Peroxidation Of Liposomes Research Articles

The interaction of superoxide radicals with active dicarbonyl compounds

The interaction of superoxide radical anion (O2 •−) with active dicarbonyls (methylglyoxal, glyoxal, and malonic dialdehyde) was studied. It was demonstrated that glyoxal and methylglyoxal inhibited superoxide-dependent accumulation of formazan; however, malonic dialdehyde stimulated this process. The formation of O2 •− in these experiments occurred during the decomposition of the SOTS-1 azo initiator. On the other hand, all of the studied dicarbonyls in this system of O2 •− generation competed for superoxide with the TIR ON spin trap. These compounds also inhibited luminal-dependent chemiluminescence during the AIBN azo initiator-induced peroxidation of liposomes from the egg phosphatidylcholine. A mechanism for the antiradical and antioxidant effects of the studied dicarbonyls, assuming the production of free radical intermediates in their reactions with O2 •− or its protonated form, is proposed.

Methylglyoxal as a scavenger for superoxide anion-radical.

Methylglyoxal at a concentration of 5 mM caused a significant inhibition of superoxide anion radical (O2 (·-)) comparable to the effect of Tirone. In the process of O2 (·-) generation in the system of egg phosphatidylcholine liposome peroxidation induced by the azo-initiator AIBN, a marked inhibition of chemiluminescence in the presence of 100 mM methylglyoxal was found. At the same time, methylglyoxal did not inhibit free radical peroxidation of low-density lipoprotein particles, which indicates the absence of interaction with methylglyoxal alkoxyl and peroxyl polyenoic lipid radicals. These findings deepen information about the role of methylglyoxal in the regulation of free radical processes.

Peroxidation of Lipoprotein and of Certain Liposomal Lipids Depend Similarly on the Physico-Chemical Properties of the Aggregated Lipids

In view of the complexity of biological membranes, the dependence of peroxidation on the physicochemical properties of membranes and on different antioxidants have been studied in liposomes made of different compositions, under different conditions. low molecular weight antioxidants and the dependence of these effects on various factors have also been studied. In 007, we reviewed the literature regarding these issues. Recent information clarifies several aspects of the peroxidation in these model membranes, including the promotion of copper-induced peroxidation by inclusion of negatively charged phospholipids (PA and PS), inhibition of the peroxidation of PS-containing liposomes by nano-molar concentrations of Tocopherol and other 12 (out of 37 tested) phenols of possible semi-quinone structures and promotion of copper induced peroxidation by soluble “antioxidants. Several new studies support our previous interpretation that the dependence of peroxidation on the packing of the PUFA in the bilayer (and the microviscosity of the liposomal membranes) is due to the consequent change of the rate of lateral diffusion of the PUFA-bearing phospholipids. Recent study ruled out this interpretation on the basis of the similar effects of “fluidizing” and “rigidifying” non-oxidizable phospholipids. Nonetheless, the data is insufficient to rule out the previous interpretation because both the propagation and termination of the free radical chain reaction depend similarly on the micro-viscosity (and the rate of lateral diffusion), while the results of a change in the rate of these two reactions are opposite. New data also show how peroxidation affect continuous peroxidation and demonstrate that liposomes are affected by antioxidants similar to LDL only when they contain PS.

A Study on Biological Activity of the Polyphenol Fraction from Fruits of Rosa Rugosa Thunb

This study aimed to determine the biological activity of Rosa rugosa Thunb. extracts in the four versions: methanolic (ME), diethyl ether (EE), ethyl acetate (EA) and water (WE). We studied the antioxidant activity of the extracts in relation to phospholipid membranes, their association constants with the membrane, and antiradical and anti-inflammatory activity. Percentage of their liposomal encapsulation was also assessed. R. rugosa ME and EA extracts were observed to decrease peroxidation of PC liposomes (UVC-induced) to similar levels (ME) or lower (EA) compared with the activity of Rosa canina L. The other two extracts (EE and WE) of R. rugosa showed significantly lower antioxidant activity than extracts of R. canina. R. rugosa quenched DPPH• and inhibited COX activity. R. rugosa EA has the highest association constant, whereas the association constants of EE, ME, and WE are approximately 1–2 orders of magnitude smaller. EA extract also showed the highest percentage of encapsulation in liposomal carriers. Practical Applications Literature reports attest to the healing properties of extract of R. canina (Litozin, AXELLUS, Denmark). Our studies have demonstrated that extracts of R. rugosa have strong antioxidant (in relation to UV-induced oxidation of phosphatidylcholine liposomes) and antiradical (in relation to DPPH•) properties, indicating a potential use of these extracts as natural antioxidants, e.g., in the food, pharmaceutical and beauty industry, in particular for protecting peroxidation sensitive components such as unsaturated fatty acids. The ability of R. rugosa extract (the EA version) to strongly inhibit oxidation of arachidonic acid, induced by COX-1 and COX-2, indicates a possibility of application of this extract in prevention and/or elimination of arthritis. The determined high level of encapsulation of R. rugosa EA extract in liposomal carriers may increase the bioavailability of the extract supplied to the body in the form of liposomal nanocapsules and thus be instrumental in effective treatment of inflammatory conditions.

Effect of defatted sesame meal extract on phospholipid peroxidation in liposomes

We compared the protective effect of defatted sesame meal extracts (DSMEs; prepared either after roasting sesame seeds at 200°C for 60 min or without roasting) on phospholipid peroxidation of liposomes, with the protective effect of natural antioxidants (ascorbic acid and α-tocopherol). We investigated phospholipid peroxidation after induction with either a hydrophilic radical generator (2,2′-azobis [2-amidinopropane]) or a hydrophobic radical generator (2,2′-azobis [2,4-dimethylvaleronitrile]), for 6 h at 37 and 50°C. Added antioxidants prevented peroxidation of phospholipids in liposomes, and roasted DSME showed a significantly stronger antioxidant activity than unroasted DSME in all cases. The results indicate that heat treatment significantly increases the protective effect of DSME on lipid peroxidation in phospholipid bilayers under both hydrophilic and hydrophobic conditions, regardless of the hydrophobicity of the radical generators.

Effect of UV radiation and hydrogen peroxide on the antiradical and antioxidant activities of DOPA-melanin and melanosomes from retinal pigment epithelial cells

The effect of continuous UV radiation and hydrogen peroxide on destruction and antioxidant properties of synthetic DOPA-melanin (prepared by oxidation of 3,4-dihydroxyphenylalanine (DOPA)) and melanosomes isolated from cells of the retinal pigment epithelium (RPE) was investigated. The kinetics of melanin destruction was recorded based on the accumulation of fluorescent low-molecular-weight reaction products, the antiradical activity of melanin was determined by chemiluminescence method, the concentration of free radical products was measured by electron paramagnetic resonance, and the antioxidant activity of melanins was estimated by their inhibitory effect on lipid peroxidation. It was shown that UVC—UVA irradiation (up to 5 hours) of DOPA-melanin and melanosomes of retinal pigment epithelium decreased neither the latency period of luminol chemiluminescence nor the inhibitory action of pigments on Fe2+- and UV-induced peroxidation of cardiolipin liposomes. However, very long UV irradiation gave rise to fluorescent destruction products, decreased the concentration of paramagnetic centers in the pigment (especially light-dependent ones), and decreased the antiradical and antioxidant activities. For example, UV irradiation of DOPA-melanin during 52 h resulted in approximately a 2-fold decrease in the concentration of paramagnetic centers and decline of antiradical and antioxidant activities. However, even with such a hard irradiation the pigment retained significant inhibitory activity against lipid peroxidation. The oxidative destruction of DOPA-melanin in the presence of hydrogen peroxide in the dark resulted in complete destruction of the polymer and loss of its protective properties. It is assumed that destruction of RPE cell melanin is caused mainly by oxidative processes.

PH dependent antioxidant activity of lettuce (L. sativa) and synergism with added phenolic antioxidants

Influence of pH on the antioxidant activities of combinations of lettuce extract (LE) with quercetin (QC), green tea extract (GTE) or grape seed extract (GSE) was investigated for both reduction of Fremy’s salt in aqueous solution using direct electron spin resonance (ESR) spectroscopy and in l-α-phosphatidylcholine liposome peroxidation assay measured following formation of conjugated dienes. All examined phenolic antioxidants showed increasing radical scavenging effect with increasing pH values by using both methods. QC, GTE and GSE acted synergistically in combination with LE against oxidation of peroxidating liposomes and with QC showing the largest effect. The pH dependent increase of the antioxidant activity of the phenols is due to an increase of their electron-donating ability upon deprotonation and to their stabilization in alkaline solutions leading to polymerization reaction. Such polymerization reactions of polyphenolic antioxidants can form new oxidizable –OH moieties in their polymeric products resulting in a higher radical scavenging activity.

Insulin glycation coupled with liposomal lipid peroxidation and microglial cell death

Type 2 diabetes is characterized jointly by hyperglycemia and hyperinsulinemia, which make insulin prone to glycation then fibrillation.

Prevention of peroxidation of cardiolipin liposomes by quinol-based antioxidants

In mammalian mitochondria, cardiolipin molecules are the primary targets of oxidation by reactive oxygen species. The interaction of oxidized cardiolipin molecules with the constituents of the apoptotic cascade may lead to cell death. In the present study, we compared the effects of quinol-containing synthetic and natural amphiphilic antioxidants on cardiolipin peroxidation in a model system (liposomes of bovine cardiolipin). We found that both natural ubiquinol and synthetic antioxidants, even being introduced in micro- and submicromolar concentrations, fully protected the liposomal cardiolipin from peroxidation. The duration of their action, however, varied; it increased with the presence of either methoxy groups of ubiquinol or additional reduced redox groups (in the cases of rhodamine and berberine derivates). The concentration of ubiquinol in the mitochondrial membrane substantially exceeds the concentrations of antioxidants we used and would seem to fully prevent peroxidation of membrane cardiolipin. In fact, this does not happen: cardiolipin in mitochondria is oxidized, and this process can be blocked by amphiphilic cationic antioxidants (Y. N. Antonenko et al. (2008) Biochemistry (Moscow), 73, 1273-1287). We suppose that a fraction of mitochondrial cardiolipin could not be protected by natural ubiquinol; in vivo, peroxidation most likely threatens those cardiolipin molecules that, being bound within complexes of membrane proteins, are inaccessible to the bulky hydrophobic ubiquinol molecules diffusing in the lipid bilayer of the inner mitochondrial membrane. The ability to protect these occluded cardiolipin molecules from peroxidation may explain the beneficial therapeutic action of cationic antioxidants, which accumulate electrophoretically within mitochondria under the action of membrane potential.

The initiation of free radical peroxidation of low-density lipoproteins by glucose and its metabolite methylglyoxal: a common molecular mechanism of vascular wall injure in atherosclerosis and diabetes.

It was found that glucose in the range of concentrations 12.5-100mM stimulated Cu(2+)-mediated free radical peroxidation of low-density lipoproteins (LDL) from human blood plasma. Considering the kinetic parameters of LDL peroxidation we proposed that intensification of this process may be caused by formation of free radical intermediates of glucose auto-oxidation. Addition of SOD to the medium inhibited LDL oxidation, indicating the formation of superoxide anion-radicals under autoxidation of glucose. Similarly, SOD inhibited free radical peroxidation of liposomes from egg lecithin in the presence of glucose that confirms the generation of superoxide radicals under co-oxidation of unsaturated lipids and glucose. Normalization of glucose level in the blood of patients with type 2 diabetes mellitus during therapy was accompanied by a significant decrease in LDL oxidation in vivo (the decrease in primary and secondary lipoperoxidation products). The formation of superoxide anion-radicals was observed during interaction of aminoacid L-lysine with a product of glucose oxidative metabolism-methylglyoxal, but not with a product of lipoperoxidation malonyldialdehyde. In accordance with the foregoing the administration of sugar-lowering drug metformin, which binds and utilizes methylglyoxal, caused a stronger inhibition of LDL peroxidation in the blood of patients with diabetes mellitus, probably due to decrease in methylglyoxal-dependent generation of superoxide anion-radicals. Based on the results we set out the hypothesis about autocatalytic mechanism of free radical reactions involving natural dicarbonyls and suppose the common molecular mechanism of vascular wall injury in atherosclerosis and diabetes.

Antioxidant activity of flavanoids on basis of vegetation raw material of Kazakhstan (794.2)

The most important of flavanoids in metabolism in vegetable cell is connected with its ability to make up tough chelate complexes with various ions of metals. Also the ability to interact with free radicals, to participate in electrons’ transport, to connect with various enzymes, changing their activity. Flavanoids are able to penetrate into cell membranes where the flavanoids destroy the primary and by‐productive of lipids peroxidation (PLP). As we know pathological state in human’s body in that or another degree is closely connected with the processes PLP. We have made research of the comparative study of individual compounds of routine, quercertin’s influence and total medicines on the basis of hawthorn blood‐red, Russian thistle and saw‐wort upon the dynamics of lipids peroxidation under strict gycopsy at laboratory rats. About the PLP’s condition we have judged by the maintenance of malonic dialdehyde (MDA). Antioxidant activity we have valued by changing catalase activity in serum blood but antioxidant effect by inhibition peroxidation of liposomes at presence exogenous substances. It should be in mind that changes of catalase activity adequate antioxidant effect of having researched medicines. More marked antioxidant activity was expressive quercetin which can be explained by two factors: by the same doze of medicines, the less molar mass quercetin rises the concentration of substances.

Semiquinone glucoside derivative (SQGD) isolated from Bacillus sp. INM-1 protects against gamma radiation-induced oxidative stress

In the present study, radioprotective potential of Semiquinone glucoside derivative (SQGD) isolated from radioresistant bacterium Bacillus sp. INM-1 was evaluated. γ-Radiation induced protein carbonylation, plasmid DNA damage, enzyme functional impairment, lipid peroxidation, HO radicals generation and their protection by SQGD was assessed. As a result of SQGD treatment, significant inhibition (p<0.05) in protein carbonylation was observed with BSA. SQGD treatment was found to restore supercoiled (∼70±3.21%) form of irradiated plasmid DNA against γ-irradiation. SQGD protects enzymes (EcoR1 and BamH1) against radiation-induced dysfunctioning. SQGD significantly inhibited (p<0.05) lipid peroxidation in liposomes, brain and liver homogenate. Higher HO• radicals-averting activity of SQGD was observed in the serum and liver homogenate of C57BL/6 mice against H2O2-induced oxidative stress. In conclusion, SQGD demonstrates excellent radical-scavenging activity towards bio-macromolecules in irradiated environment and can be developed as an ideal radioprotector against radiation-induced oxidative stress in future.

Membrane lipid peroxidation by the peroxidase-like activity of magnetite nanoparticles.

We report that Fe3O4 nanoparticles are able to catalyse lipid peroxidation in liposomes at acidic but not neutral pH. The oxidation is dependent on either pre-existing lipid peroxides or hydrogen peroxide as a substrate. This is the first evidence for metal oxide nanoparticles as peroxidase mimetics oxidising biomolecules in a relevant environment.

Effects of Different Phases of Cigarette Smoke on Lipid Peroxidation and Membrane Structure in Liposomes

This paper discloses for the first time the effects of the gas phase (GP) and the tar of cigarette smoke on lipid peroxidation (LPO) and on the structure of different lipid regions in liposomes. The LPO development was analysed in terms of the total unsaturation of lipids (double-bond, DB, content) and the formation of dienic conjugates (DC), ketodienes (KD), and malonic dialdehyde (MDA). As expected, the exposure of liposomes to either the GP or the tar led to a significant decrease in the DB content. However, the formation of oxidation products revealed different dynamics: MDA generation was inhibited, while the formation of DC and KD increased during the first few hours of the LPO development followed by its inhibition. The smoke constituents exhibited opposite effects on the structure of the lipid bilayer of liposomes: the GP markedly enhanced the microviscosity of liposomal membranes, whereas the tar caused a drastic lowering of microviscosity.

Iron‐mediated peroxidation in marine emulsions and liposomes studied by dissolved oxygen consumption

Low molecular weight (LMW) iron is present in most food in traces or significant amounts. Upon contact with unsaturated lipids LMW iron acts as a prooxidant. This creates oxidative stability problems for products containing marine polyunsaturated lipids. In this work, LMW iron‐mediated oxidation in fish oil emulsions stabilized with phospholipids and Tween and in liposomes made from phospholipids was studied. Marine and non‐marine sources of phospholipids were used. The aim was to evaluate how physicochemical factors and lipid properties affect the prooxidant activity of LMW iron. The oxidation was followed by measuring the rate of the dissolved oxygen consumption by fatty acids. Red‐ox cycling of iron (Fe3+/Fe2+) aided by decomposition of the pre‐formed lipid hydroperoxides was the major prooxidation mechanism in both emulsions and liposomes. The elimination of the pre‐formed lipid hydroperoxides prevented LMW iron‐mediated oxidation. The oxygen uptake rates were highest for various liposomes/emulsions at pH 4–5 making this an optimum pH for iron activity. The pro‐oxidant effect of iron was reduced by using less unsaturated phospholipids, specific amounts of emulsifiers, a presence of chloride anions, or xanthan gum. This work suggests that the iron ions are tightly associated with the phosphate groups within the phospholipids heads. The measurement of oxygen consumption is a good tool for systematic oxidation studies in emulsions and liposomes, and may be useful for assessment of optimal conditions for reduction of LMW iron‐mediated oxidation in emulsion‐like systems.Practical applications: For a successful addition of marine polyunsaturated lipids into processed food, it is important to understand the pro‐oxidation mechanisms of ubiquitous LMW iron, as well as how physicochemical conditions affect the pro‐oxidative activity or LMW iron. This work addresses these issues in emulsions and liposomes and the knowledge may help to characterize effective hurdles for pro‐oxidant activity of LMW iron. There is a need for quick and inexpensive oxidation assays in order to be able to screen and model the effects of different factors on the oxidative stability of lipids. The measurement of dissolved oxygen in emulsions and liposomes has been used in this study for evaluating the effect of the different psychochemical and chemical factors on iron‐mediated oxidation. This method has a potential as a tool for assessment of optimal conditions for prevention of LMW iron‐mediated lipid oxidation in emulsion type systems and understanding the possible mechanisms.

Antioxidative Capacity of Proanthocyanidins from China Bitter Humulus Lupulus in Vitro

Seven proanthocyanidin fractions (PAs, P0–P6) were obtained from China bitter hop Tsingtao (Humulus lupulus) by organic solvent extraction, macroporous resin AB-8 adsorption, and the classification of oligomers/polymers. The average degrees of polymerization (ADP) for fractions P0–P6 were 2.22, 2.59, 3.54, 3.88, 2.63, 3.02, and 5.57, respectively. Antioxidative activities including the reducing power (RP), inhibition capability of lipid peroxidation (ICLP), antioxidant in linoleic acid system (ALAS), ability of scavenging hydroxyl free radical [HO·] (ASHFR), and thiobarbituric acid (TBA) value (carbonyl compounds in beer could react with TBA and produce a typical color at 530 nm) were observed in this study. The antioxidation of PAs was related to its structure and the reaction system and there was no obvious correlation between ICLP (ALAS/ASHFR) and the ADP of PAs. The RP (the reducing power of antioxidants by converting Fe3+ in potassium ferricyanide into Fe2+) of PA fractions from strong to weak were Pn fractions (n = 0–5) > (+)-catechin > Vc (the reference substance of antioxidants, ascorbic acid) > P6 fractions (ADP = 5.57). A considerable concentration effect was observed in the inhibition of both the liposome peroxidation process and the linoleic system. However, the ICLP was above 20% at 234 nm and 532 nm when the addition of P3 or P4 fraction was 30 μg/mL, and the inhibition effect of P2 fraction was the strongest in the linoleic acid system. The distinct inhibition difference of PAs in autoxidation of linoleic acid system existed when the addition was 10 μg/mL, and Pn fractions (n = 3–6) showed strong antioxidation compared to Vc in this situation. The ASHFR gradually strengthened with concentration. When the concentration reached 50 μg/mL, the ASHFR were all above 5% and increased with the ADP of all PA fractions, and the ASHFR of P6 fraction (ADP = 5.57) exceeded 20%. During wort boiling process, P3 and P4 fractions had powerful antioxidative ability, which could significantly inhibit the formation of aging precursors.

Chemical composition and biological activities of Hypericum pamphylicum

The chemical composition of methanolic extract of Hypericum pamphylicum (HPM) was analyzed by using high performance liquid chromatography–diode array detector (HPLC-DAD). The plant contained hypericin (0.00002±0.000001%), hyperforin (trace), chlorogenic acid (5.58±1.59%), hyperoside (0.46±0.05%), isoquercitrin (0.134±0.02%) and quercetin (0.087±0.008%). The antioxidant activity of HPM was evaluated by using several antioxidant tests. HPM exhibits inhibitory capacity against phosphatidylcholine liposome peroxidation, induced with iron and ascorbic acid, scavenge DPPH and superoxide radicals and act as reductants. The cytotoxic activity of HPM was also determined by using MTT cell viability assay on HeLa and NRK-52E cell lines. HPM demonstrated no cytotoxicity against HeLa and NRK-52E cell lines with IC50 values of 1.89mg/ml and 1.05mg/ml, respectively. The different crude extracts of H. pamphylicum were analyzed using a microdilution assay for antimicrobial activity against several microorganisms. The most active extract was HPM which showed activity against Staphylococcus aureus. It is observed that nearly all extracts showed activity against Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella thypi, Shigella flexneri, Proteus mirabilis and Candida albicans.

Screening of the antioxidant potential of the leaves and flowers from Rosa horrida Fischer

Rosa species are widely distributed in Turkey. Ethanolic extracts from flowers and leaves of R. horrida, one of the 25 species of Rosa used as a traditional Turkish medicine as tonic, diuretic, expectorant, stomachic and for the treatment of dyspepsia, diarrhea and nephritis were tested in vitro for their ability to scavenge DPPH·, ABTS·+ and superoxide radicals, reduce Fe (III) to Fe (II) and to inhibit peroxidation of phophatidylcholine liposomes induced with ascorbate/Fe (III) in order to establish a relashionship between their antioxidant potency and the content of phenolic compounds. Flowers and leaves extracts showed high DPPH radical scavenging activity as shown by the lowest value of EC50, indicating the strong hydrogen-donating ability. The extracts showed similar metal chelation ability in Fe2+-ferrozin test, reducing power and ability to scavenge ABTS·+ radical cation as shown by the small differences of the EC50 values, since the EC50 values were found to be highest in TBA test, suggesting that the extracts were less effective in protection of liposomes from lipid peroxidation. The activities correlated well with total phenolic and flavonoid contents, indicating that the extracts may play an important role in chemical protection from oxidative damage by possessing endogenious antioxidants, such as phenolics and flavonoids. Key words: Rosa horrida, leaves, flowers, polyphenol content, antioxidant potential.

The modified action of triphenyllead chloride on UVB-induced effects in albumin and lipids

Previously we have shown a toxic effect of the organometallic compound triphenyllead (TPhPb) on cells. In the present study we evaluated the destructive effect of TPhPb on model systems – serum albumin and liposome membranes – alone and under UVB irradiation. UVB irradiation of bovine serum albumin results in protein SS bond reduction, free SH and CO group formation and decrease in fluorescence intensity of tryptophans. Triphenyllead chloride alone and under UVB irradiation did not induce protein oxidation, measured as formation of carbonyl groups, in serum albumin; however, it decreased the content of SH groups in both cases (alone and under UVB radiation) in a dose-dependent manner. It was found that triphenyllead chloride alone did not induce lipid peroxidation of liposomes but increased their fluidity. However, under UVB irradiation TPhPb dramatically enhances the pro-oxidant action of UVB in a manner dependent on concentration and intensity of radiation, and these effects were suppressed by Trolox. These results suggest that the toxicity of TPhPb under UVB irradiation is due to formation of radical forms of the compound and its disordered effects on the membrane structure.

Antioxidant synergism between ethanolic Centella asiatica extracts and α-tocopherol in model systems

The synergistic antioxidant effects of ethanolic extracts of Centella asiatica (CE), and α-tocopherol have been studied. The types of interactions exhibited by CE and α-tocopherol combined at different ratios were measured using three assays: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) radical-scavenging capacity, the β-carotene bleaching system and liposome peroxidation assays. Fixed-fraction isobolographic analysis was used to detect any inducement of the antioxidant activity compared with the individual activities of CE and α-tocopherol. Of all synergistic combinations of CE and α-tocopherol, only fraction 2/3 showed the synergistic combination that fits well in three different assays and can be explained by the regeneration of α-tocopherol by CE despite the interaction effect of β-carotene present in the analytical assay. This phenomenon involved complex interactions between CE and α-tocopherol to exhibit different degrees of interactions that eventually increased antioxidant activity.