Novel and colourful flavonoids - characterisation, secondary structures and properties

This dissertation focuses on isolation and structural elucidation of flavonoid pigments. The properties of these pigments have been explored, in terms of their chemical properties, especially regarding secondary structures in neutral solvents. Liverworts (Marchantiophyta) are basal land plants considered the probable most recent common ancestor of land plants and algae. The liverwort Marchantia polymorpha produces red flavonoid pigmentation in response abiotic stress, bound to the cell wall, that is proposed to be early evolved anthocyanidin forms. Taking advantage of transgenic marchantia plants overexpressing flavonoid production, two red pigments were isolated and their structure elucidated to be 2,3,6,8-tetrahydroxybenzofuro[3,2-b]chromen-5-ium-6-O-(2-O-α-rhamnopyranosyl-β-glucopyranoside (1), and its aglycone (2). In this work we show that these pigments represent a previously unreported flavonoid class named auronidins, for their similarities in structure to anthocyanidins and to aurones. Chemically, auronidins are capable of expressing a wide range of colours depending on the pH similar to anthocyanins. They also express strong fluorescence under UV light, in contrast to the majority of known anthocyanins. The chemical properties of 1 have been compared to both an aurone (aureusidin 4-O-glucoside, 4) and an anthocyanin (cyanidin 3-O-glucoside, 3). We have also shown that auronidins comes from the flavonoid biosynthetic pathway, which is a part of the larger phenylpropanoid pathway, but from a path distinct from that leading to anthocyanins. A summary of their work is presented here. Peat moss (Sphagnum) is another basal land plant that are more evolutionary advanced than liverwort. Several species of peat moss produce red pigmentation bound to the cell walls in response to abiotic stress. These pigments have been isolated from Sphagnum sp. and Sphagnorubin C (5) have been structurally elucidated. This anthocyanin lacks glycosidic substitution, have an extended aromatic skeleton, and lacks the characteristic hydroxyl group in 3-position (3-deoxyanthocyanidin) featured in the vast majority of anthocyanins. In addition to the flavylium cation form, the trans-chalcone form of 5 have been elucidated, which in pure DMSO is stable and is fully reversible to the flavylium cation form by the addition of acid. No signs of neither hemiketal forms nor the cis-chalcone form was observed. This was the first full structural elucidation of a trans-chalcone form of a naturally occurring anthocyanin. In addition to the pigments isolated from basal land plants, several new sources of anthocyanins have been discovered. Isolation and structural elucidation of anthocyanins from the mauve flowers of Erlangea tomentosa provided this thesis with two new anthocyanins, based on the novel anthocyanidin erlangidin, the first reported natural anthocyanidin having C-ring methoxylation (erlangidin 5-O-(4-(E-caffeoyl)-6-(malonyl)-β-glucopyranoside)-3’-O-(6-(3-(β-glucopyranosyl)-E-caffeoyl)-β-glucopyranoside), 6, and erlangidin 5-O-(6-(malonyl)-β-glucopyranoside)-3’-O-(6-(3-(β-glucopyranosyl)-E-caffeoyl)-β-glucopyranoside), 7). Additionally, new sources of petanin (8) and negretein (9) were discovered, including ‘Sun black’ tomato (Solanum lycopersicum), a tomato cultivar bred to produce deep purple to black anthocyanin-rich skin, and several cultivars of blue potato (Solanum tuberosum).Doktorgradsavhandlin

Characterization of a Natural, Stable, Reversible and Colourful Anthocyanidin Network from Sphagnum Moss Based Mainly on the Yellow Trans‐Chalcone and Red Flavylium Cation Forms

Anthocyanins with various functions in nature are one of the most important sources of colours in plants. They are based on anthocyanidins or 3-deoxyanthocyanidins having in common a C15-skeleton and are unique in terms of how each anthocyanidin is involved in a network of equilibria between different forms exhibiting their own properties including colour. Sphagnorubin C (1) isolated from the cell wall of peat moss (Sphagnum sp.) was in fairly acidic and neutral dimethyl sulfoxide characterized by nuclear magnetic resonance (NMR) and ultraviolet–visible (UV–vis) absorption techniques. At equilibrium, the network of 1 behaved as a two–component colour system involving the reddish flavylium cationic and the yellow trans–chalcone forms. The additional D- and E-rings connected to the common C15-skeleton extend the π-conjugation within the molecule and provide both bathochromic shifts in the absorption spectra of the various forms as well as a low isomerization barrier between the cis- and trans-chalcone forms. The hemiketal and cis-chalcone forms were thus not observed experimentally by NMR due to their short lives. The stable, reversible network of 1 with good colour contrast between its two components has previously not been reported for other natural anthocyanins and might thus have potential in future photochromic systems. This is the first full structural characterization of any naturally occurring anthocyanin chalcone form.publishedVersio

Nutraceutical characterization of anthocyanin-rich fruits produced by «Sun Black» tomato line

Tomato (Solanum lycopersicum L.) is one of the most cultivated vegetable in the world and it represents a large source of bioactive compounds, including carotenoids and polyphenols (phenolic acids and flavonoids). However, the concentration of flavonoids in tomato is considered sub-optimal, particularly because anthocyanins are not generally present. Therefore, this crop has been the object of an intense metabolic engineering in order to obtain anthocyanin-enriched tomatoes by using either breeding or transgenic strategies. Some wild tomato species, such as S. chilense and S. cheesmaniae, biosynthesize anthocyanins in the fruit sub-epidermal tissue, and some alleles from those genotypes have been introgressed into a new developed purple tomato line, called “Sun Black” (SB). It is a tomato line with a purple skin color, both in green and in red fruit stages, due to the biosynthesis of anthocyanins in the peel, and a normal red color pulp, with a taste just like a traditional tomato. SB is the result of a breeding programme and it is not a genetically modified (GM) product. We report the chemical characterization and structure elucidation of the attractive anthocyanins found in the peel of SB tomato, as well as other bioactive compounds (carotenoids, polyphenols, vitamin C) of the whole fruit. Using one- and two-dimensional NMR experiments, the two main anthocyanins were identified to be petunidin 3-O-[6″-O-(4‴-O-E-p-coumaroyl-α-rhamnopyranosyl) -β-glucopyranoside]-5-O-β-glucopyranoside (petanin) and malvidin 3-O-[6″-O-(4‴-O-E-p-coumaroyl-α-rhamnopyranosyl)-β-glucopyranoside]-5-O-β-glucopyranoside (negretein). The total anthocyanins in the whole ripe fruit was 1.2 mg/g dry weight (DW); 7.1 mg/100 g fresh weight (FW). Chlorogenic acid (the most abundant phenolic acid) was 0.6 mg/g DW; 3.7 mg/100 g FW. The main flavonol, rutin was 0.8 mg/g DW; 5 mg/100 g FW. The total carotenoid content was 211.3 μg/g DW; 1,268 μg/100 g FW. The total phenolic content was 8.6 mg/g DW; 52.2 mg/100 g FW. The vitamin C content was 37.3 mg/100 g FW. The antioxidant activities as measured by the TEAC and ORAC assays were 31.6 and 140.3 μmol TE/g DW, respectively (193 and 855.8 μmol TE/100 g FW, respectively). The results show the unique features of this new tomato genotype with nutraceutical properties.publishedVersio

Novel and colourful flavonoids - characterisation, secondary structures and properties

This dissertation focuses on isolation and structural elucidation of flavonoid pigments. The properties of these pigments have been explored, in terms of their chemical properties, especially regarding secondary structures in neutral solvents. Liverworts (Marchantiophyta) are basal land plants considered the probable most recent common ancestor of land plants and algae. The liverwort Marchantia polymorpha produces red flavonoid pigmentation in response abiotic stress, bound to the cell wall, that is proposed to be early evolved anthocyanidin forms. Taking advantage of transgenic marchantia plants overexpressing flavonoid production, two red pigments were isolated and their structure elucidated to be 2,3,6,8-tetrahydroxybenzofuro[3,2-b]chromen-5-ium-6-O-(2-O-α-rhamnopyranosyl-β-glucopyranoside (1), and its aglycone (2). In this work we show that these pigments represent a previously unreported flavonoid class named auronidins, for their similarities in structure to anthocyanidins and to aurones. Chemically, auronidins are capable of expressing a wide range of colours depending on the pH similar to anthocyanins. They also express strong fluorescence under UV light, in contrast to the majority of known anthocyanins. The chemical properties of 1 have been compared to both an aurone (aureusidin 4-O-glucoside, 4) and an anthocyanin (cyanidin 3-O-glucoside, 3). We have also shown that auronidins comes from the flavonoid biosynthetic pathway, which is a part of the larger phenylpropanoid pathway, but from a path distinct from that leading to anthocyanins. A summary of their work is presented here. Peat moss (Sphagnum) is another basal land plant that are more evolutionary advanced than liverwort. Several species of peat moss produce red pigmentation bound to the cell walls in response to abiotic stress. These pigments have been isolated from Sphagnum sp. and Sphagnorubin C (5) have been structurally elucidated. This anthocyanin lacks glycosidic substitution, have an extended aromatic skeleton, and lacks the characteristic hydroxyl group in 3-position (3-deoxyanthocyanidin) featured in the vast majority of anthocyanins. In addition to the flavylium cation form, the trans-chalcone form of 5 have been elucidated, which in pure DMSO is stable and is fully reversible to the flavylium cation form by the addition of acid. No signs of neither hemiketal forms nor the cis-chalcone form was observed. This was the first full structural elucidation of a trans-chalcone form of a naturally occurring anthocyanin. In addition to the pigments isolated from basal land plants, several new sources of anthocyanins have been discovered. Isolation and structural elucidation of anthocyanins from the mauve flowers of Erlangea tomentosa provided this thesis with two new anthocyanins, based on the novel anthocyanidin erlangidin, the first reported natural anthocyanidin having C-ring methoxylation (erlangidin 5-O-(4-(E-caffeoyl)-6-(malonyl)-β-glucopyranoside)-3’-O-(6-(3-(β-glucopyranosyl)-E-caffeoyl)-β-glucopyranoside), 6, and erlangidin 5-O-(6-(malonyl)-β-glucopyranoside)-3’-O-(6-(3-(β-glucopyranosyl)-E-caffeoyl)-β-glucopyranoside), 7). Additionally, new sources of petanin (8) and negretein (9) were discovered, including ‘Sun black’ tomato (Solanum lycopersicum), a tomato cultivar bred to produce deep purple to black anthocyanin-rich skin, and several cultivars of blue potato (Solanum tuberosum)

Characterization of a Natural, Stable, Reversible and Colourful Anthocyanidin Network from Sphagnum Moss Based Mainly on the Yellow Trans-Chalcone and Red Flavylium Cation Forms

Anthocyanins with various functions in nature are one of the most important sources of colours in plants. They are based on anthocyanidins or 3-deoxyanthocyanidins having in common a C15-skeleton and are unique in terms of how each anthocyanidin is involved in a network of equilibria between different forms exhibiting their own properties including colour. Sphagnorubin C (1) isolated from the cell wall of peat moss (Sphagnum sp.) was in fairly acidic and neutral dimethyl sulfoxide characterized by nuclear magnetic resonance (NMR) and ultraviolet–visible (UV–vis) absorption techniques. At equilibrium, the network of 1 behaved as a two–component colour system involving the reddish flavylium cationic and the yellow trans–chalcone forms. The additional D- and E-rings connected to the common C15-skeleton extend the π-conjugation within the molecule and provide both bathochromic shifts in the absorption spectra of the various forms as well as a low isomerization barrier between the cis- and trans-chalcone forms. The hemiketal and cis-chalcone forms were thus not observed experimentally by NMR due to their short lives. The stable, reversible network of 1 with good colour contrast between its two components has previously not been reported for other natural anthocyanins and might thus have potential in future photochromic systems. This is the first full structural characterization of any naturally occurring anthocyanin chalcone form

Characterization of a Natural, Stable, Reversible and Colourful Anthocyanidin Network from Sphagnum Moss Based Mainly on the Yellow Trans‐Chalcone and Red Flavylium Cation Forms

Anthocyanins with various functions in nature are one of the most important sources of colours in plants. They are based on anthocyanidins or 3-deoxyanthocyanidins having in common a C15-skeleton and are unique in terms of how each anthocyanidin is involved in a network of equilibria between different forms exhibiting their own properties including colour. Sphagnorubin C (1) isolated from the cell wall of peat moss (Sphagnum sp.) was in fairly acidic and neutral dimethyl sulfoxide characterized by nuclear magnetic resonance (NMR) and ultraviolet–visible (UV–vis) absorption techniques. At equilibrium, the network of 1 behaved as a two–component colour system involving the reddish flavylium cationic and the yellow trans–chalcone forms. The additional D- and E-rings connected to the common C15-skeleton extend the π-conjugation within the molecule and provide both bathochromic shifts in the absorption spectra of the various forms as well as a low isomerization barrier between the cis- and trans-chalcone forms. The hemiketal and cis-chalcone forms were thus not observed experimentally by NMR due to their short lives. The stable, reversible network of 1 with good colour contrast between its two components has previously not been reported for other natural anthocyanins and might thus have potential in future photochromic systems. This is the first full structural characterization of any naturally occurring anthocyanin chalcone form

Radical Scavenging and Anti-Inflammatory Activities of Representative Anthocyanin Groupings from Pigment-Rich Fruits and Vegetables

Anthocyanins, the naturally occurring pigments responsible for most red to blue colours of flowers, fruits and vegetables, have also attracted interest because of their potential health effects. With the aim of contributing to major insights into their structure–activity relationship (SAR), we have evaluated the radical scavenging and biological activities of selected purified anthocyanin samples (PASs) from various anthocyanin-rich plant materials: two fruits (mahaleb cherry and blackcurrant) and two vegetables (black carrot and “Sun Black” tomato), differing in anthocyanin content (ranging from 4.9 to 38.5 mg/g DW) and molecular structure of the predominant anthocyanins. PASs from the abovementioned plant materials have been evaluated for their antioxidant capacity using Trolox Equivalent Antioxidant Capacity (TEAC) and Oxygen Radical Absorbance Capacity (ORAC) assays. In human endothelial cells, we analysed the anti-inflammatory activity of different PASs by measuring their effects on the expression of endothelial adhesion molecules VCAM-1 and ICAM-1. We demonstrated that all the different PASs showed biological activity. They exhibited antioxidant capacity of different magnitude, higher for samples containing non-acylated anthocyanins (typical for fruits) compared to samples containing more complex anthocyanins acylated with cinnamic acid derivatives (typical for vegetables), even though this order was slightly reversed when ORAC assay values were expressed on a molar basis. Concordantly, PASs containing non-acylated anthocyanins reduced the expression of endothelial inflammatory antigens more than samples with aromatic acylated anthocyanins, suggesting the potential beneficial effect of structurally diverse anthocyanins in cardiovascular protection

GM1 structure determines SV40-induced membrane invagination and infection

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