A method to measure the oxidizability of both the aqueous and lipid compartments of plasma.

The lipophilic radical initiator (MeO-AMVN) and the fluorescent probe C11BODIPY581/591 (BODIPY) were used to measure the lipid compartment oxidizability of human plasma. Aqueous plasma oxidizability was initiated by the aqueous peroxyl radical generator, AAPH, and 2',7'-dichlorodihydrofluorescein (DCFH) was employed as the marker of the oxidative reaction. The distribution in aqueous and lipid compartments of the two radical initiators was determined by measuring the rate of consumption of the plasma hydrophilic and lipophilic endogenous antioxidants. In the presence of AAPH (20 mM), the order of consumption was: ascorbic acid > alpha-tocopherol > uric acid > beta-carotene, indicating a gradient of peroxyl radicals from the aqueous to the lipid phase. When MeO-AMVN was used (2mM), beta-carotene was consumed earlier than uric acid and almost at the same time as alpha-tocopherol, reflecting the diffusion and activation of MeO-AMVN in the lipophilic phase. The rate of BODIPY oxidation (increase in green fluorescence) significantly increased after the depletion of endogenous alpha-tocopherol and beta-carotene, whereas it was delayed for 180 min when AAPH was used instead of MeO-AMVN. The measurement of lipid oxidation in plasma was validated by adding to plasma the two lipophilic antioxidants, alpha-tocopherol and beta-carotene, whose inhibitory effects on BODIPY oxidation were dependent on the duration of the preincubation period and hence to their lipid diffusion. DCFH oxidation induced by AAPH only began after uric acid, the main hydrophilic plasma antioxidant, was consumed. In contrast, when MeO-AMVN was used, DCFH oxidation was delayed for 120 min, indicating its localization in the aqueous domain. In summary, the selective fluorescence method reported here is capable of distinguishing the lipophilic and hydrophilic components of the total antioxidant capacity of plasma.     

Total antioxidant performance: a validated fluorescence assay for the measurement of plasma oxidizability

The antioxidant capacity of human plasma was determined by following the oxidation kinetics of the lipid-soluble fluorescent marker BODIPY using 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) as the lipophilic radical initiator. The results are expressed as a total antioxidant performance (TAP) value based on the inhibition of BODIPY oxidation, as determined by the appearance of green fluorescence, with respect to a control sample (phosphatidylcholine with or without delipidized human serum). The suitability of the assay was evaluated on the basis of its precision, reproducibility, and specificity. The intra- and interassay coefficients of variation both were less than 5%. The addition of a representative substrate of plasma peroxidation, phosphatidylcholine, up to 750mug/ml did not induce significant changes in the TAP value. Also, BODIPY photooxidation was not observed during the experimental time course (220min). The TAP values of 6 plasma samples from healthy donors were measured and correlated with the main plasma water- and lipid-soluble antioxidants (uric acid and ascorbic acid, alpha-tocopherol, and carotenoids) and lipid profiles. Significant correlations were found between TAP and uric acid (R=0.97, P<0.05) and cholesterol-adjusted alpha-tocopherol (R=0.93, P<0.01). The results confirm that the TAP assay is suitable to measure the antioxidant activity of plasma antioxidants localized in both the lipophilic and hydrophilic compartments.     

(-)-Epigallocatechin-3-gallate prevents oxidative damage in both the aqueous and lipid compartments of human plasma

When human plasma was exposed to the hydrophilic radical initiator, AAPH, (-)-epigallocatechin-(3)-gallate (EGCG) dose-dependently inhibited the aqueous compartment oxidation (IC(50)=0.72 microM) (monitored by DCFH oxidation) and spared the lipophilic antioxidants, alpha-tocopherol, and carotenoids, but not ascorbic acid. When radicals were selectively induced in the lipid compartment by the lipophilic radical initiator, MeO-AMVN, EGCG spared alpha-tocopherol, but not carotenoids and inhibited the lipid compartment oxidation (monitored by BODIPY 581/591) with a potency lower than that found in the aqueous compartment (IC(50)=4.37 microM). Our results indicate that EGCG, mainly localized in the aqueous compartment, effectively quenches aqueous radical species, thus limiting their diffusion into the lipid compartment and preventing lipid-soluble antioxidant depletion. Further, ESR experiments confirmed that EGCG recycled alpha-tocopherol through a H-transfer mechanism at the aqueous/lipid interface affording an additional protective mechanism to the lipid compartment of plasma.     

Action of DCFH and BODIPY as a probe for radical oxidation in hydrophilic and lipophilic domain

Fluorogenic probes such as 2',7'-dichlorofluorescin (DCFH) have been extensively used to detect oxidative events and to measure antioxidant capacity. At the same time, however, the inherent drawbacks of these probes such as non-specificity towards oxidizing species have been pointed out. The present study was carried out to analyze the action and dynamics of 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY) and DCFH as a fluorescent probe in the free radical-mediated lipid peroxidation in homogeneous solution, aqueous suspensions of liposomal membranes and LDL and plasma. The rate constant for the reaction of BODIPY with peroxyl radicals was estimated as 6.0 x 10(3) M(-1) s(-1), which makes BODIPY kinetically an inefficient probe especially in the presence of potent radical-scavenging antioxidants such as tocopherols, but a convenient probe for lipid peroxidation. On the other hand, the reactivity of DCFH toward peroxyl radicals was as high as Trolox, a water-soluble analogue of alpha-tocopherol. Thus, DCFH is kinetically more favored probe than BODIPY and could scavenge the radicals within lipophilic domain as well as in aqueous phase. The partition coefficients for BODIPY and DCFH were obtained as 4.57 and 2.62, respectively. These results suggest that BODIPY may be used as an efficient probe for the free radical-mediated oxidation taking place in the lipophilic domain, especially after depletion of alpha-tocopherol, while it may not be an efficient probe for detection of aqueous radicals.     

Action of DCFH and BODIPY as a Probe for Radical Oxidation in Hydrophilic and Lipophilic Domain

Fluorogenic probes such as 2',7'-dichlorofluorescin (DCFH) have been extensively used to detect oxidative events and to measure antioxidant capacity. At the same time, however, the inherent drawbacks of these probes such as non-specificity towards oxidizing species have been pointed out. The present study was carried out to analyze the action and dynamics of 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY) and DCFH as a fluorescent probe in the free radical-mediated lipid peroxidation in homogeneous solution, aqueous suspensions of liposomal membranes and LDL and plasma. The rate constant for the reaction of BODIPY with peroxyl radicals was estimated as 6.0×10₃ M_-1s_-1, which makes BODIPY kinetically an inefficient probe especially in the presence of potent radical-scavenging antioxidants such as tocopherols, but a convenient probe for lipid peroxidation. On the other hand, the reactivity of DCFH toward peroxyl radicals was as high as Trolox, a water-soluble analogue of α-tocopherol. Thus, DCFH is kinetically more favored probe than BODIPY and could scavenge the radicals within lipophilic domain as well as in aqueous phase. The partition coefficients for BODIPY and DCFH were obtained as 4.57 and 2.62, respectively. These results suggest that BODIPY may be used as an efficient probe for the free radical-mediated oxidation taking place in the lipophilic domain, especially after depletion of α-tocopherol, while it may not be an efficient probe for detection of aqueous radicals.     

A method to measure antioxidant activity in organic media: application to lipophilic vitamins

The 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) radical (ABTS(.+)) can be generated by the enzymatic system formed by hydrogen peroxide and horseradish peroxidase in an organic medium. The ABTS radical is easily generated in acidified ethanol medium in about 100 s with a stability of 1.7 x 10(-3) (-deltaabs/min) monitored at 730 nm. Other organic solvents, such as methanol or acetone, have lower radical generation times but the radical is less stable. The addition of Trolox or a lipophilic antioxidant such as alpha-tocopherol or beta-carotene produces a decrease in absorbance that can be used to estimate antioxidant capacity. Using a spectrophotometric end-point method and microplate-reader equipment, we have developed a method that estimates the antioxidant activity of different lipophilic vitamins. The use of Trolox as an antioxidant standard led to a limit of detection of 0.08 nmoles and limit of quantitation of 0.28 nmoles, while similar values were obtained for alpha-tocopherol and beta-carotene. The relative antioxidant activity values obtained by different antioxidants showed that alpha-tocopherol has a similar antioxidant potential to Trolox and that beta-carotene has 2.6 times the antioxidant potential of Trolox. In our opinion, this method can be useful for estimating the antioxidant activity in lipophilic samples and as a complement to other methods that measure antioxidant activity in aqueous media.     

Evaluation of activity of selected antioxidants on proteins in solution and in emulsions

Protection against protein oxidation by lipophilic and hydrophilic antioxidants in model systems using bovine serum albumin (BSA) in solution alone, or in an emulsion with linolenic acid methyl ester (LnMe) was found to be strongly dependent on the oxidation initiator. Tocopherol, Trolox, or the carotenoids astaxanthin and canthaxanthin were incubated with BSA or BSA/LnMe and oxidation was initiated either with the water-soluble azo-initiator 2,2' azo-bis-(2-amidinopropane) hydrochloride (AAPH), or FeCl₃ and ascorbate, or the Fenton system using FeCl₂/EDTA/H₂O₂, or with the singlet oxygen generating species anthracene-9,10-dipropionic acid disodium 1,4 endoperoxide (NDPO₂).

The results show that all the antioxidants tested were inefficient in the system with FeCl₃/ascorbate. However, with the other initiating agents, the hydrophilic antioxidant, Trolox, was the most effective in preventing both protein and lipid oxidation. In contrast the lipophilic antioxidants were ineffective in preventing oxidation of BSA in aqueous solution, but did show some moderate antioxidative activity on protein and lipid in the BSA/LnMe system. Using the singlet oxygen generating system it was also demonstrated that Trolox always provided better protection of the protein than tocopherol and the carotenoids in both the BSA and the BSA/LnMe systems. In conclusion, prevention of protein oxidation using a water-soluble antioxidant has a protective effect on the lipid fraction and this approach deserves further attention in complex biological systems.     

Evaluation of activity of selected antioxidants on proteins in solution and in emulsions

Protection against protein oxidation by lipophilic and hydrophilic antioxidants in model systems using bovine serum albumin (BSA) in solution alone, or in an emulsion with linolenic acid methyl ester (LnMe) was found to be strongly dependent on the oxidation initiator. Tocopherol, Trolox, or the carotenoids astaxanthin and canthaxanthin were incubated with BSA or BSA/LnMe and oxidation was initiated either with the water-soluble azo-initiator 2,2′ azo-bis-(2-amidinopropane) hydrochloride (AAPH), or FeCl₃ and ascorbate, or the Fenton system using FeCl₂/EDTA/H₂O₂, or with the singlet oxygen generating species anthracene-9,10-dipropionic acid disodium 1,4 endoperoxide (NDPO₂).

The results show that all the antioxidants tested were inefficient in the system with FeCl₃/ascorbate. However, with the other initiating agents, the hydrophilic antioxidant, Trolox, was the most effective in preventing both protein and lipid oxidation. In contrast the lipophilic antioxidants were ineffective in preventing oxidation of BSA in aqueous solution, but did show some moderate antioxidative activity on protein and lipid in the BSA/LnMe system. Using the singlet oxygen generating system it was also demonstrated that Trolox always provided better protection of the protein than tocopherol and the carotenoids in both the BSA and the BSA/LnMe systems. In conclusion, prevention of protein oxidation using a water-soluble antioxidant has a protective effect on the lipid fraction and this approach deserves further attention in complex biological systems.     

Assessment of antioxidant capacity for scavenging free radicals in vitro: a rational basis and practical application

With increasing evidence showing the involvement of oxidative stress induced by free radicals in the development of various diseases, the role of radical-scavenging antioxidants has received much attention. Although many randomized controlled clinical trials do not support the beneficial effects of indiscriminate supplementation of antioxidants, more recent studies suggest that antioxidants such as vitamin E may be effective for prevention and treatment of some diseases when given to the right subjects at the right time. Many studies on the antioxidant capacity assessed by various available methods showed inconsistent results and the assessment of antioxidant capacity has been the subject of extensive studies and arguments. This study was performed to elucidate the basic chemistry required for the development of a reliable method for the assessment of antioxidant capacity for radical scavenging in vitro. In this study, the capacity of α-tocopherol and its related compounds, ascorbic acid, and uric acid for scavenging radicals was assessed from their effects on the rate of decay of hydrophilic and lipophilic probes with various reactivities toward free radicals induced by hydrophilic and lipophilic radicals in homogeneous solution and heterogeneous micelle systems. Fluorescein, pyranine, and pyrogallol red were used as hydrophilic probes, and BODIPY and N,N-diphenyl-p-phenylenediamine were used as lipophilic probes. We show that the rate and amount of radical scavenging by antioxidants, termed the antioxidant radical absorbance capacity, could be assessed by an appropriate combination of radical initiator and probe. This method was applied to the assessment of radical-scavenging capacity of human plasma, wine, and green tea powder.     

Antioxidant activity applying an improved ABTS radical cation decolorization assay.

A method for the screening of antioxidant activity is reported as a decolorization assay applicable to both lipophilic and hydrophilic antioxidants, including flavonoids, hydroxycinnamates, carotenoids, and plasma antioxidants. The pre-formed radical monocation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS*+) is generated by oxidation of ABTS with potassium persulfate and is reduced in the presence of such hydrogen-donating antioxidants. The influences of both the concentration of antioxidant and duration of reaction on the inhibition of the radical cation absorption are taken into account when determining the antioxidant activity. This assay clearly improves the original TEAC assay (the ferryl myoglobin/ABTS assay) for the determination of antioxidant activity in a number of ways. First, the chemistry involves the direct generation of the ABTS radical monocation with no involvement of an intermediary radical. Second, it is a decolorization assay; thus the radical cation is pre-formed prior to addition of antioxidant test systems, rather than the generation of the radical taking place continually in the presence of the antioxidant. Hence the results obtained with the improved system may not always be directly comparable with those obtained using the original TEAC assay. Third, it is applicable to both aqueous and lipophilic systems.     

Soraphinol C, a new free-radical scavenger from Sorangium cellulosum

A new compound named soraphinol C (1) was isolated from myxobacteria Sorangium cellulosum KM1001 a soil isolate, together with a structurally related known compound, 4-hydroxysattabacin (2). These compounds were isolated by silica gel column chromatography and recycling preparative HPLC, consecutively. The structures of the compounds were determined on the basis of combined spectroscopic analyses. Compounds 1 and 2 exhibited antioxidant activity as a radical scavenger in the experiment using a hydrophilic free-radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride with ORAC values of 0.956 and 0.617, respectively.     

Complex trade-offs in the pigeon (Columba livia): egg antioxidant capacity and female serum oxidative status in relation to diet quality

Despite the great deal of ecological research interest in modulators of offspring quality and consequences of reproduction on female status, we still know little about the relationships among diet quality, antioxidant capacity of egg components (yolk and albumen) and oxidative status of female birds. In this study, I compared the egg quality (egg size, albumen and yolk antioxidant capacity) and serum oxidative status (oxidative damage, total serum antioxidant capacity, and serum thiols) of female pigeons (Columba livia) fed with foods of different quality (standard quality and decreased quality). I also analysed the patterns of covariation among egg and female traits. The study focussed on the first clutch laid by the female in the breeding season and on the short-term effects of a decrease in diet quality. The treatment did not affect the egg volume, the lipophilic and hydrophilic components of antioxidant capacity (lipOXY and hydrOXY, respectively) or the antioxidant capacity of the albumen (albumOXY). However, females fed a higher quality diet were fatter and had marginally higher values of serum hydroperoxides (oxidative damage) than females fed a lower quality diet. Moreover, females that showed an increase in yolk hydrOXY and serum hydroperoxides also showed a decrease in yolk lipOXY, albumOXY and serum antioxidant capacity. These results show that the female’s oxidative status can be correlated with the antioxidant content of her eggs, but the nature of these correlations is complex, depending on the molecular component measured. The results also suggest that in the pigeon the deposition of hydrophilic and lipophilic antioxidants in the egg may trade off against each other.     

Antioxidant activities of estrogens against aqueous and lipophilic radicals; differences between phenol and catechol estrogens

Natural estrogens have much greater radical-scavenging antioxidant activity than has previously been demonstrated, with activities up to 2.5 times those of vitamin C and vitamin E. The biological significance of this finding remains to be elucidated. In this work the antioxidant activity of a range of estrogens (phenolic, catecholic and stilbene-derived) has been studied. The activity of these substances as hydrogen-donating scavengers of free radicals in an aqueous solution has been determined by monitoring their relative abilities to quench the chromogenic radical cation 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS*+). The results show that the order of reactivity in scavenging this radical in the aqueous phase is dependent on the precise estrogenic structure, with phenolic estrogens being more potent antioxidants than catecholestrogens or diethylstilbestrol. The ability of the same estrogens to scavenge lipid phase radicals has also been assessed, determined by the ex vivo enhancement of the resistance of low-density lipoprotein (LDL) to oxidation; the order of efficacy is different from that in the aqueous phase, with the phenolic estrogens estriol, estrone and 17beta-estradiol being less potent than 2-hydroxyestradiol, 4-hydroxyestradiol, or diethylstilbestrol. In this lipid-based system, phenolic estrogens were found to be unable to regenerate alpha-tocopherol from LDL subjected to oxidative stress, while at the same time 2- and 4-hydroxyestradiol significantly delayed alpha-tocopherol loss. These results indicate that the various estrogens are good scavengers of free radicals generated in both the aqueous and the lipophilic phases. The antioxidant activity of an estrogen depends not only on the hydrophilic or lipophilic nature of the scavenged radical, but also on the phenol and catechol structures of the estrogen compound.     

Caffeic acid phenolipids in the protection of cell membranes from oxidative injuries. Interaction with the membrane phospholipid bilayer

Caffeic acid (CA) has demonstrated a strong intracellular antioxidant ability by scavenging ROS, contributing to the maintenance of cell membrane structural integrity and to reduce oxidative injuries in other cell components. Nevertheless, caffeic acid has limited usage, due to its hydrophilic character. In this work, the introduction of alkyl chains in the caffeic acid molecule by esterification (methyl - C1, ethyl - C2, butyl - C4, hexyl - C6, octyl - C8 and hexadecyl - C16), significantly increased its lipophilicity. All caffeates tested showed a much higher protective activity than caffeic acid against red blood cells (RBCs) AAPH-induced oxidative stress; this protection was heavily dependent on the length of the alkyl chain of the esters, and on their concentration. At 2.5 and 5 μM, the more lipophilic compounds (C8 and C16) showed a remarkable antioxidant activity, inhibiting hemolysis; probably, their better location within the membrane leads to a better antioxidative protection; however, at 50 μM, the more hydrophilic compounds (C1-C4) showed a better activity against hemolysis than the more lipophilic ones (C8-C16). At this higher concentration, the better interaction of the more lipophilic compounds with the membrane seems to cause changes in RBC membrane fluidity, disturbing membrane integrity. Our data show that the antioxidant activity of these compounds could play an important role for the protection of different tissues and organs, by protecting cell membranes from oxidative injuries.     

Vitamin C preserves the cardio-protective paraoxonase activity of high-density lipoprotein during oxidant stress

HDL-associated paraoxonase (PON) antioxidant enzyme activity is cardio-protective. We investigated whether vitamin C prevented loss of PON activity from HDL during oxidant stress. HDL was incubated with either hydrophilic or lipophilic peroxyl radical initiators in the absence (control) or presence of vitamin C (50 and 100 micromol/L). Regardless of the type of radical, accumulation of lipid oxidation products in HDL was similar in incubations lacking vitamin C. Loss of PON activity was greater in HDL exposed to hydrophilic, in contrast to lipophilic, radicals, but addition of vitamin C maintained enzyme activity. Vitamin C's capacity to attenuate loss of the HDL ability to prevent atherogenic modification of LDL (assessed as electrophoretic mobility) was, however, modest, and appeared limited only to those incubations in which HDL was exposed to lipophilic radicals. Our results indicate that vitamin C may, under some conditions, prevent loss of cardio-protective function from HDL during oxidant stress.     

On-line antioxidant activity determination: comparison of hydrophilic and lipophilic antioxidant activity using the ABTS*+ assay

The ABTS/H(2)O(2)/HRP decoloration method is capable of determining both hydrophilic (in buffered media) and lipophilic (in organic media) antioxidant properties in complex samples. Now, we have adapted this method for on-line chromatographic determination. The easy, rapid and controlled generation of the ABTS radical and its great stability in buffered and organic media were important characteristics in the measurement of antioxidant activities. The HPLC-ABTS method used two pumps (one for isocratic eluting-phase and the other for preformed ABTS radical) and an UV-VIS diode array detector. The dual analysis of samples -- conventional (with UV-VIS detection) and ABTS-scavenging (at 600 nm) -- provided valuable on-line information about the correspondence between the presence of a determined compound and its possible antioxidant activity, and was applicable to both hydrophilic and lipophilic antioxidants (HAA and LAA). A comparison between HAA and LAA determined by the end-point method and by the on-line HPLC method is presented. The application to juices showed that both methods are suitable, sensitive and selective, gave similar values, and the HPLC-ABTS method contributed additional information about the antioxidant activity profile.     

Rational discovery and development of a mitochondria-targeted antioxidant based on cinnamic acid scaffold

A novel mitochondria-targeted antioxidant (TPP-OH) was synthesized by attaching the natural hydrophilic antioxidant caffeic acid to an aliphatic lipophilic carbon chain containing a triphenylphosphonium (TPP) cation. This compound has similar antioxidant activity to caffeic acid as demonstrated by measurement of DPPH/ABTS radical quenching and redox potentials, but is significantly more hydrophobic than its precursor as indicated by the relative partition coefficients. The antioxidant activity of both compounds was intrinsic related to the ortho-catechol system, as the methoxylation of the phenolic functions, namely in TPP-OCH(3) and dimethoxycinnamic acid, gave compounds with negligible antioxidant action. The incorporation of the lipophilic TPP cation to form TTP-OH and TPP-OCH(3) allowed the cinnamic derivatives to accumulate within mitochondria in a process driven by the membrane potential. However, only TPP-OH was an effective antioxidant: TPP-OH protected cells against H(2)O(2) and linoleic acid hydroperoxide-induced oxidative stress. As mitochondrial oxidative damage is associated with a number of clinical disorders, TPP-OH may be a useful lead that could be added to the family of mitochondria-targeted antioxidants that can decrease mitochondrial oxidative damage.     

Enhanced oxidative stability of a hydrophilic arginine-conjugated linoleic acid complex

The application of conjugated linoleic acid (CLA) as a functional food ingredient has been limited due to its high cost of purification and insolubility in water. Recently, we reported a method to produce high-purity CLA using two-step urea-inclusion crystallization and a hydrophilic arginine-CLA (Arg-CLA) complex. The present study compared the oxidative stability and antioxidant activity of CLA and Arg-CLA. Hydroperoxide production from CLA increased about 12-fold at 10 hr after heat treatment at 100(R)C, whereas Arg-CLA did not exhibit significant hydroperoxide production. Furthermore, the Arg-CLA complex showed synergistic antioxidant activity in a 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical scavenging assay. Arg-CLA at 20 mM scavenged 89% of ABTS radicals in 3 h, whereas CLA alone quenched only 48% under the same conditions. Our results indicate that a hydrophilic Arg-CLA exhibits enhanced oxidative stability and antioxidant activity, which could expand the scope of CLA application in various food industries.     

Allelopathic activity of Chara aspera

Allelopathic activity of Chara aspera was determined in agar diffusion assays using planktonic cyanobacteria as target organisms. Growth inhibition of cyanobacterial strains was observed in bioassays inoculated with living Chara aspera shoots as well as with 60% aqueous methanol extracts of Chara aspera. For further analysis, the methanol extract was fractionated into three parts: a lipophilic methanol – a butylmethylether-extract and a hydrophilic methanol extract. The bioassays indicated that major allelopathic activity was retained in the hydrophilic methanol – and the lipophilic butylmethylether-extract. Separation of the extracts by means of high performance liquid chromatography followed by fractionation of the eluant resulted in supplementary nine fractions, three from each part, respectively. Three fractions exhibited a strong growth inhibition of the target organism Anabaena cylindrica Lemmermann. The second and the third fraction of the lipophilic butylmethylether extract indicate the presence of novel allelopathic active compounds with lipophilic characteristics. The results lead to the suggestion that more than two chemical compounds in Chara aspera are responsible for the growth inhibition of cyanobacteria.     

On-line antioxidant activity determination: comparison of hydrophilic and lipophilic antioxidant activity using the ABTS•+ assay

Abstract

The ABTS/H₂O₂/HRP decoloration method is capable of determining both hydrophilic (in buffered media) and lipophilic (in organic media) antioxidant properties in complex samples. Now, we have adapted this method for on-line chromatographic determination. The easy, rapid and controlled generation of the ABTS radical and its great stability in buffered and organic media were important characteristics in the measurement of antioxidant activities. The HPLC-ABTS method used two pumps (one for isocratic eluting-phase and the other for preformed ABTS radical) and an UV-VIS diode array detector. The dual analysis of samples – conventional (with UV-VIS detection) and ABTS-scavenging (at 600 nm) – provided valuable on-line information about the correspondence between the presence of a determined compound and its possible antioxidant activity, and was applicable to both hydrophilic and lipophilic antioxidants (HAA and LAA). A comparison between HAA and LAA determined by the end-point method and by the on-line HPLC method is presented. The application to juices showed that both methods are suitable, sensitive and selective, gave similar values, and the HPLC-ABTS method contributed additional information about the antioxidant activity profile.