In vitro assay and inhibition of 9-cis-epoxycarotenoid dioxygenase (NCED) from Solanum lycopersicum and Zea mays.

The article reports methods for the expression and assay of 9-cis-epoxycarotenoid cleavage dioxygenase (NCED), an enzyme involved in the biosynthesis of phytohormone abscisic acid in plants. A method for the preparation of the unstable substrate 9'-cis-neoxanthin from fresh spinach is described. The inhibition of Solanum lycopersicum NCED by a series of aryl hydroxamic acid inhibitors is illustrated, and inhibitors D2 and D4 are assayed against NCED isozymes from Zea mays.

A Novel Phytocolorant, Neoxanthin, as a Potent Chemopreventive: Current Progress and Future Prospects.

Cancer is a general term for a group of similar diseases. It is a combined process that results from an accumulation of abnormalities at different biological levels, which involves changes at both genetic and biochemical levels in the cells. Several modifiable risk factors for each type of cancer include heredity, age, and institutional screening guidelines, including colonoscopy, mammograms, prostate-specific antigen testing, etc., which an individual cannot modify. Although a wide range of resources is available for cancer drugs and developmental studies, the cases are supposed to increase by about 70% in the next two decades due to environmental factors commonly driven by the way of living. The drugs used in cancer prevention are not entirely safe, have potential side effects and are generally unsuitable owing to substantial monetary costs. Interventions during the initiation and progression of cancer can prevent, diminish, or stop the transformation of healthy cells on the way to malignancy. Diet modifications are one of the most promising lifestyle changes that can decrease the threat of cancer development by nearly 40%. Neoxanthin is a xanthophyll pigment found in many microalgae and macroalgae, having significant anti-cancer, antioxidant and chemo-preventive activity. In this review, we have focused on the anti-cancer activity of neoxanthin on different cell lines and its cancer-preventive activity concerning obesity and oxidative stress. In addition to this, the preclinical studies and future perspectives are also discussed in this review.

l-Tryptophan synergistically increased carotenoid accumulation with blue light in maize (Zea mays L.) sprouts.

In the present study, l-tryptophan was applied in combination with blue light to modulate carotenoid biosynthesis in maize sprouts. The profiles of carotenoids, chlorophylls, and relative genes in carotenoid biosynthesis and light signaling pathways were studied. l-tryptophan and blue light both promoted the accumulation of carotenoids, and their combination further increased carotenoid content by 120%. l-tryptophan exerted auxin-like effects and stimulated PSY expression in blue light exposure maize sprouts, resulting in increased α- and ß- carotenes. l-tryptophan could also play a photoprotective role through the xanthophyll cycle under blue light. In addition, CRY in the light signaling pathway was critical for carotenoid biosynthesis. These findings provide new insights into the regulation of carotenoid biosynthesis and l-tryptophan could be used in conjunction with blue light to fortify carotenoids in maize sprouts.

Neoxanthin alleviates the chronic renal failure-induced aging and fibrosis by regulating inflammatory process.

Chronic renal failure (CRF) refers to progressive renal damage caused by chronic kidney diseases (CKD). Dialysis therapy and kidney transplantation are the important treatment for CRF. However, due to the limitation of conditions, they cannot be widely utilized. At present, the treatment of renal failure is a worldwide problem in clinic. Therefore, in the current study, we investigated the potential therapeutic effects of neoxanthin on CFR-caused aging and fibrosis. In this work, the effects of neoxanthin on CRF were studied using experimental techniques such as biochemistry, immunohistochemistry and molecular biology. In vitro, neoxanthin alleviated the aging and oxidative damage of kidney cells. In vivo, we found that Neoxanthin could alleviate adenine-induced CRF. Neoxanthin also inhibited CRF-caused renal aging, fibrosis, oxidative stress and inflammation. These findings indicate that neoxanthin could delay the progression of CRF and alleviate CRF-induced aging and fibrosis. Collectively, we found that neoxanthin shows good potential to inhibit CRF-caused kidney aging and fibrosis, suggesting that neoxanthin may be used as a drug (or a functional food) for the treatment of CRF-related diseases.

Synthetic-biological approach for production of neoxanthin in Escherichia coli.

Carotenoids are isoprenoid pigments produced typically in plants, algae, and part of bacteria and fungi. Violaxanthin, neoxanthin, and lutein are xanthophylls biosynthesized specifically in land plants and part of algae. Nowadays, it is feasible to produce violaxanthin and lutein in Escherichia coli by pathway engineering, whereas there is no report to synthesize neoxanthin in E. coli. So far, several genes have been reported to code for neoxanthin synthases, e.g., NSY (NXS), ABA4 and VDL, which were assigned to catalyze a reaction for forming neoxanthin from violaxanthin. However, neither gene of these was common in plants or algae that biosynthesize neoxanthin, nor was confirmed by the E. coli complementation system. This study showed that the algal VDL gene (PtVDL1) was functional in recombinant E. coli cells accumulating violaxanthin to produce neoxanthin, whereas the E. coli cells failed to generate neoxanthin, when the NSY or ABA4 gene was introduced there instead of VDL. This result notes that VDL is one of veritable neoxanthin synthase genes.

Neoxanthin prevents H2O2-induced cytotoxicity in HepG2 cells by activating endogenous antioxidant signals and suppressing apoptosis signals.

BACKGROUND: The liver has a solid inbuilt antioxidant defense system to regulate oxidative stress. However, exposure to an excessive level of ROS causes liver injury. This study examined the cytoprotective effect of neoxanthin, a xanthophyll antioxidant molecule isolated from Solanum trilobatum in stress-induced HepG2 cells. METHODS AND RESULTS: The cytotoxic effect of H2O2 and cytoprotective potential of ß-carotene, lutein, and neoxanthin was analyzed by WST-1 assay. The intracellular ROS level and mitochondrial membrane potential (MMP) were measured using DCFH-DA (2', 7'-dichlorofluorescin diacetate) and JC-10 MMP assay. The expression of anti-oxidant and apoptotic markers was measured by western blot analysis. Neoxanthin pretreatment exhibited better protection than ß-carotene and lutein against cell death caused by H2O2. It significantly arrested H2O2-mediated elevation of intracellular ROS levels and protected MMP. The intracellular antioxidant enzymes HO-1 and SOD-2 were upregulated by neoxanthin pretreatment. Neoxanthin also activated the protein expression of redox-sensitive transactivation factors, Nrf2 and NF-kB. The cytoprotective effect of neoxanthin was associated with increased expression of the anti-apoptotic protein, Bcl-2 and decreased pro-apoptotic protein Bax. CONCLUSIONS: For the first time, our results demonstrate that neoxanthin offers adequate protection against stress-mediated cytotoxicity in hepatocytes by activating the intracellular antioxidant defense system and blocking apoptosis.

Neoxanthin in young vegetable leaves prevents fat accumulation in differentiated adipocytes.

The present study aimed to investigate the effect of young leaves on fat accumulation in differentiated 3T3-L1 adipocytes. A potent preventive effect on fat accumulation was observed in fractions of young leaves of spinach, beet, and arugula extracted with a low-polarity solvent (hexane:acetone:ethanol:toluene = 10:6:7:6). This effect was seemingly associated with the leaf carotenoid content, including lutein, ß-carotene, and neoxanthin. Among these, only neoxanthin, with the characteristic structure of 5,6-monoepoxide and an allenic bond, significantly prevented fat accumulation in a dose-dependent manner. The preventive effect and carotenoid content, including neoxanthin, of these young leaves did not differ from those of the corresponding adult leaves. Therefore, our study demonstrated that young vegetable leaves, such as spinach, beet, and arugula leaves, contained neoxanthin, which prevented fat accumulation in adipocytes in vitro. In the future, the effectiveness of such young leaves and neoxanthin should be investigated in vivo.

Neoxanthin affects the stability of the C2 S2 M2 -type photosystem II supercomplexes and the kinetics of state transition in Arabidopsis.

Neoxanthin (Neo), which is only bound to the peripheral antenna proteins of photosystem (PS) II, is a conserved carotenoid in all green plants. It has been demonstrated that Neo plays an important role in photoprotection and its deficiency fails to impact LHCII stability in vitro and indoor plant growth in vivo. Whether Neo is involved in maintaining the PSII complex structure or adaptive mechanisms for the everchanging environment has not yet been elucidated. In this study, the role of Neo in maintaining the structure and function of the PSII-LHCII supercomplexes was studied using Neo deficient Arabidopsis mutants. Our results show that Neo deficiency had little effect on the electron transport capacity and the plant fitness, but the PSII-LHCII supercomplexes were significantly impacted by the lack of Neo. In the absence of Neo, the M-type LHCII trimer cannot effectively associate with the C2 S2 -type PSII-LHCII supercomplexes even in moderate light conditions. Interestingly, Neo deficiency also leads to decreased PSII protein phosphorylation but rapid transition from state 1 to state 2. We suggest that Neo might enforce the interactions between LHCII and the minor antennas and that the absence of Neo makes M-type LHCII disassociate from the PSII complex, leading to the disassembly of the PSII-LHCII C2 S2 M2 supercomplexes, which results in alterations in the phosphorylation patterns of the thylakoid photosynthetic proteins and the kinetics of state transition.

Photoprotective Role of Neoxanthin in Plants and Algae.

Light is a paramount parameter driving photosynthesis. However, excessive irradiance leads to the formation of reactive oxygen species that cause cell damage and hamper the growth of photosynthetic organisms. Xanthophylls are key pigments involved in the photoprotective response of plants and algae to excessive light. Of particular relevance is the operation of xanthophyll cycles (XC) leading to the formation of de-epoxidized molecules with energy dissipating capacities. Neoxanthin, found in plants and algae in two different isomeric forms, is involved in the light stress response at different levels. This xanthophyll is not directly involved in XCs and the molecular mechanisms behind its photoprotective activity are yet to be fully resolved. This review comprehensively addresses the photoprotective role of 9'-cis-neoxanthin, the most abundant neoxanthin isomer, and one of the major xanthophyll components in plants' photosystems. The light-dependent accumulation of all-trans-neoxanthin in photosynthetic cells was identified exclusively in algae of the order Bryopsidales (Chlorophyta), that lack a functional XC. A putative photoprotective model involving all-trans-neoxanthin is discussed.

The bioactive profile of lettuce produced in a closed soilless system as configured by combinatorial effects of genotype and macrocation supply composition.

The effect of cultivar and nutrient solution macrocation proportions (SK, SCa, SMg) on the bioactive content of hydroponically cultivated lettuce was evaluated on two lettuce cultivars (red and green-pigmented Salanova®) grown in a fully controlled Fitotron® chamber. Fresh weight and color attributes were superior in green Salanova and in SK-treated plants, while elevated macrocation proportions (SK, SCa, and SMg) affected the corresponding minerals, P and Na content. SCa and SMg treatments raised ascorbate concentration and reduced nitrate levels in treated plants. Chicoric and chlorogenic acids were higher in red over green Salanova. Chlorogenic acid was higher in SCa and SMg plants and chicoric acid levels were SMg > SCa > SK. The SMg-treated red Salanova contained higher concentrations of target carotenoids. In conclusion, nutrient solution management constitutes an effective cultural practice to increase bioactive properties and functional quality of hydroponically grown lettuce.

Influence of long term nitrogen limitation on lipid, protein and pigment production of Euglena gracilis in photoheterotrophic cultures.

Nitrogen limitation is considered a good strategy for enhancement of algal lipid production while conversely N repletion has been shown to result in biomass rich in proteins. In this study, the influence of long-term N limitation on Euglena gracilis fatty acid (FA), protein, chlorophyll a, and carotenoid concentrations was studied in N limited cultures. Biomass composition was analyzed from three-time points from N starved late stationary phase cultures, exposed to three different initial N concentrations in the growth medium. Total lipid content increased under N limitation in ageing cultures, but the low N content and prolonged cultivation time resulted in the formation of a high proportion of saturated FAs. Furthermore, growth as well as the production of proteins, chlorophyll a and carotenoids were enhanced in higher N concentrations and metabolism of these cellular components stayed stable during the stationary growth phase. Our findings showed that a higher N availability and a shorter cultivation time is a good strategy for efficient E. gracilis biomass production, regardless of whether the produced biomass is intended for maximal recovery of polyunsaturated FAs, proteins, or photosynthetic pigments. Additionally, we showed an increase of neoxanthin, ß-carotene, and diadinoxanthin as a response to higher N availability.

Esterified carotenoids as new food components in cyanobacteria.

Among the nutritional properties of microalgae, this study is focused in the presence of carotenoid esters in prokaryote microalgae, an event that has not been shown so far. Three carotenoid esters that accumulate in non-stressful culture conditions are identified in Aphanotece microscopica Nägeli and Phormidum autumnale Gomont, what may provide an extra value to the quality attributes of the carotenoid profile in cyanobacteria as functional foods. In addition, new data on the carotenoid characterization added quality criteria for the identification of the esterified metabolites, enabling the monitoring of these food components. Specifically, the metabolomic approach applied to the food composition analysis, has allowed to differentiate between the esters of zeinoxanthin and ß-cryptoxanthin, which were undifferentiated to date during the MS characterization of carotenoids in other food sources. We propose a new qualifier product ion specific for zeinoxanthin ester, which it is not present in the MS2 spectrum of ß-cryptoxanthin esters.

Industrial potential of carotenoid pigments from microalgae: Current trends and future prospects.

Microalgae are rich source of various bioactive molecules such as carotenoids, lipids, fatty acids, hydrocarbons, proteins, carbohydrates, amino acids, etc. and in recent Years carotenoids from algae gained commercial recognition in the global market for food and cosmeceutical applications. However, the production of carotenoids from algae is not yet fully cost effective to compete with synthetic ones. In this context the present review examines the technologies/methods in relation to mass production of algae, cell harvesting for extraction of carotenoids, optimizing extraction methods etc. Research studies from different microalgal species such as Spirulina platensis, Haematococcus pluvialis, Dunaliella salina, Chlorella sps., Nannochloropsis sps., Scenedesmus sps., Chlorococcum sps., Botryococcus braunii and Diatoms in relation to carotenoid content, chemical structure, extraction and processing of carotenoids are discussed. Further these carotenoid pigments, are useful in various health applications and their use in food, feed, nutraceutical, pharmaceutical and cosmeceutical industries was briefly touched upon. The commercial value of algal carotenoids has also been discussed in this review. Possible recommendations for future research studies are proposed.

An efficient one-step scheme for the purification of major xanthophyll carotenoids from lettuce, and assessment of their comparative anticancer potential.

The foremost problem in carotenoid research is the excessive cost and difficulty of maintaining pure carotenoid compounds. This work presents an economical, efficient, and simplified one-step scheme for the purification of four major xanthophyll carotenoids from lettuce by utilizing preparative thin layer chromatography on Hyflo-Super-Cel: MgO (Heavy): calcium sulfate hemihydrate (9:9:2 w/w) based adsorbent. The mobile phase of acetone: hexane (1:1) provided the perfect separation of major xanthophylls, resulting in 95-96% purity after just single-step separation, with no interference from chlorophylls or other minor carotenoids. The identity of carotenoids was confirmed by absorption spectroscopy, chemical tests and APCI+-MS/MS. The proposed scheme can be used to isolate the carotenoids at the analytical and preparative scale. In anticancer studies, among four xanthophylls, 9-Z-neoxanthin was found most potent for reduction of cell viability of cervical (HeLa) and lung cancer (A549) cells, with IC50 values of 3.8 and 7.5 µM, respectively.

Carotenoid profiling of yams: Clarity, comparisons and diversity.

Screening carotenoids of elite accessions of yam (Dioscorea spp.) used in the global yam breeding program has been conducted to quantitatively determine the carotenoid composition of the crop. Comparisons to previous data reporting cerotenoid levels in yam has been made, in order to deduce greater perspectives across multiple studies. Characterisation of complex species and accession -specific profiles have shown a rich base of diversity that can inform breeding strategies. Key findings include; (i) the identification of accessions rich in ß-carotene which can aid provitamin A biofortification, (ii) Data disputing the commonly held belief that yellow Guinea yam (D. cayennensis) has higher ß-carotene content than that of white Guinea yam (D. rotundata), and (iii) the tentative identification of C25-epoxy-apocarotenoid persicaxanthin with potential implications for tuber dormancy.

Nutritional composition of mungbean and soybean sprouts compared to their adult growth stage.

This study determined the level of phytonutrients in mungbean and soybean sprouts compared to mature mungbean grain and vegetable soybean. The comparison included landraces and improved mungbean and soybean varieties to assess the effect of breeding on the phytonutrient content of both crops. Sprouting mungbean enhanced vitamin C content 2.7-fold compared to mature mungbean grain. Relatively old mungbean accessions were superior in protein, calcium (Ca), iron (Fe), zinc (Zn), carotenoid and vitamin C content compared to improved mungbean lines at the fully mature stage. With regard to nutritional value, the vegetable soybean stage was superior to soybean sprouts in terms of content of protein (14% increase), Zn (45%), Ca (72%), and Fe (151%). Isoflavones, reported to have beneficial effects on human health, are found at high concentrations in soybean sprouts and could easily provide the recommended anticarcinogenic dose range from 1.5 to 2.0mg/kg of body weight per day.

Characterization of carotenoid profile of Spanish Sanguinos and Verdal prickly pear (Opuntia ficus-indica, spp.) tissues.

Carotenoid profiles of different tissues (peel, pulp and whole fruit) of Spanish Sanguinos (red) and Verdal (orange) prickly pears (Opuntia ficus-indica spp.) have been characterized in detail and quantified for the first time. Carotenoids were determined by HPLC-PDA-MS (APCI+), using a reverse phase C30 column. A total of 9 xantophylls and 4 hydrocarbon carotenes were identified. Also, minor amounts of chlorophyll a, a' and b can be observed in Opuntia peel extracts. All carotenoids were found to be present in their free form (no carotenoid esters were detected). The RAE was highest in Opuntia peels, showing values from 19.20 to 16.48µg/100g fresh weigth, for Sanguinos and Verdal Opuntia fruits, respectively. The main carotenoid in Opuntia peel extracts was (all-E)-lutein with 1132.51 and 767.98µg/100g fresh weigth, followed by (all-E)-ß-carotene with 200.40 and 173.50µg/100g fresh weigth for Sanguinos and Verdal varieties of Opuntia fruits, respectively.

Carotenoids and carotenoid esters of orange- and yellow-fleshed mamey sapote (Pouteria sapota (Jacq.) H.E. Moore & Stearn) fruit and their post-prandial absorption in humans.

Although different genotypes of mamey sapote with distinct pulp colors are consumed in countries from Central to South America, in-depth knowledge on genotype-related differences of their carotenoid profile is lacking. Since the fruit was found to contain the potentially vitamin A-active keto-carotenoids sapotexanthin and cryptocapsin, we sought to qualitatively and quantitatively describe the carotenoid profile of different genotypes by HPLC-DAD-MSn. Sapotexanthin and cryptocapsin were present in all genotypes. Keto-carotenoids such as cryptocapsin, capsoneoxanthin, and their esters were most abundant in orange-fleshed fruit, whereas several carotenoid epoxides prevailed in yellow-fleshed fruit. Differing carotenoid profiles were associated with different color hues of the fruit pulp, while the widely variable carotenoid content (3.7-8.0mg/100gFW) was mainly reflected by differences in color intensity (chroma C∗). Furthermore, the post-prandial absorption of sapotexanthin to human plasma was proven for the first time. Besides sapotexanthin, cryptocapsin was found to be resorbed.

Probing the pigment binding sites in LHCII with resonance Raman spectroscopy: The effect of mutations at S123.

Resonance Raman spectroscopy was used to evaluate the structure of light-harvesting chlorophyll (Chl) a/b complexes of photosystem II (LHCII), reconstituted from wild-type (WT) and mutant apoproteins over-expressed in Escherichia coli. The point mutations involved residue S123, exchanged for either P (S123P) or G (S123G). In all reconstituted proteins, lutein 2 displayed a distorted conformation, as it does in purified LHCII trimers. Reconstituted WT and S123G also exhibited a conformation of bound neoxanthin (Nx) molecules identical to the native protein, while the S123P mutation was found to induce a change in Nx conformation. This structural change of neoxanthin is accompanied by a blue shift of the absorption of this carotenoid molecule. The interactions assumed by (and thus the structure of the binding sites of) the bound Chls b were found identical in all the reconstituted proteins, and only marginally perturbed as compared to purified LHCII. The interactions assumed by bound Chls a were also identical in purified LHCII and the reconstituted WT. However, the keto carbonyl group of one Chl a, originally free-from-interactions in WT LHCII, becomes involved in a strong H-bond with its environment in LHCII reconstituted from the S123P apoprotein. As the absorption in the Qy region of this protein is identical to that of the LHCII reconstituted from the WT apoprotein, we conclude that the interaction state of the keto carbonyl of Chl a does not play a significant role in tuning the binding site energy of these molecules.

Intraspecific Variation in Carotenoids of Brassica oleracea var. sabellica.

Carotenoids are best known as a source of natural antioxidants. Physiologically, carotenoids are part of the photoprotection in plants as they act as scavengers of reactive oxygen species (ROS). An important source of carotenoids in European food is Brassica oleracea. Focusing on the most abundant carotenoids, we estimated the contents of ß-carotene, (9Z)-neoxanthin, zeaxanthin, and lutein as well as those of chlorophylls a and b to assess their variability in Brassica oleracea var. sabellica. Our analyses included more than 30 cultivars categorized in five distinct sets grouped according to morphological characteristics or geographical origin. Our results demonstrated specific carotenoid patterns characteristic for American, Italian, and red-colored kale cultivars. Moreover, we demonstrated a tendency of high zeaxanthin proportions under traditional harvest conditions, which accord to low-temperature regimes. We also compared the carotenoid patterns of self-generated hybrid lines. Corresponding findings indicated that crossbreeding has a high potential for carotenoid content optimization in kale.