Promotive effect of auxins on UDP-glucose: flavonol glucosyltransferase activity in Vitis sp. cell cultures

The addition of 2,4-dichlorophenoxyacetic acid (2, 4-D) to Vitis sp. cell cultures significantly enhanced the production of quercetin 3,7,4'-tri-O-glucoside, 3,7-di-O-glucoside and 3,4'-O-glucoside from quercetin. This enhancement of glucosylation by 2,4-D was also observed in cell cultures of other plant species. The activity of UDP-glucose: flavonol glucosyltransferase (UFGT) in cell-free extracts of Vitis sp. cell cultures increased approximately 10-fold, 48 h after the addition of 2,4-D to the culture medium. The UFGT activity increased linearly up to 15 h and showed a maximal response to the addition of 10-50 mg/l of 2,4-D at 48 h. The promotive effect of 2,4-D was inhibited by cycloheximide suggesting that de novo protein synthesis was involved in this phenomenon. Interestingly, similar promotive effects on the UFGT activity were observed for other phytohormones such as kinetin and several anti-auxins.     

Chemical composition and antioxidant activity of tronchuda cabbage internal leaves

A phytochemical study was undertaken on the internal leaves of tronchuda cabbage (Brassica oleracea L. var. costata DC). Seventeen phenolic compounds were characterized and quantified by reversed-phase HPLC-DAD-ESI-MS ⁿ and HPLC/DAD, respectively: quercetin 3-O-sophoroside-7-O-glucoside, 3-p-coumaroylquinic acid, kaempferol 3-O-sophoroside-7-O-glucoside, kaempferol 3-O-(caffeoyl)-sophoroside-7-O-glucoside, sinapoyl gluc-oside acid, kaempferol 3-O-(sinapoyl)-sophoroside-7-O-glucoside, kaempferol 3-O-(feruloyl)-sophoroside-7-O-glucoside, kaempferol 3-O-(p-coumaroyl)-sophoroside-7-O-glucoside, 4-p-coumaroylquinic acid, sinapic acid, kaempferol 3-O-sophoroside, 3 isomeric forms of 1,2-disinapoylgentiobiose, 1-sinapoyl-2-feruloylgentiobiose, 1,2,2-trisinapoylgentiobiose and 1,2′-disinapoyl-2-ferul-oylgentiobiose. Seven organic acids (aconitic, citric, ascorbic, malic, quinic, shikimic and fumaric acids) were also identified and quantified. The hot water extract of tronchuda cabbage internal leaves was investigated for its capacity to act as a scavenger of DPPH^• radical and reactive oxygen species (superoxide radical, hydroxyl radical and hypochlorous acid), exhibiting antioxidant capacity in a concentration dependent manner against all radicals.     

Aglycone and glycoside specificity of apple skin flavonoid glycosyltransferase

O-Glycosyltransferase(s) extracted from apple (Malus pumila Mill) fruit skin showed activity towards a range of flavonols and anthocyanins. However, no glycosylating activity was shown towards a dihyroflavonol (dihydroquercetin), a flavanone (eriodictyol) or a flavone (luteolin). The enzyme preparation glycosylated those flavonoids normally present in apple skins (quercetin and cyanidin) and in addition several other related compounds (delphinidin, fisetin, isorhamnetin, kaempferol, myricetin and pelargonidin). This enzyme(s) specifically transferred the glycosyl moiety from sugar nucleotide donors to the 3-position of the flavonoid nucleus. Only flavonoid 3-glycosides occur naturally in apple skin. Activity with different sugar donors was in the order galactose>glucose>xylose, which reflected the ratios of cyanidin and quercetin glycosides found in apple fruit skin. There were slight differences in the relative UFGT activity with quercetin and the three different sugar donors between ‘Granny Smith’ and ‘Splendour’, and this was reflected by similar differences in the ratios of endogenous quercetin glycosides. ©1997 SCI     

Effect of curing and cooking on flavonols and anthocyanins in traditional varieties of onion bulbs

The stability of the major flavonol glucosides and anthocyanins was studied in two regional varieties of Portuguese onion (a white variety “branca da Póvoa” and a red variety “vermelha da Póvoa”). White and red onions from 2007 and 2008 harvests were subjected to field curing with and without light, but the red cultivar from 2008 was also subjected to typical domestic processing, including chopping and different cooking treatments. Field curing resulted in increases in quercetin content compared to levels at lifting, especially important for all white bulbs (33–40% increase). Flavonol and anthocyanin levels in onions cured in the dark were similar to those obtained in bulbs cured in the light. The treatments chopping followed by refrigerated storage, oven roasting and frying, did practically not contribute to modify the total levels of flavonols. Moderate microwave cooking did not affect to the flavonol content, but intense microwave treatment cause flavonol losses of 16% and 18% for quercetin 3,4′-diglucoside (QdG) and quercetin 4′-glucoside (QmG), respectively. Boiling onions for 30 min leaded losses of quercetin glycosides, which leached to the boiling water without being degraded at 37% and 29% for QdG and QmG, respectively. Boiling for 60 min had more severe effects, since it caused the degradation of quercetin derivatives at 53% and 44% for QdG and QmG, respectively. For anthocyanins, the severity of the cooking treatments was in the following order: frying > boiling > roasting (microwave roasting > oven roasting).     

Identification of flavanones from peel of Citrus changshan-huyou Y. B. Chang, by HPLC–MS and NMR

High-performance liquid chromatography coupled with a UV photodiode-array detector and a mass spectrometer (HPLC–MS) was used to analyze the constituents of an extract of Citrus changshan-huyou Y. B. Chang peel. Structures of two flavanones were identified based on their abundant [M+H]⁺ ions, UV spectra, and ¹HNMR and ¹³CNMR spectrometer as 5,4′-dihydroxy flavanone-7-O-α-glucoside (naringenin-7-O-α-glucoside) and 5,3′-dihydroxy-4′-methoxy flavanone-7-O-α-glucoside (hesperetin-7-O-α-glucoside). The two flavanones were first identified from peel of Citrus changshan-huyou Y. B. Chang.     

Bioconversion of anthocyanin glycosides by Bifidobacteria and Lactobacillus

Eight strains of Lactobacillus plantarum, 6 strains of Lactobacillus casei, Lactobacillus acidophilus LA-5, and Bifidobacterium lactis BB-12 were screened for β-glucosidase activity. We then proceeded to investigate the enzymatic potential of selected strains for bioconversion of delphinidin and malvidin glycosides to their metabolites. L. plantarum and L. casei strains showed the highest cell-envelope associated β-glucosidase activity. Intracellular β-glucosidase activity from B. lactis BB-12 was up to 287-fold higher than that of the other strains. The L. acidophilus strain showed low β-glucosidase activity, both, intra and extracellularly. No aglycons were detected in bacterial extract reactions with anthocyanin glycosides. Delphinidin-3-glucoside underwent chemical degradation to form mainly gallic acid, although delphinidin-3-glucoside degradation due to B. lactis BB-12 and enzymatic activity towards chemically-formed metabolites due to L. casei LC-01 were observed. Incubation of malvidin-3-glucoside with B. lactis BB-12, L. plantarum IFPL722, and L. casei LC-01 cell-free extracts led to different patterns of gallic, homogentisic and syringic acid formation.     

Anthocyanin compositions and biological activities from the red petals of Korean edible rose (Rosa hybrida cv. Noblered)

The objectives of the present study were to investigate anthocyanin profiles and their biological properties, including antioxidant, anticancer, and antiallergic, from the red petals of Korean edible rose (Rosa hybrida cv. Noblered). The acidic methanol extract of this species showed potent biological activities at a concentration of 50 μg/mL. Its anthocyanins were characterised as cyanidin 3,5-di-O-glucoside and pelargonidin 3,5-di-O-glucoside using reversed-phase C₁₈ column chromatography, NMR spectroscopy, and HPLC-DAD-ESI/MS analysis. Cyanidin 3,5-di-O-glucoside was the predominant constituent (375 mg/100 g), representing about 85% of total content. Cyanidin 3,5-di-O-glucoside exhibited good scavenging activity against DPPH radical with IC₅₀ value of 55.2 μg/mL; pelargonidin 3,5-di-O-glucoside showed potent anticancer effects against LNCap (human prostate cell line), ACHN (human renal cell line) and MOLT-4F (human leukaemia cell line) cell cultures, with IC₅₀ values of 6.43, 18.3, and 6.78 μg/mL, respectively. Antiallergic activities were only moderate.     

A novel selective nucleoside phosphorylating enzyme from Morganella morganii

A selective nucleoside phosphorylating enzyme was purified to homogeneity from Morganella morganii NCIMB10466 crude extract. The enzyme appeared to consist of six subunits identical in molecular mass (M_r 25,000). It phosphorylated various nucleosides at the 5′-position to produce nucleoside-5′-monophosphates using pyrophosphate as the phosphate source. Energy-rich compounds, such as carbamylphosphate and acetylphosphate, were also very effective phosphate donors. The enzyme also exhibited phosphatase activity, and dephosphorylated various phosphate esters, but had a weak effect on nucleoside-3′-monophosphates. Based on the results of the kinetic study, the enzyme appeared to be an acid phosphatase. Its activity was partly inhibited by sulfhydryl reagents and heavy metal ions, but not by chelating reagents such as EDTA. Using the purified enzyme, 32.6 mM 5′-IMP was synthesized from inosine with a 41% molar yield, but the synthesized 5′-IMP was hydrolyzed back to inosine and phosphate as the reaction time was extended.     

Enzymatic de-glycosylation of rutin improves its antioxidant and antiproliferative activities

Bioavailability and biological properties of flavonoid glycosides can be improved after the enzymatic hydrolysis of specific glycosyl groups. In this study, we evaluate the antioxidant and antiproliferative potential of rutin after enzymatic hydrolysis performed by α-l-rhamnosidases (hesperidinase from Penicillium sp. and naringinase from Penicillium decumbens) previously heated at 70 °C for 30 min to inactivate the undesirable β-d-glucosidase activity. The highest in vitro antioxidant activity determined by DPPH radical scavenging was achieved with rutin hydrolyzed by hesperidinase. Rutin was predominantly bioconverted into quercetin-3-glucoside. There was no statistical difference between xanthine oxidase inhibition by rutin before and after hydrolysis. However, in vitro inhibitory activity against ten human tumor cell lines showed that hydrolyzed rutin exerted a more potent antiproliferative effect than quercetin and rutin on various cancer cell lines, specially glioma, and ovarian and breast adenocarcinomas. These results indicate that quercetin-3-glucoside could be a promising functional derivative obtained by rutin hydrolysis.     

Antioxidant and antiproliferative activities of phytochemicals from Quince (Cydonia vulgaris) peels

Fifty-nine secondary metabolites have been isolated from Cydonia vulgaris peels and characterised on the basis of their spectroscopic features. Among them, five metabolites, 3β-(18-hydroxylinoleoyl)-28-hydroxyurs-12-ene (12), 3β-linoleoylurs-12-en-28-oic acid (15), 3β-oleoyl-24-hydroxy-24-ethylcholesta-5,28(29)-diene (24), tiglic acid 1-O-β-d-glucopyranoside (35), and 6,9-dihydroxymegastigmasta-5,7-dien-3-one 9-O-β-d-gentiobioside (46), have been isolated and elucidated for the first time. All of the compounds were tested for their radical-scavenging and antioxidant activities by measuring their capacity to scavenge the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical, and anion superoxide radical and to induce the reduction of Mo(VI) to Mo(V). The antiproliferative activity of all the most abundant compounds by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) bioassay on murine B16-F1 melanoma cells has been also assessed.     

Phenolic Compounds from the Leaves of Vitis labrusca and Vitis vinifera L. as a Source of Waste Byproducts: Development and Validation of LC Method and Antichemotactic Activity

The cultivation of Vitis (Vitaceae) grape varieties is one of the most important economic activities in agribusiness in southern Brazil. Vitis varieties are rich in polyphenolic compounds with several pharmacological and biological activities, such as antioxidant action. In this context, this study analyzed qualitatively and quantitatively the anthocyans and flavonoids found in the leaves of grape varieties Vitis vinifera and Vitis labrusca. For this purpose, vine leaf extracts were prepared and the chemical profile of each was characterized by LC/MS-MS. Two high performance liquid chromatography-validated methods were performed using UV/VIS-LC-DAD detector to quantify phenolic compounds. The main anthocyanins isolated from vine leaves were cyanidin-3-O-glucoside and peonidin-3-O-glucoside. The flavonoids identified were rutin, quercetin-3-O-galactoside, quercetin-3-O-glucoside, and quercetin-3-O-glucuronide, which was the predominant compound. The Waters X-Terra® RP18 column allowed the effective separation of quercetin-3-O-glucuronide from the other flavonoids for the first time, besides the partial separation of quercetin-3-O-galactoside from quercetin-3-O-glucoside. Furthermore, another phenolic compound was confirmed by MS spectrometry, using direct infusion, as being trans-caftaric acid. The present study also investigates the antichemotactic activity in vitro of grape crude extracts, fractions, and isolated compounds. It was demonstrated that almost all fractions and isolated compounds showed increased antichemotactic effect in response to LPS with a more pronounced values of IC₅₀ for anthocyanins fraction, rutin, quercetin-3-O-galactoside, and trans-caftaric acid (0.9, 1.6, 3.7, and 5.1 ng/mL, respectively).     

Effect of selected phytochemicals on cell proliferation in A549 lung cancer cells

Effects of quercetin 3-β-d-glucoside, resveratrol, and curcumin on A549 lung cancer cell proliferation and the mechanism of these phytochemicals in regulating apoptosis and cell cycle arrest were investigated. A549 cells were treated with quercetin 3-β-d-glucoside, resveratrol, or curcumin at 37°C for 96 hr and cell viability was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Proteins related to apoptosis and cell cycle in A549 cells were quantified with Western blotting assay. Quercetin 3-β-d-glucoside, resveratrol, and curcumin inhibited A549 cell proliferation in a dose-dependent manner (p<0.05). Quercetin 3-β-Dglucoside significantly decrease the expression level of CDK4 at concentrations of 5 mM and above (p<0.05). Curcumin lowered expression level of Bcl-2, CDK4, and cyclin D1 at concentrations of 100, 50, and above, and 50 mM and above, respectively (p<0.05). These results suggest that phytochemicals, which can be found in a normal diet, inhibit lung cancer cell proliferation and regulate the expression of the proteins involved in apoptosis and cell cycle.     

Radical scavenging compounds in onion skin

Two major flavonols (F_I and F_II) in onion skin were isolated. According to the ultraviolet–visible absorption spectra, F_I and F_II fractions were thought to be 3,5,7,3′-OH and 3 (5),7,3′,4′-OH of flavonol, respectively. After acid hydrolysis of the two fractions, F_I was found to consist of quercetin and glucose, but F_II only contained quercetin. F_I and F_II were confirmed by fast atom bombardment as quercetin 4′-glucoside and quercetin, respectively. The F_I and F_II fractions exhibited potent radical scavenging activity against DPPH, superoxide anion (O₂·^-) , and hydroxyl (·OH) radicals.     

First accurate profiling of antioxidant anthocyanins and flavonols of Tibouchina urvilleana and Tibouchina mollis edible flowers aided by fractionation with Amberlite XAD-7

A purification and fractionation process of the edible flowers of Tibouchina mollis and Tibouchina urvilleana followed by the first attempt to the anthocyanin and flavonol characterisation and identification by UHPLC-DAD-ESI-MS were developed. T. urvilleana exhibited a higher monomeric anthocyanin content, mainly due to the presence of the 3-O-(6′-p-coumaroyl)-glucoside derivatives of malvidin and petunidin. Quercetin-3-O-hexoside was the major flavonol identified in T. urvilleana, and the lack of myricetin derivatives was also exhibited. The anthocyanin and flavonol profile of T. mollis was more miscellaneous, characterised by the occurrence of cyanidin-3-O-glucoside followed by the 3-O-(6′-p-coumaroyl)-glucoside and 3-O-glucoside derivatives of malvidin and petunidin as anthocyanins, and myricetin, quercetin, and 3-O-hexosides of kaemperol and quercetin as flavonol compounds. Therefore, the anthocyanin and flavonol profile, through a process based on purification and fractionation, could be a useful tool to ensure the authenticity of the Tibouchina. Furthermore, the purification process made the antioxidant activity increase, which is greatly correlated to the reduction capacity.     

Composition and content of flavonol glycosides in broccoli florets (Brassica olearacea) and their fate during cooking

The two main flavonol glycosides present in broccoli florets were identified as quercetin 3-O-sophoroside and kaempferol 3-O-sophoroside. Three minor glucosides of quercetin and kaempferol were also detected, namely isoquercitrin, kaempferol 3-O-glucoside and a kaempferol diglucoside. The sophorosides of quercetin and kaempferol were present in raw florets at a level of 65 mg kg⁻¹ and 166 mg kg⁻¹ fresh weight, respectively. The total content of quercetin and kaempferol glycosides expressed as aglycone was 43 and 94 μg g⁻¹ fresh weight, respectively, and these agree with other recently published data. During the cooking process only 14–28% of the individual glucosides were retained in the cooked tissue, the remainder being largely leached into the cooking water with only a small loss attributed to the formation of the respective aglycones. © 1998 SCI.     

α-Anomer-selective glucosylation of (+)-catechin by the crude enzyme, showing glucosyl transfer activity, of Xanthomonas campestris WU-9701

α-Anomer-selective glucosylation of (+)-catechin was carried out using the crude enzyme, showing α-glucose transferring activity, of Xanthomonas campestris WU-9701 with maltose as a glucosyl donor. When 60 mg of (+)-catechin and 50 mg of the enzyme (5.25 units as maltose hydrolysing activity) were incubated in 10 ml of 10 mM citrate-Na₂HPO₄ buffer (pH 6.5) containing 1.2 M maltose at 45°C, only one (+)-catechin glucoside was selectively obtained as a product. The (+)-catechin glucoside was identified as (+)-catechin 3′-O-α- -glucopyranoside (α-C-G) by ¹³C-NMR, ¹H-NMR and two-dimensional HMBC analysis. The reaction at 45°C for 36 h under the optimum conditions gave 12 mM α-C-G, 5.4 mg/ml in the reaction mixture, and the maximum molar conversion yield based on the amount of (+)-catechin supplied reached 57.1%. At 20°C, the solubility in pure water of α-C-G, of 450 mg/ml, was approximately 100 fold higher than that of (+)-catechin, of 4.6 mg/ml. Since α-C-G has no bitter taste and a slight sweet taste compared with (+)-catechin which has a very bitter taste, α-C-G may be a desirable additive for foods, particularly sweet foods.     

A new flavone from antioxidant extracts of Pistacia terebinthus

Acetone and methanol extracts of the fruits of Pistacia terebinthus L. subsp. terebinthus L. were studied for their antioxidant activity by investigating their total phenolic and flavonoid contents, β-carotene bleaching potential, DPPH radical scavenging effect, scavenging activity on superoxide anion radical, reducing power, and metal chelating effect on ferrous ion. Both extracts showed very similar chemical profile by checking on TLC plates, and exhibited high scavenging activity on superoxide anion radical and DPPH radical. Due to these similarities they were combined and fractionated on a silica gel column for their constituents, and the most active three fractions in DPPH assay were purified to afford a new flavone 6′-hydroxyhypolaetin 3′-methyl ether (1) besides a group of known flavonoids apigenin, luteolin, luteolin 7-O-glucoside, quercetin, quercetagetin 3-methyl ether 7-O-glucoside, isoscutellarein 8-O-glucoside. Their structures were established by UV, UV shift reagents, and ¹H NMR spectroscopic techniques. Antioxidant activity of the new flavone was investigated by β-carotene bleaching and DPPH radical scavenging activity methods, and it showed a high activity in the first system, but not so good in the latter.     

Anti-melanogenesis properties of quercetin- and its derivative-rich extract from Allium cepa

In an effort to find a new whitening agent, we have found that the methanol extract of the dried skin of Allium cepa showed inhibition of melanin formation. Bioassay-guided fractionation led to the isolation of quercetin (1) and quercetin 4’-O-β-glucoside (3) from A. cepa as the inhibitors of melanin formation in B16 melanoma cells with IC₅₀ values of 26.5 and 131 μM, respectively. In addition, we evaluated the effect of some quercetin derivatives, such as isoquercitrin (2), quercetin 3,4’-O-diglucoside (4), rutin (5) and hyperin (6) on B16 melanoma cells. These quercetin derivatives did not show any inhibition of melanin formation. Furthermore, the ORAC values of compounds 1–6 were 7.64, 8.65, 4.82, 4.32, 8.17 and 9.34 μmol trolox equivalents/μmol, respectively. Dried skin of red onion showed inhibitory activity against melanin formation in B16 melanoma cells, as well as antioxidant properties.     

Antihypertensive properties of flavonoid-rich apple peel extract

Hypertension is a major public health problem rising across the globe. Inhibition of angiotensin converting enzyme (ACE) is identified as a main therapeutic target in controlling high blood pressure. The present study investigated the ACE inhibitory property of a flavonoid-rich apple peel extract (FAE), its constituents, selected flavonoids and some quercetin metabolites using a biochemical assay of ACE inhibition and a human umbilical vein endothelial cell (HUVEC) model. FAE, all the tested flavonoids except genistein, and two quercetin metabolites (quercetin-3-O-glucuronic acid and quercetin-3-O-sulfate) significantly (p < 0.05) inhibited ACE. Enzyme kinetic analysis revealed that flavonoids are competitive inhibitors of ACE. In the HUVEC model, FAE, quercetin-3-O-glucoside and quercetin-3-O-glucuronic acid inhibited significantly (p < 0.05) ACE activity. Overall, FAE and most of the flavonoids tested showed ACE inhibition in vitro which needs further investigations using animal and human clinical trials.     

Analysis of the Major Flavonol Glycosides Present in Four Varieties of Onion (Allium cepa) and Changes in Composition Resulting from Autolysis

The major flavonoids of mature onion bulb were confirmed as the 3,4′-O-diglucoside (Qdg) and 4′-O-monoglucoside (Qmg) of quercetin using a combination of chromatographic comparisons, mass spectrometry and nuclear magnetic resonance spectroscopy. These two components account for over 85% of the total flavonoids in three varieties of onion with Qdg as the main component. Quercetin is detected in these long stored onions but only at low levels of less than 2% of the total. The remaining flavonoid fraction (approx 15%) comprises upto 17 different components of which quercetin-3-O-glucoside and isorhamnetin glucoside are prominent members although each contribute less than 1% of the total flavonoid fraction. There are significant differences in the levels of Qdg and Qmg between the four different onion varieties analysed; Qdg varying from 50–1300 mg kg^-1 fresh onion tissue and Qmg from 36–394 mg kg^-1. Maceration of the tissue for the three varieties tested led to a loss of Qdg and the appearance of Qmg and free quercetin. In the variety Rijnsburger 50% of the Qdg was degraded in 5 h and had completely disappeared after 24 h. These changes in Qdg can be quantitatively explained by increases in Qmg and free quercetin. The possible significance of quercetin glycosides in the diet is discussed. © 1997 SCI