Monoamine oxidase B and free radical scavenging activities of natural flavonoids in Melastoma candidum D. Don.

Monoamine oxidase type B (MAO-B) activity and free radicals are elevated in certain neurological diseases. Four natural flavonoids, quercitrin, isoquercitrin, rutin, and quercetin, were isolated for the first time from the leaves of Melastoma candidum D. Don. They exhibited an inhibitory effect on MAO-B. These potent flavonoids were purified using bioassay-guided fractionation and were separated by Diaion, Sephadex LH-20, and MCI CHP20P columns. The IC(50) values of the four potent flavonoids, quercitrin, isoquercitrin, rutin, and quercetin on monoamine oxidase were 19.06, 11.64, 3.89, and 10.89 microM and enzyme kinetics analysis revealed apparent inhibition constants (K(i)) of 21.01, 2.72, 1.83, and 7.95 microM, respectively, on the substrate, benzylamine. The four potent compounds also exhibited hydroxyl radical scavenging activity as determined using a spin trapping electron spin resonance method. This suggests that the four flavonoids from M. candidum possess both MAO-B inhibitory and free radical scavenging activities. These important properties may be used for preventing some neurodegenerative diseases in the future.     

Simple assessment of radical scavenging capacity of beverages

The radical-scavenging antioxidants play an important role against oxidative stress in the defense system in vivo. The beneficial effects of antioxidants contained in foods and beverages have been well-accepted, and their antioxidant capacity has been assessed by various methods. In the present study, a simple method is proposed in which the total radical scavenging capacity is assessed from the bleaching of pyranine and pyrogallol red induced by free radicals generated from azo initiator. The total content of antioxidants contained in red wine, green tea, and cassis drink and their reactivities toward peroxyl radicals were measured from the lag phase and rate of bleaching using pyranine and pyrogallol red as a probe, respectively. It was found that this method to follow the bleaching of two probes by visible light spectrophotometer is convenient and applicable for assessment of total radical scavenging capacity of both content and activity of the antioxidants contained in beverages.     

pH-Dependent radical scavenging capacity of green tea catechins

The effect of pH on the radical scavenging capacity of green tea catechins was investigated using experimental as well as theoretical methods. It was shown that the radical scavenging capacity of the catechins, quantified by the TEAC value, increases with increasing pH of the medium. Comparison of the pKa values to theoretically calculated parameters for the neutral and deprotonated forms indicates that the pH-dependent increase in radical scavenging activity of the catechins is due to an increase of electron-donating ability upon deprotonation. The data also reveal that the radical scavenging activity of the catechins containing the pyrogallol (or catechol) and the galloyl moiety over the whole pH range is due to an additive effect of these two independent radical scavenging structural elements. Altogether, the results obtained provide better insight into the factors determining the radical scavenging activity of the catechins and reveal that the biological activity of green tea catechins will be influenced by the pH of the surrounding medium or tissues.     

Assessment of radical scavenging capacity and lipid peroxidation inhibiting capacity of antioxidant

The role of radical scavenging antioxidants against oxidative stress has received much attention, and the antioxidant capacity has been assessed by various methods. Among them, a method that measures the effect of antioxidant on decay of the probe is one of the most widely used methods. The present study was performed to compare the two methods to assess the antioxidant capacity, one to follow the decay of the probe and the other to measure lipid peroxidation products in human plasma. It was shown that the method following probe decay was suitable for assessment of radical scavenging capacity of antioxidant, but not for the capacity to inhibit lipid peroxidation in plasma. This is true whether a hydrophilic or lipophilic probe is used. Such different results arise from the fact that the efficacy of inhibition of lipid peroxidation by antioxidants depends on the fate of antioxidant-derived radical and interaction between antioxidants as well as the capacity of free radical scavenging. Thus, the capacity of antioxidants for inhibition of lipid peroxidation should be assessed from the effect on the extent of oxidation, not from the effect on probe decay.     

High-throughput quantitation of peroxyl radical scavenging capacity in bulk oils

Autoxidation of methyl linoleate (8:2 mixture with decane, 37 degrees C) was induced by 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN, 17.7 mM) and the kinetics of oxygen consumption monitored using a 96-well microplate coated with an oxygen-sensitive fluorescence probe, a ruthenium dye, embedded in a silicone matrix at the bottom of the microplate. The probe does not participate in the reaction; instead, its fluorescence intensity is inversely proportional to the solution oxygen concentration as it changes during oxidation. In the absence of antioxidants, the oxidation rate has a linear relationship with the square root of the initiator concentrations. This is in agreement with theoretical autoxidation kinetics equations. In the presence of tocopherol-type antioxidants, a sharp lag phase appears. The quantitation of the antioxidant capacity is achieved using the area under the curve (AUC) approach. The assay has a 2 h running time, a linearity range from 1.56 to 18.7 microM (Trolox), and a limit of quantitation at 2.7 microM Trolox equivalency. The peroxyl radical scavenging capacities of several cold-pressed and organically grown plant seed oils were quantified along with the tocopherol concentrations of the oils. Tocopherols contribute only a fraction of the peroxyl radical scavenging capacity of the oils, and there is poor correlation between total tocopherol concentrations and radical scavenging capacity, suggesting that the antioxidant capacity of oils is due not only to tocopherols but also to other lipid-soluble antioxidants.     

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.     

Conjugated linoleic acid isomers differ in their free radical scavenging properties

Conjugated linoleic acid (CLA) isomers were investigated for free radical scavenging properties against the stable 2,2-diphenyl-1-picryhydrazyl radical (DPPH(*)) by electron spin resonance (ESR) spectrometry and spectrophotometric methods. ESR measurements confirmed that both c9,t11-CLA and t10,c12-CLA directly reacted with and quenched DPPH radicals, whereas spectrophotometric analysis demonstrated that c9,t11-CLA and t10,c12-CLA differed in their kinetic and thermodynamic properties in reacting with DPPH radicals. t10,c12-CLA was shown to exhibit a greater initial velocity in CLA-DPPH radical reactions at levels of 2.5-80 mg/mL, and c9,t11-CLA scavenged more DPPH radicals at steady state. Similar dose and time relationships were observed for both isomers. In addition, a mixture of c9,t11- and t10,c12-CLA isomers demonstrated a greater initial velocity in quenching DPPH radicals than either isomer alone on the same concentration basis, suggesting that a synergistic effect between CLA isomers existed in their reactions with DPPH radicals. These results support the conclusion that individual CLA isomers differ in their biological actions and indicate that interaction(s) between isomers may contribute to their beneficial effects.     

Electron spin resonance estimation of hydroxyl radical scavenging capacity for lipophilic antioxidants

Hydroxyl radical scavenging capacity estimation for lipophilic antioxidants is a challenge due to their poor solubility in aqueous radical generating and measuring systems. In this study, an electron spin resonance (ESR) method was developed and validated for its application in estimating the relative hydroxyl radical (HO*) scavenging capacity for lipophilic antioxidants under physiological pH using a Fenton Fe2+/H2O2 system for radical generation and acetonitrile as a solvent. The Fenton Fe2+/H2O2 system generates a constant flux of pure HO* under the assay conditions. The method was validated by linearity, precision, and reproducibility using selected known lipophilic antioxidants including alpha-tocopherol, lutein, beta-carotene, and BHT. The potential effects of commonly used water-miscible and water-immiscible organic solvents on the Fenton Fe2+/H2O2 HO* generating system as well as their possible interactions with the fluorescent and spectroscopic probes were also reported. In addition, the limitation of the ESR assay was described.     

Determination of peroxyl radical scavenging activity of flavonoids and plant extracts using an automatic potentiometric titrator

A novel potentiometric method for evaluation of peroxyl radical scavenging activity of flavonoids and plant extracts was developed. The oxidation of potassium iodide (KI) was performed in acetonitrilephosphate buffer (1:1) containing antioxidant using 2,2'-azobis(2-amidinopropane) dihydrochloride as a peroxyl radical generator. The amount of iodine released from KI during a 20-min free radical oxidation was determined quantitatively using an automatic potentiometric titrator with sodium thiosulfate. The radical scavenging activity of the sample was expressed as the inhibition ratio for iodine release of the control group mediated by the radical. The results obtained from some authentic polyphenols correlated well with those of previous reports. This is a simple, time-saving method requiring less than 30 min and is useful in assessing the radical scavenging activity of antioxidants in plant extracts. We describe the radical scavenging activities of various flavonoids including 21 kinds of tea catechins and vegetable extracts by this method.     

Contribution of proanthocyanidins to the peroxy radical scavenging capacity of some Italian red wines

Highly reactive radicals, ROO(*), were generated from 2, 2'-azobis[2-(2-imidazolin-2-yl)propane] and linoleic acid. The ROO(*) scavenging capacity of some Italian red wines was evaluated following the changes in oxygen consumption. Under the experimental conditions the time course of oxygen consumption shows two typical behaviors: trolox-like (class I) and gallic acid-like (class II). Usually the time course of wine was similar to that of gallic acid. The rate of oxygen consumption was found to decrease exponentially with the amount of wine or gallic acid added to the test solution. On this basis the capacity of red wines to scavenge peroxy radicals was expressed as content of gallic acid (S(GA)). The S(GA) values were found to be correlated to the amount of total proanthocyanidins and total polyphenols of some Italian red wines (p < 0.01). The proanthocyanidins extracted from seeds were shown to make a major contribution to the peroxy radical scavenging capacity of red wines, whereas, interestingly, the chemical class of the low molecular weight tannins reactive to vanillin did not correlate with the S(GA) values.     

Preservation of kombucha tea-effect of temperature on tea components and free radical scavenging properties

Kombucha tea is sugared black tea fermented with a consortium of acetic acid bacteria and yeasts (tea fungus) for 14 days. The tea tastes slightly sweet and acidic. The formation of tea fungal biofilms during storage is a big problem when kombucha tea is being stored and commercialized. Various thermal treatments have been tried for long-term storage of kombucha tea. The present study revealed the influence of heat on the biochemical constituents and the free radical scavenging properties of kombucha tea. Heat treatment at 60, 65, and 68 degrees C for 1 min controlled biofilm formation in kombucha tea without changing its clarity, taste, and flavor. However, tea polyphenols and black tea quality parameters showed varying stability during the storage period. A decrease in free radical scavenging properties was also found during the storage period. Because the biological activities of kombucha tea depended on the biochemical constituents, it was concluded that heat treatment was not a suitable method for kombucha tea preservation.     

Stable free radicals and peroxyl radical trapping capacity in red wines

Thirty-two experimental red wines, obtained from eight cultivars and aged in bottles for 2 and 7 years, were examined for the presence of stable free radicals (SFR), for the peroxyl radical trapping capacity (PRTC), and for the concentrations of some important polyphenol families. Aging significantly increases SFR, polyphenol polymers with n > or = 5 (HMWP), and PRTC and is accompanied by a strong decrease of free anthocyanins. Multivariate regression analyses show that HMWP and SFR are independently associated with PRTC while HMWP and anthocyanins are independently associated with the formation of SFR. These results indicate that polymeric polyphenols generated from anthocyanins and proanthocyanidins during wine aging are able to convert highly reactive free radicals into nonreactive radicals through electron delocalization. The strict correlation between SFR and antioxidant activity that we found suggests that these characteristics are related to the functional properties of food.     

Antioxidant activity of South African red and white cultivar wines: free radical scavenging

The free radical scavenging activity of South African red (n = 46) and white (n = 40) cultivar wines was determined using 2,2'-azinobis(3-ethylbenzothialozinesulfonic acid) radical cations (ABTS(.+)) and 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH.). The total antioxidant activities (TAA) of red and white wines using ABTS(.+) were 14.916 and 0.939 mM Trolox, respectively, at corresponding total phenol (TP) contents of 2339.0 and 273.8 mg of gallic acid equiv/L. Ruby Cabernet wines had the lowest TAA(ABTS) (13.177 mM Trolox) of the red wines, whereas the TAA(ABTS) values of Chardonnay and Chenin blanc wines were the highest (1.060 mM Trolox) and lowest (0.800 mM Trolox) of the white wines. The TAA(DPPH) values were of the same magnitude as the TAA(ABTS) values, and similar trends were observed. TAA correlated (P < 0.001) with total phenol content of red (r = 0.935) and white (r = 0.907) wines, as well as flavanol content of red wines (r = 0.866) and tartaric acid ester content of white wines (r = 0.767). Canonical discriminant analysis using phenolic composition and antioxidant activity was applied to differentiate between red and white cultivar wines.     

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.     

Neuroprotective effects of resveratrol on MPTP-induced neuron loss mediated by free radical scavenging

Resveratrol is a natural polyphenol and possesses many biological functions such as anti-inflammatory activity and protection against atherosclerosis and myocardial infraction. Parkinson's disease is a common progressive neurodegenerative disease. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is the most useful neurotoxin to induce Parkinsonism. The present study was carried out to elucidate the neuroprotective effect and possible mechanism of resveratrol on MPTP-induced striatal neuron loss. Sixty adult Balb/c mice were divided into four groups: sham operation, MPTP treatment (30 mg/kg, i.p.), MPTP combined with resveratrol administration (20 mg/kg, i.v.), and resveratrol treatment alone. Microdialysis and high-performance liquid chromatography were used to analyze dihydroxybenzoic acid (DHBA) that reflected the hydroxyl radical level. In the present study, we found MPTP chronic administration significantly induced motor coordination impairment in mice. After MPTP administration, the hydroxyl radical levels in substantia nigra were also significantly elevated and animals displayed severe neuronal loss. Resveratrol administration significantly protected mice from MPTP-induced motor coordination impairment, hydroxyl radical overloading, and neuronal loss. Our results demonstrated that resveratrol could elicit neuroprotective effects on MPTP-induced Parkinsonism through free radical scavenging.     

Fenton-dependent damage to carbohydrates: free radical scavenging activity of some simple sugars

The antioxidant properties of simple carbohydrates were studied in a chemical system. Hydroxyl radicals generated by a Fenton reaction induce damage on simple carbohydrates with a consequent free radical scavenging activity. Carbohydrate activities were measured by different methods as spin-trapping of hydroxyl radical and electron paramagnetic resonance detection and 1,1-diphenyl-2-picrylhydrazyl quenching. Carbohydrate damage was evaluated in a Fenton system by measuring the reactive substances to thiobarbituric acid, by their decreased detection with an HPLC test, and by a gas chromatographic determination of formic acid from sugar oxidation. Different intensities of damage and scavenging were found according to molecular structure, and some hyphotheses on the carbohydrate action against free radicals were attempted. The assayed disaccharides were shown to be more active toward and less damaged by hydroxyl radical than monosaccharides.     

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.     

Anthocyanin quantification and radical scavenging capacity of Concord, Norton, and Marechal Foch grapes and wines

The anthocyanin content and the radical scavenging capacity of three non-Vitis vinifera grapes (Marechal Foch, Norton, and Concord varieties) were determined. Analyses of anthocyanins in the skin (S) and wine (W) of these grape varieties were performed by spectrophotometry, HPLC with electrochemical detection, and matrix-assisted laser desorption ionization (MALDI). The total anthocyanin contents of S samples were 258 +/- 37 mg/100 g of wet weight for Foch, 888 +/- 78 mg/100 g for Norton, and 326 +/- 5.9 mg/100 g for Concord grapes. The malvidin 3,5-diglucoside content quantified by HPLC indicated that Norton S had the highest amount of the compound (327 +/- 110 mg/100 g). The MALDI mass spectrometric analysis indicated an abundance of malvidin glucosides in W of Foch grapes and in S and W of Norton grapes and of cyanidin aglycon in S and W of Concord grapes. S samples were subjected to a radical scavenging capacity test using the 2,2-diphenyl-1-picrylhydrazyl radical and compared to Trolox. The radical scavenging capacity for Foch S was 0.78 mM Trolox equiv, that of Concord S, 0.80 Trolox equiv, and that of Norton S was highest at 0.95 Trolox equiv. The higher concentrations of malvidin 3,5-diglucoside in S of grape varieties were associated with greater radical scavenging capacity.     

Peroxyl radical scavenging capacity, polyphenolics, and lipophilic antioxidant profiles of mulberry fruits cultivated in southern China

Twenty-seven cultivars of mulberry fruits ( Morus atropurpurea Roxb) were analyzed for their total phenolic content, total anthocyanin content, and peroxyl radical scavenging capacities. The proanthocyanidin contents of the fruit were also quantified using 4-dimethylamino-cinnamaldehyde assay, and characterization was attempted using electrospray ionization mass spectra. The phenolic compounds of mulberry fruits were characterized using HPLC with ESI-MS and diode array detection. Results showed that the content of mulberry fruits varied with different cultivars with total phenolic content, total anthocyanin content, total proanthocyanidin content, and peroxyl radical scavenging capacities ranging from 0.060-0.244, 0.001-0.056, 0.001-0.015, and 0.301-1.728, respectively. Good correlations were observed among the phenolic, anthocyanin, and proanthocyanidin contents and the radical scavenging capacities of mulberry fruits. Mulberry fruits were found to contain low amount of proanthocyanidins. The high total phenolic content of mulberry fruits were mainly contributed by anthocyanins, rutin, and chlorogenic acids. The lipid soluble antioxidants are profiled by an HPLC method developed in-house, and the results of selected mulberry fruits revealed significant amounts of lutein and delta- and gamma-tocopherols but low alpha-tocopherol. Our results provide useful antioxidant nutritional information of a mulberry cultivar that has potential for large scale plantations.     

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.