Copigments in the blueing of sepal colour of Hydrangea macrophylla

From blue sepals of Hydrangea macrophylla, copigments which show a blueing effect on the hydrangea anthocyanin were isolated and identified as 3-p-coumaroylquinic acid and 3-caffeoylquinic acid. 5-Caffeoylquinic acid (chlorogenic acid) which was also found in the blue sepals, however, did not show such a blueing effect though it acted as a copigment. Likewise, the 4-esters of p-coumaroyl- and caffeoylquinic acids (not found in sepals) produced purple rather than blue colours. The facts suggest that the stereostructures of 3-p-coumaroyl- and 3-caffeoylquinic acids are effective for molecular interaction between the p-coumaroyl or caffeoyl residue in the compounds and the anthocyanin. The anthocyanin in red and blue sepals of hydrangea was confirmed to be delphinidin 3-monoglucoside.     

The chemical mechanism for Al complexing with delphinidin: A model for the bluing of hydrangea sepals

The blooms of many hydrangea cultivars can be red or blue, with the color depending on the soil pH. This dependence reflects the availability of Al³⁺ to the plant under acidic conditions, as Al³⁺ changes the color of the anthocyanin pigment in hydrangea sepals from red to blue. A chemical model, Al³⁺ and delphinidin in acidic ethanol, was developed to understand the spectral characteristics and bluing of the hydrangea sepals. Delphinidin as its flavylium cation leads to red solutions in the model system. In the presence of Al³⁺, the Al³⁺ removes H⁺ ions from delphinidin, transforming delphinidin's flavylium cation to its blue quinoidal base anion which complexes with the Al³⁺. To further stabilize this complex, a second flavylium cation stacks on top of the complexed quinoidal base anion, creating a bathochromic shift of the cation's spectral signature and accentuating the blue color. This Al³⁺-delphinidin entity forms in adequate concentration for bluing only if there is a sufficient excess of Al³⁺, the exact excess being a function of pH and concentration. The role of Al³⁺ in bluing is not just to form a primary complex with delphinidin, but also to create a template for the stacking of delphinidin (or possibily co-pigments).     

Sepal color variation of Hydrangea macrophylla and vacuolar pH measured with a proton-selective microelectrode

Sepal color of hydrangea varies with the environmental conditions. Although chemical and biological studies on this color variation have a long history, little correct knowledge has been generated about color development. All colored sepals contain the same anthocyanin, delphinidin 3-glucoside. Thus, there must be some other system for developing the wide variety of colors. In hydrangea sepals the cells of the epidermis are colorless and only the second layer of cells contain pigment. We prepared protoplasts without any color change during enzyme treatment of sepals and measured the vacuolar pH of each of the colored cells. We could correlate the color of a single hydrangea cell with its vacuolar pH using a combination of micro-spectrophotometry and a proton-selective microelectrode. Values for the vacuolar pH of blue (lambda vismax: 589 nm) and red cells (lambda vismax: 537 nm) were 4.1 and 3.3, respectively, the vacuolar pH of blue cells being significantly higher.     

Blueing of sepal colour of Hydrangea macrophylla

Blue and red sepals of Hydrangea macrophylla were quantitatively analyzed for aluminium, anthocyanin (delphinidin 3-glucoside) and copigments (caffeoyl- and p-coumaroylquinic acids). All the blue sepals examined contained both Al and copigments (especially 3-caffeoylquinic acid) in considerable amounts. In in vitro experiments using 3- and 5-caffeoylquinic acids, Al and delphinidin 3-glucoside, it was shown that 3-caffeoylquinic acid and Al formed a blue complex with the anthocyanin. Absorption spectra of the blue complex were practically identical with those of the blue solutions obtained from blue hydrangea sepals by extraction with 4 M NACl. In contrast, 5-caffeoylquinic acid (chlorogenic acid) which was also present in hydrangea sepals gave only a red-purple colour with Al and the anthocyanin. Neither 3-caffeoylquinic acid nor Al independently produced blue colour when mixed with the anthocyanin in the mole ratios of 1–30, this being the range that the compounds were found in blue sepals. These results suggest that blue colour of hydrangea sepals is due mainly to the blue complex of delphinidin 3-glucoside-aluminium-3-caffeoylquinic acid. The role of aluminium may be to stabilize an interaction between the quinic ester and the anthocyanin.     

Role of aluminum in red-to-blue color changes in <Emphasis Type="Italic">Hydrangea macrophylla</Emphasis> sepals

Red, purple, and blue sepals on selected cultivars of Hydrangea macrophylla were analyzed for their aluminum content. This content was determined to be a function of the sepal color with red sepals possessing 0–10 μg Al/g fresh sepal, purple sepals having 10–40 μg Al/g fresh sepal, and blue sepals containing greater than 40 μg Al/g fresh sepal. Accordingly, the threshold aluminum content needed to change H. macrophylla sepals from red to blue was about 40 μg Al/g fresh sepal. Higher aluminum concentrations were incorporated into the sepals, but this additional aluminum did not affect the intensity or hue of the blue color. These observations agreed with a chemical model proposing that the concentration of the blue Al³⁺-anthocyanin complex reached a maximum when a sufficient excess of aluminum was present. In addition, the visible absorbance spectra of harvested red, purple, and blue sepals were duplicated by Al³⁺ and anthocyanin (delphinidin-3-glucoside) mixtures in this model chemical system.     

Semisynthetic lysogalactolipids of plant origin

Starting from the natural mono- and digalactosyl diglycerides, 1′-O-acyl-3′-O-β-d-galactopyranosyl-sn-glycerol and 1′-O-acyl-3′-O-(6-O-α-d-galactopyranosyl-β-d-galactopyranosyl)-sn-glycerol were synthesized. In an attempt to prepare the 2′-O-acyl-isomer, only a mixture of the 1′-and 2′-O-acyl-isomers was obtained.     

Anthocyanins in Caprifoliaceae

The qualitative and relative quantitative anthocyanin content of 19 species belonging to the genera Sambucus, Lonicera and Viburnum in the family Caprifoliaceae has been determined. Altogether 12 anthocyanins were identified; the 3-O-glucoside (2), 3-O-galactoside (5), 3-O-(6″-O-arabinosylglucoside) (7), 3-O-(6″-O-rhamnosylglucoside) (9), 3-O-(2″-O-xylosyl-6″-O-rhamnosylglucoside) (10), 3-O-(2″-O-xylosylgalactoside) (11), 3-O-(2″-O-xylosylglucoside) (12), 3-O-(2″-O-xylosylglucoside)-5-O-glucoside (14), 3-O-(2″-O-xylosyl-6″-O-Z-p-coumaroylglucoside)-5-O-glucoside (15) and 3-O-(2″-O-xylosyl-6″-O-E-p-coumaroylglucoside)-5-O-glucoside (16) of cyanidin, in addition to the 3-O-glucosides of pelargonidin and delphinidin (1 and 3). Pigment 7 is the first complete identification of the disaccharide vicianose, 6″-O-α-arabinopyranosyl-β-glucopyranose, linked to an anthocyanidin.     

Sodium borohydride reduction of anthocyanidins

The reduction of anthocyanidins with NaBH₄ in EtOH or MeOH produces inter alia racemates of epicatechins. Thus, the (±) racemates of 3′,5′-di-O-methylepigallocatechin, 3′-O-methylepigallocatechin, and 3′-O-methylepicatechin have been identified as the reduction products of malvidin, petunidin, and peonidin, respectively, by their UV, MS and NMR spectra.     

Light filtering by epidermal flavonoids during the resistant response of cotton to Xanthomonas protects leaf tissue from light-dependent phytoalexin toxicity

2,7-Dihydroxycadalene and lacinilene C, sesquiterpenoid phytoalexins that accumulate at infection sites during the hypersensitive resistant response of cotton foliage to Xanthomonas campestris pv. malvacearum, have light-dependent toxicity toward host cells, as well as toward the bacterial pathogen. Adaxial epidermal cells surrounding and sometimes covering infection sites turn red. The red cells exhibited 3-4-fold higher absorption at the photoactivating wavelengths of sunlight than nearby colorless epidermal cells. Red epidermal cells protected underlying palisade mesophyll cells from the toxic effects of 2,7-dihydroxycadalene plus sunlight, indicating a role for epidermal pigments in protecting living cells that surround infection sites from toxic effects of the plant’s own phytoalexins. A semi-quantitative survey of UV-absorbing substances extracted from epidermal strips from inoculated and mock-inoculated cotyledons indicated that the principal increase in capacity to absorb the photoactivating wavelengths was due to a red anthocyanin and a yellow flavonol, which were identified as cyanidin-3-O-β-glucoside and quercetin-3-O-β-glucoside, respectively.     

Isolation and structure elucidation of 5′-O-β-d-glucopyranosyl-dihydroascorbigen from Cardamine diphylla rhizome

From the methanol extract of Cardamine diphylla rhizome, 5′-O-β-d-glucopyranosyl-dihydroascorbigen (1) and 6-hydroxyindole-3-carboxylic acid 6-O-β-d-glucopyranoside (2) were isolated. The structures of the compounds were elucidated using spectroscopic methods. This is the second report on the presence of a glucosylated indole ascorbigen in plants.     

Cyanosalvianin, a supramolecular blue metalloanthocyanin, from petals of Salvia uliginosa

A metalloanthocyanin, cyanosalvianin, was found in blue petals of Salvia uliginosa. Cyanosalvianin consisted of 3-O-(6-O-p-coumaroylglucopyranosyl)-5-O-(4-O-acetyl-6-O-malonylglucopyranosyl) delphinidin, 7,4′-di-O-glucopyranosylapigenin and magnesium ion. We reproduced the same blue color as the petals by mixing the three components together. An ESI-MS measurement gave a molecular weight of 9014 indicating the composition of cyanosalvianin to be six molecules of the anthocyanin component, six molecules of the flavone component and two magnesium ions. The special arrangement of the organic components in cyanosalvianin was analyzed by CD and 2D-NMR spectroscopy. It was clarified that cyanosalvianin has a similar structure to that of commelinin, a metalloanthocyanin isolated from blue dayflower, Commelina communis.     

Efficient synthesis of 3-O-thia-cPA and preliminary analysis of its biological activity toward autotaxin

The efficient synthesis of 3-O-thia-cPAs (4a-d), sulfur analogues of cyclic phosphatidic acid (cPA), has been achieved. The key step of the synthesis is an intramolecular Arbuzov reaction to construct the cyclic thiophosphate moiety. The present synthetic route enables the synthesis of 4a-d in only four steps from the commercially available glycidol. Preliminary biological experiments showed that 4a-d exhibited a similar inhibitory effect on autotaxin (ATX) as original cPA.     

The chemotaxonomic and medicinal significance of phenolic acids in Arctopus and Alepidea (Apiaceae subfamily Saniculoideae)

The occurrence of (R)-3′-O-β-d-glucopyranosylrosmarinic acid, rosmarinic acid and caffeic acid in two important South African medicinal plants is reported for the first time. (R)-3′-O-β-d-Glucopyranosylrosmarinic acid and rosmarinic acid were isolated and identified in several samples from three species of the genus Arctopus L. (sieketroos) and three species of the genus Alepidea F. Delaroche (ikhathazo), both recently shown to be members of the subfamily Saniculoideae of the family Apiaceae. The compounds occur in high concentrations (up to 15.3 mg of (R)-3′-O-β-d-glucopyranosylrosmarinic acid per g dry wt) in roots of Arctopus. Our results provide a rationale for the traditional uses of these plants, as the identified compounds are all known for their antioxidant activity, with rosmarinic acid further contributing to a wide range of biological activities. Furthermore, we confirm the idea that (R)-3′-O-β-d-glucopyranosylrosmarinic acid is a useful chemotaxonomic marker for the subfamily Saniculoideae.     

The blue anthocyanin pigments from the blue flowers of Heliophila coronopifolia L. (Brassicaceae)

Six acylated delphinidin glycosides (pigments 1-6) and one acylated kaempferol glycoside (pigment 9) were isolated from the blue flowers of cape stock (Heliophila coronopifolia) in Brassicaceae along with two known acylated cyanidin glycosides (pigments 7 and 8). Pigments 1-8, based on 3-sambubioside-5-glucosides of delphinidin and cyanidin, were acylated with hydroxycinnamic acids at 3-glycosyl residues of anthocyanidins. Using spectroscopic and chemical methods, the structures of pigments 1, 2, 5, and 6 were determined to be: delphinidin 3-O-[2-O-(β-xylopyranosyl)-6-O-(acyl)-β-glucopyranoside]-5-O-[6-O-(malonyl)-β-glucopyranoside], in which acyl moieties were, respectively, cis-p-coumaric acid for pigment 1, trans-caffeic acid for pigment 2, trans-p-coumaric acid for pigment 5 (a main pigment) and trans-ferulic acid for pigment 6, respectively. Moreover, the structure of pigments 3 and 4 were elucidated, respectively, as a demalonyl pigment 5 and a demalonyl pigment 6. Two known anthocyanins (pigments 7 and 8) were identified to be cyanidin 3-(6-p-coumaroyl-sambubioside)-5-(6-malonyl-glucoside) for pigment 7 and cyanidin 3-(6-feruloyl-sambubioside)-5-(6-malonyl-glucoside) for pigment 8 as minor anthocyanin pigments. A flavonol pigment (pigment 9) was isolated from its flowers and determined to be kaempferol 3-O-[6-O-(trans-feruloyl)-β-glucopyranoside]-7-O-cellobioside-4′-O-glucopyranoside as the main flavonol pigment.On the visible absorption spectral curve of the fresh blue petals of this plant and its petal pressed juice in the pH 5.0 buffer solution, three characteristic absorption maxima were observed at 546, 583 and 635 nm. However, the absorption curve of pigment 5 (a main anthocyanin in its flower) exhibited only one maximum at 569 nm in the pH 5.0 buffer solution, and violet color. The color of pigment 5 was observed to be very unstable in the pH 5.0 solution and soon decayed. In the pH 5.0 solution, the violet color of pigment 5 was restored as pure blue color by addition of pigment 9 (a main flavonol in this flower) like its fresh flower, and its blue solution exhibited the same three maxima at 546, 583 and 635 nm. On the other hand, the violet color of pigment 5 in the pH 5.0 buffer solution was not restored as pure blue color by addition of deacyl pigment 9 or rutin (a typical flower copigment). It is particularly interesting that, a blue anthocyanin-flavonol complex was extracted from the blue flowers of this plant with H₂O or 5% HOAc solution as a dark blue powder. This complex exhibited the same absorption maxima at 546, 583 and 635 nm in the pH 5.0 buffer solution. Analysis of FAB mass measurement established that this blue anthocyanin-flavonol complex was composed of one molecule each of pigment 5 and pigment 9, exhibiting a molecular ion [M+1] ⁺ at 2102 m/z (C₉₃H₁₀₅O₅₅ calc. 2101.542). However, this blue complex is extremely unstable in acid solution. It really dissociates into pigment 5 and pigment 9.     

O-Methylated mannogalactan from the microalga Coccomyxa mucigena, symbiotic partner of the lichenized fungus Peltigera aphthosa

A structural study of the carbohydrates from Coccomyxa mucigena, the symbiotic algal partner of the lichenized fungus Peltigera aphthosa, was carried out. It produced an O-methylated mannogalactan, with a (1 → 6)-linked β-galactopyranose main-chain partially substituted at O-3 by β-Galp, 3-OMe-α-Manp or α-Manp units. There were no similarities with polysaccharides previously found in the lichen thallus of P. aphthosa. Moreover, the influence of lichenization in polysaccharide production by symbiotic microalgae and the nature of the photobiont in carbohydrate production in lichen symbiosis are also discussed.     

C-glycosylanthocyanidins synthesized from C-glycosylflavones

Nine C-glycosyldeoxyanthocyanidins, 6-C-β-glucopyranosyl-7-O-methylapigeninidin, 6-C-β-glucopyranosyl-7-O-methylluteolinidin, 6-C-β-(2″-O-β-glucopyranosylglucopyranosyl)-7-O-methylapigeninidin, 6-C-β-(2″-O-β-glucopyranosylglucopyranosyl)-7,4′-di-O-methylapigeninidin, 8-C-β-glucopyranosylapigeninidin, 8-C-β-(2″-O-α-rhamnopyranosylglucopyranosyl)apigeninidin, 8-C-β-(2″-O-α-(4″′-O-acetylrhamnopyranosyl)glucopyranosyl)apigeninidin, 6,8-di-C-β-glucopyranosylapigeninidin (8), 6,8-di-C-β-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.     

Novel synthesis of methyl 4,6-O-benzylidenespiro[2-deoxy-α-d-arabino-hexopyranoside-2,2′-imidazolidine] and its homologue and sugar-γ-butyrolactam derivatives from methyl 4,6-O-benzylidene-α-d-arabino-hexopyranosid-2-ulose

Novel methyl 4,6-O-benzylidenespiro[2-deoxy-α-d-arabino-hexopyranoside-2,2′-imidazolidine] and its homologue methyl 4,6-O-benzylidene-3′,4′,5′,6′-tetrahydro-1′H-spiro[2-deoxy-α-d-arabino-hexopyranoside-2,2′-pyrimidine] have been synthesized in good yields by reaction of methyl 4,6-O-benzylidene-α-d-arabino-hexopyranosid-2-ulose with 1,2-diaminoethane and 1,3-diaminopropane. The results are completely different from the reaction with arylamines or alkylamines. One-pot synthesis of novel (E)-methyl 4-[hydroxy (methoxy)methylene]-5-oxo-1-alkyl-(4,6-O-benzylidene-2-deoxy-α-d-glucopyranosido)[3,2-b]pyrrolidines has been achieved by the reaction of alkylamines with the butenolide-containing sugar, derived from the aldol condensation of methyl 4,6-O-benzylidene-α-d-arabino-hexopyranosid-2-ulose with diethyl malonate. These sugar-γ-butyrolactam derivatives are potential GABA receptor ligands.