The individual variation in carotenoid content of the lichen species Dirinaria applanata (Fée) Awasthi

Column and thin‐layer chromatography revealed the presence of the following carotenoids in thalli of Dirinaria applanata from 13 different sites: α‐carotene, β‐carotene, β‐cryptoxanthin, lutein, 3′‐epilutein, zeaxanthin, antheraxanthin, canthaxanthin, astaxanthin, violaxanthin, mutatoxanthin, neoxanthin, capsochrome, fucoxanthinol, paracentrone and apo‐6′‐lycopenal. In the thalli of all 13 specimens of Dirinaria applanata β‐carotene, lutein, astaxanthin and violaxanthin were found as constant carotenoids. The total content of carotenoids ranged from 21.0 (from Mexico) to 54.9 μg g⁻¹ dry weight (from Antilles).     

4‐Oxo‐β‐apo‐13‐carotenone from the Cyanobacterium Anabaena cylindrica PCC 7122

Apocarotenoids are widely distributed among living organisms (bacteria, fungi, algae, plants and even animals) and have been associated with several signaling functions. These compounds are generated by the activity of carotenoid cleavage dioxygenases (CCDs), whose diversity greatly contributes to the large number of apocarotenoids that have been described so far. It is nevertheless expected that a considerable diversity of these molecules is yet to be discovered. In this work, we describe the isolation and structural elucidation of the apocarotenoid 4‐oxo‐β‐apo‐13‐carotenone from the cultured freshwater cyanobacterium Anabaena cylindrica PCC 7122, corresponding to the first report of this compound from natural sources.     

Evolutionary aspects and enzymology of metazoan carotenoid cleavage oxygenases

The carotenoids are terpenoid fat-soluble pigments produced by plants, algae, and several bacteria and fungi. They are ubiquitous components of animal diets. Carotenoid cleavage oxygenase (CCO) superfamily members are involved in carotenoid metabolism and are present in all kingdoms of life. Throughout the animal kingdom, carotenoid oxygenases are widely distributed and they are completely absent only in two unicellular organisms, Monosiga and Leishmania. Mammals have three paralogs 15,15′-β-carotene oxygenase (BCO1), 9′,10′-β-carotene oxygenase (BCO2) and RPE65. The first two enzymes are classical carotenoid oxygenases: they cleave carbon‑carbon double bonds and incorporate two atoms of oxygen in the substrate at the site of cleavage. The third, RPE65, is an unusual family member, it is the retinoid isomerohydrolase in the visual cycle that converts all-trans-retinyl ester into 11-cis-retinol. Here we discuss evolutionary aspects of the carotenoid cleavage oxygenase superfamily and their enzymology to deduce what insight we can obtain from their evolutionary conservation.     

Sioxanthin,a novel glycosylated carotenoid,reveals an unusual subclustered biosynthetic pathway

Members of the marine actinomycete genus Salinispora constitutively produce a characteristic orange pigment during vegetative growth. Contrary to the understanding of widespread carotenoid biosynthesis pathways in bacteria, Salinispora carotenoid biosynthesis genes are not confined to a single cluster. Instead, bioinformatic and genetic investigations confirm that four regions of the Salinispora tropica CNB‐440 genome, consisting of two gene clusters and two independent genes, contribute to the in vivo production of a single carotenoid. This compound, namely (2′S)‐1′‐(β‐D‐glucopyranosyloxy)‐3′,4′‐didehydro‐1′,2′‐dihydro‐φ,ψ‐caroten‐2′‐ol, is novel and has been given the trivial name ‘sioxanthin’. Sioxanthin is a C₄₀‐carotenoid, glycosylated on one end of the molecule and containing an aryl moiety on the opposite end. Glycosylation is unusual among actinomycete carotenoids, and sioxanthin joins a rare group of carotenoids with polar and non‐polar head groups. Gene sequence homology predicts that the sioxanthin biosynthetic pathway is present in all of the Salinispora as well as other members of the family Micromonosporaceae. Additionally, this study's investigations of clustering of carotenoid biosynthetic genes in heterotrophic bacteria show that a non‐clustered genome arrangement is more common than previously suggested, with nearly half of the investigated genomes showing a non‐clustered architecture.     

Total Tocopherols,Carotenoids, and Fatty Acids Content Variation of Pistacia atlantica from Different Organs’ Crude Oils and Their Antioxidant Activity during Development Stages

The current study investigated the effect of developmental stages on the chemical composition and the antioxidant activity of fifteen crude oil samples obtained from Pistacia atlantica Desf. leaves, galls, and fruits. Twelve fatty acids were detected by GC/FID, linolenic acid (C18 : 3) was the major fatty acid detected in leaves crude oils that registered [41.73 % (P<0.05)] on the last stage. The best content of tocopherols and carotenoids was recorded at the last stage for leaves and galls oils, respectively, with values of [1.530±0.01, 0.52±0.01 (P<0.05) mg α‐tocopherol equivalent/g DW] and [86.60±0.95, 69.15±0.13 (P<0.05) μg β‐carotene equivalent/g DW]. For fruits oils, the content varied depending on the levels of fruits maturation. The results from DPPH, FRAP, and ABTS assays revealed that the antioxidant activity increased with the increasing content of tocopherols and carotenoids in leaves and galls oils during development stages, and varied for fruits oils depending on the ripening stages. Moreover, according to PCA analysis, the best phytoconstituent content and antioxidant activity were attributed to P. atlantica Desf. fruit's crude oils. Also, a strong relationship was found between the antioxidant activity and bioactive phytochemical components, such as tocopherols, carotenoids, and omega‐three fatty acid, which confirmed that P. atlantica Desf. crude oils present a valuable source of natural antioxidant that could be used for pharmaceutical and food industries purposes.     

Fruit carotenoid‐deficient mutants in tomato reveal a function of the plastidial isopentenyl diphosphate isomerase (IDI1) in carotenoid biosynthesis

Isoprenoids consist of a large class of compounds that are present in all living organisms. They are derived from the 5C building blocks isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP). In plants, IDP is synthesized in the cytoplasm from mevalonic acid via the MVA pathway, and in plastids from 2‐C‐methyl‐d ‐erythritol‐4‐phosphate through the MEP pathway. The enzyme IDP isomerase (IDI) catalyzes the interconversion between IDP and DMADP. Most plants contain two IDI enzymes, the functions of which are characteristically compartmentalized in the cells. Carotenoids are isoprenoids that play essential roles in photosynthesis and provide colors to flowers and fruits. They are synthesized in the plastids via the MEP pathway. Fruits of Solanum lycopersicum (tomato) accumulate high levels of the red carotene lycopene. We have identified mutations in tomato that reduce overall carotenoid accumulation in fruits. Four alleles of a locus named FRUIT CAROTENOID DEFICIENT 1 (fcd1) were characterized. Map‐based cloning of fcd1 indicated that this gene encodes the plastidial enzyme IDI1. Lack of IDI1 reduced the concentration of carotenoids in fruits, flowers and cotyledons, but not in mature leaves. These results indicate that the plastidial IDI plays an important function in carotenoid biosynthesis, thus highlighting its role in optimizing the ratio between IDP and DMADP as precursors for different downstream isoprenoid pathways.     

Identification of the gene responsible for torulene cleavage in the Neurospora carotenoid pathway

Torulene, a C₄₀ carotene, is the precursor of the end product of the Neurospora carotenoid pathway, the C₃₅ xanthophyll neurosporaxanthin. Torulene is synthesized by the enzymes AL-2 and AL-1 from the precursor geranylgeranyl diphosphate and then cleaved by an unknown enzyme into the C₃₅ apocarotenoid. In general, carotenoid cleavage reactions are catalyzed by carotenoid oxygenases. Using protein data bases, we identified two putative carotenoid oxygenases in Neurospora, named here CAO-1 and CAO-2. A search for novel mutants of the carotenoid pathway in this fungus allowed the identification of two torulene-accumulating strains, lacking neurosporaxanthin. Sequencing of the cao-2 gene in these strains revealed severe mutations, pointing to a role of CAO-2 in torulene cleavage. This was further supported by the identical phenotype found upon targeted disruption of cao-2. The biological function was confirmed by in vitro assays using the purified enzyme, which cleaved torulene to produce β-apo-4′-carotenal, the corresponding aldehyde of neurosporaxanthin. The specificity of CAO-2 was shown by the lack of γ-carotene-cleaving activity in vitro. As predicted for a structural gene of the carotenoid pathway, cao-2 mRNA was induced by light in a WC-1 and WC-2 dependent manner. Our data demonstrate that CAO-2 is the enzyme responsible for the oxidative cleavage of torulene in the neurosporaxanthin biosynthetic pathway.     

PHOTOSYNTHETIC PIGMENT COMPOSITION IN THE PRIMITIVE GREEN ALGA MESOSTIGMA VIRIDE (PRASINOPHYCEAE): PHYLOGENETIC AND EVOLUTIONARY IMPLICATIONS1

The photosynthetic pigment composition of Mesostigma viride Lauterborn, a primitive green alga, was determined. This alga contained chl a and b, lycopene, lutein, siphonaxanthin, γ‐carotene, β‐carotene, antheraxanthin, violaxanthin, neoxanthin, and two novel carotenoid fatty acid esters, siphonaxanthin C12:0 ester and siphonaxanthin C14:0 ester. The esters were saturated, whereas all previously identified siphonaxanthin and loroxanthin esters have been mono‐unsaturated (trans‐Δ2). Neoxanthin was the all‐trans form. This is the first such case detected in the chloroplasts of green plants. The 9′‐cis form of neoxanthin is believed to be universally present in the chloroplasts of green plants (Streptophyta and Chlorophyta) and is a precursor of abscisic acid. However, the 9′‐cis form was not found in M. viride. Based on these results, we discuss the phylogenetic implications and early evolution of the antenna pigment system in green plants.     

Chemical Composition,Antibacterial, Antioxidant and in Vitro Antidiabetic Activities of Essential Oils from Eruca vesicaria

This work describes the study of the chemical composition and bioactivity of the essential oils (EOs) of the different organs (leaves, flowers, stems and roots) from Eruca vesicaria. According to the GC and GC/MS analysis, all the EOs were dominated by erucin (4‐methylthiobutyl isothiocyanate) with a percentage ranging from 17.9 % (leaves) to 98.5 % (roots). The isolated EOs were evaluated for their antioxidant (DPPH, ABTS and β‐carotene/linoleic acid), antibacterial and inhibitory property against α‐amylase and α‐glucosidase. Most EOs exhibited an interesting α‐glucosidase and α‐amylase inhibitory potential. The roots essential oil was found to be the most active with IC₅₀ values of 0.80±0.06 and 0.11±0.01 μg mL^?1, respectively. The essential oil of roots exhibited the highest antioxidant activity (DPPH, PI=92.76±0.01 %; ABTS, PI=78.87±0.19; and β‐carotene, PI=56.1±0.01 %). The isolated oils were also tested for their antibacterial activity against two Gram‐positive and three Gram‐negative bacteria. Moderate results have been noted by comparison with Gentamicin used as positive control.     

Non‐endogenous ketocarotenoid accumulation in engineered Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 is a model species commonly employed for biotechnological applications. It is naturally able to accumulate zeaxanthin (Zea) and echinenone (Ech), but not astaxanthin (Asx), which is the highest value carotenoid produced by microalgae, with a wide range of applications in pharmaceutical, cosmetics, food and feed industries. With the aim of finding an alternative and sustainable biological source for the production of Asx and other valuable hydroxylated and ketolated intermediates, the carotenoid biosynthetic pathway of Synechocystis sp. PCC 6803 has been engineered by introducing the 4,4′ β‐carotene oxygenase (CrtW) and 3,3′ β‐carotene hydroxylase (CrtZ) genes from Brevundimonas sp. SD‐212 under the control of a temperature‐inducible promoter. The expression of exogenous CrtZ led to an increased accumulation of Zea at the expense of Ech, while the expression of exogenous CrtW promoted the production of non‐endogenous canthaxanthin and an increase in the Ech content with a concomitant strong reduction of β‐carotene (β‐car). When both Brevundimonas sp. SD‐212 genes were coexpressed, significant amounts of non‐endogenous Asx were obtained accompanied by a strong decrease in β‐car content. Asx accumulation was higher (approximately 50% of total carotenoids) when CrtZ was cloned upstream of CrtW, but still significant (approximately 30%) when the position of genes was inverted. Therefore, the engineered strains constitute a useful tool for investigating the ketocarotenoid biosynthetic pathway in cyanobacteria and an excellent starting point for further optimisation and industrial exploitation of these organisms for the production of added‐value compounds.     

Drought-induced senescence is characterized by a loss of antioxidant defences in chloroplasts

The relationship between drought, oxidative stress and leaf senescence was evaluated in field‐grown sage (Salvia officinalis L.), a drought‐susceptible species that shows symptoms of senescence when exposed to stress. Despite the photoprotection conferred by the xanthophyll cycle, drought‐stressed senescing leaves showed enhanced lipid peroxidation, chlorophyll loss, reduced photosynthetic activity and strong reductions of membrane‐bound chloroplastic antioxidant defences (i.e. β‐carotene and α‐tocopherol), which is indicative of oxidative stress in chloroplasts. H₂O₂ accumulated in drought‐stressed senescing leaves. Subcellular localization studies showed that H₂O₂ accumulated first in xylem vessels and the cell wall and later in the plasma membrane of mesophyll cells, but not in chloroplasts, indicating reactive oxygen species other than H₂O₂ as direct responsible for the oxidative stress observed in the chloroplasts of drought‐stressed senescing leaves. The strong degradation of β‐carotene and α‐tocopherol suggests an enhanced formation of singlet oxygen as the putative reactive oxygen species responsible for oxidative stress to senescing chloroplasts. This study demonstrates that oxidative stress in chloroplasts mediates drought‐induced leaf senescence in sage growing in Mediterranean field conditions.     

Chemical Profile and Antioxidant Properties of Extracts and Essential Oils from Citrus × limon (L.) Burm. cv. Femminello Comune

Citrus × limon cv. Femminello Comune (Rutaceae) from Rocca Imperiale (Italy), one of the six Protected Geographical Indication (PGI) Italian lemon crops, has been recently received renewed interest. In this work, fresh and dried peels and leaves were extracted by hydrodistillation, supercritical fluid extraction (SFE), and Soxhlet apparatus. Chemical profile was assessed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). Except for leaves extracts obtained by Soxhlet apparatus, the monoterpene hydrocarbons fraction dominated. Limonene, γ‐terpinene, and β‐pinene were the main identified compounds. The antioxidant activity was investigated using different in vitro assays namely 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), ABTS, ferric reducing ability power (FRAP), and β‐carotene bleaching test. In DPPH test, the essential oil obtained by hydrodistillation of fresh peel exhibited the highest activity (IC₅₀ of 1.17 mg/ml). Leaves extracted by SFE showed a good activity in both DPPH and β‐carotene bleaching test with IC₅₀ values of 2.20 and 6.66 mg/ml, respectively. Monoterpene hydrocarbons fraction exhibited a positive Pearson's correlation coefficient with all antioxidant assays. Leaves, often considered waste material, should be considered from a different point because they represent a matrix of indisputable interest.     

Effects of β‐carotene on antioxidant status in rats with chronic alcohol consumption

This study examined the effects of β‐carotene on antioxidant status in rats with chronic alcohol consumption. At the beginning of experiment (week 0), according to both the plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, rats (n = 24) were divided into 3 groups and fed with a standard diet (group C), a diet containing ethanol (group E), or a diet containing ethanol and β‐carotene (group E+B). After 10 weeks, plasma AST and ALT, fat accumulation in the liver, antioxidant enzyme activities in erythrocytes and the liver, malondialdehyde (MDA), and α‐tocopherol and retinol in plasma and hepatic samples were analyzed. The chronic alcohol diet significantly increased AST and ALT levels in plasma, and these changes were prevented by supplementing the diet with β‐carotene. Glutathione (GSH) in erythrocytes and in the liver was significantly elevated in rats fed with a diet containing β‐carotene. The results indicate that β‐carotene supplementation can prevent ethanol‐induced liver damage and increase GSH concentrations in erythrocytes and the liver. Copyright © 2009 John Wiley & Sons, Ltd.     

Phenolic compounds from Selaginella moellendorfii

Chemical investigation of the leaves and roots of Selaginella moellendorfii Hieron has resulted in the isolation and characterization of two new flavone glucosides, 7‐O‐(β‐glucopyranosyl(1→2)‐[β‐glucopyranosyl(1→6)]‐β‐glucopyranosyl)flavone‐3′,4′,5,7‐tetraol ( 1 ) and 7‐O‐(β‐glucopyranosyl(1→2)‐[β‐glucopyranosyl(1→6)]‐β‐glucopyranosyl)flavone‐4′,5,7‐triol ( 2 ), two new biflavonoids, 2,3‐dihydroflavone‐5,7,4′‐triol‐(3′→8″)‐flavone‐5″,6″,7″,4′′′‐tetraol ( 3 ) and 6‐methylflavone‐5,7,4′‐triol‐(3′→O→4′′′)‐6″‐methylflavone‐5″,7″‐diol ( 4 ), two new lignans, (7′E)‐3,5,3′,5′‐tetramethoxy‐8 : 4′‐oxyneolign‐7′‐ene‐4,9,9′‐triol ( 5 ) and 3,3′‐dimethoxylign‐8′‐ene‐4,4′,9‐triol ( 6 ), together with two known monolignans, four known lignans, and four known biflavonoids. Their structures were established by spectroscopic means and by comparison with literature values.     

Genetic variation in the β, β‐carotene‐9′, 10′‐dioxygenase gene and association with fat colour in bovine adipose tissue and milk

β, β‐carotene‐9′, 10′‐dioxygenase (BCO2) plays a role in cleaving β‐carotene eccentrically, and may be involved in the control of adipose and milk colour in cattle. The bovine BCO2 gene was sequenced as a potential candidate gene for a beef fat colour QTL on chromosome (BTA) 15. A single nucleotide base change located in exon 3 causes the substitution of a stop codon (encoded by the A allele) for tryptophan⁸⁰ (encoded by the G allele) (c. 240G>A, p.Trp80stop, referred to herein as SNP W80X). Association analysis showed significant differences in subcutaneous fat colour and beta‐carotene concentration amongst cattle with different BCO2 genotypes. Animals with the BCO2 AA genotype had more yellow beef fat and a higher beta‐carotene concentration in adipose tissues than those with the GA or GG genotype. QTL mapping analysis with the BCO2 SNP W80X fitted as a fixed effect confirmed that this SNP is likely to represent the quantitative trait nucleotide (QTN) for the fat colour‐related traits on BTA 15. Moreover, animals with the AA genotype had yellower milk colour and a higher concentration of beta‐carotene in the milk.