Inhibitions of the autoxidation of linoleic acid by flavonoids in micelles.

The activities of five flavonoids as chain-breaking antioxidants have been studied for the autoxidation of linoleic acid in cetyl trimethylammonium bromide (CTAB) micelles at 37 degrees C. Flavonols such as quercetin, rutin and morin exhibited antioxidant activities, while two flavanones, naringin and hesperidin, did not suppress the oxidation appreciably. The ratio of rate constants for inhibition and propagation kinh/kp and stoichiometric factor n were determined.     

The correlation between active oxygens scavenging and antioxidative effects of flavonoids

The abilities of 15 flavonoids as a scavenger of active oxygens (hydroxyl radical and superoxide anion) were studied. Hydroxyl radical (^.OH) was generated by the Fenton system, and assayed by the determination of methanesulfinic acid (MSA) formed from the reaction of dimethyl sulfoxide (DMSO) with ^.OH. (+)-Catechin, (−)-epicatechin, 7,8-dihydroxy flavone, and rutin showed the ^.OH scavenging effect 100–300 times superior to that of mannitol, a typical ^.OH scavenger. The other flavonoids showed no ^.OH scavenging effect at their concentrations up to 50 μM. Baicalein, quercetin, morin, and myricetin unexpectedly increased the ^.OH production in the Fenton system. The flavonoids tested now, except monohydroxy flavones, were more or less inhibitive to the superoxide anion (O₂) generation in the hypoxanthine-xanthine oxidase system. A great part of this inhibitory effect was likely owing to suppression of xanthine oxidase activity by the flavonoids. The flavonoids, which scavenged ^.OH or O₂⁻, were necessarily antioxidants to the peroxidation of methyl linoleate. However, there was a type of flavonoid such as morin, which have neither ^.OH nor O₂⁻ scavenging effect, but was a strong antioxidant.     

Comparative mutagenesis of plant flavonoids in microbial systems.

The plant flavonoids quercetin (3,5,7,3',4'-pentahydroxyflavone), morin (3,5,7,2',4'-pentahydroxyflavone), kaempferol (3,5,7,4'-tetrahydroxyflavone), chrysin (5,7-dihydroxyflavone), fisetin (3,7,3',4'-tetrahydroxyflavone), myricetin (3,5,7,3',4',5'-hexahydroxyflavone), myricitrin (myricetin-3-rhamnoside), hesperetin (3',5,7-trihydroxy-4'-methoxyflavanone), quercitrin (quercetin-3-L-rhamnoside), rutin (quercetin-3-rhamnosylglucoside or quercetin-3-rutinoside), and hesperidin (hesperetin-7-rutinoside) have been assayed for mutagenicity in the Salmonella/microsomal activation system. Quercetin, morin, kaempferol, fisetin, myricetin, quercitrin and rutin were mutagenic in the histidine reversion system with the frameshift strain TA98. The flavonols quercetin and myricetin are mutagenic without metabolic activation, although more effective when a rat liver microsomal preparation (S-9) is included; all others require metabolic activation. Flavonoids are common constituents of higher plants, with extensive medical uses. In addition to pure compounds, we have examined crude extracts of tobacco (snuff) and extracts from commonly available nutritional supplements containing rutin. Mutagenic activity can be detected and is correlated with the flavonoid content.     

Rutin-enhanced antibacterial activities of flavonoids against Bacillus cereus and Salmonella enteritidis

The antibacterial activities of flavonoids were found by the paper disk method to be enhanced by combining or mixing them. The combinations of quercetin and quercitrin, quercetin and morin, and quercetin and rutin were much more active than either flavonoid alone. Although rutin did not show activity in itself, the antibacterial activities of quercetin and morin were enhanced in the presence of rutin. The antibacterial activities of flavonoids, in combination with morin and rutin, were evaluated, based on the minimum inhibition concentration (MIC) in a liquid culture, by using Salmonella enteritidis and Bacillus cereus as the test bacteria. The activities of galangin, kaempherol, myricetin and fisetin were each enhanced in the presence of rutin when S. enteritidis was used as the test bacterium. The MIC value for kaempherol was markedly decreased by the addition of rutin. Morin inhibited DNA synthesis, and this effect was promoted by rutin at a concentration of 25 microg/ml.     

New potent antioxidative hydroxyflavanones produced with Aspergillus saitoi from flavanone glycoside in citrus fruit

Potent antioxidative hydroxyflavanones were produced with Aspergillus saitoi from hesperidin or naringin, which are flavanone glycosides in citrus fruit with weak antioxidative activity. The hydroxyflavanone produced from hesperidin was identified as 8-hydroxyhesperetin (8-HHE), a novel substance, and those from naringin were identified as carthamidin (6-hydroxynaringenin) and isocarthamidin (8-hydroxynaringenin) by FAB-MS, 1H-NMR and 13C-NMR analyses. The antioxidative activity of these hydroxyflavanones was examined by using the free radical-scavenging system of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the methyl linoleate oxidation system. The hydroxyflavanones (8-HHE, carthamidin, and isocarthamidin) exhibited stronger activity than the flavanone glycosides (hesperidin or naringin) and their aglycones (hesperetin or naringenin). The activity of 8-HHE and isocarthamidin was comparable to that of alpha-tocopherol, and that of carthamidin was weaker than that of isocarthamidin. The hydroxyflavanones, which were hydroxylated on A ring of flavanone by Aspergillus saitoi, were obtained as potent antioxidants.     

Inhibition of Ca-transport ATPase from synaptosomal vesicles by flavonoids

The inhibitory action of the flavonoid quercetin has been examined on the calcium-transport ATPase of synaptosomal vesicles and compared to that of two other flavonoids, morin and rutin. We have found that while quercetin caused a 50% inhibition of calcium transport at a concentration of 15 μM, morin and rutin had similar effects at concentrations of about 200 μM. A similar order of potency was observed also for ATP hydrolysis, though at higher concentrations. Quercetin also strongly inhibited phosphorylation of membrane proteins by ATP in synaptosomal vesicles. Rutin and morin had an almost negligible effect on membrane protein phosphorylation. The order of inhibitory potency of the flavonoids on the Ca²⁺-transport ATPase from synaptosomal vesicles: quercetin > morin > rutin, could be linked to their possible solubility in the membrane lipid phase since: (1) it paralleled their partitioning between a mixture of oil and water; (2) it paralleled their uptake from the reaction mixture by synaptosomal vesicles and phosphatidylcholine liposomes; (3) they had almost equal potency as inhibitors of the water soluble system of histone phosphorylation by protein kinase.     

Inhibition of Ca2+-transport ATPase from synaptosomal vesicles by flavonoids

The inhibitory action of the flavonoid quercetin has been examined on the calcium-transport ATPase of synaptosomal vesicles and compared to that of two other flavonoids, morin and rutin. We have found that while quercetin caused a 50% inhibition of calcium transport at a concentration of 15 μM, morin and rutin had similar effects at concentrations of about 200 μM. A similar order of potency was observed also for ATP hydrolysis, though at higher concentrations. Quercetin also strongly inhibited phosphorylation of membrane proteins by ATP in synaptosomal vesicles. Rutin and morin had an almost negligible effect on membrane protein phosphorylation. The order of inhibitory potency of the flavonoids on the Ca²⁺-transport ATPase from synaptosomal vesicles: quercetin > morin > rutin, could be linked to their possible solubility in the membrane lipid phase since: (1) it paralleled their partitioning between a mixture of oil and water; (2) it paralleled their uptake from the reaction mixture by synaptosomal vesicles and phosphatidylcholine liposomes; (3) they had almost equal potency as inhibitors of the water soluble system of histone phosphorylation by protein kinase.     

Superoxide scavenging by polyphenols: effect of conjugation and dimerization

The superoxide anion scavenging capacity of two flavonols (quercetin and kaempferol) and some of their conjugates (quercetin-3-rhamnoglucoside, quercetin-3-sophoroside, quercetin-3-sulphate, quercetin-3-glucuronide, kaempferol-3-sophoroside, kaempferol-3-glucuronide) and of several hydroxycinnamic acids (caffeic acid, ferulic acid, 5-5 diferulic acid, 8-O-4 diferulic acid and 8-8 diferulic acid) were studied. Superoxide anions were generated non-enzymatically in a phenazine methosulphate-NADH system and assayed by reduction of nitro-blue tetrazolium. Among the flavonols examined, the most effective scavengers of superoxide anions were the sophoroside, glucuronide and rhamnoglucoside conjugates. Conversely, quercetin-3-sulphate and the flavonol aglycones, exhibited some pro-oxidant activity at the range of concentrations tested (0.5-10 microM). These results show that conjugation has a marked effect on the scavenging capacity of flavonols and that the type of conjugate at the 3-position determines the final superoxide scavenging capacity. Caffeic acid and ferulic acid showed no effect on the generation of superoxide anions by phenazine methosulphate-NADH. However, dimerization of ferulic acid enhanced the superoxide scavenging capacity of this hydroxycinnamic acid, but this depended on the type of linkage between the monomers. The order, from highest to lowest, of superoxide radical scavenging capacity for the dimers of ferulic acid was: 5-5-diferulic acid > 8-O-4-diferulic acid > 8-8-diferulic acid.     

Anti- and pro-oxidative effects of flavonoids on metal-induced lipid hydroperoxide-dependent lipid peroxidation in cultured hepatocytes loaded with α-linolenic acid

Lipid hydroperoxide (LOOH)–dependent lipid peroxidation was induced in α-linolenic acid (LNA)-loaded hepatocytes by adding Fe, Cu, V, or Cd ions at concentrations from 20 to 500 μM. The effects of structurally related flavonoids at concentrations from 10 to 500 μM on the lipid peroxidation were examined. The results with regard to each flavonoid subclass are as follows: (i) Flavonols such as myricetin, quercetin, fisetin, and kaempferol, but not morin, showed dose-dependent antioxidative activity against metal-induced lipid peroxidation at all metal concentrations. Myricetin, quercetin, and fisetin were the most effective antioxidants, although their efficacies depended on the metal ion. Kaempferol and morin had antioxidative activity equal to the other flavonols in the presence of Cu ions, but were much less effective for the other three metal ions. (ii) Flavones, luteolin, apigenin, and chrysin were antioxidative at low Fe concentrations, but were pro-oxidative at high Fe concentrations. Luteolin exhibited antioxidative activity similar to that of catechol-containing flavonols in the presence of the other three metal ions. Apigenin and chrysin also acted as pro-oxidants with V or with all metal ions, respectively. (iii) Taxifolin, a flavanone, also showed both anti- and prooxidative activity, depending on Fe concentrations, but with other metal showed only antioxidative activity ions. (iv) Epigallocatechin, a flavanol, was antioxidative with all metal ions, and its activity was similar to that of catechol-containing flavonols. The various effects of flavonoids on metal-induced lipid peroxidation in LNA-loaded hepatocytes is discussed with regard to the change in redox potential of flavonoid–metal complexes.     

DNA adducts with antioxidant flavonoids: morin, apigenin, and naringin

Flavonoids have recently attracted a great interest as potential therapeutic drugs against a wide range of free-radical-mediated diseases. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. While the antioxidant activity of these natural polyphenolic compounds is well known, their bindings to DNA are not fully investigated. This study was designed to examine the interactions of morin (Mor), naringin (Nar), and apigenin (Api) with calf thymus DNA in aqueous solution at physiological conditions, using constant DNA concentration (6.25 mM) and various drug/DNA(phosphate) ratios of 1/40 to 1. FTIR and UV-Vis spectroscopic methods were used to determine the ligand binding modes, the binding constant, and the stability of DNA in flavonoid-DNA complexes in aqueous solution. Spectroscopic evidence shows both intercalation and external binding of flavonoids to DNA duplex with overall binding constants of K(morin) = 5.99 x 10(3) M(-1), K(apigenin) = 7.10 x 10(4) M(-1), and K(naringin) = 3.10 x 10(3) M(-1). The affinity of ligand-DNA binding is in the order of apigenin > morin > naringin. DNA aggregation and a partial B- to A-DNA transition occurs upon morin, apigenin, and naringin complexation.     

A New Proteolytic Enzyme from Achromobacter lyticus M497-1

Potent antioxidative hydroxyflavanones were produced with Aspergillus saitoi from hesperidin or naringin, which are flavanone glycosides in citrus fruit with weak antioxidative activity. The hydroxyflavanone produced from hesperidin was identified as 8-hydroxyhesperetin (8-HHE), a novel substance, and those from naringin were identified as carthamidin (6-hydroxynaringenin) and isocarthamidin (8-hydroxynaringenin) by FAB-MS, ¹H-NMR and ¹³C-NMR analyses. The antioxidative activity of these hydroxyflavanones was examined by using the free radical-scavenging system of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the methyl linoleate oxidation system. The hydroxyflavanones (8-HHE, carthamidin, and isocarthamidin) exhibited stronger activity than the flavanone glycosides (hesperidin or naringin) and their aglycones (hesperetin or naringenin). The activity of 8-HHE and isocarthamidin was comparable to that of α-tocopherol, and that of carthamidin was weaker than that of isocarthamidin. The hydroxyflavanones, which were hydroxylated on A ring of flavanone by Aspergillus saitoi, were obtained as potent antioxidants.     

Flavonoids: efficient protectors of glucose-6-phosphate dehydrogenase from ultrasonic cavitation-induced inactivation

Seven structurally diverse flavonoids have been shown to decrease glucose-6-phosphate dehydrogenase (G6PDH) inactivation in 0.1 M phosphate buffer (pH 7.4), induced by exposure to a high temperature (44 degrees C), or by a low-frequency ultrasound (27 kHz, 60 Wt/cm2). The activity of the compounds was assessed by their ability to change effective first-order rate constants characterizing the total (thermal and ultrasonic), thermal, and ultrasonic inactivation of 2.5 nM G6PDH (k(in), k(in)* [Russian characters: see text] kin(us), respectively). The value dependences of these constants on flavonoid concentrations (0.01-50 microM) were obtained. Rank order of potency exhibited by the compounds in protecting G6PDH appeared as follows: hesperidin > morin > silibin > naringin = quercetin > kampferol > astragalin. The data obtained confirm the crucial role of free radicals formed in the field of ultrasonic cavitation (HO* and O2*-) in G6PDH inactivation in solutions.     

Pro-oxidative properties of flavonoids in human lymphocytes

The pro-oxidative properties of the four flavonoids, quercetin, morin, naringenin and hesperetin, in human lymphocyte system were investigated. Naringenin and hesperetin accelerated the oxidation of deoxyribose induced by Fe(3+)/H(2)O(2) in a concentration range of 0-200 microM, but quercetin and morin decreased it when the concentration was greater than 100 microM. The generation of hydrogen peroxide and the superoxide anion and the production of TBARS in lymphocytes were increased with increasing concentration of a flavonoid. Cell membrane protein thiols of the lymphocytes decreased when treated with the four flavonoids. Quercetin and hesperetin had no significant effect (p>0.05) on the activity of glutathione reductase, but morin and naringenin could inhibit the activity of the enzyme at a concentration of 200 microM, when compared to the control group. The glutathione S-transferase activity was slightly decreased by treatment with each of the four flavonoids only at a concentration of 200 microM. Therefore, the DNA damage in lymphocytes induced by the flavonoids in the model system might have been due to their stimulation of oxidative stress in the lymphocytes, which resulted in the decrease of cell membrane protein thiols, increase of lipid peroxidation in cell membrane and in the influence of the antioxidative enzyme activities.     

Flavonoids suppresses the enhancing effect of beta-carotene on DNA damage induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A549 cells

This study investigated the individual and combined effects of beta-carotene with a common flavonoid (naringin, quercetin or rutin) on DNA damage induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-related carcinogen in human. A human lung cancer cell line, A549, was pre-incubated with beta-carotene, a flavonoid, or both for 1h followed by incubation with NNK for 4 h. Then, we determined DNA strand breaks and the level of 7-methylguanine (7-mGua), a product of NNK metabolism by cytochrome P450 (CYP). We showed that beta-carotene at 20 microM significantly enhanced NNK-induced DNA strand breaks and 7-mGua levels by 90% (p < 0.05) and 70% (p < 0.05), respectively, and that the effect of beta-carotene was associated with an increased metabolism of NNK by CYP because the concomitant addition of 1-aminobenzotriazole, a CYP inhibitor, with beta-carotene to cells strongly inhibited NNK-induced DNA strand breaks. In contrast to beta-carotene, incubation of cells with naringin, quercetin or rutin added at 23 microM led to significant inhibition of NNK-induced DNA strand breaks, and the effect was in the order of quercetin > naringin > rutin. However, these flavonoids did not significantly affect the level of 7-mGua induced by NNK. Co-incubation of beta-carotene with any of these flavonoids significantly inhibited the enhancing effect of beta-carotene on NNK-induced DNA strand breaks; the effects of flavonoids were dose-dependent and were also in the order of quercetin > naringin > rutin. Co-incubation of beta-carotene with any of these flavonoids also significantly inhibited the loss of beta-carotene incorporated into the cells, and the effects of the flavonoids were also in the order of quercetin > naringin > rutin. The protective effects of these flavonoids may be attributed to their antioxidant activities because they significantly decreased intracellular ROS, and the effects were also in the order of quercetin > naringin > rutin. These in vitro results suggest that a combination of beta-carotene with naringin, rutin, or quercetin may increase the safety of beta-carotene.     

Effects of flavonoids on nonenzymatic lipid peroxidation: structure-activity relationship

The in vitro effects of several flavonoids on nonenzymatic lipid peroxidation in the rat brain mitochondria was studied. The lipid peroxidation was indexed by measuring the MDA production using the 2-thiobarbituric acid TBA test. The flavonoids, apigenin, flavone, flavanone, hesperidin, naringin, and tangeretin promoted the ascorbic acid-induced lipid peroxidation, the extent of which depended upon the concentration of the flavonoid and ascorbic acid. The other flavonoids studied, viz., quercetin, quercetrin, rutin, taxifolin, myricetin, myricetrin, phloretin, phloridzin, diosmetin, diosmin, apiin, hesperetin, naringenin, (+)-catechin, morin, fisetin, chrysin, and 3-hydroxyflavone, all showed varying extents of inhibition of the nonenzymatic lipid peroxidation, induced by either ascorbic acid or ferrous sulfate. The flavonoid aglycones were more potent in their antiperoxidative action than their corresponding glycosides. Structure-activity analysis revealed that the flavonoid molecule with polyhydroxylated substitutions on rings A and B, a 2,3-double bond, a free 3-hydroxyl substitution and a 4-keto moiety, would confer upon the compound potent antiperoxidative properties.     

Fast atom bombardment and tandem mass spectrometry for structure determination of steroid and flavonoid glycosides

The combination of fast atom bombardment (FAB) and tandem mass spectrometry (MS-MS) was tested for its applicability to generate useful structural information for steroid and flavonoid glycosides. The following compounds were investigated: quercetin, myricitrin, apigetrin, fraxin, rutin, neohesperidin, hesperidin, naringin, apiin, cymarin, digoxin, digitoxin, xanthorhamnin, and frangulin. Upon FAB, the sample molecules are desorbed as (M + H)+, (M - H)-, or as (M + Na)+ or (M + K)+. Collisional activation of (M + H)+ or (M - H)- ions in the MS-MS experiment leads to sequential losses of glycoside moieties in a manner which permits the sequence of glycosides to be established. Some glycosides occur as mixtures of homologs. Proper interpretation of the MS-MS or collisional activation decomposition spectra often allows the homology to be located. In addition to the simple and highly selective fragmentations observed in this combined experiment, FAB and MS-MS also remove interference caused by the ubiquitous matrix ions which are desorbed by FAB.     

Differential effects of flavonoids on bovine kidney low molecular mass protein tyrosine phosphatase

Among the structurally related flavonoids tested on the bovine kidney low molecular weight protein tyrosine phosphatase (LMrPTP) activity, quercetin activated by about 2.6-fold the p-nitrophenyl-phosphate (p-NPP)-directed reaction, in contrast to morin that acted as a competitive inhibitor, with Ki values of 87, 73 and 50 μM for p-NPP, FMN, and tyrosine-phosphate, respectively. Other related flavonoids, such as rutin, kaempferol, catechin, narigin, phloretin and taxifolin did not significantly affect the LMrPTP activity.

The positions of the hydroxyl groups in the structures of the flavonoids were important for their distinct effects on LMrPTP activity. The hydroxyl groups at C3′ and C4′ and the presence of a double bond at C2 and C3 were essential for the activating effect of quercetin. The absence of the 3′-OH (kaempferol), absence of the double bond (taxifolin) and the presence of the sugar rutinose at the 3-OH (rutin) suppressed the effect of quercetin. The C2′- and C4′-hydroxyl groups, the presence of the double bond, and a C4-ketone group were important requirements for the inhibitory effects of morin.     

Inhibition of Ca2+-transport ATPase from synaptosomal vesicles by flavonoids

The inhibitory action of the flavonoid quercetin has been examined on the calcium-transport ATPase of synaptosomal vesicles and compared to that of two other flavonoids, morin and rutin. We have found that while quercetin caused a 50% inhibition of calcium transport at a concentration of 15 microM, morin and rutin had similar effects at concentrations of about 200 microM. A similar order of potency was observed also for ATP hydrolysis, though at higher concentrations. Quercetin also strongly inhibited phosphorylation of membrane proteins by ATP in synaptosomal vesicles. Rutin and morin had an almost negligible effect on membrane protein phosphorylation. The order of inhibitory potency of the flavonoids on the Ca2+-transport ATPase from synaptosomal vesicles: quercetin greater than morin greater than rutin, could be linked to their possible solubility in the membrane lipid phase since: (1) it paralleled their partitioning between a mixture of oil and water; (2) it paralleled their uptake from the reaction mixture by synaptosomal vesicles and phosphatidylcholine liposomes; (3) they had almost equal potency as inhibitors of the water soluble system of histone phosphorylation by protein kinase.     

DPPH radical scavengers from dried leaves of oregano (Origanum vulgare)

1,1-Dipehnyl-2-picrylhydrazyl (DPPH) radical scavenging activities were found in the extract of dried leaves of oregano (Origanum vulgare). The water-soluble active ingredients were isolated, and their structures were determined to be 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate and 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl 4-O-methylprotocatechuate by (1)H-, (13)C-NMR, DEPT, HMQC, and HMBC spectral analyses, and by NOE experiments. The DPPH radical scavenging activities of these compounds were compared with those of rutin, quercetin and rosmarinic acid at a concentration of 2 x 10(-5) M. The scavenging activity of 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate was almost the same as that of quercetin and rosmarinic acid, but that of 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybennzyl 4-O-methylprotocatechuate was less than that of quercetin, rosmarinic acid and 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate. The amount of 4'-O-beta-D-glucopyranosyl-3',4'-dihydroxybenzyl protocatechuate was estimated to be 3.8 mg/1 g of dried leaves by an HPLC analysis.     

Substituted aminophenols and flavonoids as potential components for test-systems for total antioxidant activity

Kinetics of ortho-phenylenediamine (PDA) oxidation has been investigated in the “pseudoperoxidase” system, Methemalbumin-H₂O₂, in the presence of 2-amino-4-tert-butylphenol (ATBP), 2-amino-4,6-ditert-butylphenol (ADTBP) and its four N-acyl derivates, as well as flavonoids (quercetin, morin, silibin, hesperidin and naringin). Under standard experimental conditions (20°C, phosphate buffered saline, pH 7.4, containing 5.25% ethanol and DMF) ATBP, ADTBP and two its N-acyl-derivatives and also all five flavonoids inhibited PDA oxidation with different efficiency, which was characterized in terms of the inhibition constants, K_i, M, or the percent of inhibition degree at the maximal concentrations of these inhibitors. Quercetin (K_i = 7.7 × 10^?5 M) and ATBP (K_i = 1.26 × 10^?4 M) were the most effective antioxidants. Using these characteristics and other required criteria, the pairs PDA-quercetin and PDA-ATBP were proposed for a practical application in the test-systems for evaluation of total antioxidant activity of biological objects.