Isolation of carotenoids

Aman, R., Isolation of carotenoids from plant materials and dietary supplements by high-speed counter-current chromatography, J. Chromat., 1074, 107, 2005. 

Stewart, L, and Wheaton, T.A., Conversion of 3-citraurin to reticulaxanthin and 3-apo-8 -carotenal to citranaxanthin during isolation of carotenoids from citrus. Phytochemistry, 12, 2947, 1973. 

Matsui, T., and Maruhashi, K., Isolation of Carotenoid-Deficient Mutant From Alkylated Dibenzothiophene Desulfurizing Nocardioform Bacteria, Gordonia Sp TM414. Current Microbiology, 2004. 48(2) pp. 130-134. 

Basic Protocol 1 Solvent Extraction and Isolation of Carotenoids F2.1.1... 

Following extraction, an efficient way of initiating the isolation of carotenoids is to saponify the extract. This removes many of the unwanted lipids present in the sample as well as chlorophyll. The saponification by-products, which to a great extent are sodium or potassium salts, are easily separated by an aqueous solution of a highly polar salt. The addition of water also helps wash off excess alkali and other water-soluble and water-complexed compounds. This procedure hydrolyzes xantho-phyll esters to form the hydroxylated carotenoid. 

Babu, C. M., Chakrabarti, R., and Sambasivarao, K. R. S. (2008). Enzymatic isolation of carotenoid-protein complex from shrimp head waste and its use as a source of carotenoids. LWT 41, 227-235. 

Supercritical fluid extraction has been suggested as an alternative method for selective one-step isolation of carotenoids without degradations (but see Britton et al., 1995). For instance, Favati et al. (1988) isolated (3-carotene... 

For the isolation of carotenoids from animal sources, the tissue is extracted with propanone and a similar work-up procedure adopted as for plant carotenoids. 

The isolation of carotenoids still presents many problems, due in part to the close similarity between isomers differing only in the position or stereochemistry of one double bond. Several reports claim improved methods for the thin-layer chromatographic separation or even gas-chromatographic separation of carotenoids. Analytical methods have been reviewed and extensive tables for the identification of carotenoids published. ... 

In the early part of this century, the first procedures for the isolation of carotenoids and, subsequently, the first methods for systematic structure elucidation were developed. The initial synthetic procedures were limited to simple chemical modifications of the isolated carotenoids. These methods gradually lost their importance for preparative purpose because of the poor yields of such chemical transformations carried out on a chemically labile C40-polyene and the necessity to purify the products by extensive chromatography. 

Algae are also superbly suited for the isolation of carotenoids. Thus, the freshwater green alga Haematococcus pluvialis is used to obtain astaxanthin. When a pond starts to dry up, or nutrients become limited, the algae form a protective cell wall, encyst and enter a dormant phase. Massive amounts of astaxanthin are... 

Saponification of the extracts is generally desirable to remove unwanted lipid materials. However, this step is omitted in the isolation of carotenol esters, since these are hydrolyzed by this procedure. It is also omitted in the isolation of carotenoids such as fucoxanthin and peridinin, which are alkali-labile. If acetone has been used in the initial extraction, it is essential that all traces be removed before saponification. The general procedure used involves dissolving the total lipid fraction in an alcoholic (ethanol or methanol) solution of potassium hydroxide. The mixture is then either heated for a short period of time while kept in the dark, or left in the dark at room temperature for 12-16 h. There has been considerable discussion of the merits of these two procedures. Which method is used is dependent on the nature of the samples being analyzed and the requirements of the analysis (Davies, 1976 Liaaen-Jensen, 1971). After saponification, water is added, and neutral lipids (the unsaponifiable fraction) are extracted with diethyl ether or hexane. Acidic carotenoids remain in the alkaline phase and are extracted with diethyl ether or hexane after acidification with acetic acid. The unsaponifiable fraction usually contains sterols as well as carotenoids. If desired, sterol contaminants can be removed by precipitation from cold (- 10°C) petroleum ether or by precipitation of these compounds as their digitonides. 

The isolation of carotenoid genes is now making progress, with the isolation of the crti gene from Rhodobacter and the cloning of the al-3... 

Carotenoids are natural pigments characterized by a tail to tail linkage between two C20 units and an extended conjugated system of double bonds They are the most widely dis tributed of the substances that give color to our world and occur m flowers fruits plants insects and animals It has been estimated that biosynthesis from acetate produces approximately a hundred million tons of carotenoids per year The most familiar carotenoids are lycopene and (3 carotene pigments found m numerous plants and easily isolable from npe tomatoes and carrots respectively... 

Some prospective and case-control studies also investigated the relationship of carotenoids and the evolution of CCA-IMT. Although the EVA study showed no association between total carotenoids and IMT, others like the ARIC study, the Los Angeles Atherosclerosis Study, " and the Kuopio Ischaemic Heart Disease Risk Factor Study demonstrated the protective role of isolated carotenoids such as lycopene, lutein, zeaxanthin, and P-cryptoxanthin on IMT. Thus, findings from prospective and case-control studies have suggested that some carotenoids such as lycopene and P-carotene may present protective effects against CVD and particularly myocardial infarcts and intima media thickness, a marker of atherosclerosis. 

This in vitro approach thus has a great potential for studying the intestinal absorption processes of carotenoids and other pigments. It is important to note the existence of several clones isolated from the parent Caco-2 cell line that can be used for studying... 

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... 

Pfister, S. et al.. Isolation and structure elucidation of carotenoid-glycosyl esters in gardenia fruits Gardenia jasminoides Ellis) and saffron (Crocus sativus Linne), J. Agric. Food Chem., 44, 2612, 1996. 

CO2 extraction has been prevalent for the isolation of essential oils and other natural lipophilic pigments like carotenoids. Hot water and superheated water extraction methods are used for analytical preparation of polar pigments. The technique is commonly referred to as subcritical water extraction because the practitioners of this approach come from SEE backgrounds. 

Unicelluar algal and bacterial genes were the first to be isolated and characterized and led to the isolation of most of the higher plant genes involved in carotenoid biosynthesis. Carotenogenic gene clusters from bacteria and algae" - - - contributed immensely to the elucidation of the carotenoid pathway. 

Linden, H., Vioque, A., and Sandmann, G., Isolation of a carotenoid biosynthesis gene coding for z-carotene dasaturase from Anabaena PCC 7120 by heterologous complementation, FFMS Microbiol. Lett. 106, 99, 1993. 

When the aim is isolation for identification by direct probe insertion mass spectrometry (MS), plastic materials, filter papers, and blenders should be avoided to prevent contamination during extraction and chromatography. It is also very important to avoid the cis-trans isomerization of carotenoids in solution, which is accelerated by heat, light, acids, and active surfaces. Therefore, a pure carotenoid or even a crude extract should never be stored in solution it should be kept completely dry in an inert atmosphere at low temperature. 

Esterbauer et al. (1991) have demonstrated that /3-carotene becomes an effective antioxidant after the depletion of vitamin E. Our studies of LDL isolated from matched rheumatoid serum and synovial fluid demonstrate a depletion of /8-carotene (Section 2.2.2.2). Oncley et al. (1952) stated that the progressive changes in the absorption spectra of LDL were correlated with the autooxidation of constituent fatty acids, the auto-oxidation being the most likely cause of carotenoid degradation. The observation that /3-carotene levels in synovial fluid LDL are lower than those of matched plasma LDL (Section 2.2.2) is interesting in that /3-carotene functions as the most effective antioxidant under conditions of low fOi (Burton and Traber, 1990). As discussed above (Section 2.1.3), the rheumatoid joint is both hypoxic and acidotic. We have also found that the concentration of vitamin E is markedly diminished in synovial fluid from inflamed joints when compared to matched plasma samples (Fairburn etal., 1992). This difference could not be accounted for by the lower concentrations of lipids and lipoproteins within synovial fluid. The low levels of both vitamin E and /3-carotene in rheumatoid synovial fluid are consistent with the consumption of lipid-soluble antioxidants within the arthritic joint due to their role in terminating the process of lipid peroxidation (Fairburn et al., 1992). 

NPQ (Rakhimberdieva et al. 2004) exactly matches the absorption spectrum of the carotenoid, 3 -hydrox yech i nenone (Polivka et al. 2005) in the OCP. The OCP is now known to be specifically involved in the phycobilisome-associated NPQ and not in other mechanisms affecting the levels of fluorescence such as state transitions or D1 damage (Wilson et al. 2006). Studies by immunogold labeling and electron microscopy showed that most of the OCP is present in the interthylakoid cytoplasmic region, on the phycobilisome side of the membrane, Figure 1.2 (Wilson et al. 2006). The existence of an interaction between the OCP and the phycobilisomes and thylakoids was supported by the co-isolation of the OCP with the phycobilisome-associated membrane fraction (Wilson et al. 2006, 2007). 

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