Spinach chlorophyll

Chlorophyll. Chemically pure chlorophyll is difficult to prepare, since it occurs mixed with other colored substances such as carotenoids. Commercially it is solvent extracted from the dried leaves of various plants such as broccoli or spinach. Chlorophyll is water-iosoluble. It has none of the characteristics of a dye in that it has no aflinity for the usual libers such as cotton or wool. Chlorophyll is properly classified as a pigment tCI Natural Green 3 Cl 75810), As such. It finds use lor coloring soaps, waxes, inks. fats, or nils. Chlorophyll is an ester composed of an acidic pint, chlorophyllin, esterilied by an aliphatic alcohol known as phylol. Hydrolysis of chlorophyll using sodium hydroxide produces the moderately water-soluble sodium salts of chlorophyllin. phytol. and methanol. The magnesium in chlorophyllin may be replaced by copper. The sodium copper chlorophyllin salt is heat-stable, and is ideal for coloring foods where heat is involved, such as in canning. 

Often, but not always, this is accompanied by an inaease in chlorophyll a/b ratio arising from a decline in LHCIIb content. The size ofthe pool, as a % total carotenoid varies from 15% to 30%. The upper limit might be defined by the number of binding sites on LHCII. It has been estimated that PS II in spinach (chlorophyll a/b = 3.5) has a binding site capacity of 18-21 xanthophyll cycle carotenoids Ruban et al., 1999). Measurements indicate... 

A chromatographic column filled in three sections with ground sugar, chalk, and alumina. When a petroleum extract of spinach leaves is run onto the top of the column, ihe extract spreads down the column, but not uniformly bands of green chlorophylls stop near the top. yellow xanthophyll further down, and red carotene near the bottom. 

In a clinical trial performed in China, the administration of 300 mg/day of copper chlorophyllin to humans who had detectable levels of serum aflatoxin due to unavoidable food contamination resnlted in a 50% reduction of median urinary levels of aflatoxin-DNA adducts. If health benefits from consuming natural chlorophylls were confirmed, it wonld be easy to add green leafy vegetables to a daily diet to obtain the benefit. Since leafy vegetables contain usually up to 200 mg chloro-phylls/100 g fresh weight, the intake of approximately 1 to 2 cups of raw spinach/day... 

Diets high in red meat and low in green vegetables have been associated with increased colon cancer risk and the opposite has been postulated for diets rich in green vegetables. A plausible explanation for an increased colon cancer risk is that dietary haem is metabolized in the gut to a factor that increases colonic cytotoxicity and hyperproliferation, which are considered important risk factors in the development of cancer. In this sense, it has been shown that spinach and isolated natural chlorophyll, but not sodium-copper chlorophyUin, prevented the proliferation of colonic cells and may therefore reduce colon cancer risk. It has been speculated that haem and chlorophylls, due to their hydrophobicity, form a complex, thus preventing the metabolism of haem. ... 

Ferruzzi, M.G., Failla, M.L., and Schwartz, S.J., Assessment of degradation and intestinal cell uptake of carotenoids and chlorophyll derivatives from spinach puree using an in vitro digestion and Caco-2 cell model, J. Agric. Food Chem., 49, 2082, 2001. 

Lopez-Ayerra, B., Mnrcia, M.A., and Garcia-Carmona, R, Lipid peroxidation and chlorophyll levels in spinach dnring refrigerated storage and after indnstrial processing, Food Chem., 61, 113, 1998. 

Chlorophyll catabolism has been intensively studied in some plants, e.g., rape-seed, barley, spinach, tobacco, Cercidiphyllum japonicum, Lolium temulentum, Liq-quidambar styraciflua and Arabidopsis thaliana, which present all NCC catabolites with similar basic structures. " This suggests a uniform breakdown of chlorophyll in which the oxidative opening of pheophorbide a seems to be a key step. Structural differences among the compounds have been related to at least six basic types of peripheral transformations. Some of them seem to operate either in sequence or in parallel, depending on the plant species, which caused the appearances of different... 

Despite the difficulties in extracting and identifying colorless catabolic products that are extremely labile and detectable only in trace amounts, several of the mysteries of chlorophyll catabolism have been revealed and about 14 non-fluorescent chlorophyll catabolytes (NCCs) from higher plants, mainly in senescent leaves, have been detected and analyzed structurally. Among them, NCCs from rapeseed (Bms-sica napus) from Liquidambar styraciflua, from Cercidiphyllum japonicum, five NCCs from degreened leaves of spinach Spinacia oleracea) and, more recently, two NCCs from tobacco Nicotiana rusticd) and five NCCs from Arabidopsis thaliana have been identified. 

Schwartz, S.J., Woo, S.L., and Von Elbe, J.H., High-performance liquid chromatography of chlorophylls and their derivatives in fresh and processed spinach, J. Agric. Food Ghem., 29, 533, 1981. 

Oberhuber, M. et al.. Chlorophyll breakdown — on a nonfluorescent chlorophyll catabohte from spinach, Helv. Chim. Acta, 84, 2615, 2001. 

FIGURE 7.2 Kubelka-Munk densitogram of a spinach leaf extract taken at 305 nm. The two peaks at 16-mm and 17-mm distances are due to chlorophyll b and chlorophyll a. 

Experiment 36 Solid-Liquid Extraction Chlorophyll from Spinach Leaves... 

The purpose of this experiment is to test four organic solvents of varying polarity to determine which solvent is more effective in extracting chlorophyll from spinach leaves. The solvents are ethyl acetate, dichloromethane, acetone, and ethanol. 

Using the individual solvents as blanks, acquire a visible absorption spectrum of each sample. Superimpose the four spectra, and determine which solvent is the most effective solvent for extracting chlorophyll from spinach leaves. 

Fig. 2.132. Chromatogram of spinach, stored frozen until analysis by HPLC (A) and after acidifying the same pigment extract with 0.2ml M HC1 per 10 ml extract and exposure to air and light for 15 h at 20°C (B). Zinc-phtalocyanine was used as an internal standard (IS). Peak identification 1 = chlorophyll-b 2 = chlorophyll-a x = unknown degradation product 3 = IS 4 = pheophytin-b 5 = pheophytin-a 6 = chlorophyll-b 7 = chlorophyll-a 8 = pheophytin-b 9 = pheophytin-a. Reprinted with permission from T. Bohn et al. [303].

Chloroplasts. Intact chloroplasts were isolated from freshly harvested growth chamber-grown spinach (Splnacla oleracea L.) as described by Lilley and Walker (10). Thylakoids were prepared by the method of Armond t al. (11). Chlorophyll concentrations were determined by the method of MacKinney (12). Photochemical reactions yere conducted at 25°C with a photon fluence rate of 750... 

The biological functions of chloroplast ferredoxins are to mediate electron transport in the photosynthetic reaction. These ferredoxins receive electrons from light-excited chlorophyll, and reduce NADP in the presence of ferredoxin-NADPH reductase (23). Another function of chloroplast ferredoxins is the formation oT" ATP in oxygen-evolving noncyclic photophosphorylation (24). With respect to the photoreduction of NADP, it is known that microbial ferredoxins from C. pasteurianum (16) are capable of replacing the spinach ferredoxin, indicating the functional similarities of ferredoxins from completely different sources. The functions of chloroplast ferredoxins in photosynthesis and the properties of these ferredoxin proteins have been reviewed in detail by Orme-Johnson (2), Buchanan and Arnon (3), Bishop (25), and Yocum et al. ( ). 

---