Biological evaluation of novel derivatives of the orange pigments from Monascus sp. as inhibitors of melanogenesis

Nitrogenous derivatives of the two orange pigments from Monascus sp. with anti-melanogenic activities were prepared using fermentation and chemical synthesis. The pigments were produced in a 5 l jar fermentor. A total of 33 derivatives were synthesized via incorporation of L-amino acids and amines into the pigments. Two derivatives with high inhibitory melanin-synthesizing activities and low cell toxicities were selected based on testing using B16F10 cells. Glutamic acid and (S)-(+)-1-amino-2-propanol derivatives showed high inhibitory activities against melanogenesis. Both the reaction and expression of tyrosinase, an important enzyme in the melanin-synthesizing pathway, were inhibited by the glutamic acid derivative in a dose-dependent manner. The (S)-(+)-1-amino-2-propanol derivative inhibited expression of tyrosinase in cells, but not the tyrosinase reaction. TRP1 and TRP2, other important proteins in melanin-synthesis, were not affected by the two derivatives.     

Isolation and Characterization of Carotenoid Pigments of Micrococcus roseus

In addition to canthaxanthin, seven pigment fractions were isolated from Micrococcus roseus. They were purified by solvent partitioning and by column and thin-layer chromatography. Visible absorption spectra, chromatographic behavior, and partition coefficients of the pigments and derivatives prepared from the pigments were used in characterizing them. Both alpha- and beta-carotene derivatives were present. The structure of one pigment was suggested as phoenicoxanthin (3-hydroxy-4,4'-diketo-beta-carotene). Four other pigments were tentatively characterized as a dihydroxy-3,4-dehydro-alpha-carotene, a dihydroxy-alpha-carotene, a diketo-alpha-carotene, and a polyhydroxy-beta-carotene. Two pigments were isolated in trace amounts and could not be characterized. All the pigments studied were isolated as mixtures of cis-trans isomers and all except the diketo-alpha-carotene were isolated as esters from M. roseus. Quantitation of the pigments showed that canthaxanthin (4,4'-diketo-beta-carotene) represented 85% of the pigment recovered from extracts. Three of the other pigments contributed a significant proportion of the remaining pigments, whereas the other four were present in only small amounts. beta-Carotene derivatives comprised 96% and alpha-carotene derivatives 4% of the pigments recovered from extracts.     

A Unique pattern of anthocyanins in Daucus carota and other umbelliferae

Three cyanidin glycosides have been identified in the black carrot: the known 3-lathyroside and two new pigments, a 3-xylosylglucosylgalactoside and its ferulyl derivative. The same pigments, together with the sinapyl derivative of the triglycoside, occur variously in other tissues of Daucus carota. Ferulyl and sinapyl derivatives of cyanidin 3-glucosylgalactoside occur exceptionally in stem of one subspecies, maritimus. One or other of the same pigments have been found to occur variously in 20 of 22 other umbellifer species surveyed. Both ferulyl and sinapyl derivatives occur in stem of Conium maculatum and Foeniculum vulgare. A further novel acylated pigment based on p-coumaric acid was found in wild celery, Apiurn graveolens. The systematic significance of these various findings is discussed.     

Acylated betacyanins from Drosanthemum floribundum

Four new acylated betacyanins have been isolated from flowers of Drosanthemum floribundum (Haw.) Schwant. These pigments were characterized as hydroxycinnamoyl derivatives of betanin and isobetanin.     

Carotenoids from plankton and photosynthetic bacteria in sediments as indicators of trophic changes in Lake Lobsigen during the last 14000 years

Previous examination of drilling cores showed that the majority of the carotenes and carotenoids originally present in the various planktonic organisms and phototrophic bacteria are preserved in sediments. The indicator pigments for algae phyla are: Lutein for Chlorophyta, Myxoxanthophylls and their derivatives for Cyanophyta, Fucoxanthin for Chrysophyta, and Alloxanthin for Cryptophyta.The pigments in the deepest sediment sample (late glacial time, clay, 12.83 m) consists primarily of Alloxanthin, secondly of Lutein and -carotene and thirdly of traces of Okenone, Speroidenone and other such bacterial carotenoids. The first plankton organisms were thus Cryptophyta and some Chlorophyta. The presence of the phototrophic bacteria pigments indicates that at the time of sediment formation, anaerobic conditions prevailed at the lake bottom.The Holocene era commences at a depth of 8.55 m and is characterized by the first occurrence of Myxoxanthophyll and Echinenone from Cyanophyta, as well as by a rapid increase of -carotene, Lutein and Alloxanthin. The pronounced occurrence of Oscillatoria rubescens (blood of the Burgundies), characterized by Oscillaxanthin at 8.21 m must be considered for Swiss lakes as a very surprising discovery.The intensive plankton production again lead to stringent anaerobic conditions. Predominant among phototrophic bacteria pigments were Okenone, Spheroidene and Rhodopin.     

Graciliformin and its acetates in Cladonia graciliformis

From the lichen Cladonia graciliformis, three new pigments A, B and C were isolated along with bellidiflorin. Pigment A was an epimer of (+)-rugulosin diacetate and pigment B and C its corresponding deacetylated derivatives. Bellidiflorin is probably an iron complex compound of A. The chemical constituents of the mycobionts of Cl. graciliformis and Cl. bellidiflora were also studied.     

A reduction in temperature induces bioactive red pigment production in a psychrotolerant Penicillium sp. GEU_37 isolated from Himalayan soil

Filamentous fungi are being globally explored for the production of industrially important bioactive compounds including pigments. In the present study, a cold and pH tolerant fungus strain Penicillium sp (GEU_37), isolated from the soil of Indian Himalaya, is characterized for the production of natural pigments as influenced by varying temperature conditions. The fungal strain produces a higher sporulation, exudation, and red diffusible pigment in Potato Dextrose (PD) at 15 °C as compared to 25 °C. In PD broth, a yellow pigment was observed at 25 °C. While measuring the effect of temperature and pH on red pigment production by GEU_37, 15 °C and pH 5, respectively, were observed to be the optimum conditions. Similarly, the effect of exogenous carbon and nitrogen sources and mineral salts on pigment production by GEU_37 was assessed in PD broth. However, no significant enhancement in pigmentation was observed. Chloroform extracted pigment was separated using thin layer chromatography (TLC) and column chromatography. The two separated fractions i.e., fractions I and II with Rf values 0.82 and 0.73, exhibited maximum light absorption, λ_max, at 360 nm and 510 nm, respectively. Characterization of pigments using GC–MS showed the presence of the compounds such as phenol, 2,4-bis (1,1-dimethylethyl) and eicosene from fraction I and derivatives of coumarine, friedooleanan, and stigmasterole in fraction II. However, LC-MS analysis detected the presence of derivatives of compound carotenoids from fraction II as well as derivative of chromenone and hydroxyquinoline as major compounds from both the fractions along with other numerous important bioactive compounds. The production of such bioactive pigments under low temperature conditions suggest their strategic role in ecological resilience by the fungal strain and may have biotechnological applications.     

Chlorophyll analysis by high-performance liquid chromatography

The separation and determination of chlorophylls by high-performance liquid chromatography (HPLC) is described. Chlorophylls and their derivatives were separated by reversed-phase HPLC based on hydrophobic interaction between solute and support, using an octadecyl silica column and elution with 100% methanol. Separated pigments were detected fluorometrically with a sensitivity in the picomole range: the fluorescence response was linear over a wide pigment concentration range. Resolution of five chlorophylls a and four protochlorophyll species esterified with different alcohols was achieved within 22 min in a single experiment. This method can be used for the determination of chlorophyll b, bacteriochlorophyll a esters and products synthesized from chlorophyll, but not for nonesterified pigments, i.e., chlorophyllide, protochlorophyllide and chlorophyll c. The chromatographic mobility of chlorophyll a esterified with different alcohols increases with increasing number of carbon atoms in the esterifying alcohols. The plots obtained from the logarithm of the capacity factor (k′) of these pigments versus the numbers of carbon atoms of the alcohol molecule gave a straight line, thus permitting the estimation of the chain length of unknown pigment esterifying alcohols. This HPLC separation technique did not cause the formation of artifacts. The deviation of the individual retention time for each pigment is less than ±0.5%, thus making this method suitable for the rapid identification and quantification of unknown pigments.     

Formation of water-solubleMonascus red pigments by biological and semi-synthetic processes

Summary New water-soluble red pigments were produced byMonascus sp. in a chemically defined fermentation medium containing glutamate as nitrogen source. They were isolated and characterized as glutamate derivatives of the well-known orangeMonascus pigments (monascorubrin and rubropunctatin). The new pigments have several advantages over the known redMonascus pigments (rubropunctamine and monascorubramine) including very high water-solubility, higher absorption coefficient, and greater resistance to decoloration by light. Adding glutamate, glycine or leucine to a resting-cell system led to the formation of specific water-soluble red pigments corresponding to the exogenous amino acid. The water-soluble red pigments produced by resting-cells have retention times identical to those of the corresponding red derivatives made chemically from the orange pigments in methanol-phosphate buffer at pH 7. The hydrophobicities of the amino acid sources correspond to the HPLC retention times of the red pigments derived from them.     

Formation of stereoisomeric mixtures of naphthoquinone derivatives in Echium lycopsis callus cultures

Callus cultures of Echium lycopsis were shown to produce a large amount of a mixture of red pigments consisting of five esterified derivatives of 5,8-dihydroxy-2-(1-hydroxy-4-methyl-3-pentenyl)-1,4-naphthoquinone. Examination of the absolute configuration of these compounds revealed that the cultures produced both the R-form (shikonin) and the S-form (alkannin) in various ratios depending upon the esterified derivative, although the overall ratio for the total derivatives was ca 1:1. On the other hand, all the corresponding derivatives produced by Lithospermum cultures were primarily of the R-form. It was also demonstrated that pigment formation in Echium cultures was inhibited by either white or blue light as well as by the synthetic auxin 2,4-D as in the case of Lithospermum cultures.     

Morpho-physiological and biochemical responses of muskmelon genotypes to different degree of water deficit

Morpho-physiological and biochemical analyses were carried out in eight diverse indigenous muskmelon (Cucumis melo L.) genotypes exposed to different degrees of water deficit (WD). The ability of genotypes MM-7, and especially MM-6, to counteract better the negative effect of WD was associated with maintaining higher relative water content (RWC), photosynthetic rate, efficiency of PSII, and photosynthetic pigments compare to other genotypes. Furthermore, MM-6 showed a better ability to maintain cellular homeostasis than the others. It was indicated by a stimulated antioxidative defense system, i.e., higher activities of antioxidant enzymes, accumulation of nonenzymatic antioxidants together with lower concentration of reactive oxygen species and malondialdehyde. However, the genotypes MM-2 and MM-5 suffered greatly due to WD and showed reduced RWC, photosynthetic rates, pigment content, and exhibited higher oxidative stress observed as lower antioxidant enzyme activities.     

Near edge X-ray absorption fine structure spectroscopy (NEXAFS) of pigment-protein complexes: peridinin-chlorophyll a protein (PCP) of Amphidinium carterae

Peridinin-chlorophyll a protein (PCP) is a unique water soluble antenna complex that employs the carotenoid peridinin as the main light-harvesting pigment. In the present study the near edge X-ray absorption fine structure (NEXAFS) spectrum of PCP was recorded at the carbon K-edge. Additionally, the NEXAFS spectra of the constituent pigments, chlorophyll a and peridinin, were measured. The energies of the lowest unoccupied molecular levels of these pigments appearing in the carbon NEXAFS spectrum were resolved. Individual contributions of the pigments and the protein to the measured NEXAFS spectrum of PCP were determined using a “building block” approach combining NEXAFS spectra of the pigments and the amino acids constituting the PCP apoprotein. The results suggest that absorption changes of the pigments in the carbon near K-edge region can be resolved following excitation using a suitable visible pump laser pulse. Consequently, it may be possible to study excitation energy transfer processes involving “optically dark” states of carotenoids in pigment-protein complexes by soft X-ray probe optical pump double resonance spectroscopy (XODR).     

Alleviation of drought stress of marigold (Tagetes erecta) plants by using arbuscular mycorrhizal fungi

The effect of an arbuscular mycorrhizal fungus “AMF” (Glomus constrictum Trappe) on growth, pigments, and phosphorous content of marigold (Tagetes erecta) plant grown under different levels of drought stress was investigated. The applied drought stress levels reduced growth vigor (i.e. plant height, shoot dry weight, flower diameter as well as its fresh and dry weights) of mycorrhizal and non-mycorrhizal plant as compared to control plant (non-drought stressed plant). The presence of mycorrhizal fungus, however, stimulated all growth parameters of the treated plant comparing to non-mycorrhizal treated plant. The photosynthetic pigments (carotene in flowers and chlorophylls a and b in leaves) were also stimulated by the mycorrhizal fungi of well-watered as well as of water-stressed plants. The total pigments of mycorrhizal plants grown under well-watered conditions were higher than those of non-mycorrhizal ones by 60%. In most cases, drought-stressed mycorrhizal plants were significantly better than those of the non-mycorrhizal plants. So, the overall results suggest that mycorrhizal fungal colonization affects host plant positively on growth, pigments, and phosphorous content, flower quality and thereby alleviates the stress imposed by water with holding.     

Tolerance to Heterodera glycines in Soybean

Fifty-four susceptible soybean, Glycine max, cultivars or plant introductions were evaluated for tolerance to H. glycines, the soybean cyst nematode (SCN). Seed yields of genotypes were compared in nematicide-treated (1,2-dibromo-3-chloropropane, 58 kg a.i./ha) and nontreated plots at two SCN-infested locations over 3 years. Distinct and consistent levels of tolerance to SCN were observed among soybean genotypes. PI 97100, an introduction from Korea, exhibited the highest level of tolerance with an average tolerance index ([yield in nontreated plot ÷ yield in nematicide-treated plot] × 100) of 96 over 2 years. Coker 156 and Wright had moderate levels of tolerance (range in index values 68 to 95) compared to the intolerant cuhivars Bragg and Coker 237 (range in index values 33 to 68). Most of the soybean genotypes evaluated were intolerant to SCN. The rankings of five genotypes for tolerance to SCN and Hoplolaimus columbus were similar. Tolerance for seed yield was more consistently correlated with tolerance for plant height (r = 0.55 to 0.64) than for seed weight (r = 0.23 to 0.65) among genotypes.     

A TLC-GLC procedure for the quantitation of algal photosynthetic pigments and their degradation products

A procedure employing and GLC techniques for the analysis of algal chlorophylls and their degradation products has been developed and evaluated. Algal pigments were separated on MN 300 cellulose plates developed in an ascending solvent system of hexane saturated with acetonitrile and n-propanol (100:0,4, v:v). Quantitation of chlorophyll a, b and pheophytin a, b were accomplished by GLC analysis of their phytol, following alkaline methanolic hydrolysis of the individual pigments. The amount of chlorophyllides/pheophorbides in a sample was estimated by its free phytol content. This technique is especially valuable for the evaluation of the pigment contents of near sediment phytoplankton and periphyton samples, where large quantities of chlorophyll degradation products and/or carotenoid pigments are generally present which may interfere significantly with the routine analytical methods.     

Toxicity,of extracts of barley kernels inoculated withFusarium spp. toArtemia salina

Toxicity toA. salina, of the Fusarium metabolites: deoxynivalenol (DON), its acetylated derivatives (3- and 15-AcDON), zearalenone (ZON), neosolaniol (NEO), nivalenol (NIV), T-2, HT-2 toxins, has been examined and compared with toxicity of extracts of barley kernels (8 cultivars and 4 lines) inoculated withFusarium culmorum, F. graminearum andF. sporotrichioides respectively. Estimated LC₅₀ values were expressed as relative toxicity (RT) in mg DON/kg for samples inoculated withF. culmorum, F. graminearum or in mg T-2/kg forF. sporotrichioides inoculations. Toxicity of extracts of the same genotype/line kernels was compared among different pathogens used for inoculation and differences in Fusarium head blight susceptibility of different genotypes/lines inoculated with the sameFusarium strain were found. Significant correlation between toxicity of extracts (LC₅₀, RT) and toxic metabolites concentration was found ( $\bar r = 0.82$ ; P = 0.01). Bioassays withA. Salina offer a fast, easy and inexpensive method to examine cereal genotypes susceptibility to Fusarium head blight and mycotoxins accumulation in kernels.     

Shift in birch leaf metabolome and carbon allocation during long-term open-field ozone exposure

Current and future ozone concentrations have the potential to reduce plant growth and increase carbon demand for defence and repair processes, which may result in reduced carbon sink strength of forest trees in long‐term. Still, there is limited understanding regarding the alterations in plant metabolism and variation in ozone tolerance among tree species and genotypes. Therefore, this paper aims to study changes in birch leaf metabolome due to long‐term realistic ozone stress and to relate these shifts in the metabolism with growth responses. Two European white birch (Betula pendula Roth) genotypes showing different ozone sensitivity were growing under 1.4–1.7 × ambient ozone in open‐field conditions in Central Finland. After seven growing seasons, the trees were analysed for changes in leaf metabolite profiling, based on 339 low molecular weight compounds (including phenolics, polar and lipophilic compounds, and pigments) and related whole‐tree growth responses. Genotype caused most of the variance of metabolite concentrations, while ozone concentration was the second principal component explaining the metabolome profiling. The main ozone caused changes included increases in quercetin‐phenolic compounds and compounds related to leaf cuticular wax layer, whereas several compounds related to carbohydrate metabolism and function of chloroplast membranes and pigments (such as chlorophyll‐related phytol derivatives) were decreasing. Some candidate compounds such as surface wax‐related squalene, 1‐dotriacontanol, and dotriacontane, providing growth‐related tolerance against ozone were demonstrated. This study indicated that current growth‐based ozone risk assessment methods are inadequate, because they ignore ecophysiological impacts due to alterations in leaf chemistry.     

Near edge X-ray absorption fine structure spectroscopy (NEXAFS) of pigment–protein complexes: Peridinin–chlorophyll a protein (PCP) of Amphidinium carterae

Peridinin–chlorophyll a protein (PCP) is a unique water soluble antenna complex that employs the carotenoid peridinin as the main light-harvesting pigment. In the present study the near edge X-ray absorption fine structure (NEXAFS) spectrum of PCP was recorded at the carbon K-edge. Additionally, the NEXAFS spectra of the constituent pigments, chlorophyll a and peridinin, were measured. The energies of the lowest unoccupied molecular levels of these pigments appearing in the carbon NEXAFS spectrum were resolved. Individual contributions of the pigments and the protein to the measured NEXAFS spectrum of PCP were determined using a “building block” approach combining NEXAFS spectra of the pigments and the amino acids constituting the PCP apoprotein. The results suggest that absorption changes of the pigments in the carbon near K-edge region can be resolved following excitation using a suitable visible pump laser pulse. Consequently, it may be possible to study excitation energy transfer processes involving “optically dark” states of carotenoids in pigment–protein complexes by soft X-ray probe optical pump double resonance spectroscopy (XODR).