Chemical Hemisynthesis of Sulfated Cyanidin-3-O-Glucoside and Cyanidin Metabolites.

The metabolism of anthocyanins in humans is still not fully understood, which is partly due to the lack of reference compounds. It is known that sulfation is one way of the complex phase II biotransformation mechanism. Therefore, cyanidin-3-O-glucoside and the cyanidin aglycone were chemically converted to their sulfates by reaction with sulfur trioxide-N-triethylamine complex in dimethylformamide. The reaction products were characterized by UHPLC coupled to linear ion trap and IMS-QTOF mass spectrometry. Based on MS data, retention times, and UV-Vis spectra, the compounds could tentatively be assigned to A-, C-, or B-ring sulfates. Analysis of urine samples from two volunteers after ingestion of commercial blackberry nectar demonstrated the presence of two sulfated derivatives of the cyanidin aglycone and one sulfated derivative of the cyanidin-3-O-glucoside. It was found that both the A ring and the B ring are sulfated by human enzymes. This study marks an important step toward a better understanding of anthocyanin metabolism.

Regiospecific 7-O-prenylation of anthocyanins by a fungal prenyltransferase.

Anthocyanins are a type of well-known natural flavonoids for their various beneficial health effects. However, prenylated anthocyanins are not discovered in nature although prenylation is believed to generally enhance the biological accessibility of flavonoids. In this article, we demonstrate the first example for prenylation of anthocyanins. A chemo-enzymatic approach was achieved for the synthesis of a series of 7-O-prenylated anthocyanins, using the fungal prenyltransferase CdpC3PT from Neosartorya fischeri.

Improvement of Naturally Derived Food Colorant Performance with Efficient Pyranoanthocyanin Formation from Sambucus nigra Anthocyanins Using Caffeic Acid and Heat.

Consumers and regulations encourage the use of naturally derived food colorants. Anthocyanins (ACN), plant pigments, are unstable in foods. In aged red wines, ACN with a free hydroxyl group at C-5 condenses to form pyranoanthocyanins (PACN), which are more stable but form inefficiently. This study attempted to produce PACN efficiently using high cofactor concentration and heat. Elderberry anthocyanins were semi-purified and caffeic acid (CA) was dissolved in 15% ethanol and diluted with a buffer to achieve ACN:CA molar ratios of 1:50, 1:100, 1:150, and 1:200, then incubated at 65 °C for 5 days. The effect of temperature was tested using ACN samples incubated with or without CA at 25 °C, 50 °C, and 75 °C for 7 days. Compositional changes were monitored using uHPLC-PDA-MS/MS. Higher CA levels seemed to protect pigment integrity, with ACN:CA 1:150 ratio showing the highest tinctorial strength after 48 h. PACN content growth was fastest between 24 and 48 h for all ACN:CA ratios and after 120 h, all ACN had degraded or converted to PACN. PACN formed faster at higher temperatures, reaching ~90% PACN in 24 h and ~100% PACN in 48 h at 75 °C. These results suggest that PACN can form efficiently from elderberry ACN and CA if heated to produce more stable pigments.

Hemisynthesis of Anthocyanin Phase II Metabolites by Porcine Liver Enzymes.

The aim of this work was to obtain phase II metabolites of cyanidin-3- O-glucoside and its aglycone using porcine liver enzymes. For this purpose, anthocyanins extracted from blackberry concentrate and containing mostly cyanidin-3- O-glucoside were incubated with the S9, microsomal, and cytosolic fractions of porcine liver. The reactions were targeted to the direction of the respective phase II transformation by the addition of activated cofactors. LC-MS n and LC-IMS-QTOF-MS analyses showed that one methylated, three glucuronidated and three sulfated metabolites of cyanidin-3- O-glucoside were generated. The aglycone, cyanidin, was sulfated and glucuronidated by the liver enzymes. In addition, both were glucuronidated and methylated simultaneously. The detected compounds and the generated data like exact masses, mass spectra, and CCS values may serve as a basis in the search for metabolites formed in vivo. As their effects are largely unexplored, the described synthesis may contribute to a better understanding of the metabolism of anthocyanins.

Mechanistic investigation of anthocyanidin derivatives as α-glucosidase inhibitors.

Eight anthocyanidin derivatives (1-8) were evaluated as potential inhibitors of the catalysis of α-glucosidase. Among them, compounds 4 and 8 had the highest levels of inhibitory activity at 100 µM (IC50 values of 14.4 ±â€¯0.1 and 29.7 ±â€¯1.2 µM) and acted in a dose-dependent manner. Enzyme kinetic analysis further revealed that these inhibitors interacted with α-glucosidase in a mixed noncompetitive mode. Moreover, fluorescence quenching studies provided parameters for calculating the binding mechanism between receptor and ligand. On the basis of these studies, and in silico simulations, we determined that the ligand was likely docked in the receptor. Thus, compounds 4 and 8 are excellent potential targets for in vitro cell-based and in vivo assays related to treatment of diabetes.

Design, Synthesis, Pharmacological Evaluation and Vascular Effects of Delphinidin Analogues.

BACKGROUND: Among polyphenolic compounds suggested to prevent cardiovascular diseases (CVDs) and to explain the "French paradox", the anthocyanidin delphinidin (Dp) has been reported to support at least partly the vascular beneficial effects of dietary polyphenolic compounds including those from fruits and related products as red wine. It has also been highlighted that Dp interacts directly with the active site of estrogen receptor α (ERα), leading to activation of endothelial NO synthase (eNOS) pathway thus contributing to the prevention of endothelial dysfunction in mice aorta. However, anthocyanidins have very low bioavailability and despite a well described in vitro efficacy, the very high hydrophilicity and physicochemical instability of Dp might explain the lack of in vivo reported effects. OBJECTIVE: The aim of this study was to identify new Dp analogues with increased lipophilicity and vasorelaxation potential by a chemical modulation of its structure and to characterize the signaling pathway notably in relation with ERα signaling and nitric oxide (NO) production. METHOD: OCH3-substituted delphinidin analogues were obtained through the coupling of the corresponding acetophenones with substituted benzaldehydes. Prediction of resorption of the flavylium derivatives was performed with the calculated logP and induction of vasorelaxation was performed by myography on WT and ERαKO mice thoracic aorta rings and compared to Dp. NO production was evaluated in vitro on human primary endothelial cells. RESULTS: Eight Dp analogues were synthesized including four new flavylium derivatives. Two compounds (9 and 11) showed a strong increase of vasorelaxation potential and a theoretically increased bioavailability compared to Dp. Interestingly, 9 and 11 induced increased O2 - or NO endothelial production respectively and revealed a novel NO-dependent ERα-independent relaxation compared to Dp. We suggested that this mechanism may be at least in part supported by the inhibition of vascular cyclic nucleotide phosphodiesterase (PDEs). CONCLUSION: The current study demonstrated that pharmacomodulation of the Dp backbone by replacement of OH groups by OCH3 groups of the A and B rings led to the identification and characterization of two compounds (9 and 11) with enhanced physio-chemical properties that could be associated to higher permeability capability and pharmacological activity for the prevention of CVDs compared to Dp.

Delphinidin inhibits BDNF-induced migration and invasion in SKOV3 ovarian cancer cells.

Brain-derived neurotrophic factor (BDNF), the TrkB ligand, is associated with aggressive malignant behavior, including migration and invasion, in tumor cells and a poor prognosis in patients with various types of cancer. Delphinidin is a diphenylpropane-based polyphenolic ring structure-harboring compound, which exhibits a wide range of pharmacological activities, anti-tumor, anti-oxidant, anti-inflammatory, anti-angiogenic and anti-mutagenic activity. However, the possible role of delphinidin in the cancer migration and invasion is unclear. We investigated the suppressive effect of delphinidin on the cancer migration and invasion. Thus, we found that BDNF enhanced cancer migration and invasion in SKOV3 ovarian cancer cell. To exam the inhibitory role of delphinidin in SKOV3 ovarian cancer migration and invasion, we investigated the use of delphinidin as inhibitors of BDNF-induced motility and invasiveness in SKOV3 ovarian cancer cells in vitro. Here, we found that delphinidin prominently inhibited the BDNF-induced increase in cell migration and invasion of SKOV3 ovarian cancer cells. Furthermore, delphinidin remarkably inhibited BDNF-stimulated expression of MMP-2 and MMP-9. Also, delphinidin antagonized the phosphorylation of Akt and nuclear translocation of NF-κB permitted by the BDNF in SKOV3 ovarian cancer cells. Taken together, our findings provide new evidence that delphinidin suppressed the BDNF-induced ovarian cancer migration and invasion through decreasing of Akt activation.

Semisynthesis and spectral characterization of 5-methylpyranopelargonidin and 4-methylfuropelargonidin and their separation and detection in strawberry fruit wine.

Condensation of anthocyanins and their aglycons with small organic molecules yields more stable natural dyes, e.g. pyranoanthocyanins arising spontaneously in various food products. Reaction of pelargonidin with acetone produces two isomeric anthocyanidin dyes - 5-methylpyranopelargonidin and 4-methylfuropelargonidin. A robust semipreparative liquid chromatographic method was developed to isolate both derivatives from a simple aged solution of pelargonidin in methanol: acetone: 37% aqueous hydrochloric acid (1:1:0.008, v/v/v). Detailed interpretation of mass and nuclear magnetic resonance spectra allowed to assign structures of both dyes in isolated fractions. A fast UHPLC-MS method was optimized for the control of their production in the reaction mixture. Use of reversed stationary phase and acidic mobile phases in gradient mode allowed separation of both isomers in less than one minute. Fragmentation of both dyes after collision activated dissociation in collision cell was studied comprehensively and the observed processes were compared with data from quantum calculations (computational chemistry utilizing DFT methods). When comparing both isomers, retro-Diels-Alder fragmentation appears preferred in furo-derivative, while small losses (i.e. methane and water molecules) were more pronounced in pyrano-derivative. Both studied isomeric dyes were found in laboratory prepared strawberry fruit wine and their content was compared with major present anthocyanins and their derivatives.

Synthesis of 8-Aryl-O-methylcyanidins and Their Usage for Dye-Sensitized Solar Cell Devices.

Anthocyanins as natural pigments are colorful and environmentally compatible dyes for dye-sensitized solar cells (DSSCs). To increase the efficiency, we designed and synthesized unnatural O-methylflavonols and O-methylcyanidins that possess an aryl group at the 8-position. We synthesized per-O-methylquercetin from quercetin, then using selective demethylation prepared various O-methylquercetins. Using the Suzuki-Miyaura coupling reaction, 8-arylation of per-O-methylquercetin was achieved. Using a LiAlH4 reduction or Clemmensen reduction, these flavonols were transformed to the corresponding cyanidin derivatives in satisfactory yields. Using these dyes, we fabricated DSSCs, and their efficiency was investigated. The efficiency of tetra-O-methylflavonol was 0.31%. However, the introduction of the 8-aryl residue increased the efficiency to 1.04%. In comparison to these flavonols, O-methylcyanidins exhibited a lower efficiency of 0.05% to 0.52%. The introduction of the 8-aryl group into the cyanidin derivatives did not result in a remarkable increase in the efficiency. These phenomena may be due to the poor fit of the HOMO-LUMO level of the dyes to the TiO2 conduction band.

Synthetic Anthocyanidins from Natural Benzopyrans.

A hydride abstraction strategy can be used to make anthocyanidins and isoflavylium salts from benzopyrans in good yields.

Antiradical and reductant activities of anthocyanidins and anthocyanins, structure-activity relationship and synthesis.

Eight anthocyanidins, seven anthocyanins and two synthesized 4'-hydroxy flavyliums were examined as hydrogen donors to DPPH, ABTS and hydroxyl radicals, and as electron donors in the FRAP assay. Most compounds gave better activities than trolox and catechol. A structure-activity relationship (SAR) study showed that, in the absence of the 3-OH group, radicals of the 4, 5 or 7-OH groups can only be stabilized by resonance through pyrylium oxygen, while 3-OH group improved hydrogen atom donation because of the stabilization by anthocyanidin semiquinone-like resonance. Electron donation was also enhanced by the 3-OH group. Both anthocyanidins and their respective anthocyanins showed similar trends and close activities. Different types of sugar unit bonded to the 3-OH group or counter ion had minor effect on activities. The catechol structure improved both hydrogen and electron donation. Compounds lacking the catechol structure had a decreasing order of H-atom and electron donation (Mv>Pn>Pg>Ap>4'-OH-flavylium) consistent with the decreasing number of their hydroxyl and/or methoxy groups.

Synthesis, characterisation and antioxidant features of procyanidin B4 and malvidin-3-glucoside stearic acid derivatives.

The acylation of procyanidin B4 with a saturated fatty acid chloride containing 18 carbon atoms was studied in order to obtain procyanidin B4 3-O-di-stearic acid conjugate. This compound was structurally characterised by mass spectrometry and 1D and 2D NMR techniques. Derivatization of malvidin-3-glucoside using stearoyl chloride in acetonitrile was also performed yielding mono-, di- and tri-stearic ester derivatives. The novel derivatives obtained revealed significant antioxidant activity, although lower than the respective precursors. However, the chemical modification of anthocyanins and procyanidins (water soluble pigments) to more lipophilic compounds has the advantage of increased bioavailability in biological matrices, and to potentiate their application in food matrices and cosmetic products.

Novel pyrano and vinylphenol adducts of deoxyanthocyanidins in sorghum sourdough.

This study determined the fate of deoxyanthocyanidins in sorghum sourdoughs. Sourdoughs prepared from the red sorghum variety Town were fermented with the caffeic acid-decarboxylating strains Lactobacillus plantarum FUA3171 and the decarboxylase negative L. casei FUA3166. Deoxyanthocyanidins were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Apigeninidin and methoxyapigeninidin were the major deoxyanthocyanidins prior to fermentation. During fermentation, novel deoxyanthocyanidins were formed. Purification by preparative LC, followed by NMR analysis and high-resolution MS identified two of the compounds as 6-deoxyanthocyanidin-vinylphenol and pyrano-3-deoxyanthocyanidin. To identify pathways for their formation, sorghum was fermented with single strains, L. plantarum or L. casei. 6-Deoxyanthocyanidin-vinylphenol and pyrano-3-deoxyanthocyanidin were formed only during fermentation with L. plantarum FUA3171, indicating a role of vinylphenol in their formation. Chemical synthesis confirmed that 6-deoxyanthocyanidin-vinylphenol and pyrano-3-deoxyanthocyanidin are formed from apigeninidin with vinylphenol but not with p-coumaric acid as reactants. In conclusion, the products of microbial decarboxylation of hydroxycinnamic acids convert apigeninidin and methoxyapigeninidin to pyrano-3-deoxyanthocyanidins and vinylphenol adducts.

Thermodynamics, kinetics, and photochromism of oaklins: a recent family of deoxyanthocyanidins.

Two oaklins guaiacylcatechinpyrylium (GCP) and syringylcatechinpyrylium (SCP) and a model compound deoxypeonidin (DOP) were synthesized, and the rate and equilibrium constants of the respective pH dependent network of chemical reactions were calculated. In contrast to anthocyanins, the three compounds possess a small cis-trans isomerization barrier and hence the rate of the trans-chalcone formation follows a bell-shaped curve as a function of pH. The three compounds exhibit photochromism obtained by irradiation of the trans-chalcone, which, depending on pH, leads to the colored species flavylium cation and quinoidal base. The flash photolysis together with pH jumps followed by UV-vis absorption and stopped flow is a very useful tool to achieve the rate and equilibrium constants of the network of chemical reactions followed by these molecules. Oaklin compounds which are formed in wine aged in oak barrels present physical-chemical properties more similar to simpler deoxyanthocyanidins rather than anthocyanins and may play a significant role in color changes observed in wine aging. Given their higher stability, they may be regarded as potential food colorants.

Synthesis, isolation, structure elucidation, and color properties of 10-acetyl-pyranoanthocyanins.

Grape anthocyanins reacted with diacetyl, a secondary metabolite of microorganisms involved in winemaking, to form 10-acetyl-pyranoanthocyanins, a type of anthocyanin-derived pigment similar to other vitisin-type pyranoanthocyanins found in red wines. The structures of 10-acetyl-pyranomalvidin-3-ß-O-glucoside and 10-acetyl-pyranopeonidin-3-ß-O-glucoside were confirmed by spectroscopic methods (UV-vis, MS/MS, and NMR) after their synthesis and isolation. In contrast to other vitisin-type pyranoanthocyanins, the newly described 10-acetyl-pyranoanthocyanins exhibited differentiated color-related properties. They showed an important tendency to occur as colorless hemiacetals at C-10 under wine pH conditions, while co-occurrence of flavylium cation and quinoidal base yielded a broad visible absorbance band around 510-520 nm. Moreover, they easily reacted with bisulfite in acidic aqueous solution (pH 2.0), but the expected bleaching was not observed. Bisulfite bonded to the carbonyl of 10-acetyl substituent instead of the expected C-10 position of the pyranoanthocyanin core, thus giving rise to a red pigment hypsochromically shifted toward orangish nuances (maximum absorbances at 487-491 nm).

A gram scale synthesis of a multi-13C-labelled anthocyanin, [6,8,10,3',5'-13C5]cyanidin-3-glucoside, for use in oral tracer studies in humans.

The major dietary anthocyanin, cyanidin-3-glucoside, was prepared on a 4 g scale from three units of diethyl [2-(13)C]malonate and one unit of [1,3-(13)C(2)]acetone, such that five isotope locations were distributed throughout the molecule to provide a penta-(13)C(5)-labelled anthocyanin, [6,8,10,3',5'-(13)C(5)]cyanidin-3-glucoside chloride, for use in human stable-isotope tracer studies.

Establishment of the chemical equilibria of different types of pyranoanthocyanins in aqueous solutions: evidence for the formation of aggregation in pyranomalvidin-3-O-coumaroylglucoside-(+)-catechin.

The chemical equilibria of the pyranomalvidin-3-glucosides linked to (+)-catechin, (-)-epicatechin, and catechol moieties (and the respective coumaroylglucoside compounds) were established by means of UV-vis spectroscopy. The conjugated double bonds among pyranic rings C and D provide a higher electronic delocalization that prevents the nucleophilic attack of water at position 2. Consequently, besides flavylium cation (AH(+)), the bases A, A(-), and A(2-) have been identified by increasing pH, and the respective acidity constants were determined by spectrophotometry. The formation of dimers at higher concentration was observed for pyranomalvidin-3-O-coumaroylglucoside-(+)-catechin, and the respective data treated by the exciton model suggests the formation of a dimer where the monomers form J-type aggregates with the dipolar moments in opposite directions and rotated by 174° at a distance of 5.2 Å (from the center).

Structural properties of anthocyanins: rearrangement of C-glycosyl-3-deoxyanthocyanidins in acidic aqueous solutions.

Seven C-glycosyl-3-deoxyanthocyanidins were made from their corresponding C-glycosylflavones. The structures of their rearrangement products, which were formed in acidic aqueous solutions, were elucidated. Rotameric conformers were detected for all of the 8-C-glycosyldeoxyanthocyanidins but were absent for their isomeric 6-C-analogues in acidified methanolic NMR solvent. A correlation method based on HPLC-DAD and NMR integration of similar samples made it possible for the first time to determine accurately the proportions of two isomeric 6-C- and 8-C-glycosylflavonoids occurring in mixtures. Each of the C-glycosyldeoxyanthocyanidins established fixed equilibrium proportions with their corresponding A-ring isomer in aqueous solutions, even under relatively strong acidic conditions (pH approximately 1), whether one started with pure 6-C- or 8-C-glycosyl-3-deoxyanthocyanidin. The nature of the aglycone, C-glycosyl moiety, and temperature were found to affect the equilibrium proportions. Increased water content (to a certain level) and temperatures were shown to increase the isomerization rates. The flavylium cations were the only equilibrium forms present at detectable quantities. The significance of rotation of the A-ring during isomerization was confirmed by lack of rearrangement of both 6-C- and 8-C-glycosyl-3-deoxy-5-carboxypyranoanthocyanidins. The intermediary C-ring open forms of the C-glycosyldeoxyanthocyanidins experience fast ring closure to their cyclic forms, which may reduce irreversible degradation reported for open chalcone forms of the common anthocyanins. The stable C-glycosyl-3-deoxyanthocyanidins may thus attract interest as possible colorants in the food industry, etc.

Sorghum 3-deoxyanthocyanins possess strong phase II enzyme inducer activity and cancer cell growth inhibition properties.

3-Deoxyanthoxyanins (3-DXA) possess unique chemical and biochemical properties and may be useful in helping reduce incidence of gastrointestinal cancer. This study tested sorghum extracts rich in 3-DXA as well as isolated and synthetic 3-DXA for potential to induce activity of phase II enzymes in murine hepatoma cells using the NAD(P)H:quinone oxidoreductase (NQO) assay and to inhibit proliferation of the HT-29 human colon cancer cells using MTT and PicoGreen assays. Crude black sorghum extract that contained high levels of methoxylated 3-DXA was a strong inducer of NQO activity (3.0 times at 50 microg/mL), compared to red or white sorghum extracts with low or no methoxylated 3-DXA (1.6 times at 200 microg/mL). All sorghum extracts had strong antiproliferative activity against HT-29 cells after 48 h of incubation (IC(50) = 180-557 microg/mL). Among isolated fractions, nonmethoxylated 3-DXA were very effective against HT-29 cell growth (IC(50) = 44-68 microM at 48 h), but were noninducers of NQO. On the other hand, the methoxylated 3-DXA had both strong antiproliferative activity (IC(50) < 1.5-53 microM) and NQO inducer activity (2-3.7 times). Dimethoxylated 3-DXA were more potent than monomethoxylated analogues. Methoxylation of 3-DXA is essential for NQO activity and also enhances tumor cell growth inhibition.

C-glycosylanthocyanidins synthesized from C-glycosylflavones.

Nine C-glycosyldeoxyanthocyanidins, 6-C-beta-glucopyranosyl-7-O-methylapigeninidin, 6-C-beta-glucopyranosyl-7-O-methylluteolinidin, 6-C-beta-(2''-O-beta-glucopyranosylglucopyranosyl)-7-O-methylapigeninidin, 6-C-beta-(2''-O-beta-glucopyranosylglucopyranosyl)-7,4'-di-O-methylapigeninidin, 8-C-beta-glucopyranosylapigeninidin, 8-C-beta-(2''-O-alpha-rhamnopyranosylglucopyranosyl)apigeninidin, 8-C-beta-(2''-O-alpha-(4'''-O-acetylrhamnopyranosyl)glucopyranosyl)apigeninidin, 6,8-di-C-beta-glucopyranosylapigeninidin (8), 6,8-di-C-beta-glucopyranosyl-4'-O-methylluteolinidin (9), have been synthesized from their respective C-glycosylflavones (yields between 14% and 32%) by the Clemmensen reduction reaction using zinc-amalgam. The various precursors (C-glycosylflavones) of the C-glycosylanthocyanidins were isolated from either flowers of Iris sibirica L., leaves of Hawthorn 'Crataegi Folium Cum Flore', or lemons and oranges. This is the first time C-glycosylanthocyanidins have been synthesized. The structures of all flavonoids including the flavone rotamers were elucidated by 2D NMR techniques and high-resolution electrospray MS. The distribution of the various structural forms of 8 and 9 are different at pH 1.1, 4.5, and 7.0, however, the two pigments undergoes similar structural transformations at the various pH values. Pigments 8 and 9 with C-C linkages between the sugar moieties and the aglycone, were found to be far more stable towards acid hydrolysis than pelargonidin 3-O-glucoside, which has the typical anthocyanidin C-O linkage between the sugar and aglycone. This stability may extend the present use of anthocyanins as nutraceuticals, pharmaceuticals or colorants.