Antioxidants, carotenoid extraction

In a previous work, we studied the possibility of extracting antioxidants from microalgae Spirulina platensis using ASE with different solvents (33-34). Likewise, other authors have studied the carotenoids extraction from microalgae Haematococcus pluvialis and Dunaliella salina using ethanol as solvent and ASE (35). 

Fernandez, G.A., Butz, P., and Tauscher, B. 2001b. Effects of high-pressure processing on carotenoid extractability, antioxidant activity, glucose diffusion, and water binding of tomato puree (Lycopersicon esculentum MUl.). Journal of Food Science 66 1033-1038. 

Sanchez, M.C., Plaza, L., Ancos, B., and Cano, M.P. 2004. Effect of combined treatments of high-pressure and natural additives on carotenoid extractability and antioxidant activity of tomato puree (Lycopersicum esculentum Mill.). European Food Research and Technology 219 151-160. 

Gj-S transition in the cell cycle and suggests that muscadine grape skin extracts contain chemopreventive phytochemicals other than resveratrol. Lycopene (Fig. 4), a potent antioxidant carotenoid in tomatoes and other fruits, protects against prostate (Dahan et al., 2008) and other cancers (Seren et al., 2008). Plant sterol-enriched foods are an effective dietary adjuvant in reducing cardiovascular risk by lowering total cholesterol and low density lipoprotein-cholesterol (LDL-C) in serum by up to 15% (Patch et al., 2006 Jones et al., 2000). 

Many different methods have been used to evaluate the antioxidant capacities of isolated molecules, carotenoids, and other natural antioxidants and of foods and food extracts containing antioxidants. It is not the purpose of this chaper to review all the methods, but some general points can be made. First, when using only one test to evaluate the antioxidant capacities of carotenoids, one should be very careful in the interpretation of obtained data. Indeed, different results can be obtained with different tests applied to the same molecules. At least two different methods should be used to evaluate the antioxidant activity of a molecule or a food extract. " Second, lipophilicity is an important factor to consider in testing the antioxidant activities... 

Experimental evidence in humans is based upon intervention studies with diets enriched in carotenoids or carotenoid-contaiifing foods. Oxidative stress biomarkers are measured in plasma or urine. The inhibition of low density lipoprotein (LDL) oxidation has been posmlated as one mechanism by which antioxidants may prevent the development of atherosclerosis. Since carotenoids are transported mainly via LDL in blood, testing the susceptibility of carotenoid-loaded LDL to oxidation is a common method of evaluating the antioxidant activities of carotenoids in vivo. This type of smdy is more precisely of the ex vivo type because LDLs are extracted from plasma in order to be tested in vitro for oxidative sensitivity after the subjects are given a special diet. 

This method is also used to measure ex vivo low-density lipoprotein (LDL) oxidation. LDL is isolated fresh from blood samples, oxidation is initiated by Cu(II) or AAPH, and peroxidation of the lipid components is followed at 234 nm for conjugated dienes (Prior and others 2005). In this specific case the procedure can be used to assess the interaction of certain antioxidant compounds, such as vitamin E, carotenoids, and retinyl stearate, exerting a protective effect on LDL (Esterbauer and others 1989). Hence, Viana and others (1996) studied the in vitro antioxidative effects of an extract rich in flavonoids. Similarly, Pearson and others (1999) assessed the ability of compounds in apple juices and extracts from fresh apple to protect LDL. Wang and Goodman (1999) examined the antioxidant properties of 26 common dietary phenolic agents in an ex vivo LDL oxidation model. Salleh and others (2002) screened 12 edible plant extracts rich in polyphenols for their potential to inhibit oxidation of LDL in vitro. Gongalves and others (2004) observed that phenolic extracts from cherry inhibited LDL oxidation in vitro in a dose-dependent manner. Yildirin and others (2007) demonstrated that grapes inhibited oxidation of human LDL at a level comparable to wine. Coinu and others (2007) studied the antioxidant properties of extracts obtained from artichoke leaves and outer bracts measured on human oxidized LDL. Milde and others (2007) showed that many phenolics, as well as carotenoids, enhance resistance to LDL oxidation. 

This assay has been used by some authors to evaluate the in vitro effects of antioxidant extracts on LDL oxidation (Viana and others 1996 Cirico and Omaye 2006 Kedage and others 2007 Vayalil 2002 Garcfa-Alonso and others 2004 Tarwadi and Agte 2005). Oboh and others (2007) confirmed that hot pepper prevents in vitro lipid peroxidation in brain tissues. Indeed, Bub and others (2000) demonstrated that a moderate intervention with vegetable products rich in carotenoids reduces lipid peroxidation in men. Nicolle and others (2003) evaluated the effect of carrot intake on antioxidant status in cholesterol-fed rats. Later on, they showed that lettuce consumption improves cholesterol metabolism and antioxidant status in rats (Nicole and others 2004). 

Pellegrini N, Re R, Yang M and Rice-Evans C. 1999. Screening of dietary carotenoids and carotenoid-rich fruit extracts for antioxidant activities applying 2,2 -azinobis(3-ethylenebenzothiazohne-6-sulfonic acid) radical cation decolorization assay. Methods Enzymol 299 379-389. 

Ethoxyquin, a synthetic antioxidant, is not generally allowed for human consumption in foods, but it is being added to animal feed and to fruits as an antiscald agent (94,143). Ethoxyquin is also used in the spice industry to prevent carotenoid loss during postharvest handling. However, ethoxyquin-treated paprika is unacceptable for some markets and some consumers (129). Perfetti et al. (130) described a method for determination of ethoxyquin in paprika and chili powder. Ethoxyquin was extracted from the spice with hexane and partitioned into 0.3 N HC1. After adjusting the solution to pH 13-14, ethoxyquin was extracted into hexane, and the hexane layer was evaporated to dryness. An acetonitrile solution of the residue was then analyzed by reversed-phase HPLC, with detection at 254 nm. The mobile phase was water/acetonitrile with ammonium acetate buffer. Recoveries from samples fortified at 50, 100, and 200 ppm averaged 92%, with a coefficient of variation of 2.3%. The method was applied to a number of commercial samples of paprika and chili powder. Ethoxyquin was found in paprika samples at levels up to 63 ppm and in chili powder samples at levels up to 20 ppm. 

Carotenoids should be extracted from tissues as rapidly as possible. If an immediate extraction is not possible, samples should be stored below — 18°C until required. For the extraction the exactly weighed sample and the solvent are transferred into a blender, where the sample is simultaneously grinded and extracted. Since fresh tissues contain a high percentage of water, and carotenoids are lipo-soluble, the first organic solvent must be miscible with water (e.g., acetone, ethanol, methanol). After one or two extraction steps, water-immiscible solvents (e.g., diethyl ether, benzene) can be applied. Dried materials may be also extracted with water-immiscible solvents, but carotenoid recovery is usually better if the tissue is first treated with a little water and then extracted with water-miscible solvents. Prior to the extraction of fruits the addition of antioxidants [e.g.,... 

Another group of methods relies on the destruction of an indicator by an oxidant, leading to a decrease of its characteristic absorbance or fluorescence. Usually this decrease is not linear with time, so the method of choice for the analysis of data is the either the lag time measurement or the area-under-curve approach, that is, comparing the areas under curves of absorbance or fluorescence versus time for a reference sample and a sample studied. Antioxidants protecting the oxidizable substrate increase the area under the kinetic curve (Fig. 4). An absorptiometric method used the bleaching of the carotenoid crocin induced by ABAP (L23, T7). However, crocin is rather expensive and the recommended method of its preparation (by extraction of saffron) may lead to extracts of different composition and properties varying from batch to batch. 

The correlation between dietary carotenoids and the carotenoids rontinely found in the extracts from human serum/plasma has revealed that only selected groups of carotenoids make their way into the human bloodstream. Some of these carotenoids are absorbed intact and others such as lutein, zeaxanthin, and lycopene are presumably converted to several metabolites. - In addition to their antioxidant mechanism... 

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