Inhibition of protein kinase CK2 by flavonoids and tyrphostins. A structural insight

Sixteen flavonoids and related compounds have been tested for their ability to inhibit three acidophilic Ser/Thr protein kinases: the Golgi apparatus casein kinase (G-CK) recently identified with protein FAM20C, protein kinase CK1, and protein kinase CK2. While G-CK is entirely insensitive to all compounds up to 40 μM concentration, consistent with the view that it is not a member of the kinome, and CK1 is variably inhibited in an isoform-dependent manner by fisetin and luteolin, and to a lesser extent by myricetin and quercetin, CK2 is susceptible to drastic inhibition by many flavonoids, displaying with six of them IC(50) values < 1 μM. A common denominator of these compounds (myricetin, quercetin, fisetin, kaempferol, luteolin, and apigenin) is a flavone scaffold with at least two hydroxyl groups at positions 7 and 4'. Inhibition is competitive with respect to the phospho-donor substrate ATP. The crystal structure of apigenin and luteolin in complex with the catalytic subunit of Zea mays CK2 has been solved, revealing their ability to interact with both the hinge region (Val116) and the positive area near Lys68 and the conserved water W1, the two main polar ligand anchoring points in the CK2 active site. Modeling experiments account for the observation that luteolin but not apigenin inhibits also CK1. The observation that luteolin shares its pyrocatechol moiety with tyrphostin AG99 prompted us to solve also the structure of this compound in complex with CK2. AG99 was found inside the ATP pocket, consistent with its mode of inhibition competitive with respect to ATP. As in the case of luteolin, the pyrocatechol group of AG99 is critical for binding, interacting with the positive area in the deepest part of the CK2 active site.     

Altered regioselectivity of a poplar O-methyltransferase, POMT-7

O-Methylated flavonoids are biosynthesized by regioselective flavonoid O-methyltransferases (OMTs), which may account for the limited number of naturally occurring flavonoids in nature. It was previously shown that poplar POMT-7 regioselectively methylates the 7-hydroxyl group of flavones, whereas rice ROMT-9 regioselectively methylates the 3'-hydroxyl group of the substrate. We co-expressed both OMT genes (POMT-7 and ROMT-9) in E. coli and carried out biotransformation experiments of some flavonoids with the transformed E. coli strain. Contrast to the predicted regioselectivity of both POMT-7 and ROMT-9, unexpected methylation reaction products, i.e. 3',4'-O-methylated flavonoids, in addition to the predicted ones, were obtained with luteolin (5,7,3',4'-tetrahydroxyflavone) and quercetin (3,5,7,3',4'-pentahydroxyflavone) as substrates. Reactions using the 3'-O-methyl derivative of luteolin and quercetin by POMT-7 revealed that the enzyme has altered its regioselectivity from the 7- to the 4'-hydroxyl groups. These results are discussed in terms of molecular modeling of POMT-7 in relation to its methyl donor.     

Four glucosyltransferases from rice: cDNA cloning, expression, and characterization

Four UDP-dependent glucosyltransferase (UGT) genes, UGT706C1, UGT706D1, UGT707A3, and UGT709A4 were cloned from rice, expressed in Escherichia coli, and purified to homogeneity. In order to find out whether these enzymes could use flavonoids as glucose acceptors, apigenin, daidzein, genistein, kaempferol, luteolin, naringenin, and quercetin were used as potential glucose acceptors. UGT706C1 and UGT707A3 could use kaempferol and quercetin as glucose acceptors and the major glycosylation position was the hydroxyl group of carbon 3 based on the comparison of HPLC retention times, UV spectra, and NMR spectra with those of corresponding authentic flavonoid 3-O-glucosides. On the other hand, UGT709A4 only used the isoflavonoids genistein and daidzein and transferred glucose onto 7-hydroxyl group. In addition, UGT706D1 used a broad range of flavonoids including flavone, flavanone, flavonol, and isoflavone, and produced at least two products with glycosylation at different hydroxyl groups. Based on their substrate preferences and the flavonoids present in rice, the in vivo function of UGT706C1, UGT706D1, and UGT707A3 is most likely the biosynthesis of kaempferol and quercetin glucosides.     

Systematic implications of flavonoids in Hazardia

The flavonoid patterns in Hazardia species support species delimitations and relationships based on morphology and geography. The compounds thus far elucidated are glycosides of quercetin, kaempferol, isorhamnetin, luteolin, and apigenin, glycoflavones of apigenin, and methoxylated flavonol aglycones.     

Peroxidative metabolism of apigenin and naringenin versus luteolin and quercetin: glutathione oxidation and conjugation

GSH was readily depleted by a flavonoid, H(2)O(2), and peroxidase mixture but the products formed were dependent on the redox potential of the flavonoid. Catalytic amounts of apigenin and naringenin but not kaempferol (flavonoids that contain a phenol B ring) when oxidized by H(2)O(2) and peroxidase co-oxidized GSH to GSSG via a thiyl radical which could be trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) to form a DMPO-glutathionyl radical adduct detected by ESR spectroscopy. On the other hand, quercetin and luteolin (flavonoids that contain a catechol B ring) or kaempferol depleted GSH stoichiometrically without forming a thiyl radical or GSSG. Quercetin, luteolin, and kaempferol formed mono-GSH and bis-GSH conjugates, whereas apigenin and naringenin did not form GSH conjugates. MS/MS electrospray spectroscopy showed that mono-GSH conjugates for quercetin and luteolin had peaks at m/z 608 [M + H](+) and m/z 592 [M + H](+) in the positive-ion mode, respectively. (1)H NMR spectroscopy showed that the GSH was bound to the quercetin A ring. Spectral studies indicated that at a physiological pH the luteolin-SG conjugate was formed from a product with a UV maximum absorbance at 260 nm that was reducible by potassium borohydride. The quercetin-SG conjugate or kaempferol-SG conjugate on the other hand was formed from a product with a UV maximum absorbance at 335 nm that was not reducible by potassium borohydride. These results suggest that GSH was oxidized by apigenin/naringenin phenoxyl radicals, whereas GSH conjugate formation involved the o-quinone metabolite of luteolin or the quinoid (quinone methide) product of quercetin/kaempferol.     

The systematic significance of flavonoids in Persea of the Southeastern United States

The systematic significance of flavonoid distribution in the southeastern United States taxa of Persea is discussed. The compounds thus far elucidated are glycosides based on quercetin, kaempferol, apigenin or luteolin.     

Molecular cloning, expression and characterization of a glycosyltransferase from rice

Secondary plant metabolites undergo several modification reactions, including glycosylation. Glycosylation, which is mediated by UDP-glycosyltransferase (UGT), plays a role in the storage of secondary metabolites and in defending plants against stress. In this study, we cloned one of the glycosyltransferases from rice, RUGT-5 resulting in 40–42% sequence homology with UGTs from other plants. RUGT-5 was functionally expressed as a glutathione S-transferase fusion protein in Escherichia coli and was then purified. Eight different flavonoids were used as tentative substrates. HPLC profiling of reaction products displayed at least two peaks. Glycosylation positions were located at the hydroxyl groups at C-3, C-7 or C-4′ flavonoid positions. The most efficient substrate was kaempferol, followed by apigenin, genistein and luteolin, in that order. According to in vitro results and the composition of rice flavonoids the in vivo substrate of RUGT-5 was predicted to be kaempferol or apigenin. To our knowledge, this is the first time that the function of a rice UGT has been characterized.     

A new kaempferol triglycoside from Fagonia taeckholmiana: cytotoxic activity of its extracts

In addition to apigenin, apigenin 7-O-glucoside, kaempferol 3-O-glucoside, kaempferol 3,7-di-O-rhamnoside, quercetin, and quercetin 3-O-glucoside, the methanolic extract of Fagonia taeckholmiana afforded a new compound identified as kaempferol 3-O-beta-l-arabinopyranosyl-(1-->4)-alpha-l-rhamnopyranoside-7-O-alpha-l-rhamnopyranoside. Identification of the isolated compounds was based on chemical and spectroscopic analyses including UV, FABMS, (1)H, (13)C and 2D NMR, and DEPT. The cytotoxic activities of the compounds against several cancer cell lines were determined.     

Structure-activity relationships of flavonoids for vascular relaxation in porcine coronary artery

Flavonoids are polyphenolic compounds that are widespread in the plant kingdom, and structure-activity relationships (SAR) for vascular relaxation effects were examined for 17 of them using porcine coronary arteries. Density functional theory was employed to calculate the chemical parameters of these compounds. The order of potency for vascular relaxation was as follows: flavones (apigenin and luteolin) >or= flavonols (kaempferol and quercetin)>isoflavones (genistein and daidzein)>flavanon(ol)es (naringenin)>chalcones (phloretin)>anthocyanidins (pelargonidin)>flavan(ol)es ((+)-catechin and (-)-epicatechin). SAR analysis revealed that for good relaxation activity, the 5-OH, 7-OH, 4'-OH, C2=C3 and C4=O functionalities were essential. Comparison of rutin with quercetin, genistin with genistein, and puerarin with daidzein demonstrated that the presence of a glycosylation group greatly reduced relaxation effect. Total energy and molecular volume were also predictive of their relaxation activities. Our findings indicated that the most effective relaxing agents are apigenin, luteolin, kaempferol and genistein. These flavonoids possess the key chemical structures demonstrated in our SAR analysis.     

Flavonoids of Helichrysum chasmolycicum and its antioxidant and antimicrobial activities

From the aerial parts of Helichrysum chasmolycicum P.H Davis, which is an endemic species in Turkey, the flavonoids apigenin, luteolin, kaempferol, 3,5-dihydroxy-6,7,8-trimethoxyflavone, 3,5-dihydroxy-6,7,8,4′-tetramethoxyflavone, apigenin 7-O-glucoside, apigenin 4′-O-glucoside, luteolin 4′-O-glucoside, luteolin 4′,7-O-diglucoside, kaempferol 3-O-glucoside, kaempferol 7-O-glucoside and quercetin 3-O-glucoside were isolated. The methanol extract of the aerial parts of H. chasmolycicum showed antioxidant activity by DPPH method (IC₅₀ 0.92 mg/mL). Antimicrobial activity test was performed on the B, D, E extracts and also 3,5-dihydroxy-6,7,8-trimethoxyflavone and kaempferol 3-O-glucoside which were the major flavonoid compounds obtained from aerial parts of H. chasmolycicum by microbroth dilutions technique. The E (ethanol-ethyl acetate) extract showed moderate antimicrobial activity against Pseudomonas aeruginosa, B (petroleum ether-60% ethanol-chloroform) extract and 3,5-dihydroxy-6,7,8-trimethoxyflavone showed moderate antifungal activity against Candida albicans.     

Characterization of flavonoid 7-O-glucosyltransferase from Arabidopsis thaliana

Most flavonoids found in plants exist as glycosides, and glycosylation status has a wide range of effects on flavonoid solubility, stability, and bioavailability. Glycosylation of flavonoids is mediated by Family 1 glycosyltransferases (UGTs), which use UDP-sugars, such as UDP-glucose, as the glycosyl donor. AtGT-2, a UGT from Arabidopsis thaliana, was cloned and expressed in Escherichia coli as a gluthatione S-transferase fusion protein. Several compounds, including flavonoids, were tested as potential substrates. HPLC analysis of the reaction products indicated that AtGT-2 transfers a glucose molecule into several different kinds of flavonoids, eriodictyol being the most effective substrate, followed by luteolin, kaempferol, and quercetin. Based on comparison of HPLC retention times with authentic flavonoid 7-O-glucosides and nuclear magnetic resonance spectroscopy, the glycosylation position in the reacted flavonoids was determined to be the C-7 hydroxyl group. These results indicate that AtGT-2 encodes a flavonoid 7-O-glucosyltransferase.     

Influence of dietary flavonoids on the glycation of plasma proteins

It has been suggested that the increasing glycation in diabetes can influence the ability of plasma proteins to bind to small molecules. Herein, the influence of flavonoids on the glycation of plasma proteins was investigated. After being incubated with glucose at 37 °C, the levels of glycated albumin (HGA) were significantly improved in healthy human plasma proteins (HPP). The inhibitory effects of flavonoids against the formation of advanced glycation products (AGEs) in HPP were determined as: galangin > apigenin > kaempferol ≈ luteolin > myricetin > quercetin. After being combined with 20 μmol L?1 of quercetin for 11 days, the fresh plasma with δ-glucose caused 323.05-32.07% inhibition of HGA formation in type II diabetes plasma proteins (TPP). Luteolin showed weak inhibition of HGA formation in TPP. However, kaempferol, galangin and apigenin hardly inhibited the formation of HGA in TPP. These results showed that more hydroxyl groups on ring B of flavonoids will enhance the inhibitory effects on the HGA formation in TPP.     

楮叶中香豆素和黄酮类化学成分研究

本文对楮树(Broussonetia papyrifera(L.)Vent.)叶中的香豆素和黄酮类成分进行研究,利用柱层析、重结晶、色谱技术等分离手段,分离得到了10个化合物,经理化性质和波谱数据分析鉴定为伞形花内酯(1)、七叶内酯(2)、芹菜素(3)、木犀草素(4)、山柰酚(5)、牡荆素(6)、芹菜素-7-O-β-D-葡萄糖醛酸(7)、芫花素(8)、槲皮素(9)、二氢槲皮素(10).化合物1～7均为首次从该植物中分离得到.     

Distribution of flavonoids and their systematic significance in Clematis subsection Viornae

Fifteen flavonoids were identified in the eight taxa of Clematis subsection Viornae. Flavonols, flavones, and C-glycosyl flavones were present. The compounds were based primarily on luteolin, apigenin, quercetin, and kaempferol. The systematic significance of the distribution of these compounds among the taxa is discussed.     

乌饭树树叶的黄酮类成分研究

研究了乌饭树树叶中黄酮类化合物。利用色谱分离技术从中分离出5个黄酮类化合物,通过波谱数据分别鉴定为槲皮素、白杨黄素、芹黄素、山奈酚和木犀草素。其中除槲皮素外的4个化合物为首次发现。     

Foliar flavonoids of Annonaceae from Brazil: taxonomic significance

Foliar flavonoids of 31 species of the Annonaceae native to Brazil, amounting to 76 compounds, were isolated and identified. All phenols found were glycosides of either flavones (apigenin, scutellarein, hispidulin and luteolin) or flavonols (kaempferol, rhamnocitrin, 6-hydroxyrhamnocitrin, quercetin, isorhamnetin and rhamnetin), with the latter predominating. Some members of the tribe Bocageeae are distinctive for accumulating 6-oxygenated flavones and flavonols, in addition to 7-O-methylated flavonols, a feature possibly linked to the assumed advanced condition of the tribe within the family. Members of Duguetia stand out for the apparent absence of quercetin glycosides. Anaxagorea dolichocharpa seemingly lacks flavones and flavonols entirely. A UPGMA analysis based on the distribution of flavonoids does not group the analyzed species according to the available tribal division of the Annonaceae. However, several taxonomically meaningful groupings emerged through the multivariate analysis.     

Mechanism of oxidative DNA damage induced by quercetin in the presence of Cu(II)

Quercetin, one of flavonoids, has been reported to be carcinogenic. There have been no report concerning carcinogenicity of kaempferol and luteolin which have structure similar to quercetin. DNA damage was examined by using DNA fragments obtained from the human p53 tumor suppressor gene. Quercetin induced extensive DNA damage via reacting with Cu(II), but kaempferol and luteolin induced little DNA damage even in the presence of Cu(II). Excessive quercetin inhibited copper-dependent DNA damage induced by quercetin. Bathocuproine, a Cu(I)-specific chelator, catalase and methional inhibited the DNA damage by quercetin, whereas free hydroxyl radical scavengers did not. Site specificity of the DNA damage was thymine and cytosine residues. The site specificity and the inhibitory effects suggested that DNA-copper-oxygen complex rather than free hydroxyl radical induced the DNA damage. Formation of 8-oxodG by quercetin increased extensively in the presence of Cu(II), whereas 8-oxodG formation by kaempferol or luteolin increased only slightly. This study suggests a good relationship between carcinogenicity and oxidative DNA damage of three flavonoids. The mechanism of DNA damage by quercetin was discussed in relation to the safety in cancer chemoprevention by flavonoids.     

Glycosylation of flavonoids with a glycosyltransferase from Bacillus cereus

Microbial glycosyltransferases can convert many small lipophilic compounds such as phenolics, terpenoids, cyanohydrins and alkaloids into glycons using uridine-diphosphate-activated sugars. The main chemical functions of glycosylation processes are stabilization, detoxification and solubilization of the substrates. The gene encoding the UDP-glycosyltransferase from Bacillus cereus, BcGT-1, was cloned by PCR and sequenced. BcGT-1 was expressed in Escherichia coli BL21 (DE3) with a his-tag and purified using a His-tag affinity column. BcGT-1 could use apigenin, genistein, kaempferol, luteolin, naringenin and quercetin as substrates and gave two reaction products. The enzyme preferentially glycosylated at the 3-hydroxyl group, but it could transfer a glucose group onto the 7-hydroxyl group when the 3-hydroxyl group was not available. The reaction products made by biotransformation of flavonoids with E. coli expressing BcGT-1 are similar to those produced with the purified recombinant enzyme. Thus, this work provides a method that might be useful for the biosynthesis of flavonoid glucosides and for the glycosylation of related compounds.     

The chemosystematics of Egyptian Euphorbia species

Nineteen Euphorbia species were studied for their flavonoid patterns. Flavonoids identified were kaempferol, quercetin, apigenin and luteolin. Major glycosylation patterns are discussed.     

Inhibitory effects of dietary flavonoids on purified hepatic NADH-cytochrome b5 reductase: structure-activity relationships

The structure-activity relationships of flavonoids with regard to their inhibitory effects on NADH-cytochrome b5 reductase (E.C. 1.6.2.2), a clinically and toxicologically important enzyme, are not known. In the present study, the inhibitory effects of fourteen selected flavonoids of variable structure on the activity of purified bovine liver cytochrome b5 reductase, which shares a high degree of homology with the human counterpart, were investigated and the relationship between structure and inhibition was examined. Of all the compounds tested, the flavone luteolin was the most potent in inhibiting b5 reductase with an IC50 value of 0.11 μM, whereas naringenin, naringin and chrysin were inactive within the concentration range tested. Most of the remaining flavonoids (morin, quercetin, quercitrin, myricetin, luteolin-7-O-glucoside, (-)-epicatechin, and (+)-catechin) produced a considerable inhibition of enzyme activity with IC50 values ranging from 0.81 to 4.5 μM except apigenin (36 μM), rutin (57 μM) and (+)-taxifolin (IC50 not determined). The magnitude of inhibition was found to be closely related to the chemical structures of flavonoids. Analysis of structure-activity data revealed that flavonoids containing two hydroxyl groups in ring B and a carbonyl group at C-4 in combination with a double bond between C-2 and C-3 produced a much stronger inhibition, whereas substitution of a hydroxyl group at C-3 was associated with a less inhibitory effect. The physiologically relevant IC50 values for most of the flavonoids tested regarding b5 reductase inhibition indicate a potential for significant flavonoid-drug and/or flavonoid-xenobiotic interactions which may have important therapeutic and toxicological outcomes for certain drugs and/or xenobiotics.