SUPPRESSION OF LIPID PHOTOPEROXIDATION BY QUERCETIN AND ITS GLYCOSIDES IN SPINACH CHLOROPLASTS

Abstract— Quercetin, quercitrin and rutin suppressed lipid photoperoxidation in spinach chloroplasts in the presence of 100 μ M carbonylcyanide m -chlorophenylhydrazone (CCCP) or 100 μ M methyl viologen (MV). Fifty percent inhibition of lipid peroxidation by quercetin was observed between 30 and 50 μ M . Concentrations of quercetin and rutin higher than 100 μ M were required to obtain 50% inhibition. Ouercitrin was more effective than rutin in the suppression of lipid photoperoxidation.
Photooxidation of the flavonols by chloroplasts in the presence of MV was suppressed by superoxide dismutase (SOD) more than 90%, and the rates of the oxidation decreased in order of quercetin, quer citrin and rutin suggesting that the reactivity of the flavonols with O₂-decreased in that order. The photooxidation of the flavonols by CCCP-poisoned chloroplasts was partially suppressed by SOD. Radicals generated in the course of lauroyl peroxide degradation also oxidized the flavonols and the oxidation was insensitive to SOD. In these experiments, oxidation rate of quercetin was faster than those of its glycosides. The results obtained suggest that flavonols can function as antioxidants in chloroplasts by scavenging both O₂-and the radicals formed during lipid peroxidation.     

Flavonoids of the cotton plant and plants close to it

This review gives information on the distribution of flavonoids (flavonol and anthocyanin glycosides, catechins, and proanthocyanidins in plants of the family Malvaceae, namely the various species of the genera Gossypium, Hibiscus, Altheae, etc. It is shown that the flavonols are represented by glycosides of kaempferol, quercetin, myricetin, herbacetin. gossypetin, and hibiscetin, the most widespread being glycosides of quercetin and kaempferol. Characteristic for the majority of representatives of the families that were studied are flavonols having a hydroxy group in the C-8 position. Anthocyanins are represented by glycosides of cyanidin and of delphinidin. However, together with these, glycosides of methoxylated anthocyanins have also been detected — peonidin, petinidin, and malvidin. Sources of a food dye have been revealed among representatives of the family. Literature sources up to 1991 have been included.A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax 627071. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 3–19, January–February, 1994.     

Antioxidative effects of flavonols and their glycosides against the free-radical-induced peroxidation of linoleic acid in solution and in micelles

The antioxidative effect of flavonols and their glycosides against the peroxidation of linoleic acid has been studied in homogeneous solution (tBuOH/H(2)O, 3:2) and in sodium dodecyl sulfate and cetyl trimethylammonium bromide micelles. The peroxidation was initiated thermally by the water-soluble initiator 2,2'-azobis(2-methylpropionamidine) dihydrochloride, and the reaction kinetics were studied by monitoring the formation of linoleic acid hydroperoxides. The synergistic antioxidant effect of the flavonols with alpha-tocopherol (vitamin E) was also studied by following the decay kinetics of alpha-tocopherol and the alpha-tocopheroxyl radical. Kinetic analysis of the antioxidative process demonstrates that the flavonols are effective antioxidants in solution and in micelles, either alone or in combination with alpha-tocopherol. The antioxidative action involves trapping the initiating radicals in solution or in the bulk-water phase of the micelles, trapping the propagating lipid peroxyl radicals on the surface of the micelles, and regenerating alpha-tocopherol by reducing the alpha-tocopheroxyl radical. It was found that the antioxidant activity of the flavonols and their glycosides depends significantly on the position and number of the hydroxy groups, the oxidation potential of the molecule, and the reaction medium. The flavonols bearing ortho-dihydroxy groups possess significantly higher antioxidative activity than those without such functionalities, and the glycosides are less active than their parent aglycones. The activity of the flavonols is higher in micelles than in solution, while the activity of alpha-tocopherol is lower in micelles than in solution. This is because the predominant factor for controlling the activity is the hydrogen-bonding interaction of the antioxidant with the micellar surface in the case of hydrophilic flavonols, while it is the inter- and intramicellar diffusion in the case of lipophilic alpha-tocopherol.     

绿茶多酚对自由基诱导的红细胞膜过氧化的抑制作用

采用水溶性偶氮引发剂2,2'-偶氮二(2-脒基丙烷)二盐酸盐(AAPH)在37引发入血红细胞膜的过氧化,通过测定氧气吸收及维生素E的消耗研究了过氧化过程的动力学,并对从绿茶中提取的主要多酚类化合物的抗氧化活性做了定量研究。使用的绿茶多酚有:(-)-表儿茶素(EC),(-)-表儿茶素(EGC),(-)-表儿茶素酸酯(ECG)和(-)-表儿茶素培酸酯(EGCG)。结果表明,这些绿茶多酚能够显著缩短过氧化反应的动力学链长,有效地抑制红细胞膜的过氧化。抗氧化活性顺序为:EC〉GCG〉EGCG〉EGC。     

Comparison of Electrochemical Oxidation of Flavonols and Calculated Proton Affinity and Electron Transfer Enthalpy in Water

Flavonols, a class of flavonoids, are present in flowers, fruits and vegetables. They are jointly responsible for antioxidant activity as free radical acceptors. The redox behaviour of myricetin, quercetin, isorhamnetin, fisetin, morin and kaempferol is investigated using cyclic and differential pulse voltammetry. Quantum chemical calculations of proton affinities and electron transfer enthalpies were performed to identify possible reactive sites and radical species to compare them with measured oxidation potentials of the flavonols. Regarding to their chemical structure, these flavonols showed an oxidation order: myricetin > quercetin > isorhamnetin > fisetin > morin > kaempferol.     

Study of the reaction products of flavonols with 2,2-diphenyl-1-picrylhydrazyl using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry

The products obtained after the reaction between flavonols and the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH(*)) in both methanol and acetonitrile were characterized using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) and NMR spectroscopy. The flavonols studied were quercetin, kaempferol and myricetin. In methanol, two reaction products of oxidized quercetin were identified using LC/ESI-MS/MS and NMR. Quercetin was oxidized through a transfer of two H-atoms to DPPH(*) and subsequently incorporated either two CH(3)OH molecules or one CH(3)OH- and one H(2)O molecule giving the products 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-2,3-dimethoxy-2,3-dihydrochromen-4-one and 2-(3,4-dihydroxyphenyl)-3,3,5,7-tetrahydroxy-2-methoxy-2,3-dihydrochromen-4-one, respectively. LC/ESI-MS/MS analysis revealed that in methanol, kaempferol and myricetin also gave rise to methoxylated oxidation products similar to that identified for quercetin. Kaempferol, in addition, also exhibited products where a kaempferol radical, obtained by a transfer of one H-atom to DPPH(*), reacted with CH(3)OH through the addition of CH(3)O(*), yielding two isomeric products. When the reaction took place in acetonitrile, LC/ESI-MS/MS analysis showed that both quercetin and myricetin formed stable isomeric quinone products obtained by a transfer of two H-atoms to DPPH(*). In contrast, kaempferol formed two isomeric products where a kaempferol radical reacted with H(2)O through the addition of OH(*), i.e. similar to the reaction of kaempferol radicals with CH(3)OH.     

Inhibitory effects of flavonoids on free radical-induced hemolysis and their oxidative effects on hemoglobin

To investigate the effects of flavonoids on free radical-mediated biological membrane damage and the interaction of flavonoids with heme proteins, we studied the effects of quercetin, its glycosides (rutin and quercetin-3-O-glucoside), morin and (-)epicatechin on the hemolysis of the bovine erythrocytes induced by the hydrophilic free radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), and their interaction with hemoglobin. These flavonoids retarded the onset of the hemolysis dose-dependently. The effects of quercetin and other flavonoids were much greater than those of dihydric-phenols studied previously. The inhibitory effects of quercetin and its glycosides were stronger than those of morin and (-)epicatechin. In the absence of AAPH, the relatively hydrophobic flavonoids quercetin and morin induced the oxidation of oxyhemoglobin to methemoglobin. Oxidation by quercetin was especially marked. However, this oxidation did not induce hemolysis. These findings indicate that relatively hydrophobic flavonoids penetrate the cytoplasm of erythrocytes, interact with hemoglobin, and oxidize the heme iron.     

高效液相色谱法测定野葱中黄酮类化合物

建立了测定野葱中芦丁、黄酮醇类化合物的高效液相色谱方法。80%乙醇超声提取,高效液相色谱分析野葱中芦丁和黄酮醇类化合物的含量。芦丁检测条件:V(甲醇)∶V(0.2%磷酸水)=45∶55,检测波长:360nm;槲皮素、山奈酚、异鼠李素检测条件:V(甲醇)∶V(0.2%磷酸水)=40∶60,检测波长:360 nm。结果表明,野葱中芦丁含量0.22%,槲皮素、山奈酚、异鼠李素含量依次为0.42%、0、0.23%,总黄酮醇类化合物含量为1.63%。     

Kaempferol glycosides in the flowers of carnation and their contribution to the creamy white flower color

Three flavonol glycosides were isolated from the flowers of carnation cultivars 'White Wink' and 'Honey Moon'. They were identified from their UV, MS, 1H and 13C NMR spectra as kaempferol 3-O-neohesperidoside, kaempferol 3-O-sophoroside and kaempferol 3-O-glucosyl-(1 --> 2)-[rhamnosyl-(1 --> 6)-glucoside]. Referring to previous reports, flavonols occurring in carnation flowers are characterized as kaempferol 3-O-glucosides with additional sugars binding at the 2 and/or 6-positions of the glucose. The kaempferol glycoside contents of a nearly pure white flower and some creamy white flower lines were compared. Although the major glycoside was different in each line, the total kaempferol contents of the creamy white lines were from 5.9 to 20.9 times higher than the pure white line. Thus, in carnations, kaempferol glycosides surely contribute to the creamy tone of white flowers.     

Key role of chemical hardness to compare 2,2-diphenyl-1-picrylhydrazyl radical scavenging power of flavone and flavonol O-glycoside and C-glycoside derivatives

The antioxidant activities of flavonoids and their glycosides were measured with the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH radical, DPPH(·)) scavenging method. The results show that free hydroxyl flavonoids are not necessarily more active than O-glycoside. Quercetin and kaempferol showed higher activity than apigenin. The C- and O-glycosides of flavonoids generally showed higher radical scavenging activity than aglycones; however, kaempferol C3-O-glycoside (astragalin) showed higher activity than kaempferol. In the radical scavenging activity of flavonoids, it was expected that OH substitutions at C3 and C5 and catechol substitution at C2 of B ring and intramolecular hydrogen bonding between OH at C5 and ketone at C3 would increase the activity; however, the reasons have yet to be clarified. We here show that the radical scavenging activities of flavonoids are controlled by their absolute hardness (η) and absolute electronegativity (χ) as a electronic state. Kaempferol and quercetin provide high radical scavenging activity since (i) OH substitutions at C3 and C5 strikingly decrease η of flavones, (ii) OH substitutions at C3 and C7 decrease χ and η of flavones, and (iii) phenol or o-catechol substitution at C2 of B ring decrease χ of flavones. The coordinate r(χ,?η) as the electron state must be small to increase the radical scavenging activity of flavonoids. The results show that chemically soft kaempferol and quercetin have higher DPPH radical scavenging activity than chemically hard genistein and daidzein.     

The uptake behaviors of kaempferol and quercetin through rat primary cultured cortical neurons

The objective of this study was to investigate whether kaempferol and quercetin could be transported into primary cultured cerebral neurons, to establish a practical HPLC method with UV detection for the two flavonols in the neurons, and to study the uptake and transport behaviors of them through the neurons. The present results showed that the level of kaempferol in the neurons increased linearly and then reached a plateau with incubation time at the high concentration of 10 microg/mL, but not at the other two concentrations of 1 and 0.1 microg/mL. However, the levels of quercetin in the neurons were not detected at the three incubating concentrations, and there was a new peak detected in the cell whose retention time was shorter (3.42 min) than that of quercetin (4.65 min). This phenomenon suggested that quercetin might be transported into the neurons and then metabolized quickly to some derivative. Kaempferol could be transported into the neurons in a concentration- and time-dependent manner when the neurons were incubated with the culture medium containing kaempferol at the high dose. There was an apparent correlation between the concentrations of kaempferol in the medium and in the cell, indicating that the uptake of kaempferol in the cell increased along with its dose (10 microg/mL). However, there was a negative correlation between the concentrations of quercetin in the medium and in the cell. The results suggested that kaempferol and quercetin were disposed by the neurons at different way, and this might be an important factor for their different effects on primary cultured cortical cells.     

Antioxidant secondary metabolites from Geranium lasiopus Boiss. & Heldr

Chromatographic studies on the EtOAc soluble portion of the MeOH extract of Geranium lasiopus led to the isolation of eight flavonoids (kaempferol (1), quercetin (2), quercetin 3-O-β-glucopyranoside (3), quercetin 3-O-β-galactopyranoside (4), kaempferol 3-O-α-rhamnopyranosyl-(1?→?6)-β-glucopyranoside (5), quercetin 3-O-α-rhamnopyranosyl-(1?→?6)-β-glucopyranoside (6), kaempferol 3-O-α-rhamnopyranosyl-(1?→?2)-β-glucopyranoside (7) and quercetin 3-O-α-rhamnopyranosyl-(1?→?2)-β-glucopyranoside (8)), two simple phenolic compounds (gallic acid (9) and its methyl ester (10)) and a hydrolysable tannin (pusilagin (11)). The structures of the compounds were elucidated by 1- and 2-dimensional NMR techniques ((1)H, (13)C, COSY, HMBC, HMQC) and ESI-TOF-MS spectrometry. Inhibitory effects on H(2)O(2)-induced lipid peroxidation in human red blood cells of the different extracts of G. lasiopus, as well as isolated compounds, were investigated. All tested compounds showed comparable or higher activity than that of ascorbic acid and trolox.     

Simultaneous determination of quercetin, kaempferol, and isorhamnetin in phytopharmaceuticals of Hippophae rhamnoides L. by high-performance liquid chromatography with chemiluminescence detection

A novel method based on reversed-phase high-performance liquid chromatography with chemiluminescence detection has been developed for the simultaneous determination of three flavonols including quercetin, kaempferol, and isorhamnetin. The procedure was based on the chemiluminescent enhancement by flavonols of the cerium(IV)-rhodamine 6G system in sulfuric acid medium. The effects of several parameters on the HPLC resolution and CL emission were studied systematically. Good separation was achieved with isocratic elution using a mixture of methanol and aqueous 1.0% acetic acid (37:63, v/v) within 25 min. Under optimized conditions, the linear working range covers 3 orders of magnitude with relative standard deviations below 4.5% for 11 replicate injected flavonol samples, and detection limits (S/N= 3) were 1.6 x 10(-8), 3.5 x 10(-9), and 6.5 x 10(-9) g mL(-1) for quercetin, kaempferol, and isorhamnetin, respectively. The chemiluminescence reaction was compatible with the mobile phase of high-performance liquid chromatography. The proposed method has been successfully applied to the determination of three active flavonols in phytopharmaceuticals of Hippophae rhamnoides L. After a simple extraction procedure, the repeatability and recovery were satisfactory.     

Liquid chromatography–tandem mass spectrometry reveals the widespread occurrence of flavonoid glycosides in honey,and their potential as floral origin markers

HPLC-MS–MS analysis of unifloral honey extracts has shown the occurrence of flavonoid glycosides in most of the analyzed samples. These compounds are not present in large amounts, but can reach up to 600 μg/100 g honey in canola and rapeseed honeys. Rhamnosyl-hexosides (tentatively rutinosides and neohesperidosides) and dihexosides (hexosyl(1→2)hexosides and hexosyl(1→6)hexosides) of flavonols such as quercetin, kaempferol, isorhamnetin and 8-methoxykaempferol, are the main flavonoid glycosides found in honey. However, flavonoid triglycosides and monoglycosides are also detected in some floral origins. Eucalyptus and orange blossom nectars were collected and analyzed showing that nectar flavonoid glucosides, as is the case of eucalyptus flavonoids, can be readily hydrolyzed by the bee saliva enzymes, while flavonoid rhamnosyl-glucosides, as is the case of citrus nectar flavonoids, are not hydrolyzed, and because of these reasons the flavonoid glycoside content of citrus honey is higher than that of eucalyptus honey that contains mainly aglycones. The flavonoid glycoside profiles detected in honeys suggest that this could be related to their floral origin and the results show that the HPLC-MSn ion trap analysis of flavonoid glycosides in honey is a promising analytical method to help in the objective determination of the floral origin of unifloral honeys.     

Identification of flavonoids and their glycosides by high-performance liquid chromatography with electrospray ionization mass spectrometry and with diode array ultraviolet detection

Identification of flavonoids and flavonoid glycosides was carried out on Psidium guajava Linn leaves by means of high-performance liquid chromatography ultraviolet (HPLC-UV) analysis and HPLC mass spectrometry. By using HPLC-UV, two known phenolics (gallic acid and quercetin) and five newly reported ones (procatechuic acid, chlorogenic acid, caffeic acid, kaempferol and ferulic acid) were identified in alcohol guava leaf extract. Structural information about the compounds was obtained from the retention times, the UV spectra and mass spectra without the need to isolate the individual compounds. Two flavonoids (quercetin and kaempferol) and four flavonoid glycosides (three known components, quercetin 3-O-alpha-L-arabinoside, quercetin 3-O-beta-D-glucoside and quercetin 3-O-beta-D-galactoside, along with one novel compound, kaempferol-glycoside) and three other unknown compounds have been identified in the fractions.     

Involvement of Photoprotective Compounds of a Phenolic Nature in the Response of Arabidopsis Thaliana Leaf Tissues to Low-Intensity Laser Radiation

The influence of low-intensity laser radiation (LILR) on the changes in the content of anthocyanins, kaempferol, quercetin and their glycosides in the leaves of 5-week-old plants of Arabidopsis thaliana L. was studied by means of methods of high-performance liquid chromatography and gas chromatography mass spectrometry (GC-MS). It was found that in the leaves subjected to a stimulating He-Ne laser radiation dose (3.6 J cm⁻², continuous wave radiation, wavelength—632.8 nm, exposure time—5 min), the radiation induced an increase in the content of such compounds, the most significant one being in the case of anthocyanins (9 times). The present study also revealed an increase in the antioxidant potential of kaempferol, quercetin and their glycosides as a result of laser exposure. This increase was due to the preferential synthesis of compounds with a larger number of OH-groups on the phenyl ring. Thus, the content of quercetin, which has five OH-groups in its structure, increased almost by three times as compared to the control.     

Study on the interactions of kaempferol and quercetin with intravenous immunoglobulin by fluorescence quenching, fourier transformation infrared spectroscopy and circular dichroism spectroscopy

The interactions of kaempferol and quercetin with intravenous immunoglobulin (IVIG) were studied in vitro by spectroscopic methods including fluorescence spectra, Fourier transformation infrared (FT-IR) spectra and circular dichroism (CD) spectra. The binding parameters for the reactions calculated according to the Sips equation suggested that the bindings of IVIG to kaempferol and quercetin were characterized by two binding sites with the average affinity constants K(o) at 1.032 x 10(4) M(-1) and 1.849 x 10(4) M(-1), respectively. The binding of IVIG with quercetin is stronger than that of IVIG with kaempferol. They were of non-specific and weak drug-protein interactions. Docking was used to calculate the interaction modes between kaempferol and quercetin with IVIG. The secondary structural compositions of free IVIG and its kaempferol, quercetin complexes were calculated by the FT-IR difference spectra, self-deconvolution, second derivative resolution enhancement and the curve-fitting procedures of amide I band respectively, which are in good agreement with the analyses of CD spectra. The effect of 3'-OH substituent in quercetin is distinct between the interactions of IVIG with kaempferol and quercetin for the secondary structure of the protein. The observed spectral changes indicate a partial unfolding of the protein structure, but the typical beta structural conformation of IVIG is still retentive in the presence of both drugs in aqueous solution. The average binding distances between the chromophores of IVIG with kaempferol (4.30 nm) and quercetin (4.35 nm) were obtained on the basis of the theory of F?rster energy transfer. IVIG can serve as transport protein (carrier) for kaempferol and quercetin.     

Polyphenolic profile characterization of Agrimonia eupatoria L. by HPLC with different detection devices

Liquid chromatography coupled to diode array and electrospray ionization mass spectrometry detection was used to establish the polyphenolic profile of an ethyl acetate fraction from Agrimonia eupatoria L. aqueous-alcoholic extract. Additionally, an HPLC technique with post-column derivatization by p-dimethylaminocinnamaldehyde was employed for the selective detection and quantification of flavan-3-ols. Important information was obtained by combining the data of these two HPLC techniques. Flavan-3-ols (catechin and procyanidins B1, B2, B3, B6, B7, C1, C2 and epicatechin-epicatechin-catechin), quercetin 3-O-glucoside, quercetin 3-O-galactoside, kaempferol 3-O-glucoside, kaempferol 3-O-(6'-O-p-coumaroyl)-glucoside, apigenin 6-C-glucoside and various phenolic acids were identified. Antioxidant activity of the Agrimonia eupatoria L. fraction containing these compounds was assessed through the 1,1-diphenyl-2-picrylhydrazyl, trolox equivalent antioxidant capacity and thiobarbituric acid reactive substances methods. Significant activity was observed for this fraction, where compounds with recognized antiinflammatory properties such as procyanidins, kaempferol 3-O-(6'-O-p-coumaroyl)-glucoside and quercetin glycosides were identified for the first time. These results are predictive of the beneficial effects of this fraction, or some of its compounds, in human health, as possible anti-inflammatory drug.     

BILIRUBIN-PHOTOSENSITIZED LYSIS OF RESEALED ERYTHROCYTE MEMBRANES*

Abstract— The ability of bilirubin to photosensitize biomembrane damage has been tested, using resealed human erythrocyte ghosts as a model system. Continuous irradiation of bilirubin-charged ghosts with broad-band blue light resulted in membrane lysis, as evidenced by the release of trapped markers such as Na⁺ or glucose-6-phosphate. Sodium dodecyl sulfate-gel electrophoresis of membrane proteins revealed a progressive and relatively rapid loss of spectrin (bands 1 and 2) and band 4.1 during irradiation, accompanied by the appearance of cross-linked polypeptides. The antioxidant butylated hydroxytoluene protected ghosts against lysis, but not against protein cross-linking, suggesting that lipid peroxidation is the crucial membrane disrupting event.     

Antiradical and cytotoxic activity of different Helichrysum plicatum flower extracts

Flowers of Helichrysum plicatum were extracted under different experimental conditions, and their antioxidant activity was determined by DPPH radical scavenging assay. Extracts obtained with higher concentration of ethyl acetate (90% or 100%) were found to contain the greatest amount of total phenolics (> 250 mg gallic acid equivalents/g of dried extract), and high correlation between total phenolic content and antiradical activity was observed (r = -0.79). Based on the total phenolic content and antiradical activity, some extracts were selected for investigation of cytotoxic activity toward PC3, HeLa and K562 human cancer cell lines in vitro. All tested extracts exhibited moderate activity against HeLa cells (41.9-42.1 microg/mL), whereas the extract obtained with 100% ethyl acetate was the most active against K562 and PC3 cell lines (25.9 and 39.2 microg/mL, respectively). Statistical analysis revealed significant correlation between total phenolic content and cytotoxic activity against PC3 and K562 cells. HPLC identification of phenolic compounds from the extracts indicated the presence of apigenin, naringenin and kaempferol as free aglycones, and glycosides of apigenin, naringenin, quercetin and kaempferol. Among aglycones, kaempferol displayed moderate cytostatic activity against all cell lines (24.8-64.7 microM).