Theoretical investigation of the effect of sugar substitution on the antioxidant properties of flavonoids

Natural flavonoids are secondary phenolic plant metabolites known for their bioactivity as antioxidants. The evaluation of this property is generally done by the estimation of their direct free radical-scavenging activity as hydrogen or electron donating compounds. This paper reviews experimental results available in the literature for a selection of flavonoids and compares them with calculated quantities characteristic of the hydrogen or electron donation. For that purpose, bond dissociation energies, ionization potentials and electron transfer enthalpies are computed by using DFT methods and the ONIOM procedure implemented in the ab initio program Gaussian. This process has been chosen because it can be extended to the study of large molecules. When acid dissociation and interaction with the solvent are taken into account, the results present very good concordance with experimental results, enlightening the complexity of the processes involved in the classical assays which measure the ability of compounds to scavenge the (2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt) radical cation (ABTS ⁺) or the 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH^·). This study demonstrates the good accuracy of theoretical calculations in obtaining the relative energies involved in free radical scavenging abilities and its capacity for predictive behaviour. It also highlights the necessity to take into account the pK_a of the compounds and the solvent interaction. The ability of the method to calculate the antioxidant properties of larger molecules are tested on glycosylated flavonoids and the effects of sugar substitution on the antioxidant properties of flavonoids are investigated, pointing out the importance of the charges on the oxygen atoms.     

The basic antioxidant structure for flavonoid derivatives

An antioxidant structure-activity study is carried out in this work with ten flavonoid compounds using quantum chemistry calculations with the functional of density theory method. According to the geometry obtained by using the B3LYP/6-31G(d) method, the HOMO, ionization potential, stabilization energies, and spin density distribution showed that the flavonol is the more antioxidant nucleus. The spin density contribution is determinant for the stability of the free radical. The number of resonance structures is related to the π-type electron system. 3-hydroxyflavone is the basic antioxidant structure for the simplified flavonoids studied here. The electron abstraction is more favored in the molecules where ether group and 3-hydroxyl are present, nonetheless 2,3-double bond and carbonyl moiety are facultative.     

Antioxidant activity of some polyphenol constituents of the medicinal plant Phyllanthus amarus Linn

Phyllanthus amarus Linn is a widely distributed tropical medicinal plant highly valued for its therapeutic properties. The antioxidant activity of some of its principal constituents, namely amariin, 1-galloyl-2,3-dehydrohexahydroxydiphenyl (DHHDP)-glucose, repandusinic acid, geraniin, corilagin, phyllanthusiin D, rutin and quercetin 3-O-glucoside were examined for their ability to scavenge free radicals in a range of systems including 2,2-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS)/ferrylmyoglobin, ferric reducing antioxidant power (FRAP) and pulse radiolysis. In addition, their ability to protect rat liver mitochondria against oxidative damage was determined by measuring the ROO* radical induced damage to proteins and lipids and *OH radical induced damage to plasmid DNA. The compounds showed significant antioxidant activities with differing efficacy depending on the assays employed. Amariin, repandusinic acid and phyllanthusiin D showed higher antioxidant activity among the ellagitannins and were comparable to the flavonoids, rutin and quercetin 3-O-glucoside.     

Chlorine atom substitution influences radical scavenging activity of 6-chromanol

Synthetic 6-chromanol derivatives were prepared with several chlorine substitutions, which conferred both electron-withdrawing inductive effects and electron-donating resonance effects. A trichlorinated compound (2), a dichlorinated compound (3), and three monochlorinated compounds (4, 5, and 6) were synthesized; compounds 2, 3, and 6 were novel. The antioxidant activities of the compounds, evaluated in terms of their capacities to scavenge galvinoxyl radical, were associated with the number and positioning of chlorine atoms in the aromatic ring of 6-chromanol. The activity of compound 1 (2,2-dimethyl-6-chromanol) was slightly higher than the activities of compounds 2 (2,2-dimethyl-5,7-dichloro-6-chromanol) or 3 (2,2-dimethyl-5,7,8-trichloro-6-chromanol), in which the chlorine atoms were ortho to the phenolic hydroxyl group of 6-chromanol. The scavenging activity of compound 3 was slightly higher than that of 2, which contained an additional chlorine substituted in the 8 position. The activities of polychlorinated compounds 2 and 3 were higher than the activities of any of the monochlorinated compounds (4-6). Compound 6, in which a chlorine was substituted in the 8 position, exhibited the lowest activity. Substitution of a chlorine atom meta to the hydroxyl group of 6-chromanol (compounds 2 and 6) decreased galvinoxyl radical scavenging activity, owing to the electron-withdrawing inductive effect of chlorine. Positioning the chloro group ortho to the hydroxyl group (compounds 4 and 5) retained antioxidant activity because the intermediate radical was stabilized by the electron-donating resonance effect of chlorine in spite of the electron-withdrawing inductive effect of chlorine. Antioxidant activities of the synthesized compounds were evaluated for correlations with the O-H bond dissociation energies (BDEs) and the ionization potentials. The BDEs correlated with the second-order rate constants (k) in the reaction between galvinoxyl radical and the chlorinated 6-chromanol derivatives in acetonitrile. This indicated that the antioxidant mechanism of the synthesized compounds consisted of a one-step hydrogen atom transfer from the phenolic OH group rather than an electron transfer followed by a proton transfer. The synthesized compounds also exhibited hydroxyl radical scavenging capacities in aqueous solution.     

Antioxidant activity of some polyphenol constituents of the medicinal plant Phyllanthus amarus Linn

Abstract

Phyllanthus amarus Linn is a widely distributed tropical medicinal plant highly valued for its therapeutic properties. The antioxidant activity of some of its principal constituents, namely amariin, 1-galloyl-2,3-dehydrohexahydroxydiphenyl (DHHDP)-glucose, repandusinic acid, geraniin, corilagin, phyllanthusiin D, rutin and quercetin 3-O-glucoside were examined for their ability to scavenge free radicals in a range of systems including 2,2-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS)/ferrylmyoglobin, ferric reducing antioxidant power (FRAP) and pulse radiolysis. In addition, their ability to protect rat liver mitochondria against oxidative damage was determined by measuring the ROO? radical induced damage to proteins and lipids and ?OH radical induced damage to plasmid DNA. The compounds showed significant antioxidant activities with differing efficacy depending on the assays employed. Amariin, repandusinic acid and phyllanthusiin D showed higher antioxidant activity among the ellagitannins and were comparable to the flavonoids, rutin and quercetin 3-O-glucoside.     

The influence of pH on antioxidant properties and the mechanism of antioxidant action of hydroxyflavones

The effect of the pH on antioxidant properties of a series of hydroxyflavones was investigated. The pKa of the individual hydroxyl moieties in the hydroxyflavones was compared to computer-calculated deprotonation energies. This resulted in a quantitative structure activity relationship (QSAR), which enables the estimation of pKa values of individual hydroxyl moieties, also in hydroxyflavones for which these pKa values are not available. Comparison of the pKa values to the pH-dependent antioxidant profiles, determined by the TEAC assay, reveals that for various hydroxyflavones the pH-dependent behavior is related to hydroxyl moiety deprotonation, resulting in an increase of the antioxidant potential upon formation of the deprotonated forms. Comparison of these experimental results to computer calculated O-H bond dissociation energies (BDE) and ionization potentials (IP) of the nondeprotonated and the deprotonated forms of the various hydroxyflavones indicates that especially the parameter reflecting the ease of electron donation, i.e., the IP, and not the BDE, is greatly influenced by the deprotonation. Based on these results it is concluded that upon deprotonation the TEAC value increases (radical scavenging capacity increases) because electron-, not H*-, donation becomes easier. Taking into account that the mechanism of radical scavenging antioxidant activity of the neutral form of the hydroxyflavones is generally considered to be hydrogen atom donation, this implies than not only the ease of radical scavenging, but also the mechanism of antioxidant action changes upon hydroxyflavone deprotonation.     

Synthesis and in vitro antioxidant activity study of some new piperazinyl flavone compounds

Flavones exhibit a variety of beneficial effects and are well known for their medicinal importance in several diseases, including cardiovascular, neurodegenerative and cancer. The inclusion of the piperazine ring to the flavone backbone is an important strategy in drug discovery but only a few studies have synthesized piperazinyl flavone compounds to test their biological activity. While there is a major focus on the antioxidant properties of drugs in therapy of several diseases of inflammatory origin, we synthesized a series of the novel piperazinyl flavone analogues bearing the phenyl ring with different substituents. The analogues were evaluated for in vitro antioxidant activity against superoxide anion radical, hydroxyl radical, 2,2‐diphenyl‐1‐picrylhydrazyl radical, and hydrogen peroxide scavenging properties. The total antioxidant status based on the absorbance of the 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) radical cation (ABTS^+?) and total antioxidant capacity using the Fe(III)‐ferrozine complex were also monitored. The results of the above studies showed that the compounds synthesized were found possessed moderate radical scavenging potential, and that their interaction with reactive oxygen species is complex and depends on their structural conformation and the type of substituent R in the piperazine ring being attached. Best antiradical activity were found for the compounds with methoxy groups on the phenyl ring of substituent R, whereas the presence of methoxy or trifluoromethyl groups in substituent R resulted in higher ABTS^+? and ion Fe(III) reduction. These compounds are promising molecules to be used for their antioxidant properties and may be regarded, after improvement of the antioxidant potential, to control diseases of free radical etiology.     

Solvent effects are important in elucidating radical-scavenging mechanisms of antioxidants. A case study on genistein

In two recent researches on antioxidant mechanisms of flavonoids and isoflavonoids, quantum chemical method was employed to calculate the proton dissociation energies, the bond dissociation energies and the ionization potentials for the phenols and derived radicals to help determine the radical-scavenging mechanisms. As the solvent effect was left out of consideration, the conclusion drawn from the calculation incurred some controversies. In the current study, we re-calculated the parameters for genistein and its anion by employing a B3LYP/6-311+G(d,p)//6-31G(d,p) method with solvent effect. Accordingly, a more reasonable explanation on the experimentally observed behavior of genistein as a radical scavenger was obtained. Therefore, solvent effect should be considered in the investigation of radical-scavenging mechanisms of antioxidants in polar solvents.     

Quantification of polyphenolic antioxidants and free radical scavengers in marine algae

While the health benefits of antioxidant compounds from terrestrial plants are widely accepted in Western counties, there is less recognition of the health benefits of marine algal antioxidant compounds. Oceans are an abundant source of biomaterials, with many natural antioxidants derived from marine algae being investigated as potential anti-aging, anti-inflammatory, anti-bacterial, anti-fungal, cytotoxic, anti-malarial, anti-proliferative, and anti-cancer agents. The aim of this work was to quantify and compare polyphenolic content and free radical scavenging activity of algal extracts using normal phase and reverse phase thin layer chromatography. Post-chromatographic derivatization with neutral ferric chloride (FeCl₃) solution and with 2,2-diphenyl-1-picrylhydrazyl (DPPH·) free radical were used to assess total polyphenolic content and free radical scavenging activities in algal samples. Total phenolic content quantified on normal phase plates was correlated to phenolic content established on reverse phase plates. Similarly, free radical scavenging activity established on normal phase and reverse phase plates were in good agreement. However, although free radical scavenging activities determined on normal phase plates were highly correlated with polyphenolic content, this correlation was low for reverse phase plates. Lipophilic reversed phase TLC plates do not effectively separate mixtures of highly polar compounds like flavonoids, phenolic compounds and their glucosides. Thus, although reversed phase plates are recommended for assessment of free radical scavengers, as they do not influence the free radical-antioxidant reaction, they may not provide the best separation of polar phenolic compounds, especially flavonoids, and therefore may not accurately quantify polyphenolic content and free radical scavenging potential.     

Solvent Effects are Important in Elucidating Radical-Scavenging Mechanisms of Antioxidants. A Case Study on Genistein

Abstract

In two recent researches on antioxidant mechanisms of flavonoids and isoflavonoids, quantum chemical method was employed to calculate the proton dissociation energies, the bond dissociation energies and the ionization potentials for the phenols and derived radicals to help determine the radical-scavenging mechanisms. As the solvent effect was left out of consideration, the conclusion drawn from the calculation incurred some controversies. In the current study, we re-calculated the parameters for genistein and its anion by employing a B3LYP/6–311+G(d,p)//6–31G(d,p) method with solvent effect. Accordingly, a more reasonable explanation on the experimentally observed behavior of genistein as a radical scavenger was obtained. Therefore, solvent effect should be considered in the investigation of radical-scavenging mechanisms of antioxidants in polar solvents.     

DFT-Based Quantum Chemical Studies on Conformational, Electronic and Antioxidant Properties of Isobavachalcone and 4-Hydroxyderricin

Isobavachalcone and 4-hydroxyderricin which exhibit numerous biological activities are two major chalcone constituents isolated from the roots of Angelica keiskei KOIDZUMI. The conformational and antioxidant activity properties have been investigated by quantum chemical calculations based on the density functional theory, with the aim of verifying dominant antioxidant mechanisms. Three parameters of the O-H bond dissociation enthalpy (BDE), ionization potential (IP) and acidity in the presence of an implicit solvent for methanol are computed to estimate the antioxidant capacities. Results reveal that the order of antioxidant efficacies predicted by BDE and IP, different from that predicted by acidity, is in agreement with that obtained by experimental data. This demonstrates the importance of the hydrogen atom transfer and single electron transfer mechanisms to explain their capacities to scavenge 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical.     

Theoretical investigation of the formation of a new series of antioxidant depsides from the radiolysis of flavonoid compounds

This paper deals with the formation of a series of antioxidant depsides obtained from flavonoid solutions irradiated with gamma rays. These reactions take place in radiolyzed alcohol solutions, a medium that is very rich in many different highly reactive species and that hosts specific reactions. We focus on the first step of those reactions, i.e., reactivity of the solute (flavonoid) with the alkoxy radicals CH(3)O(*) and CH(3)CH(2)O(*) formed in methanol and ethanol, respectively, and their carbon-centered isomers: the 1-hydroxy-methyl ((*)CH(2)OH) and the 1-hydroxy-ethyl (CH(3)(*)CHOH) radicals. Among the different flavonoid groups of molecules, only flavonols are transformed. To establish the structure-reactivity relationship that explains why the radiolytic transformation occurs only for those compounds, the process is rationalized theoretically, with Density Functional Theory calculations, taking into account the solvent effects by a Polarizable Continuum Model and a microhydrated environment (one or two water molecules surrounding the active center). The first redox reaction, occurring between the flavonol and the reactive species formed upon irradiation of the solvent, is studied in terms of (1) the O-H bond dissociation enthalpy of each OH group of the flavonoids and (2) electron abstraction from the molecule. We conclude that the reaction, initiated preferentially by the alkoxy radicals, first occurs at the 3-OH group of the flavonol. It is then followed by the formation of a peroxyl radical (after molecular oxygen or superoxide addition). The different cascades of reactions, which lead to the formation of depsides via C-ring opening, are discussed on the basis of the corresponding calculated energetic schemes.     

Antioxidant actions of flavonoids: Thermodynamic and kinetic analysis

The benefits of flavonoids on human health are very often ascribed to their potential ability to act diminishing free radical steady state concentration in biological systems providing antioxidant protection. This is an assumption based on the chemical structures of flavonoids that support their capacity to scavenge free radicals and chelate redox-active metals. In this paper we will use thermodynamic and kinetic approaches to analyze the interactions of flavonoids with biological material and from there, extrapolate the physiological relevance of their antioxidant actions. Thermodynamic analysis predicts that both, scavenging of oxygen-derived radicals and the sequestration of redox-active metals are energetically favored. Nevertheless, the actual concentrations reached by flavonoids in most animal and human tissues following dietary ingestion are incompatible with the kinetic requirements necessary to reach reaction rates of physiological relevance. This incompatibility becomes evident when compared to other antioxidant compounds, e.g. α-tocopherol (vitamin E), ascorbate (vitamin C), and glutathione. Alternatively, lipid-flavonoid and protein-flavonoid interactions can indirectly mediate a decrease in oxidant (free radical) production and/or oxidative damage to both cell and extracellular components. The final mechanisms mediating the antioxidant actions of flavonoid will be determined by their actual concentration in the tissue under consideration.     

Simultaneous detection of the antioxidant and pro-oxidant activity of dietary polyphenolics in a peroxidase system

The ability to reduce the peroxidase (myeloglobin/H₂O₂)-generated ABTS_•+ [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) radical cation] has been used to rank the antioxidant activity of various agents including dietary flavonoids and chalcones. Surprisingly, we found that in the presence of catalytic concentrations of the phenol B-ring containing flavonoids, apigenin, naringenin and the chalcone phloretin, the formation of the ABTS_•+ was initially increased. The enhanced formation of the ABTS_•+ was attributed to the peroxidase/H₂O₂ mediated generation of polyphenolic phenoxyl radicals that were able to co-oxidize ABTS. The relative ABTS_•+ generating ability of these dietary polyphenolics correlated with their ability to co-oxidize NADH to the NAD* radical with the resultant generation of superoxide. This pro-oxidant activity was not observed for either luteolin or eriodyctiol, which are B-ring catecholic analogues of apigenin and naringenin, respectively, suggesting that these antioxidants are incapable of the transition metal-independent generation of reactive oxygen species. This pro-oxidant activity of the polyphenolics therefore needs to be taken into account when quantifying antioxidant activity.     

Artificial protein cavities as specific ligand-binding templates: characterization of an engineered heterocyclic cation-binding site that preserves the evolved specificity of the parent protein.

Cavity complementation has been observed in many proteins, where an appropriate small molecule binds to a cavity-forming mutant. Here, the binding of compounds to the W191G cavity mutant of cytochrome c peroxidase is characterized by X-ray crystallography and binding thermodynamics. Unlike cavities created by removal of hydrophobic side-chains, the W191G cavity does not bind neutral or hydrophobic compounds, but displays a strong specificity for heterocyclic cations, consistent with the role of the protein to stabilize a tryptophan radical at this site. Ligand dissociation constants for the protonated cationic state ranged from 6 microM for 2-amino-5-methylthiazole to 1 mM for neutral ligands, and binding was associated with a large enthalpy-entropy compensation. X-ray structures show that each of 18 compounds with binding behavior bind specifically within the artificial cavity and not elsewhere in the protein. The compounds make multiple hydrogen bonds to the cavity walls using a subset of the interactions seen between the protein and solvent in the absence of ligand. For all ligands, every atom that is capable of making a hydrogen bond does so with either protein or solvent. The most often seen interaction is to Asp235, and most compounds bind with a specific orientation that is defined by their ability to interact with this residue. Four of the ligands do not have conventional hydrogen bonding atoms, but were nevertheless observed to orient their most polar CH bond towards Asp235. Two of the larger ligands induce disorder in a surface loop between Pro190 and Asn195 that has been identified as a mobile gate to cavity access. Despite the predominance of hydrogen bonding and electrostatic interactions, the small variation in observed binding free energies were not correlated readily with the strength, type or number of hydrogen bonds or with calculated electrostatic energies alone. Thus, as with naturally occurring binding sites, affinities to W191G are likely to be due to a subtle balance of polar, non-polar, and solvation terms. These studies demonstrate how cavity complementation and judicious choice of site can be used to produce a protein template with an unusual ligand-binding specificity.     

菹草提取物总黄酮、总酚酸和抗氧化活性含量研究

研究菹草提取物的有效成分,测定其总黄酮含量及总酚酸含量,并确定它的乙酸乙酯和石油醚部位的抗氧化活性成分。采用紫外可见分光光度法,以芦丁和没食子酸作为控制材料,测定菹草的石油醚部位及乙酸乙酯部位中总黄酮和总酚酸含量;采用清除DPPH自由基能力测定法、测定总还原能力铁氰化钾法和水杨酸捕捉羟基自由基法来对菹草各组分提取物的抗氧化能力进行研究。菹草中有一定量的黄酮类化合物和酚酸类化合物存在,不同溶剂提取的部位所含有的总黄酮和总酚酸含量是有差异的,石油醚部位的总黄酮含量要高于乙酸乙酯部位的总黄酮含量,并且总酚酸的含量亦是如此;石油醚部位和乙酸乙酯部位的提取物具有清除DPPH、羟基自由基和还原Fe^3+的能力,各部位清除DPPH、羟基自由基的能力和还原Fe^3+的能力随着样品浓度的增大而增大,且乙酸乙酯部位的测定结果均低于石油醚部位。     

Synthesis and Antioxidant Properties of Psoralen Derivatives

Five psoralen derivatives were synthesized and the structures of them were characterized by ¹H-NMR, ¹³C-NMR, and IR. The antioxidant properties of the compounds were tested by inhibiting the free radical-initiated DNA oxidation and scavenging the radical reaction. The results showed that the effective stoichiometric factors (n) of the compounds V and IV could reach 2.00 and 2.11 in the system of inhibiting the DNA oxidation reaction initiated by 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH). In the inhibition of ⋅OH-oxidation of the DNA system, compounds I ~ V showed antioxidant properties. The thiobarbituric acid absorbance (TBARS) percentages of compounds IV and V were 76.19 % and 78.84 %. Compounds I ~ V could also inhibit Cu²⁺/GSH-oxidation of DNA, and all compounds exhibited good antioxidant properties except compound II (94.00 %). All the five compounds were able to trap diammonium 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) salt radical (ABTS⁺⋅), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH⋅) and 2,6-di-tert-butyl-alpha-(3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-p-tolylox radical (galvinoxyl⋅). The ability of compounds I ~ V to scavenge those free radicals can be measured by the k values. The k values ranged from 0.07 to 0.82 in scavenging ABTS⁺⋅, galvinoxyl, and DPPH radicals, respectively.     

Evaluation of Selected Flavonoids as Antiangiogenic,Anticancer, and Radical Scavenging Agents: An Experimental and In Silico Analysis

Developing antiangiogenic agents using natural products has remained a significant hope in the mainstream of anticancer research. In the present investigation series of flavonoids possessing di-, tri-, tetra-, and penta-hydroxy substitutions were evaluated as antiangiogenic agents using in vivo choriallantoic membrane model. The MTT-based cytotoxicity against selected cancer cell lines was carried out to determine the anticancer potential. The kinetics of free radical scavenging activities of these compounds was demonstrated using 2,2-diphenyl-1-picryl hydrazine (DPPH) and superoxide anion radicals (SORs). To understand the possible antiangiogenic mechanism, the selected flavonoids were docked in silico onto the proangiogenic peptides such as vascular endothelial growth factor (VEGF), hypoxia inducible factor (HIF-1α), and vascular endothelial growth factor receptor-2 (VEGFR2) from human origin. The results of the study shows that amongst the tested flavonoids, genistein (87.1%), kaempferol, (86.3%), and quercetin (84.7%) were found to be effective inhibitors of angiogenesis in CAM model. The antiangiogenic, cytotoxic, and antioxidant activities are discussed in light of structure–activity relationship using in silico approach and other drug-related properties were also calculated using BioMed CAChe V. 6.1.10. The results of the present study focus the isoflavone genistein, kaempferol, and quercetin as lead molecules for designing novel anti-tumor/antioxidant agents targeting angiogenesis.