Chemical and biological study of flavonoid-related plant pigment: current findings and beyond.

Flavonoids are polyphenolic plant constituents. Anthocyanins are flavonoid pigments found in higher plants that show a wide variety of colors ranging from red through purple to blue. The blue color of the flowers is mostly attributed to anthocyanins. However, only a few types of anthocyanidin, chromophore of anthocyanin, exist in nature, and the extracted pigments are unstable with the color fading away. Therefore, the wide range and stable nature of colors in flowers have remained a mystery for more than a century. The mechanism underlying anthocyanin-induced flower coloration was studied using an interdisciplinary method involving chemistry and biology. Furthermore, the chemical studies on flavonoid pigments in various edible plants, synthetic and biosynthetic studies on anthocyanins were conducted. The results of these studies have been outlined in this review.

Differences in the content of purple pigments, catechinopyranocyanidins A and B, in various adzuki beans, Vigna angularis.

Catechinopyranocyanidins A and B (cpcA, B) are purple pigments isolated from the seed coat of the small red bean called adzuki in Japanese (Vigna angularis). CpcA and B are known to be responsible for the purple color of an-paste. The quality evaluation of the paste is based on its purple color; therefore, it is important to analyze the contents of cpcA and B in various azuki beans, which differ in the cultivar, production area, and cultivation conditions. The extraction of cpcA and B from dried beans was conducted, followed by high-performance liquid chromatography analysis. The content of cpcA and B in various cultivars from Japan, Korea, China, and Nepal was quantified. The cpcA and B content was the highest in the regular adzuki cultivar from Hokkaido Prefecture (Japan), and that of the beans from Korea and China was low. The content of the pale-colored beans from Nepal was the lowest.

Blue flower coloration of Salvia macrophylla by the metalloanthocyanin, protodelphin.

A survey of metalloanthocyanin by in vivo visible spectrum and circular dichroism suggested that blue petals of Salvia macrophylla contain metalloanthocyanins. Chemical analysis of the purified blue pigment proved that the pigment in the petals is protodelphin, which is the same pigment present in the blue petals of Salvia patens composed of malonylawobanin, apigenin 7,4'-diglucosides and Mg2+.

Single-cell analysis clarifies mosaic color development in purple hydrangea sepal.

Hydrangea sepals exhibit a wide range of colors, from red, through purple, to blue; the purple color is a color mosaic. However, all of these colors are derived from the same components: simple anthocyanins, 3-O-glycosyldelphinidins, three co-pigment components, acylquinic acids and aluminum ions (Al3+ ). We show the color mosaic is a result of graded differences in intravacuolar factors. In order to clarify the mechanisms of mosaic color, we performed single-cell analyses of vacuolar pH, and anthocyanin, co-pigment and Al3+ content. From the sepals, a protoplast mixture of various colors was obtained. The cell color was evaluated by microspectrophotometry and vacuolar pH then was recorded by using a pH microelectrode. The organic and Al3+ contents were quantified by micro-HPLC. We found that the bluer the cell, the greater the ratio of 5-O-acylquinic acids and Al3+ to anthocyanins. Furthermore, reproducing experiments were conducted by mixing the components under various pH condition; all the colors could be reproduced in the various mixing conditions. Based on the above, we provide experimental evidence for cell color variation in hydrangea. Our study demonstrates the expression of phenotypic differences without any direct genomic control.

Blue flower coloration of Corydalis ambigua requires ferric ion and kaempferol glycoside.

Corydalis ambigua (Japanese name, Ezoengosaku) flowers bloom with blue to purplish petals in early spring in Hokkaido prefecture. In this study, a mechanism for blue petal coloration by ferric ions and keampferol glycoside was elucidated. Blue petals and cell sap exhibited similar visible (Vis) spectra, with λmax at approximately 600 nm and circular dichroism (CD) with positive exciton-type Cotton effects in the Vis region. Analysis of the organic components of the petals confirmed cyanidin 3-O-sambubioside and kaempferol 3-O-sambubioside as the major flavonoids. Mg, Al, and Fe were detected in petals using atomic emission spectroscopy. Color, Vis absorption, and CD consistent with those of blue petals were reproduced by mixing cyanidin 3-O-sambubioside, kaempferol 3-O-sambubioside, and Fe3+ in a buffered aqueous solution at pH 6.5. Both Fe3+ and flavonol were essential for blue coloration.

Insight into chemical mechanisms of sepal color development and variation in hydrangea.

Hydrangea (Hydrangea macrophylla) is a unique flower because it is composed of sepals rather than true petals that have the ability to change color. In the early 20th century, it was known that soil acidity and Al3+ content could intensify the blue hue of the sepals. In the mid-20th century, the anthocyanin component 3-O-glucosyldelphinidin (1) and the copigment components 5-O-caffeoylquinic, 5-O-p-coumaroylquinic, and 3-O-caffeoylquinic acids (2-4) were reported. Interestingly, all hydrangea colors from red to purple to blue are produced by the same organic components. We were interested in this phenomenon and the chemical mechanisms underlying hydrangea color variation. In this review, we summarize our recent studies on the chemical mechanisms underlying hydrangea sepal color development, including the structure of the blue complex, transporters involved in accumulation of aluminum ion (Al3+), and distribution of the blue complex and aluminum ions in living sepal tissue.

Polyacylated Anthocyanins in Bluish-Purple Petals of Chinese Bellflower, Platycodon grandiflorum.

The bluish-purple petals of Chinese bellflower, Platycodon grandiflorum (kikyo in Japanese), contain platyconin (1) as the major anthocyanin. Platyconin (1) is a polyacylated anthocyanin with two caffeoyl residues at the 7-position, and its color is stable in a diluted, weakly acidic aqueous solutions. HPLC analysis of the fresh petal extract showed the presence of several minor pigments. Photo-diode array detection of minor pigments suggested that some of these were polyacylated anthocyanins. To establish the relationship between structure and stability of the acylated anthocyanins and to obtain information on their biosynthetic pathways, minor pigments were isolated from the petals, and their structures were determined by MS and NMR analyses. Four known (2-5) and three new anthocyanins (6-8) were identified, which contained a delphinidin chromophore, and four of these (5-8) were diacylated anthocyanins, in which the acyl-glucosyl-acyl-glucosyl chain was attached at the 7-O-position of the delphinidin chromophore. These diacylated anthocyanins exhibited a bluish-purple color at pH 6, which was stable for more than a week.

Determination of absolute configuration of photo-degraded catechinopyranocyanidin A by modified Mosher's method.

Catechinopyranocyanidins A and B (cpcA and cpcB) are two purple pigments present in the seed-coat of red adzuki bean, Vigna angularis, of which cpcA is the major pigment, containing two chiral carbons in the catechin part. Their absolute configurations were determined by comparison of their experimental and quantum chemical calculated electronic circular dichroisms (ECDs). These purple pigments are labile on light irradiation and easily decompose to photo-degraded catechinopyranocyanidins A and B (pdcpcA and pdcpcB), while retaining the stereostructure of the catechin residue. We applied modified Mosher's method for determining the chirality of the secondary alcohol in pdcpcA. Hexamethylation of pdcpcA by diazomethane followed by esterification using (S)- and (R)-MTPACl gave (R)- and (S)-MTPA esters, respectively. By analysis of the NMR spectra of (R)- and (S)-MTPA esters of tetramethylated (+)-catechin, the chirality of pdcpcA was determined to be 2R, 3S, same as the absolute configuration of cpcA.

Determination of Anthocyanins and Antioxidants in 'Titanbicus' Edible Flowers In Vitro and In Vivo.

Titanbicus (TB), a hybrid of Hibiscus moscheutos × H. coccineus (Medic.) Walt., has potential to be used as an edible flower. In this study, proximate nutritional content, anthocyanin content, total polyphenol content (TPC), and antioxidant activities in vitro and in vivo were investigated. Three cultivars of TB, namely Artemis (AR), Rhea (R), and Adonis (AD), were used as materials. Protein and carbohydrates were the primary macronutrients, while crude fat and ash were detected in trace amounts. Cyanidin 3-glucoside (Cy3-G) and cyanidin 3-sambubioside (Cy3-Sam), were identified in all TBs. The highest anthocyanin content was observed in AD (47.09 ± 1.45 mg/g extract), followed by R and AR (6.04 ± 0.20 and 2.72 ± 0.11 mg/g extract, respectively). The TPC of AD (225.01 ± 1.97 mg/g extract) was greater than that of AR and R (185.41 ± 3.24 and 144.10 ± 1.71 mg/g extract, respectively). AD exhibited the strongest in vitro antioxidant activity in hydrophilic oxygen radical absorbance capacity, compared to the other two TBs. In addition, AD extract suppressed the generation of reactive oxygen species in caudal fin of wounded zebrafish. Antioxidant activities of AD appeared to be related to its total anthocyanin content, Cy3-G, Cy3-Sam, and TPC. Our findings indicate that TB, particularly the AD cultivar, would be an attractive source of bioactive compounds with antioxidant activities, and can improve both nutritional value and appearance of food.

Direct Observation of Hydrangea Blue-Complex Composed of 3-O-Glucosyldelphinidin, Al3+ and 5-O-Acylquinic Acid by ESI-Mass Spectrometry.

The blue sepal color of hydrangea is due to a metal complex anthocyanin composed of 3-O-glucosyldelphinidin (1) and an aluminum ion with the co-pigments 5-O-caffeoylquinic acid (2) and/or 5-O-p-coumaroylquinic acid (3). The three components, namely anthocyanin, Al3+ and 5-O-acylquinic acids, are essential for blue color development, but the complex is unstable and only exists in an aqueous solution. Furthermore, the complex did not give analyzable NMR spectra or crystals. Therefore, many trials to determine the detailed chemical structure of the hydrangea-blue complex have not been successful to date. Instead, via experiments mixing 1, Al3+ and 2 or 3 in a buffered solution at pH 4.0, we obtained the same blue solution derived from the sepals. However, the ratio was not stoichiometric but fluctuated. To determine the composition of the complex, we tried direct observation of the molecular ion of the complex using electrospray-ionization mass spectrometry. In a very low-concentration buffer solution (2.0 mM) at pH 4.0, we reproduced the hydrangea-blue color by mixing 1, 2 and Al3+ in ratios of 1:1:1, 1:2:1 and 1:3:1. All solution gave the same molecular ion peak at m/z = 843, indicating that the blue solution has a ratio of 1:1:1 for the complex. By using 3, the observed mass number was m/z = 827 and the ratio of 1, 3 and Al3+ was also 1:1:1. A mixture of 1, 3-O-caffeoylquinic acid (4) and Al3+ did not give any blue color but instead was purple, and the intensity of the molecular ion peak at m/z = 843 was very low. These results strongly indicate that the hydrangea blue-complex is composed of a ratio of 1:1:1 for 1, Al3+ and 2 or 3.

Change of Petals' Color and Chemical Components in Oenothera Flowers during Senescence.

Oenothera flower petals change color during senescence. When in full bloom, the flowers of O. tetraptera are white and those of O. laciniata and O. stricta are yellow. However, the colors change to pink and orange, respectively, when the petals fade. We analyzed the flavonoid components in these petals as a function of senescence using HPLC-DAD and LC-MS. In all three species, cyanidin 3-glucoside (Cy3G) was found in faded petals. The content of Cy3G increased in senescence. In full bloom (0 h), no Cy3G was detected in any of the petals. However, after 12 h, the content of Cy3G in O. tetraptera was 0.97 µmol/g fresh weight (FW) and the content of Cy3G in O. laciniata was 1.82 µmol/g FW. Together with anthocyanins, major flavonoid components in petals were identified. Quercitrin was detected in the petals of O. tetraptera and isosalipurposide was found in the petals of O. laciniata and O. stricta. The content of quercitrin did not change during senescence, but the content of isosalipurposide in O. laciniata increased from 3.4 µmol/g FW at 0 h to 4.8 µmol/g FW at 12 h. The color change in all three Oenothera flowers was confirmed to be due to the de novo biosynthesis of Cy3G.

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.

Plasma membrane-localized Al-transporter from blue hydrangea sepals is a member of the anion permease family.

In hydrangea sepals, an aluminum complex of delphinidin-3-O-glucoside is responsible for the development of the blue color, and co-existing copigments mediate the solubilization and stabilization of the blue Al-anthocyanin complex which is localized in the sepal vacuole. In addition, hydrangeas are Al-hyperaccumulators and exhibit tolerance to acidic soils, in which the toxicity is due to soluble Al ion. Therefore, an Al-absorbing transport and storage system must exist in hydrangea. Recently, we cloned vacuolar and plasma membrane-localized Al-transporters, HmVALT, and HmPALT1, which are both members of the aquaporin family. However, HmPALT1 was only expressed in the sepals, indicating that a different Al-transporter should exist for absorption and long-distance transportation in the hydrangea plant. Using genetic information and microarray analysis, we identified an additional aluminum transporter gene, HmPALT2, which belongs to a member of the anion permease. The transcript was expressed in all tissues of hydrangea plants, and a transient expression study indicated that the gene product is localized to the plasma membrane. The results of an aluminum tolerance assay using yeast cells showed that the HmPALT2 is also involved in the transport of other metal(loid)s. The over-expression of HmPALT2 in Arabidopsis resulted in aluminum-hypersensitivity, suggesting that HmPALT2 should work as an aluminum transporter into cells in planta.

DVC1-0101 to treat peripheral arterial disease: a Phase I/IIa open-label dose-escalation clinical trial.

We here report the results of a Phase I/IIa open-label four dose-escalation clinical study assessing the safety, tolerability, and possible therapeutic efficacy of a single intramuscular administration of DVC1-0101, a new gene transfer vector based on a nontransmissible recombinant Sendai virus (rSeV) expressing the human fibroblast growth factor-2 (FGF-2) gene (rSeV/dF-hFGF2), in patients with peripheral arterial disease (PAD). Gene transfer was done in 12 limbs of 12 patients with rest pain, and three of them had ischemic ulcer(s). No cardiovascular or other serious adverse events (SAEs) caused by gene transfer were detected in the patients over a 6-month follow-up. No infectious viral particles, as assessed by hemagglutination activity, were detected in any patient during the study. No representative elevation of proinflammatory cytokines or plasma FGF-2 was seen. Significant and continuous improvements in Rutherford category, absolute claudication distance (ACD), and rest pain were observed (P < 0.05 to 0.01). To the best of our knowledge, this is the first clinical trial of the use of a gene transfer vector based on rSeV. The single intramuscular administration of DVC1-0101 to PAD patients was safe and well tolerated, and resulted in significant improvements of limb function. Larger pivotal studies are warranted as a next step.

Anti-multidrug-resistant Acinetobacter baumannii activity of DS-8587: In vitro activity and in vivo efficacy in a murine calf muscle infection model.

DS-8587 is a novel broad-spectrum fluoroquinolone with extended antimicrobial activity against both Gram-positive and Gram-negative pathogens. In this study, we evaluated the in vitro and in vivo antibacterial activity of DS-8587 against multidrug-resistant (MDR) Acinetobacter baumannii. The MIC range of DS-8587 against MDR A. baumannii was 0.25-2 mg/L. These DS-8587 MICs were a minimum of 16-fold or 8-fold more potent than ciprofloxacin or levofloxacin, respectively. Bactericidal activity, a 3 log10 reduction from the initial bacterial counts, was observed within 2 h for 1593644 and 4 h for 1593684 after exposure to DS-8587. Therapeutic efficacy of DS-8587 in the murine calf muscle model was observed at 256 mg/kg. The analysis of the pharmacokinetic and pharmacodynamic index revealed that the AUC/MIC ratio showed the best correlation with efficacy. The total and free drug AUC/MIC value required for a static effect was 29.4 and 14.1, respectively. These data indicate DS-8587 would be an effective agent against MDR A. baumannii infection.

BioKnife, a uPA activity-dependent oncolytic Sendai virus, eliminates pleural spread of malignant mesothelioma via simultaneous stimulation of uPA expression.

Malignant pleural mesothelioma (MPM) is highly intractable and readily spreads throughout the surface of the pleural cavity, and these cells have been shown to express urokinase-type plasminogen activator receptor (uPAR). We here examined the potential of our new and powerful recombinant Sendai virus (rSeV), which shows uPAR-specific cell-to-cell fusion activity (rSeV/dMFct14 (uPA2), named "BioKnife"), for tumor cell killing in two independent orthotopic xenograft models of human. Multicycle treatment using BioKnife resulted in the efficient rescue of these models, in association with tumor-specific fusion and apoptosis. Such an effect was also seen on both MSTO-211H and H226 cells in vitro; however, we confirmed that the latter expressed uPAR but not uPA. Of interest, infection with BioKnife strongly facilitated the uPA release from H226 cells, and this effect was completely abolished by use of either pyrrolidine dithiocarbamate (PDTC) or BioKnife expressing the C-terminus-deleted dominant negative inhibitor for retinoic acid-inducible gene-I (RIG-IC), indicating that BioKnife-dependent expression of uPA was mediated by the RIG-I/nuclear factor-κB (NF-κB) axis, detecting RNA viral genome replication. Therefore, these results suggest a proof of concept that the tumor cell-killing mechanism via BioKnife may have significant potential to treat patients with MPM that is characterized by frequent uPAR expression in a clinical setting.

Specific expression of the vacuolar iron transporter, TgVit, causes iron accumulation in blue-colored inner bottom segments of various tulip petals.

Several flowers of Tulipa gesneriana exhibit a blue color in the bottom segments of the inner perianth. We have previously reported the inner-bottom tissue-specific iron accumulation and expression of the vacuolar iron transporter, TgVit1, in tulip cv. Murasakizuisho. To clarify whether the TgVit1-dependent iron accumulation and blue-color development in tulip petals are universal, we analyzed anthocyanin, its co-pigment components, iron contents and the expression of TgVit1 mRNA in 13 cultivars which show a blue color in the bottom segments of the inner perianth accompanying yellow- and white-colored inner-bottom petals. All of the blue bottom segments contained the same anthocyanin component, delphinidin 3-rutinoside. The flavonol composition varied with cultivar and tissue part. The major flavonol in the bottom segments of the inner perianth was rutin. The iron content in the upper part was less than that in the bottom segments of the inner perianth. The iron content in the yellow and white petals was higher in the bottom segment of the inner perianth than in the upper tissues. TgVit1 mRNA expression was apparent in all of the bottom tissues of the inner perianth. The result of a reproduction experiment by mixing the constituents suggests that the blue coloration in tulip petals is generally caused by iron complexation to delphinidin 3-rutinoside and that the iron complex is solubilized and stabilized by flavonol glycosides. TgVit1-dependent iron accumulation in the bottom segments of the inner perianth might be controlled by an unknown system that differentiated the upper parts and bottom segments of the inner perianth.

[Bactericidal effect of levofloxacin injection in combination with meropenem against Pseudomonas aeruginosa using an in vitro simulation model with a hollow fiber system].

An in vitro human plasma concentration simulation model with a hollow fiber system was established and used to evaluate the bactericidal effect of levofloxacin (LVFX) 500mg q.d. in combination with meropenem (MEPM) 1000mg t.i.d. against Pseudomonas aeruginosa. Six clinical isolates of P. aeruginosa which had MEPM MICs of 2 - 16 microg/mL, LVFX MICs of 2 microg/mL, and fractional inhibitory concentration (FIC) indices by the in vitro checkerboard method of 0.625-1 were used. In the treatment with MEPM alone, initial viable counts (10(6)-10(7) CFU/ mL) decreased, but did not reach below the detection limit (100 CFU/mL) and the regrowth of bacteria was observed. In the treatment with LVFX alone, viable counts decreased once below the detection limit, although increased after treatment for 24 hours. On the other hand, in the treatment with LVFX-MEPM combination, more potent bactericidal effects were observed compared to LVFX or MEPM alone in all strains. Especially, in the strains with MEPM MICs of 2 and 4 microg/mL, viable counts rapidly decreased below the detection limit and no regrowth was observed until 24 hours. These results suggested that LVFX-MEPM has a potential to be an effective combination against P. aeruginosa by synergistic rapid bactericidal action in clinical settings, even in the strain against which no significant synergy is confirmed by the traditional in vitro checkerboard method.

Longitudinal Hemodynamics of Aortic Bioprosthetic Valve in Hemodialysis Patients.

We examined the hemodynamic profile of bioprosthetic aortic valves in patients on hemodialysis (HD), longitudinally, and assess the incidence of adverse changes detected by echocardiography. Of 1,146 consecutive patients with severe aortic stenosis who underwent bioprosthetic aortic valve replacement (AVR), 148 patients had end-stage renal disease requiring HD. Each patient on HD was matched one-to-one with a non-HD patient on the basis of propensity scores. The mean follow-up period was 3.3 years for the HD group and 5.9 years for the non-HD group. Follow-up information was available for 95.2%. Postoperative trends of valve hemodynamics derived from linear mixed-effect models showed significant group vs time interactions between the two groups. Stable hemodynamics was consistently observed in the non-HD group, whereas the HD group showed a decrease of -0.06 cm2/y (95% confidence interval (CI), -0.10 to -0.02) in effective orifice area, an increase of 0.8 mm Hg/year (95% CI, 0.4-1.1) in mean pressure gradient, and an increase of 0.08 m/s/year (95%CI, 0.02-0.13) in peak velocity. Cumulative incidence function of SVD more than stage 2 was significantly higher in the HD group (13.1% vs 3.1% at 5 years, Gray test p = 0.01). In a multivariable Fine-Gray analysis, diabetes was independently associated with SVD more than stage 2 in the HD group (subhazard ratio, 1.91; 95% CI, 1.25-2.89; p = 0.02). Survival free-from stenotic-type SVD was significantly lower in HD patients undergoing bioprosthetic AVR. Diabetes was independently associated with postoperative stenotic-type SVD in HD patients.

Intermolecular Copigmentation Between Delphinidin 3-O-Glucoside and Chlorogenic Acid: Taking into Account the Existence of Neutral and Negatively Charged Forms of the Copigment.

Stopped flow corroborated by UV-vis measurements allowed for the calculation of the copigmentation constants of delphinidin 3-O-glucoside with the neutral (CP) and negatively charged CP(-) forms of chlorogenic acid. Solutions of delphinidin 3-O-glucoside in the absence and presence of the copigment were equilibrated at several pH values in the acidic region, pH < 6, and reverse pH jumps monitored by stopped flow were carried out by adding sufficient acid to give flavylium cation at pH ≤ 1. This procedure allows for the separation of three contributions: (i) all flavylium cation and quinoidal base species, (ii) all hemiketal species, and (iii) all cis-chalcone species. Reverse pH jumps can also be performed at fixed pH versus copigment addition. The contribution of trans-chalcone, minor species in the present system, requires reverse pH jumps from the equilibrium followed by a common spectrophotometer. The system was also studied by UV-vis as a function of the copigment addition at different pH values. A global fitting of all experimental data allowed for determination of the copigmentation constants with flavylium cation, KAH+CP = 167 M-1, KAH+CP(-) = 338 M-1; and quinoidal base, KACP = 1041 M-1, KACP(-)= 221 M-1. No significant copigmentation was observed for hemiketal and chalcones. Computational calculations confirm different geometries for the interactions of flavylium cation and quinoidal base with the neutral or the negatively charged forms of the copigment as well as predict identical relative order for the binding energies of the four adducts.