Low-density Lipoprotein Oxidation Assay Research Articles

Optimization of microwave extraction method of phenolic compounds from red onion using response surface methodology and inhibition of lipoprotein low-density oxidation

In the present study, we have optimized by surface response methodology (BBD model) phenolic compounds recovery using microwave-assisted extraction (MAE) as an innovative technique. The extraction was carried out under different experimental conditions: ethanol/water (x1, 30–80 %), time (x2, 30–180 s), microwave power (x3, 500–900 W) and solvent to sample ratio (x4, 20:1 40:1, v/w). Total phenolics content (TPC) and antioxidant activities (AA) of onion extract were investigated on 2,2−9-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS+), 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), oxygen radical absorbance capacity (ORAC) tests, and on biological model oxidation, human low-density lipoprotein (LDL). The maximum predicted TPC under the optimal MAE conditions (70 % ethanol), 700 W microwave power, 65 s extraction time and 25 mL g–1 solvent to solid ratio), was 10.90 ± 0.88 mg GAE g–1 DW. The AA of MAE or conventional extraction (CE) extract showed similar activities to neutralize DPPH, ABTS or AAPH derived radicals. The modified Gompertz model has been successfully used to describe CDs production kinetic in LDL Cu2+-mediated oxidation. This allowed the estimation of the lag time (Tlag) and the oxidation propagation rate (Rp) with precision. LDL oxidation assay showed that red onion extract increased significantly Tlag and giving an increased protection ratio at higher concentration (2.95 μmol L−1) of 180 % and 172 % for MAE and CE methods, respectively. However, Rp and CDs production remained constant. Results showed that microwave used for extraction does not affect the composition and antioxidant properties of onion sample. Red onion may be an important source of bioactive compounds and could promote health consumers.

Preparation and antioxidant activity of tyrosol and hydroxytyrosol esters

A series of lipophilic esters of tyrosol and hydroxytyrosol, two naturally occurring phenols with interesting biological properties, have been prepared by an enzymatic synthesis and saponification procedure, using lipase from Candida antarctica as the catalyst. Their antioxidant activities were evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH), human low-density lipoprotein (LDL) oxidation, and DNA strand scission assays. All methods used for evaluating antioxidant activity revealed that tyrosol and its derivatives were less active than hydroxytyrosol and its analogues due to the influence of the catechol structure of the latter. For hydroxytyrosol, the introduction of the lipid moiety decreased its antioxidant activity. Meanwhile, an unsaturation-dependent antioxidant trend was observed for tyrosol and hydroxytyrosol esters in DNA strand scission assay, and for tyrosol esters in DPPH procedure. However, in LDL oxidation assay, possible oxidation of the polyunsaturated fatty acid moiety of tyrosol esters should also be considered.

The Potential Protective Effects of Phenolic Compounds against Low-density Lipoprotein Oxidation.

The exact mechanism(s) of atherosclerosis in humans remains elusive, but one theory hypothesizes that this deleterious process results from the oxidative modification of low-density lipoprotein (LDL). Research suggests that foods rich in dietary phenolic compounds with antioxidant activity can mitigate the extent of LDL oxidation in vivo. With regard to the different classes of flavonoids, there appears to be a structurefunction relationship between the various moieties/constituents attached to the flavonoids' three ring system and their impact at retarding LDL oxidation. This article summarizes the findings to date of both in vitro and in vivo studies using foods or phenolic extracts isolated from foodstuffs at inhibiting the incidence of LDL oxidation. Three bases: SCOPUS, Web Science, and PubMed were used for search. An often used method for the determination of antioxidant properties of natural phenolic compounds is the LDL oxidation assay. LDLs are isolated from human plasma and their oxidation is induced by Cu2+ ions or 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). The sample is incubated with a phenolic extract or individual/isolated phenolic compounds. LDL oxidation is then monitored by various chemical methods (e.g., measurement of the generation of conjugated dienes and trienes). This technique confirmed the antioxidant properties of several extracts as obtained from plant material (e.g., grapes, berries, orange, grapefruit, coffee, tea, chocolate, olives, nuts) as well as the individual phenolic compounds (e.g., luteolinidin, apigenidin, caffeic acid, chlorogenic acid, catechin, quercetin, rutin). Several studies in vivo confirmed protective effects of phenolic compounds against LDL oxidation. They covered the healthy subjects with hyperlipidaemia, overweight, obesity, metabolic syndrome, heavy smokers, patients receiving haemodialysis, patients with peripheral vascular disease, and subjects at high cardiovascular risk. The studies comprise individuals of all ages, and the number of participants in the different experiments varied widely. Properly designed double-blind, placebo-controlled randomised clinical trials offer stronger evidence as to the impact of dietary phenolics consumption at retarding LDL oxidation. More such clinical trials are needed to strengthen the hypothesis that foods rich in dietary phenolic compounds with antioxidant activity can mitigate the extent of LDL oxidation in vivo.

Malaysian brown seaweeds Sargassum siliquosum and Sargassum polycystum: Low density lipoprotein (LDL) oxidation, angiotensin converting enzyme (ACE), α-amylase, and α-glucosidase inhibition activities

Two Malaysian brown seaweeds, Sargassum siliquosum and Sargassum polycystum were first extracted using methanol to get the crude extract (CE) and further fractionated to obtain fucoxanthin-rich fraction (FRF). Samples were evaluated for their phenolic, flavonoid, and fucoxanthin contents, as well as their inhibitory activities towards low density lipoprotein (LDL) oxidation, angiotensin converting enzyme (ACE), α-amylase, and α-glucosidase. In LDL oxidation assay, an increasing trend in antioxidant activity was observed as the concentration of FRF (0.04–0.2mg/mL) and CE (0.2–1.0mg/mL) increased, though not statistically significant. As for serum oxidation assay, significant decrease in antioxidant activity was observed as concentration of FRF increased, while CE showed no significant difference in inhibitory activity across the concentrations used. The IC50 values for ACE inhibitory activity of CE (0.03–0.42mg/mL) were lower than that of FRF (0.94–1.53mg/mL). When compared to reference drug Voglibose (IC50 value of 0.61mg/mL) in the effectiveness in inhibiting α-amylase, CE (0.58mg/mL) gave significantly lower IC50 values while FRF (0.68–0.71mg/mL) had significantly higher IC50 values. The α-glucosidase inhibitory activity of CE (IC50 value of 0.57–0.69mg/mL) and FRF (IC50 value of 0.50–0.53mg/mL) were comparable to that of reference drug (IC50 value of 0.54mg/mL). Results had shown the potential of S. siliquosum and S. polycystum in reducing cardiovascular diseases related risk factors following their inhibitory activities on ACE, α-amylase and α-glucosidase. In addition, it is likelihood that FRF possessed antioxidant activity at low concentration level.

Sida rhomboidea.Roxb aqueous extract down-regulates in vivo expression of vascular cell adhesion molecules in atherogenic rats and inhibits in vitro macrophage differentiation and foam cell formation

The present study evaluates efficacy of Sida rhomboidea.Roxb (SR) leaves extract in ameliorating experimental atherosclerosis using in vitro and in vivo experimental models. Atherogenic (ATH) diet fed rats recorded significant increment in the serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), very LDL (VLDL), autoantibody against oxidized LDL (Ox-LDL), markers of LDL oxidation and decrement in high-density lipoprotein (HDL) along with increment in aortic TC and TG. The ex vivo LDL oxidation assay revealed an increased susceptibility of LDL isolated from ATH rats to undergo copper mediated oxidation. These set of changes were minimized by simultaneous co-supplementation of SR extract to ATH diet fed rats. Histopathology of aorta and immunolocalization studies recorded pronounced atheromatous plaque formation, vascular calcification, significant elastin derangements and higher expression of macrophage surface marker (F4/80), vascular cell adhesion molecule-1 (VCAM-1) and p-selectin in ATH rats. Whereas, ATH+SR rats depicted minimal evidence of atheromatous plaque formation, calcium deposition, distortion/defragmentation of elastin and accumulation of macrophages along with lowered expression of VCAM-1 and P-selectin compared to ATH rats. Further, monocyte to macrophage differentiation and in vitro foam cell formation were significantly attenuated in presence of SR extract. In conclusion, SR extract has the potency of controlling experimental atherosclerosis and can be used as promising herbal supplement in combating atherosclerosis.

Cloning, high level expression of human paraoxonase-3 in Sf9 cells and pharmacological characterization of its product

Human paraoxnase-3 (hPON3) (EC3.1.8.1) is a lipid-associated enzyme with antioxidant activity, and can inhibit the oxidation of low-density lipoprotein (LDL), thereby inhibiting early atherogenic process. In the present study, human PON3 gene was cloned from Human Fetal Liver Marathon-Ready cDNA and expressed in insect cells using baculovirus vector. Twenty-eight milligrams of purified recombinant hPON3 (rhPON3) was obtained from 1 L Sf9 cells culture. The K m and V max values of rhPON3, with respective to phenylacetate hydrolysis were 7.46 ± 4.40 mM and 89 ± 10.54 U/mg ( n = 3). The kinetic parameters of V max and K m for dihydrocoumarin hydrolysis by rhPON3 were 698 ± 248 U/mg and 0.84 ± 0.24 mM ( n = 3). LDL oxidation assay indicated that rhPON3 could effectively protect LDL against copper-induced oxidation in vitro.

Dietary Antioxidants for Prevention of Cardiovascular Disease

The traditional Japanese diet with its high intake of fruits and vegetables that are rich in antioxidants is believed to effectively ward off cardiovascular disease. Oxidative stress, related to reactive oxygen and nitrogen species produced by aerobic organisms, is responsible for the pathogenesis of most chronic diseases. Oxidative low density lipoprotein (LDL) is thought to play a key role in the pathogenesis of early atherosclerosis. There has been increasing interest in antioxidant substances derived from edible plants. It has been suggested that the measurement of LDL antioxidant activity is physiopathologically more important and informative for screening antioxidant activity to prevent atherosclerosis than other methods. We assessed by LDL oxidation assay the antioxidant characteristics of various edible plants from rural areas. The mulberry (Morus alba L.) leaf showed comparatively high antioxidant activity. We identified the antioxidant compounds and investigated compound levels in the mulberry leaf and found the antioxidant activity of mulberry leaves to be mainly attributable to quercetin 3-(6-malonylglucoside) (Q3MG). Dietary consumption of mulberry leaves and/or Q3MG may enhance resistance to oxidative modification of LDL and attenuated atherosclerotic lesion development. However, the outcome of intervention trials suggested that a single antioxidant had little effect on the risk of developing cardiovascular disease. The synergic effect of certain combinations may determine outcome. We need to widen our understanding of the synergic effect of diets and nutrient-gene interactions related to nutrient/disease risk.

Relative antioxidant activity of soybean isoflavones and their glycosides

The present study compared the antioxidant potency of genistein and daidzein with their corresponding glycosides isolated from soybean seeds. The antioxidant activity was assessed using three methods, namely human low-density lipoprotein (LDL) oxidation, the ferric reducing-antioxidant power (FRAP) and the anti-DPPH free radical assays. It was found that soybean was rich in genistein and daidzein but they were present mainly in form of glycosides. Both the FRAP and the anti-DPPH assays demonstrated that these glycosides, as well as genistin, daidzein, glycitin, malonyl glycitin and malonyl genistein possessed similar antioxidant activities to their corresponding aglycones, genistein and daidzein. In contrast, the antioxidant potency of these glycosides was much weaker than their corresponding aglycones, genistein and daidzein, in the LDL oxidation assay. The present results demonstrate that genistein and daidzein were less effective, as antioxiants in the three assays, than two well-defined antioxidants, green tea epicatechin and α-tocopherol. It is concluded that soybean isoflavones and their glycosides possess antioxidant activity but they are ineffective antioxidants compared with tea epicatechins and α-tocopherol.

Antioxidant effects of phenolic rye (Secale cereale L.) extracts, monomeric hydroxycinnamates, and ferulic acid dehydrodimers on human low-density lipoproteins.

Dietary antioxidants that protect low-density lipoprotein (LDL) from oxidation may help to prevent atherosclerosis and coronary heart disease. The antioxidant activities of purified monomeric and dimeric hydroxycinnamates and of phenolic extracts from rye (whole grain, bran, and flour) were investigated using an in vitro copper-catalyzed human LDL oxidation assay. The most abundant ferulic acid dehydrodimer (diFA) found in rye, 8-O-4-diFA, was a slightly better antioxidant than ferulic acid and p-coumaric acid. The antioxidant activity of the 8-5-diFA was comparable to that of ferulic acid, but neither 5-5-diFA nor 8-5-benzofuran-diFA inhibited LDL oxidation when added at 10-40 microM. The antioxidant activity of the monomeric hydroxycinnamates decreased in the following order: caffeic acid > sinapic acid > ferulic acid > p-coumaric acid. The antioxidant activity of rye extracts was significantly correlated with their total content of monomeric and dimeric hydroxycinnamates, and the rye bran extract was the most potent. The data suggest that especially rye bran provides a source of dietary phenolic antioxidants that may have potential health effects.

Fruit Hydroxycinnamic Acids Inhibit Human Low-Density Lipoprotein Oxidation in Vitro

Oxidative modification of low-density lipoprotein (LDL) is believed to play a crucial role in atherogenesis. The antioxidant activity of hydroxycinnamic acids typically present in fruits was investigated in vitro using a human LDL oxidation assay. The o-dihydroxy compounds caffeic, caftaric, chlorogenic, and neochlorogenic acids had highest antioxidant activities and inhibited LDL oxidation from 86 to 97% at 5 μM. Ferulic and fertaric acids had lower antioxidant activities, and p-coumaric and p-coutaric acids had the lowest. Differences in antioxidant activity were thus primarily related to the hydroxylation and methylation pattern. The esterification to tartaric acid slightly enhanced the antioxidant activity of p-coumaric and ferulic acids, but esterification of caffeic acid to quinic acid (as in chlorogenic and neochlorogenic acid) had no effect on or slightly decreased antioxidant activity. The observed differences in activities are discussed in terms of structural dissimilarities of the compounds. It...

Whole plasma oxidation assay as a measure of lipoprotein oxidizability.

Lipoprotein oxidation induced in vitro in whole plasma is expected to be a more relevant model of the lipoprotein oxidation in the arterial wall than the in vitro oxidation of single isolated lipoproteins, e.g., low density lipoprotein (LDL). However, it is unclear, whether the oxidizability of whole plasma may serve as an adequate measure of the oxidizability of plasma lipoproteins. We measured the oxidizability of whole plasma diluted 150-fold as an absorbance increase at 234 nm known to reflect the level of conjugated dienes in the samples. Plasma oxidation was induced by Cu(II), 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH), lipoxygenase or myeloperoxidase+H2O2. Oxidizability of human plasma measured in the presence of Cu(II) was found to correlate with the oxidizability of LDL measured in the common Cu(II)-based LDL oxidation assay. The plasma oxidizability also correlated positively with plasma oxidizable fatty acid and negatively with plasma antioxidant content. Supplementation of human plasma with different antioxidants (albumin, urate, ascorbate, bilirubin, alpha-tocopherol and ubiquinol-10) in vitro decreased its oxidizability. Supplementation of Watanabe heritable hyperlipidaemic rabbits with different antioxidants (vitamin E, ubiquinone-10, probucol, carvedilol) in vivo lowered the oxidizability of rabbit plasma in comparison with rabbits fed standard diet. When plasma from hyperlipidaemic patients with or without coronary heart disease and from age-matched healthy controls was studied, the plasma oxidizability was found to be highest in the patients with coronary heart disease and lowest in the controls. Taken together, these data indicate that the plasma oxidation assay (i) provides information similar to that obtained using the common LDL oxidation assay, (ii) upgrades the latter, taking into account the effect of hydrophilic antioxidants on lipoprotein oxidation and characterizing the oxidizability of all plasma lipoproteins, and (iii) offers important practical advantages, such as fast and simple sample processing, low amount of plasma required and avoidance of artefactual oxidation during lipoprotein isolation. We propose the measurement of plasma oxidizability at 234 nm as an adequate practical index of the oxidizability of plasma lipoproteins.