Reactions of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK) with the tyrosine side-chain fragment, 4-ethylphenol

The melatonin metabolite N(1)-acetyl-5-methoxykynuramine (AMK) has previously been shown to interact with various free radicals. Using the ABTS cation radical [ABTS = 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)] as an electron abstracting reactant, which does not destroy the aromate, we found that the reactive intermediate derived from AMK strongly interacts with the benzene rings of other AMK molecules to form di- and oligomers. Since oligomerization is rather unlikely at physiological concentrations, we investigated reactions with other putative reaction partners. The incubation of tyrosine or several of its structural analogs with AMK in the presence of the ABTS cation radical led to numerous products, amongst which were compounds not detected when one of the educts was incubated with the ABTS cation radical alone. With tyrosine and most of its analogs, the number of products formed in the presence of AMK and ABTS cation radical was relatively high and included numerous oligomers. To optimize the yield of products of interest as well as their separation from other compounds, especially oligomers, we investigated the interaction with 4-ethylphenol, which represents the side chain of tyrosine lacking the carboxyl and amino residues of the amino acid, which otherwise can undergo additional reactions. A prominent product was chromatographically separated and analyzed by mass spectrometry [(+)-ESI-MS, (-)-ESI-MS, (+)-HRESI-MS], (1)H-NMR, and H,H-COSY correlations. The substance was identified as N-{3-[2'-(5'-ethyl-2'-hydroxyphenylamino)-5'-methoxyphenyl]-3-oxopropyl} acetamide. This chemically novel compound represents an adduct in which the amino nitrogen of AMK is attached to the C-2 atom of 4-ethylphenol, which corresponds to the C-3 atom in the benzene ring of tyrosine. This finding suggests that, upon interaction of AMK with an electron-abstracting radical, the kynuric intermediate may modify proteins at superficially accessible tyrosine residues. In fact, protein modification by an unidentified melatonin metabolite has been observed in an earlier study. The possibility of protein AMKylation may be of interest with regard to an eventual interference with tyrosine nitration or, more importantly, with tyrosine phosphorylation.     

Antioxidant properties of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK): scavenging of free radicals and prevention of protein destruction

Abstract

In numerous experimental systems, the neurohormone melatonin has been shown to protect against oxidative stress, an effect which appears to be the result of a combination of different actions. In this study, we have investigated the possible contribution to radical scavenging by substituted kynuramines formed from melatonin via pyrrole ring cleavage. N¹-Acetyl-5-methoxykynuramine (AMK), a metabolite deriving from melatonin by mechanisms involving free radicals, exhibits potent antioxidant properties exceeding those of its direct precursor N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK) and its analog N¹-acetylkynuramine (AK). Scavenging of hydroxyl radicals was demonstrated by competition with ABTS in a Fenton reaction system at pH 5 and by competition with DMSO in a hemin-catalyzed H₂O₂ system at pH 8. Under catalysis by hemin, oxidation of AMK was accompanied by the emission of chemiluminescence. AMK was a potent reductant of ABTS cation radicals, but, in the absence of catalysts, a poor scavenger of superoxide anions. In accordance with the latter observation, AMK was fairly stable in a pH 8 H₂O₂ system devoid of hemin. Contrary to AFMK, AMK was easily oxidized in a reaction mixture generating carbonate radicals. In an oxidative protein destruction assay based on peroxyl radical formation, AMK proved to be highly protective. No prooxidant properties of AMK were detected in a sensitive biological test system based on light emission by the bioluminescent dinoflagellate Lingulodinium polyedrum. AMK may contribute to the antioxidant properties of the indolic precursor melatonin.     

Reactions of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK) with the ABTS cation radical: identification of new oxidation products

The melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK; 1), which was previously shown to be a potent radical scavenger, was oxidized using the ABTS cation radical [ABTS = 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)]. Several new oxidation products were obtained, which were separated by repeated chromatography and characterized by spectroscopic methods such as mass spectrometry (ESI-MS and ESI-HRMS), 1H-NMR and 13C-NMR, HMBC, HSQC, H,H COSY correlations and IR spectroscopy. The main products were oligomers of 1 (3 dimers, 1 trimer and 2 tetramers). In all cases, the amino group N2 was involved in the reactions. Two of the dimers turned out to be cis (2a) and trans (2b) isomers containing an azo bond. In the other dimer (3a), the nitrogen atom N2 was attached to atom C5 of the second aromatic amine, with loss of the methoxy group. In the trimer (5), one N2 formed a bridge to C5 of unit B, as in the respective dimer, while this one of C had bridged to C6 of B. One of the tetramers (6) was composed of a trimer 5 attached to N2 of a fourth 1 molecule via an azo bond as in 2a/b. In the other tetramer (7), an additional C-C bond between rings B and C in 6 is assumed. Although oligomers of AMK may only attain low in vivo concentrations, the types of reactions observed shed light on the physiologically possible metabolism of AMK once reacted with a free radical. The displacement of a methoxy group, rarely seen in the oxidation of methoxylated biomolecules, underlines the reactivity of AMK (1). Preliminary data show that, in the presence of ABTS cation radicals, AMK (1) can interact with side chains of aromatic amino acids, a finding which may be crucial for understanding to date unidentified protein modification by a melatonin metabolite detected earlier in experiments with radioactively labeled melatonin.     

Melatonin and structurally-related,endogenous indoles act as potent electron donors and radical scavengers in vitro

Summary

The radical scavenging properties of melatonin, structurally-related indoles and known antioxidants were investigated in kinetic competition studies using the specific radical trapping reagent 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS). In the presence of highly reactive radicals, ABTS is oxidized to the stable thiazoline cation radical, ABTS*⁺ which, due to its intense green color, can be measured photometrically at 420 nm absorbance. The indoles melatonin, 5-methoxytryptophol, 5-methoxyindole acetic acid and 5-methoxytryptamine as well as the phenolic and thiolic antioxidants ascorbic acid, Trolox, and glutathione inhibited ABTS cation radical formation and catalyzed ABTS radical cation reduction. Melatonin was the most potent radical scavenger and electron donor when compared with the methoxylated indole analogs and the other antioxidants tested. Melatonin, the methoxylated indole analogs and the other antioxidants tested acted as potent electron donors which scavenged initiating and propagating radicals and repaired oxidative damage due to electrophile intermediates.     

Antioxidant properties of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK): scavenging of free radicals and prevention of protein destruction

In numerous experimental systems, the neurohormone melatonin has been shown to protect against oxidative stress, an effect which appears to be the result of a combination of different actions. In this study, we have investigated the possible contribution to radical scavenging by substituted kynuramines formed from melatonin via pyrrole ring cleavage. N1-Acetyl-5-methoxykynuramine (AMK), a metabolite deriving from melatonin by mechanisms involving free radicals, exhibits potent antioxidant properties exceeding those of its direct precursor N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and its analog N1-acetylkynuramine (AK). Scavenging of hydroxyl radicals was demonstrated by competition with ABTS in a Fenton reaction system at pH 5 and by competition with DMSO in a hemin-catalyzed H2O2 system at pH 8. Under catalysis by hemin, oxidation of AMK was accompanied by the emission of chemiluminescence. AMK was a potent reductant of ABTS cation radicals, but, in the absence of catalysts, a poor scavenger of superoxide anions. In accordance with the latter observation, AMK was fairly stable in a pH 8 H2O2 system devoid of hemin. Contrary to AFMK, AMK was easily oxidized in a reaction mixture generating carbonate radicals. In an oxidative protein destruction assay based on peroxyl radical formation, AMK proved to be highly protective. No prooxidant properties of AMK were detected in a sensitive biological test system based on light emission by the bioluminescent dinoflagellate Lingulodinium polyedrum. AMK may contribute to the antioxidant properties of the indolic precursor melatonin.     

Studies on Radiation Chemistry of Halogenophenols in Aqueous Solutions

Among the γ-radiolysis products of p-bromophenol in an aqueous solution, four new oligomers were obtained. By chemical and physical techniques their structures were elucidated as ortho-, meta- and para-terphenyl in which C-4, C-2′ and C-4″ are substituted by hydroxyl groups and C-5′ by a bromine atom and as 5-bromo-2,4′,4″-trihydroxy-m-terphenyl, respectively. These oligomers may be formed by the arylation of p-bromophenol or the dimeric product with an aryl radical intermediate resulted from debromination of p-bromophenol by some radiolysis product(s) of water.     

Endogenous and Dietary Indoles: A Class of Antioxidants and Radical Scavengers in the ABTS Assay

Indoles are very common in the body and diet and participate in many biochemical processes. A total of twenty-nine indoles and analogs were examined for their properties as antioxidants and radical scavengers against 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABTS^?+ radical cation. With only a few exceptions, indoles reacted nonspecifically and quenched this radical at physiological pH affording ABTS. Indoleamines like tryptamine, serotonin and methoxytryptamine, neurohormones (melatonin), phytohormones (indoleacetic acid and indolepropionic acid), indoleamino acids like l-tryptophan and derivatives (N-acetyltryptophan, l-abrine, tryptophan ethyl ester), indolealcohols (tryptophol and indole-3-carbinol), short peptides containing tryptophan, and tetrahydro-β-carboline (pyridoindole) alkaloids like the pineal gland compound pinoline, acted as radical scavengers and antioxidants in an ABTS assay-measuring total antioxidant activity. Their trolox equivalent antioxidant capacity (TEAC) values ranged from 0.66 to 3.9?mM, usually higher than that for Trolox and ascorbic acid (1?mM). The highest antioxidant values were determined for melatonin, 5-hydroxytryptophan, trp-trp and 5-methoxytryptamine. Active indole compounds were consumed during the reaction with ABTS^?+ and some tetrahydropyrido indoles (e.g. harmaline and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid ethyl ester) afforded the corresponding fully aromatic β-carbolines (pyridoindoles), that did not scavenge ABTS^?+. Radical scavenger activity of indoles against ABTS^?+ was higher at physiological pH than at low pH. These results point out to structural compounds with an indole moiety as a class of radical scavengers and antioxidants. This activity could be of biological significance given the physiological concentrations and body distribution of some indoles.     

Lidocaine: An inhibitor in the free‐radical‐induced hemolysis of erythrocytes

Lidocaine was reported to protect erythrocytes from hemolysis induced by 2,2′‐azobis(2‐amidinopropane) dihydrochloride (AAPH). Since AAPH‐induced hemolysis was a convenient in vitro experimental system to mimic erythrocytes undergoing peroxyl radicals attack, the aim of this work was to investigate the antioxidant effect of lidocaine on AAPH‐induced hemolysis by chemical kinetics. As a result, one molecule of lidocaine can only trap 0.37 radical, much lower than melatonin. Meanwhile, lidocaine cannot protect erythrocytes from hemolysis induced by hemin, which the mechanism of hemolysis was due to the erythrocyte membrane destroyed by hemin. Accordingly, lidocaine protected erythrocytes by scavenging radicals preferentially rather than by stabilizing membrane. Moreover, the interactions of lidocaine with two radical species, including 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonate) radical cation (ABTS^+?) and 2,2′‐diphenyl‐1‐picrylhydrazyl (DPPH), indicated that lidocaine can reduce ABTS^+? with 260 µM as the 50% inhibition concentration (IC₅₀) and cannot react with DPPH. Thus, lidocaine served as a reductant rather than a hydrogen donor to interact with radicals. Finally, the quantum calculation proved that, compared with the melatonin radical, the stabilization of N‐centered radical of lidocaine was higher than the amide‐type N‐centered radical but lower than the indole‐type N‐centered radical in melatonin. These results provided basic information for lidocaine to be an antiradical drug. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:81–86, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20267     

Specific Deprotonation Reactions of the Pyrimidine Radical Cation Resulting from the Menadione Mediated Photosensitization of 2′-Deoxycytidine

Menadione(2-methyl-1, 4-naphthoquinone) was shown to sensitize 2′-deoxycytidine to near ultraviolet light according to two main mechanisms. Reaction of a water molecule with the initially photo-induced pyrimidine radical cation and subsequent addition of molecular oxygen leads to the preponderant formation of the four cis and trans diastereoisomers of 5,6-dihydroxy-5,6-dihydro-2′-deoxyuridine. Pyrimidine ring opening and rearrangement products are also generated through the intermediate 6-hydroxy-5,6-dihydro-2′-deoxyurid-5-yl radical. The competitive deprotonation reaction of the radical cation is likely to involve two sites. Loss of an amino group proton is the likely initial event to explain the formation of 2′-deoxyuridine which is resistant to further photooxidation. The second deprotonation reaction involves the osidic carbon C(1′). The resulting radical will further react with oxygen leading to the release of free cytosine with concomitant formation of 2-deoxy-D-ribono-1,4-lactone. This reaction which is not prevented by hydroxyl radical scavengers constitutes to our knowledge the first example of a pyrimidine radical which is able to initiate selective intramolecular reaction at position 1 within the sugar moiety.     

Isolation of Ciliatine (2-Aminoethylphosphonic Acid) from the Bile of the Bovine

To elucidate the formation mechanism of N,N′-dialkylpyrazine cation radical during browning reaction of sugars with amino compounds, main products in an early stage of the reaction were determined quantitatively by TLC and GLC. It was shown that the Schiff base, two-carbon fragmental product of sugar, the free radical and deoxyosone were successively produced prior to the browning. Polarographical measurements indicated that the radical formation was induced by the production of some reducing substances in the reaction mixture. These results suggest that the free radical was formed by the reduction of N,N′-dialkylpyrazinium; a compound, which demonstrated to have a strong activity to browning, might be formed by condensation of two-carbon enaminol followed by oxidation.     

Ferulic acid dehydrodimers from wheat bran: isolation,purification and antioxidant properties of 8-0-4-diferulic acid

Summary

Wheat bran contains several ester-linked dehydrodimers of ferulic acid, which were detected and quantified after sequential alkaline hydrolysis. The major dimers released were: trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid (5–8-BendiFA), (Z)-β-(4-[(E)-2-carboxyvinyl]-2-methoxy-phenoxy)-4-hydroxy-3-methoxycinnamic acid (8-O-4-diFA) and (E,E)-4,4′-dihydroxy-5,5′-dimethoxy-3,3′-bicinnamic acid (5–5-diFA). trans-7-hydroxy-1-(4-hydroxy-3methoxyphenyl)-6-methoxy-1,2-dihydro-naphthalene-2,3-dicarboxylic acid (8–8-diFA cyclic form) and 4,4′-dihydroxy-3,3′-dimethoxy-β,β'-bicinnamic acid (8–8-diFA non cyclic form) were not detected. One of the most abundant dimers, 8-O-4-diFA, was purified from de-starched wheat bran after alkaline hydrolysis and preparative HPLC. The resultant product was identical to the chemically synthesised 8-O-4-dimer by TLC and HPLC as confirmed by ¹H-NMR and mass spectrometry. The absorption maxima and absorption coefficients for the synthetic compound in ethanol were: λ_max: 323 nm, λ_min: 258 nm, ε_λmax (M^?1cm^?1): 24800 ± 2100 and ε₂₈₀ (M^?1cm^?1): 19700 ± 1100. The antioxidant properties of 8-O-4-diFA were assessed using: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes and; (b) scavenging of the radical cation of 2,2′-azinobis (3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue, Trolox C. The 8-O-4-diFA was a better antioxidant than ferulic acid in both lipid and aqueous phases. This is the first report of the antioxidant activity of a natural diferulate obtained from a plant.     

2′-DMAOE RNA: Emerging Oligonucleotides with Promising Antisense Properties

Abstract

Oligonucleotides with modifications at the carbohydrate 2′-position offer potential second-generation drug candidates¹. ISIS 13312, a chimeric compound targeting CMV retinitis, has 2′-O-methoxyethyl² (2′-MOE) modifications at the ends to offer enhanced binding affinity and nuclease resistance is an example of this trend. 2′-MOE modification offers high binding affinity and nuclease resistance presumably due to conformational constraints placed on the linkage by the oxygen-oxygen gauche effect³. On the other hand, 2′-O-aminopropyl modification (2′-AP) exhibits the highest nuclease resistance⁴, due to the presence of a cationic charge at the physiological pH. However, it lacks the binding affinity advantage of MOE due to the lack of oxygen-oxygen gauche effect. To optimize the antisense properties of both 2′-MOE and 2′-AP modifications, we have designed and constructed 2′-O-(aminooxyethyl) modification (2′-AOE)⁵ and 2′-O-(dimethylaminooxy ethyl) modification (2′-DMAOE) and synthesized oligomers having these modifications. 2′-DMAOE oligomers demonstrate higher binding affinity and nuclease resistance than 2′-MOE oligomers and stand out as promising candidates for future antisense oligonucleotide drug development.     

Benzothiazolyl Guanidines as Antibacterials

Some new N-p-chlorophenyl-N′-2-(substituted) benzothiazolyl-N″-alkyl guanidines have been synthesized. Several of these including some N-p-chlorophenyl-N′-2-(substituted)benzothiazolyl guanidines and N-p-chlorophenyl-N′-2-(substituted) benzothiazolyl-N″-methyl guanidines^* have been tested for their antibacterial activities against B. subtilis, S. aureus, S. typhi., E. coli and A. tumefaciens, and also for their antitubercular activity against M. tuberculosis (H37Rv).     

Ultrasonic absorption in aqueous solution of lysozyme.

The titration curve of ultrasonic absorption at 2.82 MHz in aqueous solutions of lysozyme measured by Zana and Lang [J. Phys. Chem., 74 , 2734 (1970)] is theoretically analyzed. The maxima at pH 3 and pH 11 are describable with proton-transfer reactions of dissociable carboxyl and amino groups by assuming that volume changes due to the reactions are 2.3 and 5.2 cm³/mole, respectively, which are appreciably smaller than those of simple amino acids. The remaining, pH-independent excess absorption over solvent is measured at frequencies ranging from 3 to 150 MHz. The absorption is ascribed to the internal loss of protein. The complex compressibility β′_p ? iβ″_p of lysozyme molecule is evaluated as β′_p = 7.2 × 10^?12 cm²/dyne and β″_P = 4.3 × 10^?14 cm²/dyne from the increments over solvent in absorption as well as in sound velocity.     

The antioxidant activities of seasonings used in Asian cooking. Powerful antioxidant activity of dark soy sauce revealed using the ABTS assay

Scavenging of the ABTS (2,2′-azinobis[3-ethylbenzothiazoline-6-sulphonate])-derived nitrogen-centred radical cation (ABTS^?+) was used to compare the total antioxidant activities of several seasonings used in Asian cooking. The results were expressed as Trolox equivalent antioxidant capacity (TEAC). The TEAC activities of dark soy sauces were found to be exceptionally high. In evaluating the TEAC of commercial products, attention must be paid to the addition of preservatives by manufacturers to the seasonings tested. Sodium benzoate (a preservative added to several seasonings) did not react significantly with ABTS^?+, but the sulphite content of certain white wines may have led to an over-estimation of their TEAC.     

Antioxidative effect of melatonin on DNA and erythrocytes against free-radical-induced oxidation

The aim of this work is to investigate the antioxidative effect of melatonin (N-acetyl-5-methoxytryptamine) on the oxidation of DNA and human erythrocytes induced by 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). First, the 50% inhibition concentration (IC50) of melatonin is measured by reacting with two radical species, i.e., 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS*+) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH). The IC50 of melatonin are 75microM and 300microM when melatonin reacts with ABTS*+ and DPPH, respectively. Especially, the reactions of melatonin with ABTS*+ and DPPH are the direct evidence for melatonin to trap radicals. Then, melatonin is applied to protect DNA and human erythrocytes against oxidative damage and hemolysis induced by 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). The presence of melatonin prolongs the occurrence of the oxidative damage of DNA and hemolysis of erythrocytes, generating an inhibition period (t(inh)). The proportional relationship between t(inh) and the concentration of melatonin ([MLT]) is treated by the chemical kinetic equation, t(inh)=(n/R(i))[MLT], in which n means the number of peroxyl radical trapped by an antioxidant, and R(i) stands for the initiation rate of the radical reaction. It is found that every molecule of melatonin can trap almost two radicals in protecting DNA and erythrocytes. Furthermore, quantum calculation proves that the indole-type radical derived from melatonin is much stable than amide-type radical. Finally, melatonin is able to accelerate hemolysis of erythrocytes induced by hemin, indicating that melatonin leads to the collapse of the erythrocyte membrane in the presence of hemin. This may provide detailed information for the usage of melatonin and helpful reference for the design of indole-related drugs.     

Biological Evaluation of Secondary Metabolites from the Root of Machilus obovatifolia

Bioassay‐guided fractionation of the root of Machilus obovatifolia led to the isolation of four new lignans, epihenricine B ( 1 ), threo‐(7′R,8′R) and threo‐(7′S,8′S)‐methylmachilusol D ( 2 and 3 ), and isofragransol A ( 4 ), along with 23 known compounds. The compounds were obtained as isomeric mixtures (i.e., 2 / 3 and 4 / 20 , resp.). The structures were elucidated by spectral analyses. Among the isolates, 1 , licarin A ( 12 ), guaiacin ( 14 ), (±)‐syringaresinol ( 21 ), and (?)‐epicatechin ( 23 ) showed ABTS (=2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) cation radical‐scavenging activity, with SC₅₀ values of 11.7±0.5, 12.3±1.1, 11.0±0.1, 10.6±0.3, and 9.5±0.2 μM in 20 min, respectively. In addition, kachirachirol B ( 17 ) showed cytotoxicity against the NCI‐H460 cell line with an IC₅₀ value of 3.1 μg/ml.     

Peroxidase-like activity of Thermobifida fusca hemoglobin: The oxidation of dibenzylbutanolide

The thermostable truncated hemoglobin from the actinomyces Thermobifida fusca (Tf-trHb) displays a robust peroxidase activity, with optimum at acidic pH values, in experiments with the redox mediator ABTS. However, typical peroxidase substrates, such as phenolic or aromatic amine compounds, appear to be poor substrates for Tf-trHb. In turn, the protein is able to catalyze a unique dehydrogenation reaction of dibenzylbutanolides, suggested intermediates in the biosynthesis of podophyllotoxin, in the presence of hydrogen peroxide. Dibenzylbutanolides with a free 4″-hydroxyl group were thus converted into the corresponding 2,7″-dehydroderivatives thus setting up the basis for an efficient biotransformation of this important precursor. In particular, Tf-trHb mediated oxidation of trans-2-(4″-hydroxy-3″,5″-dimethoxybenzyl)-3-(3′,4′-methylenedioxy-7′β-hydroxybenzyl)butanolide 1 into the corresponding benzylidene-benzoyl-γ-butyrolactone 2 was obtained at high yield and with excellent selectivity.     

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.     

Dihydrochalcone glucosides and antioxidant activity from the roots of Anneslea fragrans var. lanceolata

Bioassay-guided fractionation of the roots of Anneslea fragrans var. lanceolata led to the isolation of four dihydrochalcone glucosides, davidigenin-2′-O-(6″-O-4″′-hydroxybenzoyl)-β-glucoside (1), davidigenin-2′-O-(2″-O-4″′-hydroxybenzoyl)-β-glucoside (2), davidigenin-2′-O-(3″-O-4″′-hydroxybenzoyl)-β-glucoside (3), and davidigenin-2′-O-(6″-O-syringoyl)-β-glucoside (4), and 13 known compounds. The structures were identified by means of spectroscopic analysis. Davidigenin-2′-O-(6″-O-syringoyl)-β-glucoside (4), 1-O-3,4-dimethoxy-5-hydroxyphenyl-6-O-(3,5-di-O-methylgalloyl)-β-glucopyranoside (5), lyoniresinol (10), and syringic acid (13) showed ABTS [2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] cation radical scavenging activity, with SC₅₀ values of 52.6 ± 5.5, 26.0 ± 0.7, 6.0 ± 0.2, and 27.5 ± 0.6 μg/mL in 20 min, respectively. Lyoniresinol (10), isofraxidin (12), and syringic acid (13) also showed DPPH [1,1-diphenyl-2-picrylhydrazyl] radical scavenging activity, with SC₅₀ values of 8.4 ± 1.8, 51.6 ± 2.2, and 4.3 ± 0.7 μg/mL in 30 min, respectively.