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.     

Oxidative dimers produced from protocatechuic and gallic esters in the DPPH radical scavenging reaction

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging reactions of protocatechuic and gallic acids, and their methyl esters, have been investigated by NMR. In acetone, methyl protocatechuate was gradually converted to a Diels-Alder adduct of two molecules of the intermediate quinone in the reaction with DPPH radical, whereas methyl gallate rapidly gave a symmetrical dimer via a putative quinone precursor. Both dimers are rather unstable and their structures have been deduced by in situ NMR measurements of the reaction mixtures. Gallic acid also gave a corresponding symmetrical dimer in the same reaction as methyl gallate, although protocatechuquinone produced from protocatechuic acid did not yield a Diels-Alder adduct, unlike its methyl ester. Interestingly, these dimer formations were not observed in methanol solution.     

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。     

Isolation and identification of organosulfur compounds oxidizing canine erythrocytes from garlic (Allium sativum)

Five compounds oxidizing canine erythrocytes were isolated from an aqueous ethanol garlic extract by silica gel column chromatography and preparative thin-layer chromatography. On the basis of nuclear magnetic resonance, infrared spectroscopy, and mass spectrometry, they were identified as three known compounds: bis-2-propenyl trisulfide (1), bis-2-propenyl tetrasulfide (2), and bis-2-propenyl pentasulfide (3) as well as two novel compounds, bis-2-propenyl thiosulfonate (4) and trans-sulfuric acid allyl ester 3-allylsulfanyl-allyl ester (5). A mixture of compounds 1-3 and compounds 4 and 5 induced methemoglobin formation in canine erythrocyte suspension in vitro resulting in the oxidation of canine erythrocytes. These groups of characteristic organosulfur compounds contained in garlic probably contribute to oxidations in blood. The constituents of garlic have the potential to oxidize erythrocytes and hemoglobin, suggesting that foods containing quantities of garlic should be avoided for feeding dogs.     

Identification and quantification of phenolic compounds in bambangan (Mangifera pajang Kort.) peels and their free radical scavenging activity

Phenolic compounds and antioxidant capacity of acidified methanolic extract prepared from fully ripe bambangan (Mangifera pajang K.) peel cultivated in Sarawak, Malaysia, were analyzed. The total phenolic content (98.3 mg GAE/g) of bambangan peel powder (BPP) was determined by the Folin-Ciocalteu method. BPP showed a strong potency of antioxidant activity and was consistent with that of BHT and vitamin C as confirmed by the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity and FRAP (ferric-reducing antioxidant power) assays. Gallic acid, p-coumaric acid, ellagic acid, protocatechuic acid, and mangiferin were the major compounds among the 16 phenolics that have been identified and quantified in M. pajang peels with 20.9, 12.7, 7.3, 5.4, and 4.8 mg/g BPP, respectively. Peak identities were confirmed by comparing their retention times, UV-vis absorption spectra, and mass spectra with authentic standards. The 16 phenolic compounds identified in M. pajang K. using HPLC-DAD and TSQ-ESI-MS are reported here for the first time.     

Novel fluorometric assay for hydroxyl radical scavenging capacity (HOSC) estimation

A novel fluorometric method was developed and validated for hydroxyl radical scavenging capacity (HOSC) estimation using fluorescein as the probe. A constant flux of pure hydroxyl radical is generated under physiological pH using a Fenton-like Fe3+/H2O2 reaction. The generation of pure hydroxyl radicals under the experimental conditions was evaluated and confirmed using electron spin resonance with DMPO spin-trapping measurements. The hydroxyl radical scavenging capacity of a selected antioxidant sample is quantified by measuring the area under the fluorescence decay curve with or without the presence of the antioxidant and expressed as Trolox equivalents per unit of the antioxidant. The assay may be performed using a plate reader with a fluorescence detector for high-throughput measurements. The assay was validated for linearity, precision, accuracy, reproducibility, and its correlation with a popular peroxyl radical scavenging capacity assay using selected pure antioxidant compounds and botanical extracts. This method may provide researchers in the food, nutrition, and medical fields an easy to use protocol to evaluate free radical scavenging capacity of pure antioxidants and natural extracts in vitro against the very reactive hydroxyl radical, which may be linked to numerous degenerative diseases and conditions.     

Continuous hot pressurized solvent extraction of 1,1-diphenyl-2-picrylhydrazyl free radical scavenging compounds from Taiwan yams (Dioscorea alata)

This study investigates a semicontinuous hot pressurized fluid extraction process and the scavenging activity on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical of the extract from Taiwan yams (Dioscorea alata). Liquid-liquid extractions were preliminarily employed to generate six fractions, initially extracted by ethanol. Then, the aqueous solution of dried crude ethanol extract was sequentially fractionated by hexane, chloroform, ethyl acetate, and n-butanol. The EC50 value was defined as the UV absorption of DPPH concentrations sufficiently decreased to 50% of the original value. It was found that all peel portions have a better effect on scavenging of the DPPH free radical than meat portions, especially for the ethyl acetate partition of the peel portion of Tainung #2 yam. Its EC50 value (14.5 microg mL(-1)) was even lower than that of ascorbic acid (21.4 microg mL(-1)). Furthermore, semicontinuous hot pressurized ethanol was superior to hot pressurized water in extracting the compound scavenging the DPPH radical from the Purpurea-Roxb peel. The recovery of four unknown compounds corresponded to the scavenging ratio of DPPH free radical in the hot pressurized ethanol extract. Finally, three-level and four-factor experimental design revealed that ethanol ratio and temperature were the most effective factors in order. Conditions of 80% of aqueous ethanol, 20.0 kg/kg solid ratio, 180 psig (1.342 MPa), and 100 degrees C were preferred to extract those antioxidants from the yam peel.     

Isolation and identification of mosquito bite deterrent terpenoids from leaves of American (Callicarpa americana) and Japanese (Callicarpa japonica) beautyberry

Essential oil extracts from Callicarpa americana and Callicarpa japonica were investigated. Bioassay-guided fractionation of C. americana extracts using the yellow fever mosquito, Aedes aegypti, led to the isolation of alpha-humulene, humulene epoxide II, and intermedeol and a newly isolated terpenoid (callicarpenal). Similar work involving C. japonica resulted in the isolation of an additional compound, spathulenol, as well as the four compounds isolated from C. americana. Structure elucidation was performed on all isolated compounds using a combination of gas chromatography-mass spectrometry-electron ionization, high-resolution liquid chromatography-MS-electrospray ionization, and one- and two-dimensional NMR experiments. Heretofore, 13,14,15,16-tetranorclerodane, callicarpenal, has never been identified from natural sources. Complete (1)H and (13)C NMR assignment data are provided for this compound. In bite deterrent studies, spathulenol, intermedeol, and callicarpenal showed significant repellent activity against A. aegypti and Anopheles stephensi.     

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.     

Crocin bleaching assay (CBA) in structure-radical scavenging activity studies of selected phenolic compounds

The applicability of the crocin bleaching assay (CBA) to structure-activity relationship (SAR) studies of a great number (n = 39) of selected phenolic compounds was thoroughly investigated. The focus was on the activity of hydroxybenzoic, hydroxyphenylacetic, hydroxyphenylpropanoic, and hydroxycinnamic acids. Other assays [oxygen radical absorbance capacity (ORAC), lipid oxidation] were applied when necessary. Hydroxybenzoic acids were less active than the respective simple phenols. The position of the -COOH group relative to hydroxyl substituents was critical. The number and position of the -OH groups governed the order and size of activity within the subgroup of these acids. Gallic acid was the most active, being 1.6- and 3.4-fold superior to protocatechuic and syringic acids, respectively. The effect of proximity of the -COOH group to the phenyl ring was more distinct for 3,4-guaiacol acids (ferulic > dihydroferulic congruent with homovanillic > vanillic) than for 3,4-catechol ones (caffeic > protocatechuic > or = dihydrocaffeic congruent with homoprotocatechuic). Compounds such as vanillin, tyrosol, ferulic acid derivatives, rosmarinic acid, and quercetin were examined to reinforce discussion on the basis of physical organic chemistry principles. Taking into account the acidity of most compounds, the CBA-derived order of activity was meaningful.     

Screening of free radical scavenging compounds in water extracts of Mentha samples using a postcolumn derivatization method

An on-line high-performance liquid chromatography-1,1-diphenyl-2-picrylhydrazyl (HPLC-DPPH*) method has been improved for the detection of polar and nonpolar radical scavenging compounds in complex plant extracts. Nine water extracts were prepared from different Mentha species, varieties, hybrids, and cultivars. After the components within each extract had been separated by reverse phase chromatography using 10-100% methanol with 2% acetic acid as a mobile phase, analytes within the eluent capable of scavenging a citric acid-sodium citrate-buffered methanol 1,1-diphenyl-2-picrylhydrazyl solution were detected by postcolumn derivatization at 517 nm. The HPLC-DPPH* on-line method was applied to the qualitative and quantitative analysis of Mentha extracts. There was a strong correlation between the scavenging (negative) peak area and the concentration of the radical scavenging reference substances used. The minimum detectable concentration (microg/mL) of the antioxidant compounds was determined. Caffeic acid, eriocitrin (eriodictyol-7-O-rutinoside), luteolin-7-O-glucoside, and rosmarinic acid were identified as the dominant radical scavengers in these extracts by this method.     

Molecular identification of methane oxidizing bacteria in a Japanese rice field soil

By using cultivation-independent techniques, community changes of methane-oxidizing bacteria (MOB) in rice bulk soils were investigated under field conditions in a Japanese rice field. The representative soil samples were collected during the typical rice growing season and nonrice growing period all year round. Statistical characterization of denaturing gradient gel electrophoresis (DGGE) community patterns of MOB pmoA/amoA functional gene fragments showed that MOB community structures in the rice bulk soils remained largely unchanged throughout the investigated period. The total intensity of six common DGGE bands that appeared consistently throughout the investigated period accounted for 64% of the total intensity of all 18 different DGGE bands detected. The low squared distance of the Ward cluster analysis of the DGGE pattern and the high Sorensen similarity coefficient (81%) also implied the high similarity of the MOB community structures. The stable MOB community structure did not couple well with the wide variation of soil water contents all year round. Sequencing analysis of the nine characteristic bands including six common bands revealed the presence of Type I, Type II methanotrophs, and β-proteobacterial ammonia oxidizers in rice bulk soils. In comparison with MOB type species, three DGGE bands showed a wide variation of the highly conserved amino acid residues, implying the presence of novel MOB bacteria inhabiting the rice bulk soil. The high diversity of MOB composition suggested that rice bulk soils might serve as an ideal reservoir for the dynamic changes of MOB in a rice field ecosystem in response to environment changes.     

Isolation, identification, and quantification of roasted coffee antibacterial compounds

Coffee brew is a widely consumed beverage with multiple biological activities due both to naturally occurring components and to the hundreds of chemicals that are formed during the roasting process. Roasted coffee extract possesses antibacterial activity against a wide range of microorganisms, including Staphylococcus aureus and Streptococcus mutans, whereas green coffee extract exhibits no such activity. The naturally occurring coffee compounds, such as chlorogenic acids and caffeine, cannot therefore be responsible for the significant antibacterial activity exerted by coffee beverages against both bacteria. The very low minimum inhibitory concentration (MIC) found for standard glyoxal, methylglyoxal, and diacetyl compounds formed during the roasting process points to these alpha-dicarbonyl compounds as the main agents responsible for the antibacterial activity of brewed coffee against Sa. aureus and St. mutans. However, their low concentrations determined in the beverage account for only 50% of its antibacterial activity. The addition of caffeine, which has weak intrinsic antibacterial activity, to a mixture of alpha-dicarbonyl compounds at the concentrations found in coffee demonstrated that caffeine synergistically enhances the antibacterial activity of alpha-dicarbonyl compounds and that glyoxal, methylglyoxal, and diacetyl in the presence of caffeine account for the whole antibacterial activity of roasted coffee.     

Rapid peroxyl radical scavenging capacity (PSC) assay for assessing both hydrophilic and lipophilic antioxidants

This paper reports a simple, rapid, and sensitive assay for assessing peroxyl radical scavenging capacity (PSC) of both hydrophilic and lipophilic antioxidant compounds and food extracts. The assay is based on the degree of inhibition of dichlorofluorescin oxidation by antioxidants that scavenge peroxyl radicals, generated from thermal degradation of 2,2'-azobis(amidinopropane). For hydrophilic antioxidant activity, the dose required to cause a 50% inhibition of the reaction (EC(50)) ranged from 2.41 +/- 0.02 (EGCG) to 21.26 +/- 0.38 microM (ferulic acid). EC(50) values for the hydrophilic antioxidant activity of food extracts ranged from 309.2 +/- 3.63 (apple) to 3345.1 +/- 151.5 micromol of vitamin C equiv/100 g for wheat bran. The EC(50) values for lipophilic antioxidant activity were 1.58 +/- 0.11 (Trolox), 4.35 +/- 0.43 (alpha-tocopherol), 18.94 +/- 0.38 (BHA), and 182.69 +/- 13.7 microM (BHT). Whole grain lipophilic antioxidant activity ranged from 3.49 +/- 0.57 (wheat) to 8.79 +/- 1.98 micromol of alpha-tocopherol equiv/100 g of rice. Hydrophilic antioxidant activity contributed >98% of the total antioxidant activity (hydrophilic plus lipophilic) of whole grains tested. The PSC assay was accurate (86-108% recovery), precise (0.12-11% CV), and reproducible (12% RSD) and produced results comparable to those of similar published assays. The PSC assay can be routinely used to analyze or screen both hydrophilic and lipophilic antioxidants or food extracts and will be a valuable alternative biomarker for future epidemiological studies of chronic diseases.     

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.     

Isolation and characterization of aminopeptidase (Jc-peptidase) from Japanese cedar pollen (Cryptomeria japonica)

An aminopeptidase, Jc-peptidase, was purified from Japanese cedar pollen by seven steps, including precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration, hydrophobic interaction chromatography on phenyl-agarose, and high-performance liquid chromatography. Purified Jc-peptidease has a molecular weight of 42 kDa and hydrolyzes the synthetic substrates of L-phenylalanyl-4-methylcoumaryl-7-amide (Phe-MCA) with Km = 5 x 10(-5) M, Tyr-MCA with Km = 7 x 10(-4) M, Leu-MCA with Km = 1 x 10(-3) M, and Met-MCA with Km = 1 x 10(-3) M. Other MCA analogues such as Arg-MCA or Glu-MCA failed to serve as its substrates. The activity was inhibited in the presence of phebestin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl]-L-phenylalanine, with Ki = 4.7 x 10(-5) M, or bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, with Ki = 1.1 x 10(-4) M. According to amino acid sequence analysis, the N-terminal amino group seems to be blocked. The physiological function of the aminopeptidase (Jc-peptidase) has not been clarified in vivo.     

Isolation of phytotoxic compounds from tree-of-heaven (Ailanthus altissima swingle)

The aqueous root extract of Ailanthus altissima showed allelopathic activity against radish (Raphanus sativus L. cv. "Saxa"), garden cress (Lepidium sativum L.), and purslane (Portulaca oleracea L.) seeds. A bioassay-oriented purification of active extracts, chromatographic fractions, and compounds demonstrated dose-dependent activity on germination and radicle growth of test seeds; radish seed was the most sensitive to allelochemicals. Active compounds have been isolated: ailanthone, ailanthinone, chaparrine, and ailanthinol B (quassinoid derivatives); the alkaloid 1-methoxycanthin-6-one is not active. The compound with greatest inhibitory activity is ailanthone. The data obtained suggest a possible use of tree-of-heaven root extracts or of its active constituents as natural herbicides.