Isolation and characterization of novel benzoates, cinnamates, flavonoids, and lignans from Riesling wine and screening for antioxidant activity.

A German Riesling wine has been fractionated with the aid of countercurrent chromatography. After purification by HPLC, the structures of 101 compounds were established by mass spectrometry and NMR spectroscopy. Seventy-three of the isolated compounds exhibited a phenolic or benzylic structure. Fifty-four compounds were reported for the first time as Riesling wine constituents. New compounds identified in this work included twelve benzoic and cinnamic acid derivatives. In addition to two isomeric (E)-caffeoyl ethyl tartrates, the glucose esters of (E)-cinnamic, (E)-p-coumaric, and (E)-ferulic acid, as well as the 4-O-glucosides of (E)- and (Z)-ferulic acid, have been identified for the first time in Riesling wine. The structures of two additional phenylpropanoids were elucidated as 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-propan-1-one and 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-propan-1-one. Moreover, two ethyl esters, i.e., ethyl protocatechuate and ethyl gallate, as well as the glucose ester of vanillic acid, were newly detected in Riesling wine. Novel representatives in the flavonoid group were dihydrokaempferol, dihydroquercetin, and four dihydroflavonol glycoconjugates, i.e., the 3-O-glucosides of dihydrokaempferol and dihydroquercetin, as well as the 3-O-xyloside and the 3'-O-glucoside of dihydroquercetin. Additionally, six novel lignans, i.e., lariciresinol 4-O-glucoside, three isolariciresinol derivatives, and two secoisolariciresinols, as well as three neolignans were isolated. Structural elucidation of the newly isolated wine constituents is reported together with the determination of their antioxidant activity.     

Relationship among antioxidant activity, vasodilation capacity, and phenolic content of red wines

The relationship among antioxidant activity, based on the electron-spin resonance determination of the reduction of Fremy's radical, vasodilation activity, and phenolic content was investigated in 16 red wines. The wines were selected to provide a range of origins, grape varieties, and vinification methods. Sensitive and selective HPLC methods were used for the analysis of the major phenolics in red wine: free and conjugated myricetin, quercetin, kaempferol, and isorhamnetin; (+)-catechin, (-)-epicatechin, gallic acid, p-coumaric acid, caffeic acid, caftaric acid, trans-resveratrol, cis-resveratrol, and trans-resveratrol glucoside. Total anthocyanins were measured using a colorimetric assay. The total phenolic content of the wines was determined according to the Folin-Ciocalteu colorimetric assay and also by the cumulative measurements obtained by HPLC. The 16 wines exhibited a wide range in the values of all parameters investigated. However, the total phenol contents, measured both by HPLC and colorimetrically, correlated very strongly with the antioxidant activity and vasodilation activity. In addition, the antioxidant activity was associated with gallic acid, total resveratrol, and total catechin. In contrast, only the total anthocyanins were correlated with vasodilation activity. The results demonstrate that the different phenolic profiles of wines can produce varying antioxidant and vasodilatant activities, which opens up the possibility that some red wines may provide enhanced health benefits for the consumer.     

Isoflavone characterization and antioxidant activity of ohio soybeans

Seventeen Ohio soybeans were screened for isoflavone content and antioxidant activity. Isoflavone content was determined by C(18) reversed phase high-performance liquid chromatography coupled with a photodiode array detector. Antioxidant activities of soybean extracts were measured using 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical and photochemiluminescence (PCL) methods. The highest and lowest total isoflavone contents were 11.75 and 4.20 micromol/g soy, respectively, while the average was 7.12 micromol/g soy. Antioxidant activities of soybean extracts ranged from 7.51 to 12.18 micromol butylated hydroxytoluene (BHT) equivalent/g soy using the DPPH method. Lipid and water soluble antioxidant activities of soybean extracts ranged from 2.40 to 4.44 micromol Trolox equivalent/g soy and from 174.24 to 430.86 micromol ascorbic acid equivalent/g soy, respectively, using the PCL method.     

Isolation and characterization of antioxidant compounds from Aspergillus candidus broth filtrate

The objectives of this study were to isolate the antioxidative components in the broth filtrate of Aspergillus candidus (CCRC 31543), to characterize their antioxidative properties, and to evaluate their safety. Three major compounds were isolated and identified as 3,3' '-dihydroxyterphenyllin, 3-hydroxyterphenyllin, and candidusin B. In the linoleic acid peroxidation system, the inhibition of peroxidation in these three compounds was greater than 95% and was significantly higher than that of alpha-tocopherol but equal to that of BHA at 12.5-200 microg/mL. As measured using the Rancimat method in lard, 3,3' '-di-OH-terphenyllin exhibited a protection factor value of 7.82, which was substantially higher than those of BHA (5.58) and alpha-tocopherol (4.29) at 200 microg/mL. 3,3' '-di-OH-terphenyllin and 3-OH-terphenyllin also exhibited marked scavenging effects on the alpha,alpha-diphenyl-beta-picrylhydrazyl radicals (94.7 and 96.0%, respectively), which were similar to those of BHA and alpha-tocopherol. Safety studies showed that these three compounds were neither cyto- nor geno-toxic toward human intestine 407 (INT 407) cells, nor mutagenic toward Salmonella typhimurium TA98 and TA100.     

Isolation and characterization of an oxygen radical absorbance activity peptide from defatted peanut meal hydrolysate and its antioxidant properties

Defatted peanut meal hydrolysate (DPMH) was purified using ultrafiltration, gel filtration chromatography, and high-performance liquid chromatography. A tripeptide with strong oxygen radical absorbance capacity (ORAC) was isolated and identified as Tyr-Gly-Ser by ESI-MS/MS. It was then synthesized to measure its antioxidant properties in different systems. The ORAC value of Tyr-Gly-Ser was 3-fold higher than that of glutathione (GSH), and it displayed a stronger protective effect on linoleic acid peroxidation and H(2)O(2)-induced oxidative injury in rat pheochromocytoma line PC12 cells than GSH (p < 0.05). However, Tyr-Gly-Ser showed negligible DPPH radical scavenging activity, reducing power, and no metal chelating ability. The results suggested that Tyr-Gly-Ser displayed antioxidant activity via the hydrogen atom transfer mechanism, and the Tyr at the N-terminal was the hydrogen donor. The ORAC assay was recommended as a reliable and effective method to measure the antioxidant activity in the course of antioxidant peptide isolation.     

Isolation, characterization, and biological activity of naphthoquinones from Calceolaria andina L

Two compounds recognized as responsible for the insecticidal activity of extracts of Calceolaria andina L. (Scrophulariaceae) have been isolated and characterized as 2-(1, 1-dimethylprop-2-enyl)-3-hydroxy-1,4-naphthoquinone and the corresponding acetate, 2-acetoxy-3-(1,1-dimethylprop-2-enyl)-1, 4-naphthoquinone. Their activities against 29 pest species and 9 beneficial species of arthropod from a total of 11 orders have been determined. Activities against homopteran and acarine species are of the same order as those of established pesticides, and, significantly, no cross-resistance is observed for strains resistant to established classes of insecticide. Mammalian toxicities are low.     

Isolation and characterization of a phenolic antioxidant from the Pacific oyster (Crassostrea gigas)

Using an oxygen radical absorbance capacity (ORAC) assay, antioxidant activity was detected in the ethanol extract of the Pacific oyster, which was purified by sequential extraction with organic solvents. The ethyl acetate fraction showed the strongest antioxidant activity and was further purified, yielding a single compound [as assessed by thin-layer chromatography (TLC) and reverse-phase high-performance liquid chromatography (HPLC)]. This compound was identified as 3,5-dihydroxy-4-methoxybenzyl alcohol on the basis of (1)H and (13)C nuclear magnetic resonance (NMR), heteronuclear multiple-bond correlation (HMBC), and electrospray ionization-mass spectrometry (ESI-MS) spectral analyses, a conclusion that was confirmed by chemical synthesis. The concentration of the compound was 6.7 mg/100 g of whole oyster meat wet weight. This amphiphilic antioxidant retarded the copper-mediated oxidation of low-density lipoproteins (LDLs) and the generation of thiobarbituric acid reactive substances. Furthermore, the compound showed substantial antioxidant activity using the ORAC and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays compared to natural antioxidants. Although the same compound was previously found in brown algae, its presence in other organisms and antioxidant activity are reported here for the first time.     

Peroxynitrite scavenging and cytoprotective activity of 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether from the marine alga Symphyocladia latiuscula

Peroxynitrite (ONOO(-)), formed from the reaction of superoxide (O(2)*(-)) and nitric oxide (*NO), is a cytotoxic species that can oxidize several cellular components such as proteins, lipids, and DNA. It has been implicated in diseases such as Alzheimer's disease, rheumatoid arthritis, cancer, and atherosclerosis. Due to the lack of endogenous enzymes responsible for ONOO(-) inactivation, developing a specific ONOO(-) scavenger is of considerable importance. The aim of this study was to evaluate the ability of marine natural products to scavenge ONOO(-) and to protect cells against ONOO(-). Methanolic extracts of 17 marine alga were tested for their ONOO(-) scavenging activity. Among them, Symphyocladia latiuscula showed the potent scavenging activity. CH(2)CH(2) fraction was partitioned with CH(2)CH(2) following n-hexanal extraction from the methanol extract of S. latiuscula. It was highly effective for ONOO(-) scavenging activity. Further analysis of the active fractionated extract identified 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (TDB) as a potent ONOO(-) scavenger. The data demonstrated that TDB led to decreased ONOO(-)-mediated nitration of tyrosine through electron donation. TDB showed significant inhibition on nitration of bovine serum albumin and low-density lipoprotein by ONOO(-) in a dose-dependent manner. It also provided cytoprotection from cell damage induced by ONOO(-). TDB can be developed as an effective peroxynitrite scavenger for the prevention of the involved diseases.     

Potentiality of red sorghum for producing stilbenoid-enriched beers with high antioxidant activity

trans-Piceid and trans-resveratrol were authenticated for the first time by high-resoution mass spectrometry in red sorghum grains. A 0.4-1 mg/kg amount of trans-piceid and up to 0.2 mg/kg trans-resveratrol were quantified by reversed phase high-performance liquid chromatography-atmospheric pressure chemical ionization(+)-tandem mass spectrometry. The white sorghum samples contained only traces of trans-piceid (up to 0.1 mg/kg), and trans-resveratrol was absent. In much lower amounts than procyanidins, stilbenoids are not able to contribute significantly to the exceptional antioxidant activity of red sorghum (ORAC, 83-147 μmol TE/g; AAPH, 0.61-1.79 min/mg kg(-1)). More than 10 mg/kg of total stilbenoids have been reported in some hop varieties. Yet, as hop is a minor wort ingredient as compared to cereals, red sorghum could be the main source of trans-piceid in beer. Hop remains, however, the single source of cis-piceid.     

Isolation and structural characterization of new anthocyanin-derived yellow pigments in aged red wines

Two newly formed yellow pigments that revealed unique spectral features were detected and isolated from an aged Port red wine by TSK Toyopearl HW-40(s) gel chromatography and characterized by UV-visible spectrophotometry, 1H NMR and 13C NMR, and mass spectrometry (LC-ESI/MS). The UV-vis spectra of these pigments showed maximum absorption at 478 nm that is significantly hypsochromically shifted from those of original grape anthocyanins and other pyranoanthocyanins, exhibiting a more yellow hue in acidic solution. The structures of these pigments correspond to methyl-linked pyranomalvidin 3-glucoside and its respective coumaroyl glucoside derivative. They were shown to arise from the reaction between acetoacetic acid and genuine grape anthocyanins. Isolation and NMR identification using 1D and 2D NMR techniques are reported for the first time for this new family of anthocyanin-derived yellow pigments occurring in red wines.     

Extraction of phenolics and changes in antioxidant activity of red wines during vinification.

The moderate consumption of alcoholic beverages has been associated with protection against the development of coronary heart disease. Although alcohol itself can help prevent coronary heart disease through a number of mechanisms, red wine appears to offer protection above and beyond that attributable to alcohol alone. Red wine is a complex fluid containing grape, yeast, and wood-derived phenolic compounds, the majority of which have been recognized as potent antioxidants. The aim of this study was to investigate the major phenolic contributors to the antioxidant activity of wine. To this end, four wines were followed during the first 7-9 days of vinification. Individual phenolic compounds were quantified by HPLC, and antioxidant activity was determined by electron spin resonance spectroscopy. The extraction of the phenolics was found to be influenced by vinification procedure, grape quality, and grape variety. Although fermenting wines reached a total phenolic content comparable to that of a bottled wine after 9 days of vinification, the antioxidant activity was significantly lower than that of a finished wine. This suggests that the larger polyphenolic complexes and condensation products that appear during aging make a sizable contribution to the overall antioxidant activity of red wines.     

Antioxidant profile of red wines evaluated by total antioxidant capacity, scavenger activity, and biomarkers of oxidative stress methodologies

The study of the antioxidant capacity of foodstuffs requires the use of diverse determination methods to gain a wider picture of their multiple effects. The aim of this work was to evaluate the "antioxidant profile" of red wines applying TAC (total antioxidant capacity) methods: 2,2'-azinobis(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl, N,N-dimethyl-p-phenylenediamine dihydrochloride, oxygen radical absorbance capacity, ferric reducing/antioxidant power, hydroxyl and superoxide radical scavenger activities, and biomarkers of oxidative stress methods such as lipid peroxidation inhibition and inhibition of damage to DNA. Furthermore, levels of total polyphenols (TPP) of wines were also evaluated. Three bottles of 107 different Spanish red wines (total samples 321), made from different grape varieties, aging processes, and vintages, were analyzed. The validation of TAC methods, the first step in this work, provided a good linearity, proportionality, and low detection limits. Among these methods, the ABTS was the most satisfactory for its rapidity, cost, and precision. All wines showed an important capacity to scavenge hydroxyl radicals and were capable of blocking superoxide radicals but with 10 times lower intensity. Wines also showed important protective action on biomarkers of oxidative stress; they were much more active to inhibit lipid peroxidation than DNA oxidation. Few statistically significant correlations among levels of TPP and antioxidant properties of wines were detected. Furthermore, values of these correlations were very low.     

Polyphenol content and total antioxidant activity of vini novelli (young red wines)

Eight commercial Italian vini novelli (red wines prepared by carbonic maceration and supposed to be consumed within three months from their wine-making) were evaluated for their total antioxidant activity. The wines had an average total phenol content (1605.4 +/- 337.4 mg/L gallic acid equivalents) lower than that of wines prepared by traditional maceration and consumable after aging (2057. 3 +/- 524.0 mg/L gallic acid equivalents). The average flavanol content (424.7 +/- 121.3 mg/L catechin equivalents) and the total antioxidant activity (16.8 +/- 3.8 mmol/L Trolox equivalents) of vini novelli were higher than the corresponding values (382.7 +/- 174.5 mg/L catechin equivalents and 12.3 +/- 3.3 mmol/L Trolox equivalents) found for aged wines. Three couples of experimental wines were prepared from the same grapes by traditional or carbonic maceration. These wines showed a different phenolic pattern, anthocyanins being more abundant in vini novelli. However, the average total antioxidant activities of the wines were similar, suggesting that aging (and not the wine-making technique) is the main factor influencing the antioxidant activity of red wines.     

Isolation and characterization of antioxidant protein fractions from melinjo (Gnetum gnemon) seeds

The protein from the seeds of melinjo ( Gnetum gnemon ) was purified using a precipitation method and ion exchange chromatographic techniques to identify the potent antioxidant and free radical scavenging activities. Two antioxidant protein fractions were isolated from G. gnemon seed with molecular weights of approximately 30 kDa (Gg-AOPI) and 12 kDa (Gg-AOPII) by SDS-PAGE. The N-terminal amino acid sequence of Gg-AOPII is Gly-Asn-Gly-Lys-Ala-Thr-Val-Ala-Ile-Leu-Val-Lys-Glu-Lys-Val-Glu-Tyr-Gly-Glu-Glu, and the result of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis showed that they were distinct from each other; no protein in database matching was found to both Gg-AOPI and Gg-AOPII. The antioxidant or free radical scavenging activities of Gg-AOPs were investigated by employing in vitro assay systems including the inhibition of linoleic acid autoxidation, scavenging effect on α,α-diphenyl-β-picrylhydrazyl free radical (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), reducing power, chelating abilities of metal ions Cu(2+) and Fe(2+), and protections against hydroxyl radical-mediated DNA damages. The result showed that two protein fractions exhibited significant (p < 0.05) antioxidant activities against free radicals such as DPPH, ABTS, and superoxide anion and showed activities similar to those of glutathione (G-SH) and BHT in a linoleic acid emulsion assay system. Moreover, Gg-AOPI and Gg-AOPII also exhibited notable reducing power and strong chelating effect on Fe(2+) and protected hydroxyl radical induced oxidative DNA damage. The data obtained by the in vitro systems obviously established the antioxidant potency of Gg-AOPs.     

Effect of addition of commercial grape seed tannins on phenolic composition, chromatic characteristics, and antioxidant activity of red wine

The effect of addition of grape seed tannins on the phenolic composition, chromatic characteristics, and antioxidant activity of red wine was studied. Two highly pure commercial grape seed tannins (GSE100 and GSE300) were selected, and their phenolic compositions were determined. Two types of red wines were made with Castela?o/Tinta Miu?da (3/2, w/w) grapevine varieties by fermentation on skin using two different maceration times, which correspond to the wines rich and poor in polyphenols, respectively. Each of these wines was used for experimentation with the addition of GSE100 and GSE300 before and immediately after alcoholic fermentation. Phenolic composition, chromatic characteristics, and antioxidant activity of the finished red wines were analyzed by HPLC-DAD, CIElab 76 convention, and DPPH radical test, respectively. The results showed that the addition of grape seed tannins had obvious effects of increasing color intensity and antioxidant activity only in the wines poor in polyphenols. Although GSE300 contained much higher amounts of di- and trimer procyanidins and a lower amount of polymeric proanthocyanidins, it provided effects of increasing the color intensity and antioxidant activity of the wines poor in polyphenols similar to those of GSE100. Furthermore, GSE100 released more gallic acid to wines than GSE300, although no gallic acid was detected in GSE100. Tannins added after alcoholic fermentation had a better effect on phenolic composition of red wine than tannins added before alcoholic fermentation.     

Isolation and structural characterization of new acylated anthocyanin-vinyl-flavanol pigments occurring in aging red wines

Three newly formed pigments were detected and isolated from a 2-year-old Port wine through TSK Toyopearl HW-40(S) gel column chromatography and characterized by UV-visible spectrophotometry, NMR, and mass spectrometry (ESI/MS). (1)H NMR and (13)C NMR data for these pigments obtained using 1D and 2D NMR techniques (COSY, NOESY, gHSQC, and gHMBC) are reported for the first time. The structure of the pigments was found to correspond to the vinyl cycloadducts of malvidin 3-coumaroylglucoside bearing either a procyanidin dimer or a flavanol monomer ((+)-catechin or (-)-epicatechin). Additionally, conformational analysis was performed for one of these newly formed pigment using computer-assisted model building and molecular mechanics. A chemical nomenclature is proposed to unambiguously name this new family of anthocyanin-derived pigments.     

Simulated digestion and antioxidant activity of red wine fractions separated by high speed countercurrent chromatography

Wine is an important source of dietary antioxidants because of its phenolic compound content. The antioxidant activity (AA) of pure monomer substances present in wines, such as phenolic acids, flavanols, and anthocyanins, has already been described, but the AA of polymeric phenols is still unknown. In this study, we have fractionated a red wine by countercurrent chromatography (CCC) into four fractions: fraction 1, made up of polymeric compounds; fraction 2, containing malvidin-3-glucoside; fraction 3, containing peonidin-3-glucoside; and fraction 4, containing vitisin A. The AA of these fractions was determined by oxygen radical absorbance capacity and ferric reducing ability assays. The weight of fraction 1 was the largest, so this was the largest contributor to the AA of the wine. However, the antioxidant powers (muM Trolox/g fraction) of fractions 2-4 were similar and higher than that of fraction 1. We also determined AA before and after in vitro gastric and intestinal digestions. After gastric digestion, the AA was 100-1000 times higher than the original fraction values. Gallic acid was determined in gastric and intestinal digested fractions. After intestinal digestion, the concentrations of simple phenols, such as caffeic acid, p-coumaric acid, and protocatechualdehyde, increased as they were released from the fractions under our conditions. Protocatechuic acid was determined in more intestinal digested fractions than in gastric digested fractions. These results partly explain the increase in AA after the digestion and indicate the relevance of polymeric polyphenolic compounds as precursors of smaller molecules with biological activity.     

Determination of antioxidant activity and characterization of antioxidant phenolics in the plum vinegar extract of cherry blossom (Prunus lannesiana)

Sakura-cha (salted cherry blossom tea) is a Japanese tea that is traditionally served at celebrations such as wedding ceremonies. The production of Sakura-cha includes the immersion of cherry blossom flowers in Japanese plum vinegar, and through this process, the byproduct (plum vinegar extract of cherry blossom) is obtained. In this study, the antioxidant activity of the plum vinegar extract of cherry blossom was examined. The plum vinegar extract of cherry blossom had a greater superoxide anion scavenging activity compared with red wine, which is a well-known strong antioxidant. Liquid chromatography-mass spectrometry analysis showed that cyanidin-3-glucoside, cyanidin-3-rutinoside, and caffeic acid were the major components in the phenolic extract prepared from plum vinegar extract of cherry blossom, and they possessed superoxide anion scavenging activity. Caffeic acid is mainly responsible for the scavenging activity of phenolic extract; the contributions of cyanidin-3-glucoside and cyanidin-3-rutinoside were minor.     

Purification of a lectin from the marine red alga Gracilaria cornea and its effects on the cattle tick Boophilus microplus (Acari: Ixodidae)

A lectin was purified from the seaweed Gracilaria cornea by hydrophobic interaction chromatography on phenyl-Sepharose CL-4B followed by affinity chromatography on immobilized mucin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of G. cornea lectin (GCL) revealed a single protein band of approximately 60 kDa, whereas by gel filtration on Sephadex G-100 its native molecular mass was 66 kDa. GCL exhibited a single isoeletric point of 4.3 and a 52.5% content of neutral sugars. Furthermore, the EDTA-treated lectin did not show any significant decrease in its ability to agglutinate trypsin-treated chicken erythrocytes. These data suggest that GCL is an acidic, monomeric glycoprotein that probably does not require divalent metal ions for its hemagglutinating activity. GCL hemagglutinating activity was not inhibited by any of the mono-, di-, and trisaccharides tested but was by the complex glycoproteins fetuin and porcine stomach mucin. Exposure of engorged females of the cattle tick (Boophilus microplus) to 0.1 mg mL(-1) GCL significantly (P < 0.05) reduced the female weight after the oviposition period, the egg mass weight, the hatching period, and the mean larvae survival time.     

Total phenolic content, antioxidant activity, and cross-cultural consumer rejection threshold in white and red wines functionally enhanced with catechin-rich extracts

White and red wines spiked with catechin-rich green tea extract and grape seed extract were assessed for phenolic content, antioxidant activity, and cross-cultural consumer rejection thresholds in relation to wine as a functional food. Health functionality is an important factor in functional foods, and spiking pure compounds or plant extracts is an effective method to increase or control functionality. The total phenolic content and antioxidant activity were measured in wines spiked to different extract concentrations, namely, control and 50, 100, 200, 400, and 800 mg/L, to confirm the dose-response curves in both white and red wines. Consumer rejection thresholds (CRTs) were established for spiked wines in a Korean and in an Australian population. Our results showed that the green tea extract and grape seed extract increased the antioxidant activity dose dependently, and the CRTs varied considerably between the Korean and the Australian groups, with Koreans preferring wines spiked with green tea extract and Australians showing a preference for wines spiked with grape seed extract. These results have implications for producing wine products that are enhanced in phenolic compounds and targeted to different cultural groups.