy fluids, respectively. The results demonstrate for the first time that D-galacturonic acid and amidated pectin are stable in human saliva and simulated gastric juice. The degradation of both compounds during incubation with ileostomy effluent is highlighted, providing evidence for a considerable metabolic potential of the small intestine.

Inhibition of proteasome activity by anthocyanins and anthocyanidins.

Recent reports have demonstrated multiple benefits associated with the consumption of berry fruits, including a decreased vulnerability to oxidative stress, reduced ischemic brain damage, protection of neurons from stroke-induced damage and the reversal of age-related changes in brain and behaviour. Berry fruits contain high amounts of anthocyanins, which play a major role as free radical scavengers. The present study addresses proteasome inhibition as a further mechanism by which anthocyanins and their aglycons, the anthocyanidins, may exert health-promoting effects. HL-60 cells were incubated with 19 test substances and inhibition of the chymotrypsin-like enzyme activity was determined in a chemiluminescent assay. Anthocyanins and their aglycons achieved IC(50) values ranging from 7.8 microM for kaempferidinidin and pelargonidin, to 32.4 microM for delphinidin. Thus proteasome inhibitory properties of anthocyanins may contribute to their known anticarcinogenic, antioxidative, anti-inflammatory and neuroprotective activities, rationalizing dietary supplementations with anthocyanins in the prevention and treatment of chronic diseases, including neurodegenerative disorders.

A convenient HPLC assay for the determination of fructosamine-3-kinase activity in erythrocytes.

Fructosamine-3-kinase (FN3K) mediates the regeneration of lysine from fructosamines formed on proteins as a result of the 'early' Maillard reaction. As fructosamines and advanced glycation endproducts derived therefrom are supposed to play an adverse role in the development of diabetic complications, FN3K is discussed as a protein-repairing enzyme. In this study, a method for the determination of FN3K activity in erythrocyte lysate is described which overcomes the complexity of currently known assays. The assay is based on the FN3K-dependent conversion of the synthetic UV-active fructosamine Nalpha-hippuryl-Nepsilon-(1-deoxy-D-fructosyl)lysine (BzGFruK) to Nalpha-hippuryl-Nepsilon-(phosphofructosyl)lysine (BzGpFruK). The FN3K activity was quantified by measuring the formation of BzGpFruK using RP-HPLC with UV detection. Identification of the metabolite BzGpFruK was achieved by means of UV and mass spectroscopy. The results are related to the content of haemoglobin for standardisation. First activity measurements with a chosen number of normoglycaemic subjects confirmed the convenient applicability of the method and showed distinctly different individual activities, as already discovered recently. The new established assay needs only the equipment of a routine laboratory with HPLC instrumentation. This should facilitate further studies about a possible relationship between the FN3K activity and the development of diabetic complications.