Carotenoids in fish. XXVI. Pungitius pungitius (L.) and Gasterosteus aculeatus L. (Gasterosteidae)

The author investigated the presence of various carotenoids in the different parts of the body of Pungitius pungitius (L.) and Gasterosteus aculeatus L. by means of columnar and thin-layer chromatography. The investigations revealed the presence of the following carotenoids:

in Pungitius pungitius. α-carotene, β-carotene, β-cryptoxanthin, mutatochrome, zeaxanthin and astaxanthin;

in Gasterosteus aculeatus: β-carotene, β-cryptoxanthin, β-carotene epoxide, neothxanthin, canthaxanthin, mutatochrome, lutein, phoenicoxanthin, zeaxanthin, taraxanthin, tunaxanthin, astaxanthin, astaxanthin ester and α-doradexanthin. The total carotenoid content ranged from 2.229 to 138.504 µg/g wet weight.

     

Carotenoids of Prymnesiophyceae (Haptophyceae)

A quantitative study, including mass spectrometric identification, of the carotenoids isolated from some selected prymnesiophytes harvested in exponential growth phase has been carried out. Isochrysis galbana, Hymenomonas carterae, Prymnesium parvum, Pavlova (Monochrysis) lutheri and a Pavlova sp. all produce β,β-carotene, diatoxanthin, diadinoxanthin and fucoxanthin (major carotenoid) in various proportions, in addition to several minor carotenoids were found characteristic of each alga. 19'-Hexanoyloxyfucoxanthin, previously shown to be the major carotenoid in the prymnesiophyte Emeliana (Coccolithus) huxleyi, was not encountered in the Prymnesiophyceae studied here, and we conclude that this carotenoid should be critically looked for in other members of the Gephyrocapsaceae to which E. huxleyi belongs. We further conclude that the carotenoid complement of the Chrysophyceae (in the narrow sense) should be compared with that of the Prymnesiophyceae.     

Strong and weak plasma response to dietary carotenoids identified by cluster analysis and linked to beta-carotene 15,15'-monooxygenase 1 single nucleotide polymorphisms

The mechanisms as well the genetics underlying the bioavailability and metabolism of carotenoids in humans remain unclear. To begin to address these questions, we used cluster analysis to examine individual temporal responses of plasma carotenoids from a controlled-diet study of subjects who consumed carotenoid-rich beverages. Treatments, given daily for 3 weeks, were watermelon juice at two levels (20-mg lycopene, 2.5-mg β-carotene, n=23 and 40-mg lycopene, 5-mg β-carotene, n=12) and tomato juice (18-mg lycopene, 0.6-mg β-carotene, n=10). Cluster analysis revealed distinct groups of subjects differing in the temporal response of plasma carotenoids and provided the basis for classifying subjects as strong responders or weak responders for β-carotene, lycopene, phytoene and phytofluene. Individuals who were strong or weak responders for one carotenoid were not necessarily strong or weak responders for another carotenoid. Furthermore, individual responsiveness was associated with genetic variants of the carotenoid metabolizing enzyme β-carotene 15,15'-monooxygenase 1. These results support the concept that individuals absorb or metabolize carotenoids differently across time and suggest that bioavailability of carotenoids may involve specific genetic variants of β-carotene 15,15'-monooxygenase 1.     

Chemistry,antioxidant and antimicrobial potential of nutmeg (Myristica fragrans Houtt)

Antioxidant and antimicrobial activities of nutmeg (Myristica fragrans Houtt) seed extracts were evaluated. Seeds were extracted with acetone, ethanol, methanol, butanol and water. All the extracts have shown significant antioxidant and antimicrobial activities against the tested microorganisms. Among all extracts, acetone extract has shown the highest antioxidant activity. The acetone extract showed 93.12 ± 1.48 mg gallic acid equivalents (GAE)/100 g dry weight total phenolic content, DPPH scavenging activity of 63.04 ± 1.56%, chelating activity of 64.11 ± 2.21% and 74.36 ± 1.94% inhibition of β-carotene bleaching, at 1 mg/mL extract concentration. Out of all extracts, acetone extract was able to exert antimicrobial activity against all tested bacteria and fungi. Acetone extract has shown the strongest antibacterial and antifungal activity with Staphylococcus aureus (13.8 ± 0.42 mm) and Aspergillus niger (14.4 ± 0.37 mm), respectively. GC–MS analysis of acetone extract has revealed the presence of 32 compounds of extract representing 99.49%. Sabinene (28.61%) has shown the highest occurrence in the extract. β-Pinene (10.26), α-pinene (9.72), myristicin (4.30%), isoeugenol (2.72%), p-cymene (1.81%), carvacrol (1.54%), eugenol (0.89%) and β-caryophellene (0.82%) were reported as possible contributor for antioxidant and antimicrobial activity of nutmeg.     

Salt stress (NaCl) affects plant growth and branch pathways of carotenoid and flavonoid biosyntheses in <Emphasis Type="Italic">Solanum nigrum</Emphasis>

In this study, we set out to investigate the effect of sodium chloride (NaCl) on carotenoid and flavonoid production by the black nightshade (Solanum nigrum L.). The study was carried out under green chamber conditions using seedlings subjected to 0, 50, 100 and 150 mM NaCl for 3 weeks. The negative effect of NaCl on dry biomass production of roots and leaves were accompanied by a significant restriction in K⁺, Ca²⁺ and Mg²⁺ ion uptake and by an increase in Na⁺ ion concentrations, the effects of which were most pronounced at the highest NaCl level. Salt stress also induced oxidative stress, according to the amplified levels of thiobarbituric acid reactive substances and relative ion leakage ratio. Expression of some related carotenoid (phytoene synthase 2 and β-lycopene cyclase) and flavonoids genes (phenylalanine ammonialyase, chalcone synthase and flavonol synthase) were induced by NaCl, followed enhanced production of β-carotene, lutein, and quercetin 3-β-d-glucoside. At the highest NaCl level (150 mM NaCl), quercetin 3-β-d-glucoside synthesis came at the expense of reduced β-carotene and lutein, while salt stress treatment affected leaf antioxidant activities to a great extent relative to the control. Our data suggest that the potential antioxidant properties of carotenoids and flavonoids and their related key genes may be efficiently involved in the restriction of salt-induced oxidative damages.     

Carotenoid oxidative degradation products inhibit Na+-K+-ATPase

This study investigates the biological significance of carotenoid oxidation products using inhibition of Na⁺-K⁺-ATPase activity as an index. β-Carotene was completely oxidized by hypochlorous acid and the oxidation products were analyzed by capillary gasliquid chromatography and high performance liquid chromatography. The Na⁺-K⁺-ATPase activity was assayed in the presence of these oxidized carotenoids and was rapidly and potently inhibited. This was demonstrated for a mixture of β-carotene oxidative breakdown products, β-Apo-10′-carotenal and retinal. Most of the β-carotene oxidation products were identified as aldehydic. The concentration of the oxidized carotenoid mixture that inhibited Na⁺-K⁺-ATPase activity by 50% (IC₅₀) was equivalent to 10μM non-degraded β-carotene, whereas the IC₅₀ for 4-hydroxy-2-nonenal, a major lipid peroxidation product, was 120 μM. Carotenoid oxidation products are more potent inhibitors of Na⁺-K⁺-ATPase than 4-hydroxy-2-nonenal. Enzyme activity was only partially restored with hydroxylamine and/or β-mercaptoethanol. Thus, in vitro binding of carotenoid oxidation products results in strong enzyme inhibition. These data indicate the potential toxicity of oxidative carotenoid metabolites and their activity on key enzyme regulators and signal modulators.     

Carotene Hydroxylase Activity Determines the Levels of Both α-Carotene and Total Carotenoids in Orange Carrots

The typically intense carotenoid accumulation in cultivated orange-rooted carrots (Daucus carota) is determined by a high protein abundance of the rate-limiting enzyme for carotenoid biosynthesis, phytoene synthase (PSY), as compared with white-rooted cultivars. However, in contrast to other carotenoid accumulating systems, orange carrots are characterized by unusually high levels of α-carotene in addition to β-carotene. We found similarly increased α-carotene levels in leaves of orange carrots compared with white-rooted cultivars. This has also been observed in the Arabidopsis thaliana lut5 mutant carrying a defective carotene hydroxylase CYP97A3 gene. In fact, overexpression of CYP97A3 in orange carrots restored leaf carotenoid patterns almost to those found in white-rooted cultivars and strongly reduced α-carotene levels in the roots. Unexpectedly, this was accompanied by a 30 to 50% reduction in total root carotenoids and correlated with reduced PSY protein levels while PSY expression was unchanged. This suggests a negative feedback emerging from carotenoid metabolites determining PSY protein levels and, thus, total carotenoid flux. Furthermore, we identified a deficient CYP97A3 allele containing a frame-shift insertion in orange carrots. Association mapping analysis using a large carrot population revealed a significant association of this polymorphism with both α-carotene content and the α-/β-carotene ratio and explained a large proportion of the observed variation in carrots.     

Candidate gene sequencing and validation of SNP markers linked to carotenoid content in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Cassava is a widely grown staple in Sub-Saharan Africa and consumed as a cheap source of calories, but the crop is deficient in micronutrients including pro-vitamin A carotenoids. This challenge is currently being addressed through biofortification breeding that relies on phenotypic selection. Gene-based markers linked to pro-vitamin A content variation are expected to increase the rate of genetic gain for this critical trait. We sequenced four candidate carotenoid genes from 167 cassava accessions representing the diversity of elite breeder lines from IITA. Total carotenoid content was determined using spectrophotometer and total β-carotene was quantified by high-performance liquid chromatography. Storage root yellowness due to carotenoid pigmentation was assessed. We carried out candidate gene association analysis that accounts for population structure and kinship using genome-wide single nucleotide polymorphisms (SNPs) generated through genotyping-by-sequencing. Significant SNPs were used to design competitive allele-specific PCR assays and validated on the larger population for potential use in marker-assisted selection breeding. Candidate gene sequencing of the genes β-carotene hydroxylase (crtRB), phytoene synthase (PSY2), lycopene epsilon cyclase (lcyE), and lycopene beta cyclase (lcyB) yielded a total of 37 SNPs. Total carotenoid content, total β-carotene, and color parameters were significantly associated with markers in the PSY2 gene. The SNPs from lcyE were significantly associated with color while those of lcyB and crtRB were not significantly associated with carotenoids or color parameters. These validated and breeder-friendly markers have potential to enhance the efficiency of selection for high β-carotene cassava, thus accelerating genetic gain.     

Evaluation of the antioxidant effects of carotenoids from Deinococcus radiodurans through targeted mutagenesis,chemiluminescence, and DNA damage analyses

Deinococcus radiodurans is highly resistant to reactive oxygen species (ROS). The antioxidant effect of carotenoids in D. radiodurans was investigated by using a targeted mutation of the phytoene synthase gene to block the carotenoid synthesis pathway and by evaluating the survival of cells under environmental stresses. The colorless mutant R1ΔcrtB of D. radiodurans failed to synthesize carotenoids, and was more sensitive to ionizing radiation, hydrogen peroxide, and desiccation than the wild type, suggesting that carotenoids in D. radiodurans help in combating environmental stresses. Chemiluminescence analyses showed that deinoxanthin, a major product in the carotenoid synthesis pathway, had significantly stronger scavenging ability on H₂O₂ and singlet oxygen than two carotenes (lycopene and β-carotene) and two xanthophylls (zeaxanthin and lutein). Deinoxanthin also exhibited protective effect on DNA. Our findings suggest that the stronger antioxidant effect of deinoxanthin contribute to the resistance of D. radiodurans. The higher antioxidant effect of deinoxanthin may be attributed to its distinct chemical structure which has an extended conjugated double bonds and the presence of a hydroxyl group at C-1′ position, compared with other tested carotenoids.     

In planta cleavage of carotenoids by <Emphasis Type="Italic">Arabidopsis</Emphasis> carotenoid cleavage dioxygenase 4 in transgenic rice plants

A family of carotenoid cleavage dioxygenases (CCDs) produces diverse apocarotenoid compounds via the oxidative cleavage of carotenoids as substrates. Their types are highly dependent on the action of the CCD family to cleave the double bonds at the specific position on the carotenoids. Here, we report in vivo function of the AtCCD4 gene, one of the nine members of the Arabidopsis CCD gene family, in transgenic rice plants. Using two independent single-copy rice lines overexpressing the AtCCD4 transgene, the targeted analysis for carotenoids and apocarotenoids showed the markedly lowered levels of β-carotene (74 %) and lutein (72 %) along with the changed levels of two β-carotene (C₄₀) cleavage products, a two-fold increase of β-ionone (C₁₃) and de novo generation of β-cyclocitral (C₁₀) at lower levels, compared with non-transgenic rice plants. It suggests that β-carotene could be the principal substrate being cleaved at 9–10 (9′–10′) for β-ionone and 7–8 (7′–8′) positions for β-cyclocitral by AtCCD4. This study is in planta report on the generation of apocarotenal volatiles from carotenoid substrates via cleavage by AtCCD4. We further verified that the production of these volatiles was due to the action of exogenous AtCCD4 and not the expression of endogenous rice CCD genes (OsCCD1, 4a, and 4b).     

Antioxidant activity of carotenoids: An electron-spin resonance study on β-carotene and lutein interaction with free radicals generated in a chemical system

β-Carotene is thought to be a chain-breaking antioxidant, even though we have no information about the mechanism of its antioxidant activity. Using electron-spin resonance (ESR) spectroscopy coupled to the spin-trapping technique, we have studied the effect of β-carotene and lutein on the radical adducts of the spin-trap PBN (N-t -butyl-α-phenylnitrone) generated by the metal-ion breakdown of different tert -butyl hydroperoxide (t BOOH) concentrations in methylene chloride. The peroxyl radical, along with an oxidation product of PBN (the PBNOx), trapped at room temperature from the breakdown of high concentration of t BOOH (1 M), were quenched by β-carotene or lutein, in competition with the spin-trapping agent. However, carotenoids were not able to quench the alkoxyl and methyl radicals generated in the reaction carried out in the presence of low t BOOH concentration (1 mM). The reaction between carotenoids and the peroxyl radical was also carried out in the absence of the spin trap, at 77 K: Under these different experimental conditions, we did not detect any radical species deriving from carotenoids. In the same system, a further evidence of the peroxyl radical quenching by β-carotene and lutein was obtained. The antioxidant activity of vitamin E was also tested, for comparison with the carotenoids. In the presence of α-tocopherol, peroxyl and alkoxyl radicals were quenched, and the tocopheroxyl radical was detected. Our data provide the first direct evidence that carotenoids quench peroxyl radicals. Under our experimental conditions, we did not detect any carotenoid radical species that could derive from the interaction with the peroxyl radical. The radical-trapping activity of β-carotene and lutein demonstrated in this chemical reaction contributes to our understanding carotenoid antioxidant action in biological systems. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 299–304, 1998     

Antioxidant actions of plant foods: Use of oxidative DNA damage as a tool for studying antioxidant efficacy

Plant-food-derived antioxidants and active principles such as flavonoids, hydroxycinnamates (ferulic acid, chlorogenic acids, vanillin etc.), β-carotene and other carotenoids, vitamin E, vitamin C, or rosemary, sage, tea and numerous extracts are increasingly proposed as important dietary antioxidant factors. In this endeavor, assays involving oxidative DNA damage for characterizing the potential antioxidant actions are suggested as in vitro screens of antioxidant efficacy. The critical question is the bioavailability of the plant-derived antioxidants.     

Carotenoid synthesis and phytoene synthase activity during mating of Blakeslea trispora

Carotenoid formation was investigated in wild type and carotenogenic mutants of Blakeslea trispora after mating (−) and (+) strains. The highest yields of carotenoids, especially β-carotene was observed following mating. In vitro incorporation of geranylgeranyl pyrophosphate into phytoene and β-carotene corresponded to increased carotenogenesis in the mated strains. Immuno determination of phytoene synthase protein levels revealed that the amounts of this enzyme is concurrent with the increases in carotenoid content. In fungi, phytoene synthase together with lycopene cyclase are encoded by a fusion gene crtYB or carRA with two individual domains. These domains were both heterologously expressed in an independent manner and antisera raised against both. These antisera were used, to assess protein levels in mated and non-mated B. trispora. The phytoene synthase domain was detected as an individual soluble protein with a molecular weight of 40 kDa and the lycopene cyclase an individual protein of mass about 30 kDa present in the membrane fraction following sub-cellular fractionation. This result demonstrates a post-translational cleavage of the protein transcribed from a single mRNA into independent functional phytoene synthase and lycopene cyclase.     

Assessment of carotenoid production by Dunaliella salina in different culture systems and operation regimes

The effect of operation regime and culture system on carotenoid productivity by the halotolerant alga Dunaliella salina has been analyzed. Operation strategies tested included batch and semi continuous regime, as well as a two-stage approach run simultaneously in both, open tanks and closed reactor. The best results were obtained with the closed tubular photobioreactor. The highest carotenoid production (328.8 mg carotenoid l⁻¹ culture per month) was achieved with this culture system operated following the two-stage strategy. Also, closed tubular photobioreactor provided the highest carotenoid contents (10% of dry weight) in Dunaliella biomass and β-carotene abundance (90% of total carotenoids) as well as the highest 9-cis to all-trans β-carotene isomer ratio (1.5 at sunrise).     

Qualitative composition of carotenoids, catalase and superoxide dismutase activities in tissues of the bivalve mollusc Anadara inaequivalvis (Bruguiere, 1789)

Using high performance liquid chromatography, UV-VIS spectra and mass-spectra (FAB MS), 7 carotenoid species were identified in tissues of the bivalve mollusc Anadara inaequivalvis (Bruguiere, 1789): trans- and cis-pectenolon, alloxanthin, pectenol A, β-carotene, zeaxanthin, and diatoxanthin. Their quantitative ratio in hepatopancreas, gills and foot of animals were determined. A negative correlation (R ² is about 0.9) was revealed between tissues content of a series of carotenoids (trans- and cis-pectenolon, zeaxanthin, alloxanthin, and diatoxanthin) and activity of antioxidant enzymes (catalase and superoxide dismutase). The existence of competitive interrelations between these molecular systems is proposed and underlying causes are discussed.     

Effect of food deprivation on transformation of carotenoids in the bivalve mollusc <Emphasis Type="Italic">Anadara kagoshimensis</Emphasis> (Tokunaga, 1906)

The composition and content of carotenoids in tissues of Anadara kagoshimensis (Tokunaga, 1906) were analyzed under food deprivation over 30 days. By the end of the experiment, a little and uneven decrease in the total carotenoid level was observed in the foot, hepatopancreas, gills, and tissue remainder. Initially, β-carotine, pectenolon, zeaxanthine, diatoxanthine, alloxanthine and the ethers of the latter four carotenoids were detected in tissue extracts. The qualitative carotenoid composition in the tissues under study was similar. During the experiment, changes common to all tissues were found both in the composition and content of carotenoids. β-carotene disappeared on days 3–4, the level of pectenolon, zeaxanthine, diatoxanthine and alloxanthine ethers decreased over 30 days, while the pectenolon and alloxanthine levels increased. Tissue specificity was revealed. Specifically, in the foot and hepatopancreas, a considerable increase was observed in the pectenolon and alloxanthine levels parallel to the decreasing total level of carotenoid esthers and increasing level of diatoxanthine and zeaxanthine; in the gills and lump of the remaining tissues, at the similar increase in the pectenolon and alloxanthine levels, those of diatoxanthine and zeaxanthine remained intact or even a little below the initial level. The diagram of carotenoid transformation in Anadara kagoshimensis is supplemented.     

Carotenoid stabilized gold and silver nanoparticles derived from the Actinomycete Gordonia amicalis HS-11 as effective free radical scavengers

The Actinomycete Gordonia amicalis HS-11 produced orange pigments when cultivated on n-hexadecane as the sole carbon source. When cells of this pigmented bacterium were incubated with 1 mM chloroauric acid (HAuCl₄) or silver nitrate (AgNO₃), pH 9.0, at 25 °C, gold and silver nanoparticles, respectively, were obtained in a cell associated manner. It was hypothesized that the pigments present in the cells may be mediating metal reduction reactions. After solvent extraction and High Performance Liquid Chromatography, two major pigments displaying UV–vis spectra characteristic of carotenoids were isolated. These were identified on the basis of Atmospheric Pressure Chemical Ionization Mass Spectrometry (APCI-MS) in the positive mode as 1′-OH-4-keto-γ-carotene (Carotenoid K) and 1′-OH-γ-carotene (Carotenoid B). The hydroxyl groups present in the carotenoids were eliminated under alkaline conditions and provided the reducing equivalents necessary for synthesizing nanoparticles. Cell associated and carotenoid stabilized nanoparticles were characterized by different analytical techniques. In vitro free radical scavenging activities of cells (control, gold and silver nanoparticle loaded), purified carotenoids and carotenoid stabilized gold and silver nanoparticles were evaluated. Silver nanoparticle loaded cells and carotenoid stabilized silver nanoparticles exhibited improved nitric oxide (NO) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activities compared to their control and gold counterparts. This paper thus reports cell associated nanoparticle synthesis by G. amicalis, describes for the first time the role of carotenoid pigments in metal reduction processes and demonstrates enhanced free radical scavenging activities of the carotenoid stabilized nanoparticles.