Activity and mechanism of the antioxidant properties of cyanidin-3-O-β-glucopyranoside

In the present study, the antioxidant activity, the interaction with reactive oxygen species and the redox potential of cyanidin-3-O-β-glucopyranoside (C-3-G), the main anthocyanin present in juice of pigmented oranges, were evaluated in detail. C-3-G effects on low density lipoproteins (LDL) oxidation induced by 40 μM Cu²⁺ at a pH of 7.4 were compared with those of resveratrol and ascorbic acid, two other natural antioxidants. All cyanidin-3-O-β-glucopyranoside concentrations used (1, 2, 5, 10, 20, 50, 100 and 200 μM) inhibited malondialdehyde (MDA) generation (an index of lipid peroxidation), the inhibition being significantly higher than that obtained with equal concentrations of resveratrol and ascorbic acid (IC₅₀=6.5 μM for C-3-G, 34 μM for resveratrol and 212 μM for ascorbic acid). Experiments of LDL oxidation performed at a pH of 5.0 or 6.0 showed that C-3-G antioxidant activity is not influenced by pH variations between 5.0 and 7.4. This suggests that metal chelation, exerted by C-3-G through the eventual dissociation of its phenolic groups, plays a minor role in its protective mechanism. The presence of C-3-G produced significantly higher protective effects of pigmented orange juice (obtained from Moro cultivar) with respect to blond orange juice, when tested on copper-induced LDL oxidation. The evaluation of the direct interaction with reactive oxygen species (H₂O₂, ^-O₂, OH^·), demonstrated that C-3-G is quickly oxidized by these compounds and it is, therefore, a highly efficient oxygen free radical scavenger. The powerful C-3-G antioxidant activity is in excellent agreement with the very negative redox potential (405 mV), determined through direct current cyclic voltammetry measurements.

On the basis of these results, C-3-G should be considered as one of the most effective antioxidants that can be assumed with dietary plants; therefore, pigmented oranges represent a very relevant C-3-G source because of the high content of this anthocyanin in their juice.     

Cyanidin-3-O-beta-glucopyranoside protects myocardium and erythrocytes from oxygen radical-mediated damages

The cyanidin-3- O - β-glucopyranoside (C-3-G) antioxidant capacity towards reactive oxygen species (ROS)-mediated damages was assessed in tissue and cells submitted to increased oxidative stress. In the isolated ischemic and reperfused rat heart, 10 or 30 μM C-3-G protected from both lipid peroxidation (66.7 and 94% inhibition of malondialdehyde (MDA) generation in 10 and 30 μM C-3-G-reperfused hearts, respectively, in comparison with control reperfused hearts) and energy metabolism impairment (higher ATP concentration in 10 and 30 μM C-3-G-reperfused hearts than in control reperfused hearts). These effects were associated to C-3-G permeation within myocardial cells, as indicated by results obtained in the isolated rat heart perfused for 30 min in the recirculating Langendorff mode under normoxia with 10 and 30 μM C-3-G. Protective effects were exerted, in a dose-dependent manner, by C-3-G also in 2 mM hydrogen peroxide-treated human erythrocytes. With respect to MDA formation, an apparent IC 50 of 5.12 μM was calculated for C-3-G (the polyphenol resveratrol used for comparison showed an apparent IC 50 of 38.43 μM). The general indications are that C-3-G (largely diffused in dietary plants and fruits, such as pigmented oranges very common in the Mediterranean diet) represents a powerful natural antioxidant with beneficial effects in case of increased oxidative stress, and at pharmacological concentrations it is able to decrease tissue damages occurring in myocardial ischemia and reperfusion.     

Cyanidin-3- O -β-glucopyranoside Protects Myocardium and Erythrocytes from Oxygen Radical-mediated Damages

The cyanidin-3- O - &#103 -glucopyranoside (C-3-G) antioxidant capacity towards reactive oxygen species (ROS)-mediated damages was assessed in tissue and cells submitted to increased oxidative stress. In the isolated ischemic and reperfused rat heart, 10 or 30 &#119 M C-3-G protected from both lipid peroxidation (66.7 and 94% inhibition of malondialdehyde (MDA) generation in 10 and 30 &#119 M C-3-G-reperfused hearts, respectively, in comparison with control reperfused hearts) and energy metabolism impairment (higher ATP concentration in 10 and 30 &#119 M C-3-G-reperfused hearts than in control reperfused hearts). These effects were associated to C-3-G permeation within myocardial cells, as indicated by results obtained in the isolated rat heart perfused for 30 min in the recirculating Langendorff mode under normoxia with 10 and 30 &#119 M C-3-G. Protective effects were exerted, in a dose-dependent manner, by C-3-G also in 2 mM hydrogen peroxide-treated human erythrocytes. With respect to MDA formation, an apparent IC 50 of 5.12 &#119 M was calculated for C-3-G (the polyphenol resveratrol used for comparison showed an apparent IC 50 of 38.43 &#119 M). The general indications are that C-3-G (largely diffused in dietary plants and fruits, such as pigmented oranges very common in the Mediterranean diet) represents a powerful natural antioxidant with beneficial effects in case of increased oxidative stress, and at pharmacological concentrations it is able to decrease tissue damages occurring in myocardial ischemia and reperfusion.     

Ascorbate is particularly effective against LDL oxidation in the presence of iron(III) and homocysteine/cystine at acidic pH

Metal-catalyzed LDL oxidation is enhanced by the presence of homocysteine. In this study, the effectiveness of ascorbic acid against low-density lipoprotein (LDL) oxidation by iron(III) and copper(II) in the presence of homocysteine and the main plasma disulfide cystine was investigated. Relative to the degree of LDL oxidation reached in the absence of antioxidants, ascorbic acid was particularly effective against iron-catalyzed LDL oxidation at pH 6.0. This can be explained from its stability under acidic conditions and is likely to be important in ischemia, in inflammation and exhausting exercise. At pH 7.4, an ascorbic acid concentration at least as high as the concentration of homocysteine might be necessary to efficiently inhibit LDL oxidation by iron(III) and copper(II) in the presence of homocysteine and cystine. Histidine increased the efficiency of ascorbic acid as an antioxidant against copper-mediated oxidation in this system. The capacity of homocysteine to regenerate ascorbic acid from dehydroascorbic acid appeared to play a minor role in inhibition of ascorbic acid oxidation by copper as compared to copper chelation by homocysteine.     

The compositional characterisation and antioxidant activity of fresh juices from sicilian sweet orange (Citrus sinensis L. Osbeck) varieties

Epidemiological evidence has suggested that consumption of fruit and vegetables reduces the risk of both cancer and cardiovascular diseases, potentially through the biological actions of components such as vitamin C, vitamin E, flavonoids and carotenoids. Citrus species are extremely rich sources in vitamin C and flavanones, a class of compounds which belongs to the flavonoids family. A comparison of the phenolic compositions, the ascorbic acid contents and the antioxidant activities of fresh Sicilian orange juices from pigmented (Moro, Tarocco and Sanguinello) and non-pigmented (Ovale, Valencia and Navel) varieties of orange (Citrus sinensis L. Osbeck), was undertaken. The simultaneous characterisation and quantification of the major flavanone, anthocyanin and hydroxycinnamate components were attained by HPLC with diode array detection. Differences between varieties in terms of the flavanone glycoside content, particularly hesperidin, were observed, with the Tarocco juices reporting the highest content. Furthermore, cyanidin-3-glucoside and cyanidin-3-(6"-malonyl)-glucoside were predominant in all the pigmented varieties, but their concentration was higher in the juices of the Moro variety. Quantitatively, the major antioxidant component of all juices was ascorbic acid and its concentration was significantly correlated (r = 0.74, P < 0.001) with the total antioxidant activity of the juices, determined in vitro using the ABTS radical cation decolorization assay. Similarly, hydroxycinnamates (r = 0.73, P < 0.01) and anthocyanins (r = 0.98, P < 0.001) content showed a good correlation with the determined antioxidant capacity. Therefore orange juices, particularly those rich in anthocyanins, may represent a significant dietary source of flavonoids.     

Protective effects of cyanidin-3-O-glucoside from blackberry extract against peroxynitrite-induced endothelial dysfunction and vascular failure

Anthocyanins are a group of naturally occurring phenolic compounds as colorants in several plants, flowers and fruits. These pigments have a great importance as quality indicators, as chemotaxonomic markers and antioxidants.The content of blackberry (Rubus species) juice was investigated by HPLC/ESI/MS using narrow bore HPLC columns. Using this method we demonstrated that cyanidin-3-O-glucoside represents about 80% of the total anthocyanin contents in blackberry extract. Here we investigated antioxidant activity of the blackberry juice and cyanidin-3-O-glucoside on the endothelial dysfunction in cells and in vascular rings exposed to peroxynitrite. In human umbilical vein endothelial cells (HUVEC) in vitro, peroxynitrite caused a significant suppression of mitochondrial respiration (38 +/- 2.1% of control cells), as measured by the mitochondrial-dependent conversion of the dye MTT to formazan. Peroxynitrite caused DNA strand breakage (63 +/- 1.9% single strand vs 3 +/- 0.9% single strand in control cells), as measured by the alkaline unwinding assay, and caused an activation of PARS, as measured by the incorporation of radiolabeled NAD(+) to nuclear proteins. Blackberry juice (different dilutions that contained 80 ppm;40 ppm;14.5 ppm of cyanidin-3-O-glucoside) and cyanidin-3-O-glucoside (as chloride) (0.085 microM; 0.028 microM; 0.0085 microM) reduced the peroxynitrite-induced suppression of mitochondrial respiration, DNA damage and PARS activation in HUVECs. Vascular rings exposed to peroxynitrite exhibited reduced endothelium-dependent relaxant responses in response to acetylcholine as well as a vascular contractility dysfunction in response to norepinephrine. The development of this peroxynitrite-induced vascular dysfunction was ameliorated by the blackberry juice (different dilutions that contained 80 ppm;40 ppm;14.5 ppm of cyanidin-3-O-glucoside) and cyanidin-3-O-glucoside (as chloride) (0.085 microM;0.028 microM;0.0085 microM).In conclusion our findings clearly demonstrate that blackberry juice containing cyanidin-3-O-glucoside is a scavenger of peroxynitrite and that exert a protective effect against endothelial dysfunction and vascular failure induced by peroxynitrite.     

Synergistic interaction between the probucol phenoxyl radical and ascorbic acid in inhibiting the oxidation of low density lipoprotein.

Chain-breaking antioxidants such as butylated hydroxytoluene, alpha-tocopherol, and probucol have been shown to decrease markedly the oxidative modification of low density lipoprotein (LDL). Their mechanism of action appears to involve scavenging of LDL-lipid peroxyl radicals. The purpose of this study was to investigate the occurrence of radical reactions produced during oxidation of LDL and LDL-containing probucol initiated by lipoxygenase or copper. In addition, we have investigated the possibility of a synergistic interaction between ascorbate and probucol in inhibiting the oxidation of LDL. Incubation of LDL-containing probucol and lipoxygenase produced a composite electron spin resonance (ESR) spectrum due to the endogenous alpha-tocopheroxyl radical and probucol-derived phenoxyl radical. The spectral assignment was further verified by chemical oxidation of alpha-tocopherol and probucol. In the presence of ascorbic acid, these radicals in the LDL particle were reduced to their parent compounds with concomitant formation of the ascorbate radical. In both the peroxidation of linoleic acid and the copper-initiated peroxidation of LDL, the antioxidant activity of probucol was significantly increased by low (3-6 microM) concentrations of ascorbate. The probucol-dependent inhibition of LDL oxidation was enhanced in the presence of ascorbic acid. We conclude that the reaction between the phenoxyl radical of probucol and ascorbate results in a synergistic enhancement of the antioxidant capacity of these two compounds and speculate that such reactions could play a role in maintaining the antioxidant status of LDL during oxidative stress in vivo.     

Aggregation of LDL with chondroitin-4-sulfate makes LDL oxidizable in the presence of water-soluble antioxidants

The content of plasma and arterial interstitial fluid in water-soluble antioxidants makes it unlikely for low-density lipoprotein (LDL) to oxidize by the oxidation mechanisms most frequently discussed. By aggregation of LDL in the presence of chondroitin-4-sulfate (C-4-S), but not with chondroitin-6-sulfate or sphingomyelinase, a complex arises which can oxidize in the presence of 20 microM ascorbate and 300 microM urate. This oxidation sensitivity even persists after the gel-filtration of an LDL/C-4-S/Cu(2+) complex, indicating entrapment of Cu(2+) within. This corresponds well to the known ability of C-4-S to bind copper ions and is a potential mechanism by which LDL oxidation in the arterial intima is facilitated after prolonged retention by the extracellular matrix.     

Free radical-mediated chain oxidation of low density lipoprotein and its synergistic inhibition by vitamin E and vitamin C

The oxidation of human low density lipoprotein (LDL) initiated by free radical initiator and its inhibition by vitamin E and water-soluble antioxidants have been studied. It was found that the kinetic chain length was considerably larger than 1, suggesting that LDL was oxidized by a free radical chain mechanism. Vitamin E acted as a lipophilic chain-breaking antioxidant. Water-soluble chain-breaking antioxidants such as ascorbic acid and uric acid suppressed the oxidation of LDL initiated by aqueous radicals but they could not scavenge lipophilic radicals within LDL to break the chain propagation. Ascorbic acid acted as a synergistic antioxidant in conjunction with vitamin E.     

外源咖啡酸和阿魏酸对黑莓汁中花色苷的辅色研究

为增强黑莓汁中花色苷的稳定,添加适量咖啡酸和阿魏酸到黑莓清汁中,采用可见吸收光谱和高效液相色谱-质谱研究其对黑莓花色苷的辅色作用。研究结果表明:黑莓汁中添加咖啡酸和阿魏酸显著增加了花色苷的最大吸收值(Aλmax),最大吸收波长(λmax)红移,说明咖啡酸和阿魏酸对黑莓汁中花色苷产生了辅色作用,辅色效应随时间的延长和咖啡酸、阿魏酸浓度的增加显著增强。HPLC-DAD-MS分析发现,咖啡酸辅色产生了两种新的花色苷衍生物(矢车菊素-3-O-葡萄糖苷-4-乙烯基儿茶酚和矢车菊-3-O-草酸酐酰葡萄糖苷-4-乙烯基儿茶酚),阿魏酸辅色产生了三种新的花色苷衍生物(矢车菊素-3-O-葡萄糖苷-4-乙烯基愈创木酚、矢车菊-3-O-草酸酐酰葡萄糖苷-4-乙烯基愈创木酚和矢车菊-3-O-阿拉伯糖苷-4-乙烯基愈创木酚),这些衍生物均为羟苯基-吡喃花色苷。     

beta-Carotene inhibits the oxidative modification of low-density lipoprotein

Several lines of evidence indicate that oxidized LDL (Ox-LDL) may promote atherogenesis. Hence, the role of antioxidants in the prevention of LDL oxidation needs to be determined. beta-Carotene, in addition to being an efficient quencher of singlet oxygen, can also function as a radical-trapping antioxidant. Since previous studies have failed to show that beta-carotene inhibits LDL oxidation, we re-examined its effect on the oxidative modification of LDL. For these studies, LDL was oxidized in both a cell-free (2.5 microM Cu2+ in PBS) and a cellular system (human monocyte macrophages in Ham's F-10 medium). beta-Carotene inhibited the oxidative modification of LDL in both systems as evidenced by a decrease in the lipid peroxide content (thiobarbituric-acid-reacting substances activity), the negative charge of LDL (electrophoretic mobility) and the formation of conjugated dienes. By inhibiting LDL oxidation, beta-carotene substantially decreased its degradation by macrophages. beta-Carotene (2 microM) was more potent than alpha-tocopherol (40 microM) in inhibiting LDL oxidation. Thus, beta-carotene, like ascorbate and alpha-tocopherol, inhibits LDL oxidation and might have an important role in the prevention of atherosclerosis.     

On the role of vitamin C and other antioxidants in atherogenesis and vascular dysfunction

Oxidative stress has been implicated as an important etiologic factor in atherosclerosis and vascular dysfunction. Antioxidants may inhibit atherogenesis and improve vascular function by two different mechanisms. First, lipid-soluble antioxidants present in low-density lipoprotein (LDL), including alpha-tocopherol, and water-soluble antioxidants present in the extracellular fluid of the arterial wall, including ascorbic acid (vitamin C), inhibit LDL oxidation through an LDL-specific antioxidant action. Second, antioxidants present in the cells of the vascular wall decrease cellular production and release of reactive oxygen species (ROS), inhibit endothelial activation (i.e., expression of adhesion molecules and monocyte chemoattractants), and improve the biologic activity of endothelium-derived nitric oxide (EDNO) through a cell- or tissue-specific antioxidant action. alpha-Tocopherol and a number of thiol antioxidants have been shown to decrease adhesion molecule expression and monocyte-endothelial interactions. Vitamin C has been demonstrated to potentiate EDNO activity and normalize vascular function in patients with coronary artery disease and associated risk factors, including hypercholesterolemia, hyperhomocysteinemia, hypertension, diabetes, and smoking.     

Inhibitory effect of flavonoids on low-density lipoprotein peroxidation catalyzed by mammalian 15-lipoxygenase

Lipoxygenase-dependent low-density lipoprotein (LDL) oxidation is believed to be involved in atherogenesis. Inhibition of lipoxygenase-induced lipid peroxidation might, therefore, be an important mode to suppress the development of atherosclerosis. Because dietary antioxidants inhibit LDL oxidation in vitro and their intake is inversely associated with coronary heart diseases, we compared the inhibitory effect of three typical flavonoids-quercetin, epicatechin, and flavone-with alpha-tocopherol and ascorbic acid against human LDL oxidation catalyzed by mammalian 15-lipoxygenase. The oxidative modification of LDL was monitored by measurement of cholesteryl ester hydroperoxide (CE-OOH) formation and consumption of antioxidants by using HLPC. Quercetin and epicatechin were the strongest inhibitors of LDL oxidation catalyzed by 15-lipoxygenase; ascorbic acid was an effective inhibitor in the first 3 h of oxidation; and fivefold alpha-tocopherol-enriched LDL showed a partial inhibition of CE-OOH formation only after 4-6 h of incubation. Flavone had no effect. Quercetin, ascorbic acid, and alpha-tocopherol were consumed in the first 3 h of incubation. Consumption of LDL alpha-tocopherol was partially inhibited by ascorbic acid and quercetin, whereas epicatechin and flavone were without effect. These results emphasize the inhibitory effect of the flavonoids quercetin and epicatechin on 15-lipoxygenase-mediated LDL lipid peroxidation. At similar concentrations, they are stronger antioxidants than ascorbic acid, alpha-tocopherol, and flavone.     

α-tocopherol,ascorbic acid,and rutin inhibit synergistically the copper-promoted LDL oxidation and the cytotoxicity of oxidized LDL to cultured endothelial cells

Low-density lipoproteins (LDL) mildly oxidized by copper ions or UV radiations exhibit a cytotoxic effect to cultured endothelial cells. Rutin, a polyphenolic flavonoid, ascorbic acid, and α-tocopherol were able to inhibit the peroxidation of LDL and their subsequent cytotoxicity. The mixture of the three compounds (rutin/ascorbic acid/α-tocopherol, 4/4/1) exhibited a supra-additive antioxidant effect. The inhibition of the cytotoxic effect was well correlated with that of TBARS formation. Another important conclusion is that these antioxidants were able to prevent directly at the cellular level the cytotoxic effect of oxidized LDL, since cells preincubated with them were protected against the cytotoxic effect of previously oxidized LDL. The protective effect of antioxidants was limited because of their own toxicity. The antioxidant mixture permitted a maximal cytoprotective effect with relatively lower concentrations to be obtained and the cytotoxicity of high concentrations to be avoided. In conclusion, rutin, ascorbic acid, and α-tocopherol constitute two lines of defense in protecting cells against injury owing to oxidation of LDL (1) at the LDL level, by inhibiting the LDL oxidation and the subsequent cytotoxicity, and (2) at the cellular level, by protecting the cells directly, i.e., by increasing their resistance against the cytotoxic effect of oxidized LDL.     

The importance of lipid solubility in antioxidants and free radical generating systems for determining lipoprotein proxidation.

The oxidative modification of low-density lipoprotein (LDL) plays an important role in atherosclerosis. Protecting LDL from oxidation has been shown to reduce the risk of coronary heart disease. In this study, we compared the protective effects of two lipophilic antioxidants (vitamin E and lazaroid) with two hydrophilic antioxidants (trolox and vitamin C) in the presence of several different free radical generating systems. Vitamin E (IC50 = 5.9 microM) and lazaroid (IC50 = 5.0 microM) were more effective in inhibiting lipid peroxidation caused by a Fe-ADP free radical generating system than vitamin C (IC50 = 5.2 x 10(3) microM) and trolox (IC5 = 1.2 x 10(3) microM). Preincubation of lipoproteins with a lipophilic antioxidant increased the protective effect against various free radicals. Preincubation with hydrophilic antioxidants did not have an effect. We also tested the efficacy of the antioxidants when the free radicals were generated within the lipid or the aqueous environment surrounding the LDL. For this purpose, we used the peroxyl generating azo-compounds AMVN (2,2'-azobis(2,4-dimethylvaleronitrile)) and AAPH (2,2'azobis(2-amidinopropane) dihydrochloride). All of the antioxidants tested were more effective against free radicals generated in a water soluble medium than they were against free radicals generated in a lipid environment. In conclusion, our data demonstrate that lipid solubility is an important factor for both the antioxidant and the free radical generating systems in determining the extent of lipid peroxidation in LDL. Our data also demonstrate that antioxidant efficacy in one set of experimental conditions may not necessarily translate into a similar degree of protection in another set of conditions where lipophilicity is a variable.     

Antioxidant and Prooxidant Effects of Ascorbic Acid, Dehydroascorbic Acid and Flavonoids on LDL Submitted to Different Degrees of Oxidation

Although a high intake of antioxidants may decrease the risk of developing cardiovascular diseases, under certain circunstances they may promote free radical generation and lipid peroxidation. The objectives of the present study were to determine the antioxidant effects of ascorbic acid (AA), dehydroascorbic acid (DHA) and flavonoids on LDL submitted to different degrees of oxidation. LDL was submitted to oxidation with CuCl₂ (2.4 μM). Before or at different times after the propagation of the oxidation process, 28 μM (5 μg/ml) of either AA or DHA or 5 μg/mL flavonoids extract were added. Alpha-tocopherol, conjugated dienes, thiobarbituric acid reacting substances (TBARS) and LDL electrophoretic mobility were determined as indices of LDL oxidation. The presence of any of the three antioxidants from the onset of the incubation delayed the oxidation process. However, the addition of both DHA and flavonoids to the oxidation process when it was already initiated and alpha-tocopherol consumed, accelerated the oxidation. In contrast, AA delayed the oxidation process even when added after alpha-tocopherol was consumed. Nevertheless, it also accelerated LDL oxidation when added during the propagation phase of the oxidation process. In conclusion: although AA, DHA and flavonoids delay LDL oxidation when added before the initiation of the process, they accelerate the process if added to minimally oxidized LDL.     

Antioxidative activity on human low density lipoprotein (LDL) oxidation by pentagalloic acid

The aim of this study was to investigate the efficiency of the pentagalloic acid compound in inhibiting the metal ions and cell lines that mediate in low density lipoprotein (LDL) oxidation. Pentagalloic acid prolonged the lag time preceeding the onset of conjugated diene formation. In chemically induced LDL oxidation by Cu²⁺ plus hydrogen peroxide or peroxyl radical generated by 2, 2′-azo-bis (2-amidino propane) hydrochloride (AAPH), pentagalloic acid inhibited LDL oxidation as monitored by measuring the thiobarbituric acid reactive substances (TBARS), malondialdehyde (MDA), and gel electrophoretic mobility. The physiological relevance of the antioxidative activity was validated at the cellular level where pentagalloic acid inhibited mouse macrophage J774 and endothelial cell-mediated LDL oxidation. When compared with several other antioxidants, pentagalloic acid showed a much higher ability than naturally occuring antioxidants, α-tocopherol and ascorbic acid, and the synthetic antioxidant, probucol.     

Higher cholesterol in human LDL is associated with the increase of oxidation susceptibility and the decrease of antioxidant defence: experimental and simulation data

Increased low-density lipoprotein (LDL) cholesterol is a recognized risk factor for atherosclerosis. There is also strong evidence that oxidatively modified LDL initiates the development of this pathological process and the administration of antioxidants might have a protective effect. However, the appropriate trials did not provide completely consistent results. We found in this study that the oxidation kinetics and also the antioxidant effectiveness are different depending on the cholesterol content in LDL. Higher cholesterol in LDL causes an acceleration of its oxidation as well as an increase of resistance to the antioxidative effect of ascorbic acid. In searching for a theoretical background of this dual impact of cholesterol in LDL, computer simulation of LDL oxidation was used. It was found that the pre-existing level of lipid hydroperoxides together with the total amount of oxidizable lipid substrate associated with the cholesterol level in LDL were satisfactory prerequisites for a best fit to the experimental data. In conclusion, this study provides at least a partial explanation for some failures to arrest, by administration of antioxidants, the progression of atherosclerosis in animal and human hypercholesterolemia.     

Inhibition of human low density lipoprotein oxidation by active principles from spices

Spice components and their active principles are potential antioxidants. In this study we examined the effect of phenolic and non-phenolic active principles of common spices on copper ion-induced lipid peroxidation of human low density lipoprotein (LDL) by measuring the formation of thiobarbituric acid reactive substance (TBARS) and relative electrophoretic mobility (REM) of LDL on agarose gel. Curcurriin, capsaicin, quercetin, piperine, eugenol and allyl sulfide inhibited the formation of TBARS effectively through out the incubation period of 12 h and decreased the REM of LDL. Spice phenolic active principles viz. curcumin, quercetin and capsaicin at 10 M produced 40–85% inhibition of LDL oxidation at different time intervals while non-phenolic antioxidant allyl sulfide was less potent in inhibiting oxidation of LDL. However, allyl sulfide, eugenol and ascorbic acid showed pro-oxidant activity at lower concentrations (10 M) and antioxidant activity at higher concentrations (50 M) only. Among the spice principles tested quercetin and curcumin showed the highest inhibitory activity while piperine showed least antioxidant activity at equimolar concentration during initiation phase of oxidation of LDL. The inhibitory effect of curcumin, quercetin and capsaicin was comparable to that of BHA, but relatively more potent than ascorbic acid. Further, the effect of curcurnin, quercetin, capsaicin and BHA on initiation and propagation phases of LDL oxidation showed that curcurnin significantly inhibited both initiation and propagation phases of LDL oxidation, while quercetin was found to be ineffective at propagation phase. These data suggest that the above spice active principles, which constitute about 1–4% of above spices, are effective antioxidants and offer protection against oxidation of human LDL.     

Structure-activity relationship studies of resveratrol and its analogues by the reaction kinetics of low density lipoprotein peroxidation

Resveratrol (3,5,4'-trans-trihydroxystibene) is a natural phytoalexin present in grapes and red wine, which possesses a variety of biological activities including antioxidative activity. To find more active antioxidants, with resveratrol as the lead compound, we synthesized resveratrol analogues, i.e., 3,4,3',4'-tetrahydroxy-trans-stilbene (3,4,3',4'-THS), 3,4,4'-trihydroxy-trans-stilbene (3,4,4'-THS), 2,4,4'-trihydroxy-trans-stilbene (2,4,4'-THS), 3,3'-dimethoxy-4,4'-dihydroxy-trans-stilbene (3,3'-DM-4,4'-DHS), 3,4-dihydroxy-trans-stilbene (3,4-DHS), 4,4'-dihydroxy-trans-stilbene (4,4'-DHS), 3,5-dihydroxy-trans-stilbene (3,5-DHS) and 2,4-dihydroxy-trans-stilbene (2,4-DHS). Antioxidative effects of resveratrol and its analogues against free-radical-induced peroxidation of human low density lipoprotein (LDL) were studied. The peroxidation was initiated either by a water-soluble initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH), or by cupric ion (Cu(2+)). The reaction kinetics were monitored either by the uptake of oxygen and the depletion of alpha-tocopherol (TOH) presented in the native LDL, or by the formation of thiobarbituric acid reactive substances (TBARS). Kinetic analysis of the antioxidation process demonstrates that these trans-stilbene derivatives are effective antioxidants against both AAPH- and Cu(2+)-induced LDL peroxidation with the activity sequence of 3,4,3',4'-THS approximately 3,3'-DM-4,4'-DHS>3,4-DHS approximately 3,4,4'-THS>2,4,4'-THS>resveratrol approximately 3,5-DHS>4,4'-DHS approximately 2,4-HS, and 3,4,3',4'-THS approximately 3,4-DHS approximately 3,4,4'-THS>3,3'-DM-4,4'-DHS>4,4'-DHS>resveratrol approximately 2,4-HS>2,4,4'-THS approximately 3,5-DHS, respectively. Molecules bearing ortho-dihydroxyl or 4-hydroxy-3-methoxyl groups possess significantly higher antioxidant activity than those bearing no such functionalities.