Isolation of carotenoids from plant materials and dietary supplements by high-speed counter-current chromatography

Methods for the isolation of lipophilic pigments from crude extracts of plant materials (spinach and sweet corn) by high-speed counter-current chromatography (HSCCC) were developed. Particular attention was given to (all-E)-lutein and (all-E)-zeaxanthin. However, the concomitant pigments neoxanthin, violaxanthin and beta-carotene as well as chlorophylls a and b were also considered. Furthermore, for the first time dietary supplements containing lutein and zeaxanthin were also used as a source for the recovery of carotenoids. Due to their simple matrix (oily excipient in soft gelatine capsules), sample preparation was facilitated and consumption of solvents was minimized. The carotenoids were characterized by 1H NMR spectroscopy, by LC/APcI-MS in the positive ionization mode, and by UV-vis spectroscopy.Data showed that the target compounds were of high purity (90 - 93%). Lutein and zeaxanthin may be used as reference substances for analytical purposes.     

Effect of drying temperature and blanching on the degradation of chlorophyll a and b in mint (Mentha spicata Huds.) and basil (Ocimum basilicum): Analysis by high performance liquid chromatography with photodiode array detection

Summary The effect of drying conditions on the preservation of chlorophyll pigments in mint and basil have been investigated in order to determine the effects of drying temperature and whether or not there was a prior blanching.Pigments extracted from fresh and dried samples were analyzed by reversed phase high performance liquid chromatography coupled to a photodiode array detector; isocratic separation was performed on a Zorbax ODS C18 column.The purity of the chromatographic peaks of chlorophylls and breakdown products was investigated. The visible spectra of standard samples of chlorophylls and pheophytins were compared, using least squares normalization with those of peaks from the extracts of fresh and dried mint and basil. The study has shown that chlorophylls were better preserved when drying was preceded by a short blanching; if samples were not blanched before drying, the degradation of chlorophylls a and b was best prevented by drying at low temperatures.     

HPLC separation and characterization of chlorin derivatives with intact ring V from acid degradation products of silkworm excrement crude chlorophyll mixture

A reversed-phase HPLC system with 88% methanol in 1 M ammonium acetate buffer pH 5.35 as the mobile phase on an ODS column was used to analyse pheophorbides a, a', b, b' and pyropheophorbides a and b. Pyropheophorbides a and b were found in samples obtained from silkworm excrement but not from spinach leaves though the preparation methods used were the same. This difference suggests that chlorophylls undergo metabolism in the body of silkworm to give pyrochlorophyll derivatives.     

Determination of chlorophyll in plant samples by liquid chromatography using zinc-phthalocyanine as an internal standard

Chlorophyll analysis at high precision and accuracy is limited by the lack of suitable, commercially available internal standards for HPLC analysis. Here, the commercially available dye zinc-phthalocyanine is presented as a new internal standard to quantify chlorophylls in vegetable foods and to detect chlorophyll degradation products. The technique was applied to chlorophyll analysis of a selection of vegetable foods. Pigments were extracted with N,N-dimethylformamide from the vegetables and purified by solid phase extraction. Chlorophyll a, a', b, b', corresponding pheophytins, and zinc-phthalocyanine were separated by HPLC using a C18 reverse-phase column and fluorescence detection.     

SPECTROSCOPIC CHARACTERIZATION OF TWO FORMS OF THE D1-D2-CYTOCHROME b559 COMPLEX FROM SUGAR BEET

Two D1-D2-cytochrome b559 complex forms, called RCIIa and RCIIb, with different pigment stoichi-ometry were characterized using absorption and surface-enhanced resonance Raman scattering spectroscopy and spectral gaussian deconvolution. Electronic absorption spectra of the RCIIb at 277 K showed significant differences compared to RCIIa, i.e . a strong decrease in the absorbance due to carotenoid and chlorophyll for the same amount of pheophytin. A reduced carotenoid and chlorophyll content in RCIIb was also observed in the surface-enhanced resonance Raman scattering spectra. Spectral deconvolution elicited three main absorption bands at 680, 672 and 669–670 nm, which were ascribed to P680, pheophytin and accessory chlorophyll, respectively. In addition, a minor component around 667 nm was observed in the RCIIb, most probably due to some reaction center inactivation. Calculation of the relative area under the gaussians together with pigment stoichiometry data suggest that the 680, 672 and 669–670 nm components contain, respectively, two chlorophylls, two pheophytins and four chlorophylls for the RCIIa, and two chlorophylls, two pheophytins and two chlorophylls for the RCIIb.     

Chemical bonding of chlorophylls and plant aminic axial ligands impact harvesting of visible light and quenching of fluorescence

In the present work, we tested the mode of interaction of all three polyamines (putrescine, spermidine and spermine) with chlorophyll a and b, as well as pheophytin a and b. The results showed that all three polyamines bind to the Mg ion of chlorophyll ring as probed by Raman spectroscopy. The coordination of spermine with Chl b has the most interesting features from all pigments tested. Spermine induces reversible increases and decreases of the fluorescence yield of Chl b at about 661 nm. Interestingly, equilibrium between a high-fluorescence yield conformation and a low yield is feasible by the interaction of chlorophyll b and aminic ligands. Furthermore, absorption data for the diagnostic regions of 518 and 535 nm are provided for all combinations of pigments and ligands. The significance and consistence of these results with respect to photochemical and bioenergetic principles are discussed.     

Selective photobleaching of chlorophylls and carotenoids in photosystem I particles under high-light treatment

Photosystem I particles (PSI-200) isolated from spinach leaves were studied by means of absorbance, 77K fluorescence and resonance Raman (RR) spectroscopy. The aim was to obtain better insight into the changes of the pigment spectral properties in those particles during prolonged exposure to high-light intensities and to reveal the involvement of these pigments in the photoprotection of the PSI. During prolonged exposure to high-light intensities of spinach PSI particles, a loss of a significant amount of photosynthetic pigments was observed. It was shown that various pigments exhibited different susceptibility to photodamage. In addition to bleaching of chlorophyll a (Chl a), bleaching of carotenoids was also clearly observed. RR technique allowed us to recognize the type and conformation of photobleached carotenoid molecules. Raman data revealed a nearly full photobleaching of the long-wavelength lutein molecules. The observed similar bleaching rate of the lutein molecules and the most-red shifted long-wavelength Chl a, located in the antenna membrane protein Lhca4, suggested that these molecules are located closely. Our results showed that the photobleached antenna pigments and especially luteins and the most long-wavelength absorbing chlorophylls are involved in photoprotection of PSI core complex.     

Expanded separation technique for chlorophyll metabolites in Oriental tobacco leaf using non aqueous reversed phase chromatography

An improved separation method for chlorophyll metabolites in Oriental tobacco leaf was developed. While Oriental leaf still gives the green color even after the curing process, little attention has been paid to the detailed composition of the remaining green pigments. This study aimed to identify the green pigments using non aqueous reversed phase chromatography (NARPC). To this end, liquid chromatograph (LC) equipped with a photo diode array detector (DAD) and an atmospheric pressure chemical ionization/mass spectrometer (APCI/MSD) was selected, because it is useful for detecting low polar non-volatile compounds giving green color such as pheophytin a. Identification was based on the wavelength spectrum, mass spectrum and retention time, comparing the analytes in Oriental leaf with the commercially available and synthesized components. Consequently, several chlorophyll metabolites such as hydroxypheophytin a, solanesyl pheophorbide a and solanesyl hydroxypheophorbide a were newly identified, in addition to typical green pigments such as chlorophyll a and pheophytin a. Chlorophyll metabolites bound to solanesol were considered the tobacco specific components. NARPC expanded the number of detectable low polar chlorophyll metabolites in Oriental tobacco leaf.     

Separation and determination of minor photosynthetic pigments by reversed-phase HPLC with minimal alteration of chlorophylls.

Reversed-phase HPLC conditions for separation of chlorophyll (Chl) a, Chl a' (the C132-epimer of Chl a), pheophytin (Pheo) a (the primary electron acceptor of photosystem (PS) II), and phylloquinone (PhQ) (the secondary electron acceptor of PS 1), have been developed. Pigment extraction conditions were optimized in terms of pigment alteration and extraction efficiency. Pigment composition analysis of light-harvesting complex II, which would not contain Chl a' nor Pheo a, showed the Chl a'/Chl a ratio of 3-4 x 10(-4) and the Pheo a/Chl a ratio of 4-5 x 10(-4), showing that the conditions developed here were sufficiently inert for Chl analysis. Preliminary analysis of thylakoid membranes with this analytical system gave the PhQ/Chl a' ratio of 0.58 +/- 0.03 (n = 4), in line with the stoichiometry of one molecule of Chl a' per PS I.     

Effect of Thermal and Non-Thermal Technologies on Kinetics and the Main Quality Parameters of Red Bell Pepper Dried with Convective and Microwave–Convective Methods

The drying process preserves the surplus of perishable food. However, to obtain a good-quality final product, different pretreatments are conducted before drying. Thus, the aim of the study was the evaluation of the effect of thermal (blanching treatments with hot water) and non-thermal technologies (pulsed electric field (PEF) and ultrasound (US)) on the kinetics of the drying process of red bell pepper. The convective and microwave–convective drying were compared based on quality parameters, such as physical (water activity, porosity, rehydration rate, and color) and chemical properties (total phenolic content, total carotenoids content, antioxidant activity, and total sugars content). The results showed that all of the investigated methods reduced drying time. However, the most effective was blanching, followed by PEF and US treatment, regardless of the drying technique. Non-thermal methods allowed for better preservation of bioactive compounds, such as vitamin C in the range of 8.2% to 22.5% or total carotenoid content in the range of 0.4% to 48%, in comparison to untreated dried material. Moreover, PEF-treated red bell peppers exhibited superior antioxidant activity (higher of about 15.2–30.8%) when compared to untreated dried samples, whereas sonication decreased the free radical scavenging potential by ca. 10%. In most cases, the pretreatment influenced the physical properties, such as porosity, color, or rehydration properties. Samples subjected to PEF and US treatment and dried by using a microwave-assisted method exhibited a significantly higher porosity of 2–4 folds in comparison to untreated material; this result was also confirmed by visual inspection of microtomography scans. Among tested methods, blanched samples had the most similar optical properties to untreated materials; however non-thermally treated bell peppers exhibited the highest saturation of the color.     

(Alpha-aminoacyl)amino-substituted heterocycles and related compounds

N-Protected-(aminoacyl)benzotriazoles 1a-e, g, i, j, 1a'-c' convert heterocyclic amines of the following series: thiazoles (3a and 3a'), benzothiazoles (3b and 3b'), benzimidazoles (3c and 3c'), thiadiazoles (3d), pyrimidones (9a, b, a'), pyrazoles (11a, b), and pyridines (13a-g, 13d') under microwave irradiation, into N-substituted amides in yields of 40-98% (average 76%). N-Protected peptidoylbenzotriazoles 6a, b similarly afforded C-terminal N-protected dipeptidoyl amides 7a, b (52-60%).     

ROOM TEMPERATURE TIME-RESOLVED IN μs RANGE DELAYED LUMINESCENCE OF CHLOROPHYLL a AND CHLOROPHYLL-LUTEIN MIXTURE SOLUTIONS

Using time-resolved in μS range luminescence spectroscopy, we observed at 20°C the emission of chlorophyll a, pheophytin a and chlorophyll a-lutein mixture solutions. This delayed emission exhibits several maxima in the650–750 nm region. The positions and kinetics of decay of delayed emission bands depend on chlorophyll concentration, and vary as a result of pheophytinization and addition of lutein. Our results can be explained by supposition that upon excitation, charge transfer species are formed in various pigment complexes. The back electron transfer reactions yield chlorophyll excited singlet states contributing to observed delayed emission. Delay in emission seems to be due also to the trapping of excitation on the triplet states of various forms of pigment and its detrapping with the participation of thermal energy followed by energy transfer to the forms of pigment characterized by different decay times.     

Normal-phase HPLC separation of possible biosynthetic intermediates of pheophytin a and chlorophyll a'.

Normal-phase HPLC conditions have been developed for separating the C17(3) isoprenoid isomers, which are expected to be formed as biosynthetic intermediates of chlorophyll (Chl) a, Chl a' (C13(2)-epimer of Chl a), pheophytin (Pheo) a and protochlorophyll (PChl). The application of these conditions to pigment composition analysis of greening etiolated barley leaves allowed us to detect, for the first time, the C17(3) isomers of Chl a', a possible constituent of the primary electron donor of photosystem (PS) I, P700, and those of Pheo a, the primary electron acceptor of PS II, in the very early stage of greening. The C17(3) isomer distribution patterns were approximately the same between Chl a and Chl a', but significantly different between Pheo a and Chl a', probably reflecting the similarity and difference, respectively, in the biosynthetic pathways of these pigment pairs.     

Organometallic early lanthanide clusters: syntheses and X-ray structures of new monocyclopentadienyl complexes

The reaction of Ln(BH(4))(3)(THF)(3) or LnCl(3)(THF)(3) with 1 equiv of KCp*' ligand (Cp' = C(5)Me(4)n-Pr) afforded the new monocyclopentadienyl complexes Cp*'LnX(2)(THF)(n) (X = BH(4), Ln = Sm, n = 1, 1a, Ln = Nd, n = 2, 1b; X = Cl, Ln = Sm, n = 1, 3a) and [Cp*'LnX(2)](n') (X = BH(4), n' = 6, Ln = Sm, 2a, Ln = Nd, 2b; X = Cl, Ln = Nd, 4b). All these compounds were characterized by elemental analysis and (1)H NMR. Crystals of mixed borohydrido/chloro-bridged [Cp*'(6)Ln(6)(BH(4))(12-x))Cl(x)(THF)(n')] (x = 10, n' = 4, Ln = Sm, 2a', Ln = Nd, 2b'; x = 5, n = 2, Ln = Sm, 2a' ') were also isolated. Compounds 2a, 2b, 2a', 2b', and 2a' were structurally characterized; they all exhibit a hexameric structure in the solid state containing the [Cp*(3)Ln(3)X(5)(THF)] building block. The easy clustering of THF adducts first isolated is illustrative of the well-known bridging ability of the BH(4) group. Hexameric 2a was found to be unstable in the presence of THF vapors; this may be correlated to the opening of unsymmetrical borohydride bridges observed in the molecular structure.     

CHLOROPHYLLS IN POLYMERS. II. PHEOPHYTIN a IN POLYMERS and THE INFLUENCE OF STRETCHING ON THE STATE OF CHLOROPHYLLS IN ANHYDROUS POLYMER FILMS

Abstract— In the first part of this study the spectral properties of pheophytin a in rigid, unstretched anhydrous polyvinyl alcohol and nitrocellulose films have been studied in order to establish the influence of the central magnesium atom on the state of chlorophylls in polymer systems. The absorption, fluorescence, excitation spectra and fluorescence intensity decays in the polymer films and in the solutions from which they are cast are reported. It is shown that pheophytin a aggregate formation is influenced by the nature of the polymer system. An aggregate of pheophytin a is found in polyvinyl alcohol films over a wide concentration range. On the other hand, pheophytin a exists in the monomeric form in unstretched nitrocellulose films at concentrations below 6 × 10^-6 mol/g.
In the second part of this work, the influence of stretching of the films on the state and distribution of embedded chlorophyll pigments, is described. Here we show that the chlorophyll a molecules are found to undertake a heterogenous distribution in polyvinylalcohol matrices, since stretching partially disrupts the pocket-like structures present in unstretched films. In contrast, chlorophyll a and pheophytin a molecules can be embedded in a monomeric state in nitrocellulose matrices and moreover they remain homogeneously distributed upon stretching. The chlorophyll/nitrocellulose system is concluded to be a useful model system for studies of donor-donor energy transfer processes.     

Determination of carotenoids in two algae species from the saline water of Kapulukaya reservoir by HPLC

The local algae species, Chlorella vulgaris and Scenedesmus regularis, from a highly saline water body of Kapulukaya Reservoir were isolated to analyze their carotenoid composition and content using HPLC method. The gradient solvent system of methanol–acetonitrile–water (84:14:2, v/v/v) and methylene chloride (100%), used to resolve a range of carotenoids from the saponified cells, proved an acceptable separation as inferred from the retention factor (k) ranging between 0.75 and 7.76 and the separation factor (α) values greater than 1. Resolution peaks assigned to carotenoids, 21 for C. vulgaris extracts and 22 for S. regularis extracts, were reached within the duration time of 45?min. Main carotenoids identified either tentatively or positively were all-trans-lutein, 9- or 9′-cis-lutein, 13- or 13′-cis-lutein, cis-lutein, All-trans-α-carotene, 9- or 9′-cis-α-carotene, All-trans-β-carotene, 9- or 9′-cis-β-carotene in the species except for all-trans-β-cryptoxanthin found only in S. regularis. Auroxanthin, neochrome, neoxanthin, and cis-neoxanthin were identified as epoxy-containing compounds. Quantitatively, C. vulgaris was distinguished to have greater amount of lutein and cis-isomers (2.74?mg/g), 77.89% while S. regularis was predominated by β-carotene and cis isomers as major component, being 80.72% (5.76?mg/g) in total carotenoids (TC). In terms of total carotenoids, the species were considered to be efficient sources for further practical applications.     

An Improved Method for the Preparation of Chlorophyll by Means of Column Chromatography with Sepharose CL-6B

Abstract

An improved method for the preparation of chlorophyll “a” and chlorophyll “b” from fresh spinach leaves by means of the column chromatography with Sepharose CL-6B has been developed. A good separation of the green pigments was attained on the column (φ25 × 163 mm) with a mixed solvent program of 2, 3, 10 and 20 % 2-propanol in hexane.     

Imidazole substituent effects on oxidative reactivity of tripodal(imid)2(thioether)CuI complexes

In the search for new bis(imidazole)thioether (BIT) copper complexes that accurately mimic the electronic and reactivity features of the CuM site of copper hydroxylase enzymes, a set of tripodal BIT ligands 4a, b- 6a, b has been synthesized that vary according to the imidazole C-(Ph or H) and N-(H or Me) substituents, as well as the position (2- or 4-) of the tripodal attachment. Corresponding [(BIT)Cu(L)](PF6) complexes 7a, b', 8a, b', and 9a', b' [L=CO (a), CH3CN (b)] have been prepared and characterized spectroscopically. The IR spectra of 7a- 9a (L=CO), specifically nu(CO), show little variation (2090-2100 cm(-1)), suggesting a similar electronic character of the Cu centers. In contrast, cyclic voltammetric analysis of these compounds (L=CH3CN) reveals quasi-reversible oxidation waves with significant variation of Epa in the range of + 0.45-0.57 V vs Fc/Fc(+), depending on the imidazole substituents. Each of the [(BIT)Cu(CH 3CN)]PF6 complexes reacts with dioxygen to form [(BIT)Cu(II) 2(mu-OH) 2](PF6)2 derivatives, 10- 12, but they vary considerably in their relative reactivity, following the same trend as the ease of their electrochemical oxidation, that is, [(2-BIT (NMe))Cu(CH 3CN)](+) ( 9b')>[(4-BIT (Ph,NMe))Cu(CH3CN)](+) ( 8b')>[(2-BIT (Ph2,NMe))Cu(CH3CN)](+) (1a')>[(4-BIT (Ph,NH))Cu(CH3CN)](+) (7b'). Thus, N-Me substitution and 4-tethering on the imidazole unit increase oxidation and oxygenation reactivity, while Ph-substitution and 2-tethering decrease reactivity. PM3 and DFT calculations are employed to analyze the relative stability, the electronic features, the Cu-CO vibrtional frequency, and the electrochemical and oxidative reactivity of the complexes.     

Efficient peptide coupling involving sterically hindered amino acids

Hindered amino acids have been introduced into peptide chains by coupling N-(Cbz- and Fmoc-alpha-aminoacyl)benzotriazoles with amino acids, wherein at least one of the components was sterically hindered, to provide compounds 3a-e, (3c +3 c'), 5a-d, (5a + 5a'), 6a-c, (6b + 6b'), 8a-c, 9a-e, 10a-d, and (10a + 10a') in isolated yields of 41-95% with complete retention of chirality as evidenced by NMR and HPLC analysis. The benzotriazole activation methodology is a new route for the synthesis of sterically hindered peptides. (Note: compound numbers written within brackets represent diastereomeric mixtures or racemates; compound numbers without brackets represent enantiomers.).     

Solution structures and dynamic properties of chelated d(0) metal olefin complexes [eta(5): eta(1)-C(5)R(4)SiMe(2)N(t)Bu]Ti(OCMe(2)CH(2)CH(2)CH=CH(2))(+) (R = H, Me): Models for the [eta(5): eta(1)-C(5)R(4)SiMe(2)N(t)Bu]Ti(R')(olefin)(+) intermediates in "constrained geometry" catalysts.

To model the Ti-olefin interaction in the putative [eta(5): eta(1)-C(5)R(4)SiMe(2)N(t)Bu]Ti(R')(olefin)(+) intermediates in "constrained geometry" Ti-catalyzed olefin polymerization, chelated alkoxide olefin complexes [eta(5): eta(1)-C(5)R(4)SiMe(2)N(t)Bu]Ti(OCMe(2)CH(2)CH(2)CH=CH(2))(+) have been investigated. The reaction of [eta(5): eta(1)-C(5)R(4)SiMe(2)N(t)Bu]TiMe(2) (1a,b; R = H, Me) with HOCMe(2)CH(2)CH(2)CH=CH(2) yields mixtures of [eta(5)-C(5)R(4)SiMe(2)NH(t)Bu]TiMe(2)(OCMe(2)CH(2)CH(2)CH=CH(2)) (2a,b) and [eta(5): eta(1)-C(5)R(4)SiMe(2)N(t)Bu]TiMe(OCMe(2)CH(2)CH(2)CH=CH(2)) (3a,b). The reaction of 2a/3a and 2b/3b mixtures with B(C(6)F(5))(3) yields the chelated olefin complexes [[eta(5): eta(1)-C(5)R(4)SiMe(2)N(t)Bu]Ti(OCMe(2)CH(2)CH(2)CH=CH(2))][MeB(C(6)F(5))(3)] (4a,b; 71 and 89% NMR yield). The reaction of 2b/3b with [Ph(3)C][B(C(6)F(5))(4)] yields [[eta(5): eta(1)-C(5)Me(4)SiMe(2)N(t)Bu]Ti(OCMe(2)CH(2)CH(2)CH=CH(2))][B(C(6)F(5))(4)] (5b, 88% NMR yield). NMR studies establish that 4a,b and 5b exist as mixtures of diastereomers (isomer ratios: 4a/4a', 62/38; 4b/4b', 75/25; 5b/5b', 75/25), which differ in the enantioface of the olefin that is coordinated. NMR data for these d(0) metal olefin complexes show that the olefin coordinates to Ti in an unsymmetrical fashion primarily through C(term) such that the C=C pi bond is polarized with positive charge buildup on C(int). Dynamic NMR studies show that 4b/4b' undergoes olefin face exchange by a dissociative mechanism which is accompanied by fast inversion of configuration at Ti ("O-shift") in the olefin-dissociated intermediate. The activation parameters for the conversion of 4b to 4b' (i.e., 4b/4b' face exchange) are: DeltaH = 17.2(8) kcal/mol; DeltaS = 8(1) eu. 4a/4a' also undergoes olefin face exchange but with a lower barrier (DeltaH = 12.2(9) kcal/mol; DeltaS = -2(3) eu), for the conversion of 4a to 4a'.