Isolation and identification of DPPH radical scavenging compounds in Kurosu (Japanese unpolished rice vinegar)

Dihydroferulic acid (DFA) and dihydrosinapic acid (DSA) were isolated from Kurosu (unpolished rice vinegar) as the major constituents responsible for Kurosu's radical scavenging activity. The levels of antioxidative activity of DFA and DSA in DPPH radical scavenging were higher than those of their respective structurally related compounds, ferulic acid and sinapic acid. The concentrations of DFA and DSA were low in common rice vinegar (polished rice vinegar), suggesting that Kurosu is more advantageous than rice vinegars as an antioxidative food item. As the concentrations of DFA and DSA were low in unpolished rice, too, these acids are thought to be produced in Kurosu through the process of the fermentation from ferulic acid and sinapic acid, respectively.     

Quantitative analysis of anti-inflammatory and radical scavenging triterpenoid esters in evening primrose oil

Cold pressed, nonraffinated evening primrose oil (EPO) was recently found to contain lipophilic triterpenoidal esters with radical scavenging and anti-inflammatory properties. A simple and robust method for the quantitative analysis of these 3-O-trans-caffeoyl derivatives of betulinic, morolic, and oleanolic acid was developed and validated. Separation was achieved by normal phase chromatography on a Diol column and with hexane/ethyl acetate (50:50) as eluent. The analytes could be determined directly in the oil matrix, without need of a previous removal of the triglycerides. Normal phase LC ESI-MS with a makeup flow of polar modifier was used for checking the identity and purity of analyte peaks. Samples from 22 commercially available EPOs were analyzed. The average caffeoyl ester contents were 58 mg/100 g in cold pressed oils and 4.7 mg/100 g in partially raffinated oils. In fully raffinated EPO samples, the concentration was below the limit of detection. The influence of extraction temperature on the content of caffeoyl esters in nonraffinated EPO was investigated with seeds of Oenothera biennis and Oenothera lamarckiana, respectively. With O. lamarckiana, the concentration of caffeoyl esters in the oil increased with rising pressure and temperature, whereas no such dependency was found with O. biennis. Microscopic analysis revealed some differences in the histology of the seed testa, which may explain in part the differing behaviors in the extraction experiments. There was a difference between O. biennis and O. lamarckiana oils with respect to the relative amounts of the three esters. The temperature of the extraction process had no effect on the ratio of the compounds.     

Quantitative analysis of anti-inflammatory and radical scavenging triterpenoid esters in evening primrose seeds

Lipophilic triterpenoidal esters with radical scavenging and cyclooxygenase inhibitory properties were recently found in cold-pressed, nonraffinated evening primrose oil (EPO). A quantitative assay for the analysis of 3-O-trans-caffeoyl derivatives of betulinic, morolic, and oleanolic acid in evening primrose seeds was developed and validated. Extraction efficiency >99% was achieved by means of pressurized liquid extraction with two extraction cycles and 80% (v/v) ethanol at 120 degrees C. Analysis of esters was by normal-phase high-performance liquid chromatography on a Diol column and hexane/ethyl acetate (containing 0.1% formic acid) (65:35) as the eluent. The analytes were determined without further prepurification. Seeds from defined cultures of Oenothera biennis, Oenothera lamarckiana, and Oenothera ammophila, grown under identical conditions, were analyzed. The cultures originated from seeds from eight collections in the wild and from selections from five cultivars. The content of total triterpenoidal esters in seeds varied between 1.34 and 2.78 mg/g. Three types of qualitative patterns were observed for the triterpenoidal esters. The influence of different harvest times and plant treatments was studied with the cultivar Anothera. Variations between 1.5 and 2.3 mg/g were found.     

Free radical scavenging behavior of antioxidant compounds of sesame (sesamum indicum L.) in DPPH(*) system

The free radical scavenging capacity (RSC) of antioxidants from sesame cake extract was studied using the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH(*)()) on a kinetic model. Pure lignans and lignan glycosides isolated from methanolic extract by preparative HPLC were used in the study. To understand the kinetic behavior better and to determine the RSC of sesame antioxidants, the second-order rate constant (k(2)) was calculated for the quenching reaction with [DPPH(*)] radical. The k(2) values of the sesame antioxidants were compared with those of butylated hydroxytoluene and alpha-tocopherol. The k(2) values for sesamol, sesamol dimer, sesamin, sesamolin, sesaminol triglucoside, and sesaminol diglucoside were 4.00 x 10(-)(5), 0.50 x 10(-)(5), 0.36 x 10(-)(5), 0.13 x 10(-)(5), 0.33 x 10(-)(5), and 0.08 x 10(-)(5) microM(-)(1) s(-)(1), respectively.     

银杏花粉黄酮组分分离纯化及其清除DPPH自由基能力

为了分离纯化银杏花粉中具有潜在抗氧化活性的黄酮组分及研究其潜在抗氧化活性,该文对银杏花粉的总黄酮进行提取和分步萃取,采用1,1-diphenyl-2-picrylhydrazyl(DPPH)自由基清除率检测和组分分析锁定待纯化萃取相。通过DPPH-HPLC-PAD技术筛选目标清除自由基组分,并采用制备型液相色谱技术分离纯化这些组分,然后采用紫外可见吸收光谱、质谱、核磁共振氢谱、碳谱等技术进行结构鉴定,最后通过测定DPPH自由基清除率初步研究其潜在抗氧化活性。结果显示,银杏花粉粗提物具有清除DPPH自由基活性,对DPPH的半抑制率(IC50值)为1.32 mg/m L,乙酸乙酯相和正丁醇相的IC50值为0.46、0.84 mg/m L,分别是粗提物的34.85%和63.64%。可见,乙酸乙酯相的清除DPPH活性最高。进一步研究显示,乙酸乙酯相富集了6种主要银杏花粉黄酮,而且均具有清除DPPH活性。经纯化和结构鉴定分别为山奈酚-3,4’-双-O-β-D-葡萄糖苷(1)、山奈酚-3-O-β-D-葡萄糖基-7-O-α-L-鼠李糖苷(2)、山奈酚-3-O-β-D-葡萄糖苷(3)、山奈酚-3-O-α-L-鼠李糖苷(4)、柚皮素(5)和山奈酚(6)。其清除DPPH自由基的活性由高到低依次为:化合物6化合物2化合物3化合物4化合物1化合物5。其中山奈酚清除DPPH的IC50值为0.017 mg/m L,与维生素C及芦丁相比没有显著性差异(P0.05)。上述研究为银杏花粉功能成分的深入研究奠定了基础。     

Stability to light, heat, and hydrogen peroxide at different pH values and DPPH radical scavenging activity of acylated anthocyanins from red radish extract

The stability of red radish extract to light, heat, and hydrogen peroxide at different pH values (3, 5, and 7) was examined, in which major anthocyanins were pelargonidin glycosides acylated with a combination of p-coumaric, ferulic, or caffeic acids. The light irradiation (fluorescence light, 5000 lx; at 25 degrees C) indicated that the red radish extract was more stable at lower pH than at higher pH. The HPLC analyses revealed that diacylated anthocyanins in the extract were more stable to light at pH 3 than monoacylated anthocyanins. No significant difference in degradation rates of acylated anthocyanins at pH 5 was observed, whereas anthocyanins acylated with p-coumaric or ferulic acids were more stable at pH 7 than ones with caffeic acids. The stability to heat (at 90-95 degrees C) showed a tendency similar to that for light. The number of intramolecular acyl units contributes to stability to light and heat at lower pH, whereas the characteristics of intramolecular acyl units influence the stability at higher pH. The degradation behavior of red radish extract to H2O2 were almost the same to those of light and heat, depending on the pH. However, HPLC analyses revealed that the stability of individual acylated anthocyanins were independent of the pH. These data suggest that the characteristics, the number, and the binding site of intramolecular acyl units affect the stability of anthocyanin to H2O2. DPPH radical scavenging activity of all acylated anthocyanins was higher than those of pelargonidin and perlargonidin-3-glucoside. The activity of acylated anthocyanins mostly depended on the activity of intramolecular acyl units (caffeic acid > ferulic acid > p-coumaric acid). However, the activity was highly affected by the binding site of intramolecular acyl units even if anthocyanins have common acyl units.     

Quantitative kinetic analysis of hydrogen transfer reactions from dietary polyphenols to the DPPH radical

Diphenylpicrylhydrazyl (DPPH) is widely used for quickly assessing the ability of polyphenols to transfer labile H atoms to radicals, a likely mechanism of antioxidant protection. This popular test generally pays no attention to the kinetics of H atom transfer, which however could be even more important than the total H-atom-donating capacities (stoichiometry, EC50) typically evaluated. In the present work, a series of dietary polyphenols belonging to the most representative families (flavonols from onion, flavanol monomers and oligomers from barley, and caffeic acid and caffeoyl esters from artichoke and endive) are characterized not only by their total stoichiometries (n(tot)) but also by their rate constants of first H atom abstraction by DPPH (k(1)), deduced from the kinetic analysis of the decay of the DPPH visible band following addition of the antioxidant. The mildly reactive DPPH radical allows a good discrimation between polyphenols, as demonstrated by the relatively large ranges of k(1) (ca. 400-5000 M(-)(1) s(-)(1)) and n(tot) (ca. 1-5) values typically measured with antioxidants having a single polyphenolic nucleus. With antioxidants displaying more than one polyphenolic nucleus (procyanidin oligomers, dicaffeoyl esters), the kinetic analysis makes it possible to demonstrate significant differences in reactivity between the subunits (two distinct k(1) values whose ratio lies in the range 3-10) and nonadditive stoichiometries.     

Assessment of antioxidant activity of cane brown sugars by ABTS and DPPH radical scavenging assays: determination of their polyphenolic and volatile constituents

Seven cane brown sugars (four from La Réunion, two from Mauritius, and one from France) were investigated for their polyphenol content and volatile composition in relation to their free radical scavenging capacity determined by ABTS and DPPH assays. The thin layer coated on the sugar crystal was extracted by Soxhlet extractor with dichloromethane. The volatile compounds of brown sugars were studied by GC-MS, and 43 compounds were identified. The total phenolic content of brown sugars was determined according to the Folin-Ciocalteu method. Phenolic compounds were quantified in the brown sugar extracts by LC-UV-ESI-MS. Brown sugar aqueous solutions exhibited weak free radical scavenging activity in the DPPH assay and higher antioxidant activity in the ABTS assay at relatively high concentration. The brown sugar extracts showed interesting free radical scavenging properties despite the low concentration of phenolic and volatile compounds. Sugar is a common foodstuff traditionally used for its sweetening properties, which might be accompanied by antioxidant properties arising from molecules (polyphenols, Maillard products) other than sucrose of the cane brown sugars.     

Screening for antioxidant activity in edible plant products: comparison of low-density lipoprotein oxidation assay, DPPH radical scavenging assay, and Folin-Ciocalteu assay

Oxidation of low-density lipoprotein (LDL) has been implicated in atherogenesis. Antioxidants that prevent LDL from oxidizing may reduce atherosclerosis. This study investigated LDL antioxidant activity in edible plant products for development of dietary supplementation to prevent atherosclerosis. Fifty-two kinds of edible plants were extracted using 70% aqueous ethanol solution, and the antioxidant activity of the extracts, which inhibit human LDL oxidation induced by copper ion, was determined on the basis of the oxidation lag time and represented as epigallocatechin 3-gallate equivalent. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and total phenolic content were also measured for comparisons with antioxidant activity in LDL. Plant products showing the greatest activity in LDL oxidation assay were akamegashiwa (Mallotus japonicus) leaf, Japanese privet (Ligustrum japonicum) leaf, green tea [Camellia sinensis (L.) O. Kuntze], and astringent persimmon (Diospyros kaki). The present study revealed high levels of LDL antioxidant activity in plant products for which such activity levels are underestimated in the DPPH radical scavenging assay and Folin-Ciocalteu assay.     

Superoxide radical scavenging activity of the major polyphenols in fresh plums

The effect of polyphenols among different varieties of plums on superoxide radical scavenging activity (SRSA) was studied by an enzymatic method and their IC(50) values were determined. We found that the SRSA levels of the polyphenols were closely related to their chemical structures; cyanidin showed the lowest IC(50) among the polyphenols examined, and aglycones are more effective than their glycosides. BY 69-339 cultivar exhibited the lowest IC(50) among the eleven plum cultivars, which means the highest antioxidant activity in scavenging superoxide radicals, followed by French Damson, Cacaks Best, Beltsville Elite B70197, Empress, Castleton, Stanley, NY 6, NY 101, Mirabellier, and NY 9. IC(50) values showed a higher correlation with total flavonoids (r (2) = 0.8699) than total phenolics (r (2) = 0.8355), which indicated that flavonoids might contribute to the total SRSA more directly than other polyphenols. Anthocyanins in plums appeared to be the major contributors to the total SRSA, except for two yellow cultivars having no anthocyanins. Chlorogenic acid was the predominant phenolic acid, and it also exhibited SRSA significantly in the range of 1.0 to 94.9%. Quercetins were the major flavonols in plums. However, they showed relatively low contribution to the total SRSA.     

Free radical scavenging activity and antiulcer activity of garcinol from Garcinia indica fruit rind

Garcinol, a polyisoprenylated benzophenone derivative, was purified from Garcinia indica fruit rind, and its free radical scavenging activity was studied using electron spin resonance (ESR) spectrometry. In the hypoxanthine/xanthine oxidase system, emulsified garcinol suppressed superoxide anion to almost the same extent as DL-alpha-tocopherol by weight. In the Fenton reaction system, garcinol also suppressed hydroxyl radical more strongly than DL-alpha-tocopherol. In the H(2)O(2)/NaOH/DMSO system, garcinol suppressed superoxide anion, hydroxyl radical, and methyl radical. It was thus confirmed that this derivative is a potent free radical scavenger and able to scavenge both hydrophilic and hydrophobic ones including reactive oxygen species. Orally administered garcinol prevented acute ulceration in rats induced by indomethacin and water immersion stress caused by radical formation. These results suggested garcinol might have potential as a free radical scavenger and clinical application as an antiulcer drug.     

Free radical scavenging capacity in the aging of selected red Spanish wines

Free radical scavenging capacity by the DPPH(*) method and main physicochemical properties, polyphenols content by HPLC, color by a tristimulus colorimeter, and UV-vis spectra in the aging of selected red Spanish wines, were studied. As the wines age, they become darker (lower lightness, L) and increase their hue angle (lower red color) as well as the ratio of absorbance at 420 nm to that at 520 nm. Main polyphenolics identified in the samples were tannic acid, oenin, and gallic acid. The antiradical efficiency of the samples increased during aging, which could be related to an increase in the tannic acid concentration shown by the following correlationship: EC(50) = 1/(0.18 + 0.0011[tannic acid](mg/L)) with a correlation coefficient of 0.744.     

Influence of jam processing on the radical scavenging activity and phenolic content in berries

Six selected phenolic aglycons (caffeic and ellagic acids, kaempferol, quercetin, myricetin, and morin) in nine types of berries, and their changes as influenced by jam processing, have been evaluated using optimized HPLC with diode-array detection. The berry samples, fresh and after jam processing, were analyzed, and the total amounts of selected phenolics as aglycons were identified and determined by acid hydrolysis. Their contents in fresh and jam samples did not indicate appreciable changes; therefore, the influence of jam processing on these selected phenolics in berries was suggested to be small, and was mostly present in berries as several conjugated forms that were glycosylated, esterified, etc., in the samples. The total phenolic content of each sample was also determined by the Folin-Ciocalteu method. The three samples of each berry, namely fresh, jam, and acid hydrolysate of the berry, had similar total phenolic contents. On the other hand, the scavenging effect on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was measured, and acid hydrolysates showed stronger activity than that of the fresh and jam-processed samples for all of the berry types.     

Antioxidants and radical scavenging properties of vegetable extracts in rats fed aflatoxin-contaminated diet

The present study evaluated the protection role of garlic, cabbage, and onion extracts against the toxic effects of aflatoxin. One hundred and twenty mature male Sprague-Dawley rats were randomly assigned to eight experimental groups and treated for 15 days with extracts with or without aflatoxin. Blood samples were collected from all animals from the retro-orbital venous plexus at the end of the experimentation period for biochemical analysis. Livers and kidneys were removed at the end of the treatment period for determination of glutathione, malondialdehyde, and superoxide dismutase. The results indicated that animals treated with aflatoxin showed significant signs of aflatoxicosis. Extracts alone had insignificant effects on all parameters tested, whereas cotreatment with aflatoxin and extracts resulted in a significant improvement in all parameters; moreover, garlic extract was found to be the most effective in the prevention of aflatoxin-induced toxicity and free radical generation in rats.     

Age-induced diminution of free radical scavenging capacity in bee pollens and the contribution of constituent flavonoids

Bee-collected pollen ("bee pollen") is promoted as a health food with a wide range of nutritional and therapeutic properties. The objective of the current study is to evaluate the contribution made through the free radical scavenging capability of bee-collected floval pollens by their flavonoid/phenolics constituents, and to determine whether this capability is affected by aging. The free radical scavenging effectiveness of a bee pollen (EC(50)) as measured by the DPPH method is shown to be determined by the nature and levels of the constituent floral pollens, which can be assayed via their phenolics profiles by HPLC. Each pure floral pollen has been found to possess a consistent EC(50) value, irrespective of its geographic origin or date of collection, and the EC(50) value is determined to a large extent (ca. 50%) by the nature and the levels of the pollen's flavonoids and phenolic acids. Non-phenolic antioxidants, possibly proteins, account for the balance of the activity. Pollen aging over 3 years is demonstrated to reduce the free radical scavenging activity by up to 50% in the most active floral pollens, which tend to contain the highest levels of flavonoids/phenolic acids. It is suggested that the freshness of a bee pollen may be determined from its free radical scavenging capacity relative to that of fresh bee pollen containing the same floral pollen mix.     

Polyphenol levels and free radical scavenging activities of four apple cultivars from integrated and organic farming in different italian areas

This paper investigates the influence of cultivar (Annurca, Golden Delicious, Red Chief, and Stayman Neepling), rural practice (integrated and organic), and growing region (different Italian regions) on polyphenol composition and antiradical activity of the pulp and skin of apples, as presented to the consumer at the market. Antiradical activity of fruit was strongly related with the total polyphenolic content, determined both by the spectrophotometric Folin-Ciocalteu method ( R (2) = 0.90; P < 0.01) and by HPLC ( R (2) = 0.85; P < 0.01). Considering the edible portion of the fruit, polyphenolics contribute toward explaining approximately 90% of the overall antiradical activity, thus highlighting their important role in human health protection. Therefore, the data indirectly indicated that ascorbic acid and other antiradical molecules differing from polyphenols play a much less important role in explaining the health-protecting properties of apples. Cultivar effect was by far the most important, and Annurca and Golden Delicious were respectively the best and the worst apples from the point of view of the health-protecting attributes.     

Enhancing antioxidant, antiproliferation, and free radical scavenging activities in strawberries with essential oils

Several natural antimicrobial compounds derived from essential oils of plants were investigated for their efficacies in inhibiting decay and extending the shelf life of strawberries (Fragaria x ananassas Duch.). The severity of decay in strawberries stored at 10 degrees C was significantly reduced by treatment with thymol. Treatments with menthol or eugenol also suppressed the fungal growth, but to a lesser extent. All of these three natural antimicrobial compounds extended shelf life of strawberries as compared to the control. Strawberries treated with thymol, menthol, or eugenol also maintained better fruit quality with higher levels of sugars, organic acids, phenolics, anthocyanins, flavonoids, and oxygen radical absorbance capacity than the untreated fruits. The free radical scavenging properties of strawberry fruit were evaluated against 2,2-diphenyl-1-picryhydrazyl (DPPH(.)), hydroxyl (HO(.)), and superoxide radicals (O2(.-)) using electron spin resonance measurements. Higher radical scavenging capacities were found against DPPH(.) and HO(.) in all treated fruit, particularly in berries treated with thymol, compared to those in the control groups. In addition, strawberry extracts were evaluated for their antiproliferative activities using HT-29 colon cancer cells. Extracts from all treated fruit exhibited significantly stronger inhibition on HT-29 cell proliferation than those from the control fruit. These data provide evidence that, in addition to possessing antimicrobial activity, the essential oils also increase free radical scavenging capacity and antiproliferative activity in fruit and, in turn, enhance the resistance of fruit tissues to deterioration and spoilage.     

Effect of high-pressure treatment on the carotenoid composition and the radical scavenging activity of persimmon fruit purees

The carotenoid composition of persimmon fruit purees of two cultivars, cvs. Rojo Brillante and Sharon, grown in Spain was determined by HPLC to assess the effects of high-pressure processing on some sensory (carotenoids), nutritional (provitamin A value), and health-related (radical-scavenging capacity) parameters. Total carotenoid content was higher in untreated Rojo Brillante puree (22. 11 microg g(-)(1)) than in untreated Sharon puree (15.22 microg g(-)(1)). Purees of both untreated cultivars showed similar carotenoid patterns after saponification with beta-cryptoxanthin, beta-carotene, and zeaxanthin as the main pigments. A high content of lycopene was quantified in Rojo Brillante (5.34 microg g(-)(1)), whereas only traces were detected in Sharon. The provitamin A value, reported as retinol equivalents (RE), was in untreated Rojo Brillante puree (77 RE/100 g) similar to that of Sharon (75 RE/100 g). Scavenging free radical capacity, measured as antiradical efficiency (AE), showed in untreated Rojo Brillante puree a value (12.14 x 10(-)(3)) 8.5 times higher than that in untreated Sharon (1. 42 x 10(-)(3)). Nonuniform behavior of high-pressure treatment was detected. Pressure treatments at 50 and 300 MPa/15 min/25 degrees C for Rojo Brillante and at 50 and 400 MPa/15 min/25 degrees C for Sharon increased the amount of extractable carotenoids (9-27%), which are related with the increase of vitamin A value (75-87 RE/100 g). No correlation with the increase of AE (from 1.42 x 10(-)(3) to 16.73 x 10(-)(3) and 19.58 x 10(-)(3)) after some pressure treatments (150 and 300 MPa/15 min/25 degrees C) was found.     

New qualitative approach in the characterization of antioxidants in white wines by antioxidant free radical scavenging and NMR techniques

The aim of this study was to obtain new information on antioxidant compounds in white wines. For this purpose, white wine degradation was promoted by a forced aged protocol, and six normally aged white wines from different vintages were analyzed. Both normal and forced aged wines were sequentially extracted using hexane and ethyl acetate. Apolar antioxidants were removed using hexane, and polar antioxidants were extracted with ethyl acetate. This last residue was subject to partial re-extraction with hexane and acetone. The antioxidant capacity of the wines and of each fraction was evaluated by two free radical methods, ABTS and DPPH. Normal aging provides a decrease in the total antioxidant capacity of wines. The antioxidant activity of ethyl acetate/acetone extracts was approximately 95% higher than that found for the hexane extracts. Concerning the forced aged wines, results showed that the wine submitted to a temperature of 60 degrees C for 21 days had higher antioxidant activity than that submitted to a temperature of 20 degrees C. With regard to the ethyl acetate/acetone extracts, oxygen and temperature treatment leads to a decrease in their antioxidant activity. NMR analysis was performed in the highest antioxidant capacity organic fractions (ethyl acetate/acetone extracts) and in the aqueous fraction of the control wine (T = 20 degrees C), in order to attempt the characterization of species involved in oxygen protection. Possible structures of antioxidant compounds in white wines were proposed. Two of these are tyrosol-like structures. This molecule is a well-known phenolic compound in wine, and it is reported to have antioxidative effects.