Fruit Pigmentation Research Articles

Genetic and chemical characterization of an EMS induced mutation in Cucumis melo CRTISO gene

In order to broaden the available genetic variation of melon, we developed an ethyl methanesulfonate mutation library in an orange-flesh ‘Charentais’ type melon line that accumulates β-carotene. One mutagenized M2 family segregated for a novel recessive trait, a yellow–orange fruit flesh (‘yofI’). HPLC analysis revealed that ‘yofI’ accumulates pro-lycopene (tetra-cis-lycopene) as its major fruit pigment. The altered carotenoid composition of ‘yofI’ is associated with a significant change of the fruit aroma since cleavage of β-carotene yields different apocarotenoids than the cleavage of pro-lycopene. Normally, pro-lycopene is further isomerized by CRTISO (carotenoid isomerase) to yield all-trans-lycopene, which is further cyclized to β-carotene in melon fruit. Cloning and sequencing of ‘yofI’ CRTISO identified two mRNA sequences which lead to truncated forms of CRTISO. Sequencing of the genomic CRTISO identified an A–T transversion in ‘yofI’ which leads to a premature STOP codon. The early carotenoid pathway genes were up regulated in yofI fruit causing accumulation of other intermediates such as phytoene and ζ-carotene. Total carotenoid levels are only slightly increased in the mutant. Mutants accumulating pro-lycopene have been reported in both tomato and watermelon fruits, however, this is the first report of a non-lycopene accumulating fruit showing this phenomenon.

Whole transcriptome sequencing reveals genes involved in plastid/chloroplast division and development are regulated by the HP1/DDB1 at an early stage of tomato fruit development

The phenotype of tomato high pigment-1 (hp1) mutant is characterized by overproduction of pigments including chlorophyll and carotenoids during fruit development and ripening. Although the increased plastid compartment size has been thought to largely attribute to the enhanced pigmentation, the molecular aspects of how the HP1/DDB1 gene manipulates plastid biogenesis and development are largely unknown. In the present study, we compared transcriptome profiles of immature fruit pericarp tissue between tomato cv. Ailsa Craig (WT) and its isogenic hp1 mutant. Over 20 million sequence reads, representing > 1.6 Gb sequence data per sample, were generated and assembled into 21,972 and 22,167 gene models in WT and hp1, respectively, accounting for over 60 % official gene models in both samples. Subsequent analyses revealed that 8,322 and 7,989 alternative splicing events, 8833 or 8510 extended 5'-UTRs, 8,263 or 8,939 extended 3'-UTRs, and 1,136 and 1,133 novel transcripts, exist in WT and hp1, respectively. Significant differences in expression level of 880 genes were detected between the WT and hp1, many of which are involved in signaling transduction, transcription regulation and biotic and abiotic stresses response. Distinctly, RNA-seq datasets, quantitative RT-PCR analyses demonstrate that, in hp1 mutant pericarp tissue at early developmental stage, an apparent expression alteration was found in several regulators directly involved in plastid division and development. These results provide a useful reference for a more accurate and more detailed characterization of the molecular process in the development and pigmentation of tomato fruits.

Novel phenotypes related to the breeding of purple-fruited tomatoes and effect of peel extracts on human cancer cell proliferation

The production of anthocyanins in the tomato (Solanum lycopersicum L.) fruit is normally absent or poor, but a number of mutants or introgression lines are known to increase anthocyanin levels in vegetative and reproductive tissues. Through conventional breeding, a genetic combination was obtained with the remarkable phenotype of a deep purple fruit pigmentation, due to an accumulation of anthocyanins on the peel. Such a genotype was named Sun Black (SB) as a consequence of its sensitivity to light induction. When characterized for morpho-agronomic traits, SB plants showed increased fertility. Purple fruits displayed an arrangement of the epicarp cells different from normal tomatoes, a feature that could account for different mechanical properties and shelf-life potential. The SB genotype and, to a lesser extent, its single mutant parents showed the capacity to accumulate anthocyanins in the seedling root when grown under light. This phenotype, which was greatly improved by the addition of sucrose to the germination medium, proved to be useful as selection index and gave new insights for in vitro production of anthocyanin extracts. To assess the nutraceutical potential of purple tomatoes, we tested the activity of SB skin extracts on the proliferation of two human cancer cells lines. Cell proliferation was significantly inhibited by SB extract in a dose-dependent manner. When the bioactivity of SB extracts was compared with that of other anthocyanin-containing fruits or vegetables, a significant "Extract*Line" interaction was evidenced, suggesting a crucial role for the extract composition in terms of anthocyanidins and other eventual cell growth-inhibiting compounds.

Fruit growth dynamics, respiration rate and physico-textural properties during pomegranate development and ripening

The time course and pattern of fruit growth and changes in physical, physiological properties and texture dynamics of pomegranate fruit (cvs. ‘Bhagwa’ and ‘Ruby’) along the days after full bloom (DAFB) were studied over two different year seasons. Significant variations in fruit growth, respiration rate and physico-textural properties of the fruit were found at five maturity stages (S1–S5). Fruit lineal dimensions (length and diameter) exhibited a linear growth pattern and the fruit weight followed a similar pattern. Both fruit cultivars showed a decline in respiration rate during fruit development, with the highest respiration rate measured in immature fruits (‘Bhagwa’, 66.83mLCO2kg−1h−1; ‘Ruby’, 51.17mLCO2kg−1h−1) and declining with maturity to minimum rates in fully ripe fruit (stage 5) (S5) (‘Bhagwa’, 23.84mLCO2kg−1h−1; ‘Ruby’, 19.16mLCO2kg−1h−1). No ethylene gas was detected throughout fruit development. Fruit pigmentation increased with advancing maturity and the lowest total colour difference (TCD) between fruit peel and arils was noted in immature fruit. Textural dynamics of aril revealed increasing trend in bioyield force and elasticity with advancing maturity. Overall, the study indicated that fruit reached mature stage between the 132–139 DAFB for ‘Ruby’ and 140–165 DAFB for ‘Bhagwa’. This period could be regarded as the physiological mature stage of the fruits that would present the optimum values of harvesting properties desirable in the investigated cultivars. This information could be used as a tool to assist growers in assessing fruit readiness for harvest.

Delphinidin, a dietary antioxidant, induces human epidermal keratinocyte differentiation but not apoptosis: studies in submerged and three‐dimensional epidermal equivalent models

Delphinidin (Del), [3,5,7,3'-,4'-,5'-hexahydroxyflavylium], an anthocyanidin and a potent antioxidant abundantly found in pigmented fruits and vegetables exhibits proapoptotic effects in many cancer cells. Here, we determined the effect of Del on growth, apoptosis and differentiation of normal human epidermal keratinocytes (NHEKs) in vitro in submerged cultures and examined its effects in a three-dimensional (3D) epidermal equivalent (EE) model that permits complete differentiation reminiscent of in vivo skin. Treatment of NHEKs with Del (10-40 μm; 24-48 h) significantly enhanced keratinocyte differentiation. In Del-treated cells, there was marked increase in human involucrin (hINV) promoter activity with simultaneous increase in the mRNA and protein expressions of involucrin and other epidermal differentiation markers including procaspase-14 and transglutaminase-1 (TGM1), but without any effect on TGM2. Del treatment of NHEKs was associated with minimal decrease in cell viability, which was not associated with apoptosis as evident by lack of modulation of caspases, apoptosis-related proteins including Bcl-2 family of proteins and poly(ADP-ribose) polymerase cleavage. To establish the in vivo relevance of our observations in submerged cultures, we then validated these effects in a 3D EE model, where Del was found to significantly enhance cornification and increase the protein expression of cornification markers including caspase-14 and keratin 1. For the first time, we show that Del induces epidermal differentiation using an experimental system that closely mimics in vivo human skin. These observations suggest that Del could be a useful agent for dermatoses associated with epidermal barrier defects including aberrant keratinization, hyperproliferation or inflammation observed in skin diseases like psoriasis and ichthyoses.

Abstract 3666: Delphinidin reduces cell proliferation and induces apoptosis of non-small cell lung cancer cells in vitro and in vivo.

Abstract Lung cancer is a major health problem worldwide and accounts for approximately 28% of all cancer-related deaths in the USA. Like most cancers, lung cancer is a conglomeration of diseases of diverse etiology, broadly divided into small-cell lung cancer (SCLC, comprising 20% of lung cancers), and non-small-cell lung cancer (NSCLC, comprising 80% of lung cancers). Lung cancer has proven difficult to control with conventional therapeutic and surgical approaches, and the prognosis is poor with an overall 5-year survival rate of only 15% for NSCLC in the USA. The epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-2 (VEGFR-2) have emerged as two clinically validated targets for NSCLC. The development of dietary agents which exhibit combined EGFR/VEGFR-2 inhibitory activities may be a step forward to improve antitumor efficacy and to broaden application possibilities. In our pursuit for the identification of non-toxic dietary ingredients for prevention and treatment of cancer, we found that delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, is a potent inhibitor of both EGFR and VEGFR-2 in NSCLC cells. In the present study, we determined the effects of delphinidin on cell growth and apoptosis in vitro and on tumor growth and angiogenesis in vivo in NSCLC cells that overexpress EGFR/VEGFR-2. Delphinidin (5-60 μM) treatment of NSCLC cells inhibited the (i) activation of PI3K, and (ii) phosphorylation of AKT and MAPKs. We next determined whether inhibition of EGFR/VEGFR-2 signaling pathways by delphinidin exhibits a growth inhibitory effect. We observed that delphinidin treatment of NCI-H441, SK-MES-1 and A549 cells resulted in a dose-dependent inhibition of cell growth without having any substantial effects on normal human bronchial epithelial cells as assessed by MTT assay. Treatment of NCI-H441 and SK-MES-1 cells with delphindin (5-60 μM) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and -9, (iii) downregulation of anti-apoptotic proteins (Bcl-2, Bcl-xL and Mcl-1), (iv) upregulation of pro-apoptotic proteins (Bax and Bak), and (v) decreased expression of PCNA and cyclin D1. In athymic nude mice implanted with human NSCLC cells (NCI-H441 or SK-MES-1), delphinidin (1-2 mg, i.p. thrice-weekly) treatment caused a significant inhibition of tumor growth. There was also a decrease in the expression of markers for cell proliferation (Ki-67 and PCNA) and angiogenesis (CD31 and VEGF) as well as an induction of apoptosis in tumor tissues treated with delphinidin, when compared with tumor tissues of control mice. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be useful for the management of NSCLCs, especially those that overexpress EGFR and VEGFR-2. Citation Format: Harish C. Pal, Samriti Sharma, Craig A. Elmets, Farrukh Afaq. Delphinidin reduces cell proliferation and induces apoptosis of non-small cell lung cancer cells in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3666. doi:10.1158/1538-7445.AM2013-3666

445 Protective effect of cyanidin-3-O-beta-D-glucopyranoside fraction from mulberry fruit pigment against oidative damage in streptozotocin-induced diabetic rat bladder

445 Protective effect of cyanidin-3-O-beta-D-glucopyranoside fraction from mulberry fruit pigment against oidative damage in streptozotocin-induced diabetic rat bladder

Delphinidin inhibits IL-1 -induced activation of NF- B by modulating the phosphorylation of IRAK-1Ser376 in human articular chondrocytes

In OA, there is enhanced expression of pro-inflammatory cytokines such as IL-1β in the affected joint. Delphinidin, an anthocyanidin found in pigmented fruits and vegetables, has been shown to possess anti-inflammatory and antioxidant properties. In the present study we determined whether delphinidin would inhibit the IL-1β-induced activation of NF-κB in human chondrocytes and determined the mechanism of its action. PGE2 levels and activation of NF-κB p65 in human OA chondrocytes were determined by ELISA-based assays. Protein expression of cyclo-oxygenase-2 (COX-2) and phosphorylation of kinases was determined by western immunoblotting. Expression level of mRNAs was determined by TaqMan assays. Delphinidin inhibited IL-1β-induced expression of COX-2 and production of PGE2 in human chondrocytes. Delphinidin also inhibited IL-1β-mediated phosphorylation of IL-1 receptor-associated kinase-1(Ser376), phosphorylation of IKKα/β, expression of IKKβ, degradation of IκBα, and activation and nuclear translocation of NF-κB/p65. Phosphorylation of TGF-β-activated kinase 1 was not observed but NF-κB-inducing kinase (NIK) was phosphorylated and phosphorylation of NIK was blocked by delphinidin in IL-1β-treated human chondrocytes. These data identify delphinidin as a novel inhibitor of IL-1β-induced production of cartilage-degrading molecule PGE2 via inhibition of COX-2 expression and provide new insight into the mechanism of its action. Our results also identify inhibition of IRAK1(Ser376) phosphorylation by delphinidin in IL-1β-induced activation of NF-κB in human chondrocytes. Given the important role played by IL-1β-induced NF-κB activation, COX-2 expression and PGE2 production in OA, our results may have important implications for the development of novel therapeutic strategies for the prevention/treatment of OA.

MOLECULAR AND BIOCHEMICAL STUDIES ON PHYTONUTRIENT ACCUMULATION IN PEACH FRUIT

Carotenoids are plant metabolites which are not only essential in photosynthesis but also important quality factors in determining the pigmentation and aroma of flowers and fruits. The expression patterns of relevant carotenoid genes and metabolites were studied throughout fruit development, in order to investigate carotenoid metabolism and the relationships between ethylene emission and carotenoid production in peaches (Prunus persica L. Batsch.). Four cultivars with different flesh colour (yellow vs. white) and/or flesh type (melting vs. stony-hard) were analyzed throughout the fruit development cycle. 'Redhaven Bianca', the white-fleshed mutant of 'Redhaven', showed the highest levels of ccd1 expression at all stages of fruit development. The dioxygenase genes family, in peach as in other species, is directly involved in carotenoid degradation and the concomitant emission of volatile substances with a high odour impact. In both white-fleshed stony-hard genotypes, however, this gene was very poorly expressed in the fruit, suggesting that the stony-hard trait interacts with the metabolism/catabolism of carotenoids.

Effects of Fertigation on Pigment Pattern and Fruit Quality of cv. ‘Gala’ Apples

Fruit pigmentation in ripening cv. ‘Gala, Schniga’ apples was monitored under the influence of fertigation weekly during 21 July–31 August 2009, i.e. 34 days before and 7 days after the optimal harvest date in Slovenia. The soil was a heavy loam clay with high humus content (2.8 %) and high acidity (low pH 4.9). The spectral indices NDVI (Normalized Difference Vegetation Index) and NAI (Normalized Anthocyanin Index) were obtained by a PA 1101 on attached fruit on the transition between the ground and blush colour of the fruit surface. NAI increased slowly from 0.25 to 0.6 two weeks prior to optimum maturity. NDVI values fell from 0.9 to 0.2 before and during ripening, with significant changes starting ca. 3 weeks before and during the week after the optimum harvest date. Fertigation delayed fruit ripening viz harvest date by ca. 4 days. Fertigation delayed changes in the intensity of the fruit’s epidermal pigments, expressed as NDVI for 1 week and NAI for 4 weeks, respectively, but had no significant effect on fruit colour or NDVI and NAI values at the optimal harvest date. At this time, those fruits from the fertigated trees were firmer with a lower starch index compared with the un-fertigated control. Statistically, highly significant correlations were found (p ≤ 0.01) (r = 0.35 to r = 0.91, R2 = 0.11–0.83) between all the studied spectral and standard parameters i.e. fruit fresh firmness, soluble solids content and starch index.

Protective effect of cyanidin‐3‐O‐β‐D‐glucopyranoside fraction from mulberry fruit pigment against oxidative damage in streptozotocin‐induced diabetic rat bladder

To determine whether cyanidin-3-O-β-D-glucopyranoside (C3G) fraction from mulberry fruit pigment has protective effects against bladder dysfunction on streptozotocin-induced diabetic rats Sprague-Dawley rats were divided into three groups (n = 12 in each): normal, diabetes (DM), and DM treated with C3G fraction (DM + C3G). The DM and DM + C3G groups received a single injection of streptozotocin (50 mg/kg) intraperitoneally. Four weeks after the induction of diabetes, the DM + C3G group was treated with daily oral C3G (10 mg/kg) dissolved in water, for 8 weeks. After 12 weeks of streptozotocin injections, rats in each group underwent cystometrography and bladders were used for evaluation of apoptosis and oxidative stress. The DM group showed a markedly lower maximal intravesical pressure than that observed in the control group, whereas rats in the DM + C3G group showed improved maximum intravesical pressure associated with minimization of apoptosis, and increased levels of Akt and Bad phosphorylation, implying inhibition of pro-apoptotic stimuli. The level of 8-hydroxy-2-deoxyguanosine, a marker of oxidative stress, was significantly greater in the DM group compared to the control group and it was significantly reduced in the C3G treated group. Immunoblotting revealed a significant decrease in the levels of the superoxide dismutase protein and nerve growth factor in the DM group compared with the control group; however, these proteins were upregulated in the DM + C3G group compared with the DM group. The study is the first to suggest that C3G fraction have a potency to protect the bladder under conditions of diabetes-induced oxidative stress.

The Tomato FRUITFULL Homologs TDR4/FUL1 and MBP7/FUL2 Regulate Ethylene-Independent Aspects of Fruit Ripening

Tomato (Solanum lycopersicum) contains two close homologs of the Arabidopsis thaliana MADS domain transcription factor FRUITFULL (FUL), FUL1 (previously called TDR4) and FUL2 (previously MBP7). Both proteins interact with the ripening regulator RIPENING INHIBITOR (RIN) and are expressed during fruit ripening. To elucidate their function in tomato, we characterized single and double FUL1 and FUL2 knockdown lines. Whereas the single lines only showed very mild alterations in fruit pigmentation, the double silenced lines exhibited an orange-ripe fruit phenotype due to highly reduced lycopene levels, suggesting that FUL1 and FUL2 have a redundant function in fruit ripening. More detailed analyses of the phenotype, transcriptome, and metabolome of the fruits silenced for both FUL1 and FUL2 suggest that the genes are involved in cell wall modification, the production of cuticle components and volatiles, and glutamic acid (Glu) accumulation. Glu is responsible for the characteristic umami taste of the present-day cultivated tomato fruit. In contrast with previously identified ripening regulators, FUL1 and FUL2 do not regulate ethylene biosynthesis but influence ripening in an ethylene-independent manner. Our data combined with those of others suggest that FUL1/2 and TOMATO AGAMOUS-LIKE1 regulate different subsets of the known RIN targets, probably in a protein complex with the latter.

Solution structure of the strawberry allergen Fra a 1

The PR10 family protein Fra a 1E from strawberry (Fragaria x ananassa) is down-regulated in white strawberry mutants, and transient RNAi (RNA interference)-mediated silencing experiments confirmed that Fra a 1 is involved in fruit pigment synthesis. In the present study, we determined the solution structure of Fra a 1E. The protein fold is identical with that of other members of the PR10 protein family and consists of a seven-stranded antiparallel β-sheet, two short V-shaped α-helices and a long C-terminal α-helix that encompass a hydrophobic pocket. Whereas Fra a 1E contains the glycine-rich loop that is highly conserved throughout the protein family, the volume of the hydrophobic pocket and the size of its entrance are much larger than expected. The three-dimensional structure may shed some light on its physiological function and may help to further understand the role of PR10 proteins in plants.

Synergestic effect of lycopene and Melatonin against the genesis of oxidative stress induced by cyclophosphamide in rats

Cyclophosphamide (CP) is an alkylating agent that has been considered effective for cancer treatment. Lycopene, the pigment in tomato fruits, has beneficial effect in the treatment of some diseases. The goal of this study is to evaluate the protective effect of lycopene alone or combined with melatonin (Mel) in inhibiting the oxidative stress and toxic effect of CP in rats. Five groups of rats were included in this study; Group I served as the control. Rats in groups II-V were administrated with single dose of CP (150 mg/kg B.W) interperitoneally for 3 days. On the same day of CP administration, the rats in group III were fed a diet supplemented with lycopene (50 mg/kg of diet), rats in group IV were administered with a dose of 2.5 mg Mel/kg body weight (bw) injected subcutaneously and rats in group V were supplemented with lycopene and a dose of 2.5 mg Mel/kg bw injected subcutaneously. After 15 days the blood samples were collected. Results obtained showed that CP exerted its toxic effect by increasing the free radicals and reactive oxygen species that causes lipid peroxidation and cell damage, and this in turn is detected by elevation in nitric oxide (NO) and malondialdehyde (MDA), while the activities of antioxidants enzymes including (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)) were significantly decreased as compared with the control rats. The combined treatment (Lyco + Mel) group showed potential reduction in these parameters more than those treated with lyco alone. The activities of SOD, CAT and GPx were found significantly high than lyco alone treated rats. A positive significant correlation between NO and MDA (r = .81). In conclusion, these results suggested that supplementation of diet with lycopene and Mel provided antioxidant defense with strong chemopreventive activity against Cp-induced cytotoxicity.

Carotenoid Content in Field-grown versus Greenhouse-grown Peppers: Different Responses in Leaf and Fruit

The carotenoid content of fresh fruits, like chiles or peppers (Capsicum annuum L.), is a desirable fruit quality trait because these compounds increase the nutritional value of the fruit. Carotenoids in general serve as antioxidants, whereas specific carotenoids are pro-vitamin A types and yet others are necessary for retinal pigments. In the plant, carotenoids function to harvest light energy during photosynthesis, act as antioxidants in multiple cell types, and pigment fruit and flowers to attract pollinators and seed dispersal agents. All of these cells presumably accumulate carotenoids through the same biosynthetic pathway. We investigated the relationship between light levels in the growth environment and the carotenoid levels that accumulated in mature fruit and leaves. Three chile cultivars with orange fruit, ‘Fogo’, ‘Orange Grande’, and ‘NuMex Sunset’, were grown under three different light conditions, shaded greenhouse, unshaded greenhouse, and field in Las Cruces, NM. Foliar carotenoid increased approximately twofold with increased light, whereas carotenoid content in fruit decreased two- to threefold with increased light. All cultivars showed identical trends with light despite having cultivar-specific carotenoid accumulation patterns in their fruit.

THE EFFECT OF COLOUR PLASTIC FILMS ON THE GROWTH, YIELD AND PLANT PIGMENT CONTENT OF TOMATOES

Experiments were carried out to investigate the effect of colour plastic films on the growth, development yield formation and yield quality of tomatoes. Red, yellow and blue colour polyethylene plastic films were used. The control was a colourless polyethylene film. The transparency of films during the vegetation period was detected at the wave length of 400-700 nm (photosynthetically active radiation). The ratio between red (600-700 nm) and blue (400-500 nm) transmission of the new plastic films is 1.01 for the transparent cover, 1.04 for the yellow, 1.08 for the red and 0.95 for the blue plastic film. During the vegetation period plastic film transparency and the ratio between the red and blue spectrum was changed. The tomatoes ‘Maximka’ was used for the experiments. Plants were grown in vegetation pots placed in a greenhouse or in the high tunnels covered with plastic film. Plant length, the number of leaves and leaf pigment content was determined during plant growth. A tomatoes fresh fruit weight and carotenoid content were determined. Experiments showed the influence of plastic film colour on plant growth and fruit pigment content. An increase of chlorophyll content as an effect of red and yellow colour plastic film was observed in the plant leaves. An increase of lycopene in tomato fruits under a yellow plastic film was detected. No effect from plastic film colour on the number of leaves was observed. There was a decrease in the tomato yield under blue coloured plastic film was shown.

Secagem por atomização de polpa de amora-preta usando maltodextrina como agente carreador

O objetivo deste trabalho foi avaliar a influência da temperatura do ar de secagem (160 ou 180 ºC) e da concentração de maltodextrina (5, 15 ou 25%) sobre as características físico-químicas do suco de amora-preta em pó, produzido por spray drying. O produto final foi analisado quanto ao teor de umidade e de antocianinas, higroscopicidade, atividade de água, cor, distribuição do tamanho das partículas e microestrutura. Temperaturas de secagem mais altas resultaram na redução da umidade, da atividade de água e do conteúdo de antocianinas dos pós, além da formação de partículas maiores e mais higroscópicas. A temperatura teve um efeito significativo na diminuição dos valores dos parâmetros de cor L* e C* somente nos ensaios realizados com 25% de maltodextrina. Em relação à microestrutura, as partículas produzidas à temperatura de 180 ºC apresentaram superfície lisa e uma maior uniformidade em relação às amostras secas a 160 ºC. O aumento da concentração de maltodextrina resultou na formação de partículas com menor umidade e higroscopicidade. Além disso, esses pós apresentaram menor teor de antocianinas, em função da diluição dos pigmentos da fruta, o que levou ao aumento da luminosidade e à redução do croma e do ângulo de tom das amostras. O ensaio realizado à temperatura de 160 ºC com 5% de maltodextrina foi o mais efetivo na manutenção do teor de antocianinas dos pós, visto que a retenção desses pigmentos foi em torno de 80%.

Carotenoid Profiling and Biosynthetic Gene Expression in Flesh and Peel of Wild-Type and hp-1 Tomato Fruit under UV-B Depletion

Although light is recognized as one of the main factors influencing fruit carotenogenesis, the specific role of UV-B radiation has been poorly investigated. The present work is addressed to assess the molecular events underlying carotenoid accumulation in presence or absence of ultraviolet-B (UV-B) light in tomato fruits of wild-type and high pigment-1 (hp-1), a mutant characterized by exaggerated photoresponsiveness and increased fruit pigmentation. Gene expression analyses indicated that in wild-type fruits UV-B radiation mainly negatively affects the carotenoid biosynthetic genes encoding enzymes downstream of lycopene both in flesh and peel, suggesting that the down-regulation of genes CrtL-b and CrtL-e and the subsequent accumulation of lycopene during tomato ripening are determined at least in part by UV-B light. In contrast to wild-type, UV-B depletion did not greatly affect carotenoid accumulation in hp-1 and generally determined minor differences in gene expression between control and UV-B-depleted conditions.

Abstract 2565: Delphinidin: A dual tyrosine kinase inhibitor of EGFR and VEGFR-2 reduces cell proliferation and induces apoptosis of non-small-cell lung cancer cells

Abstract Lung cancer has proven difficult to control with conventional therapeutic and surgical approaches, and the prognosis is poor with an overall 5-year survival rate of only 15% for non-small-cell lung cancer (NSCLC) in the USA. Over the last decade, the notions of targeting tyrosine kinase receptors for prevention and treatment of cancers have gained considerable attention. The epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-2 (VEGFR-2) have emerged as two clinically validated targets for NSCLC. Dual specific tyrosine kinase receptor inhibitors, which target two different classes of growth factor receptors, represent an elegant way to avoid multiple treatment modalities when used alone and in combination with various anti-signaling agents. The development of dietary agents with combined EGFR/VEGFR-2 inhibitory activities in the same molecule may be a step forward to improve antitumor efficacy and to broaden application possibilities. In our pursuit for identification of non-toxic dietary ingredients for prevention and treatment of cancer, we found that delphinidin, an anthocyanidin, present in pigmented fruits (such as pomegranate, berries, and dark grapes) and vegetables (such as egg plant, tomato, carrot and red onion), is a potent inhibitor of EGFR and VEGFR-2 in NSCLC cells. Employing EGFR and VEGFR-2 over expressing NSCLC cells (NCI-H441 and SK-MES-1); we evaluated the effect of delphinidin on EGFR/VEGFR-2 downstream signaling pathways. Delphinidin (5-60 μM) treatment of both NCI-H441 and SK-MES-1 cells inhibited the (i) activation of PI3K, and (ii) phosphorylation of AKT and mitogen activated protein kinases (such as ERK1/2, JNK1/2 and p38). We next determined whether inhibition of EGFR/VEGFR-2 signaling pathways by delphinidin exhibits growth inhibitory effect. We observed that delphinidin treatment of NCI-H441, SK-MES-1 and A-549 cells resulted in a dose-dependent inhibition of cell growth without having any substantial effect on normal human bronchial epithelial cells as assessed by an MTT assay. Treatment of NCI-H441 and SK-MES-1 cells with delphindin (5-60 μM) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and-9, (iii) downregulation of anti-apoptotic proteins (Bcl-2, Bcl-xL and Mcl-1), (iv) upregulation of pro-apoptotic proteins (Bax, Bid and Bak), and (v) decrease expression of PCNA and cyclin D1. In addition, we found that delphinidin was effective in reducing the invasive potential of NSCLC cells. This study identifies an abundant fruits and vegetables based anthocyanidin delphinidin as an effective blocker of EGFR and VEGFR-2 in lung cancer cells. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be useful for the management of NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2565. doi:1538-7445.AM2012-2565

Delphinidin blocks IL-1β-induced activation of NF-κB by modulating the IKKβ gene expression and the activation of NIK in human chondrocytes

Purpose: Osteoarthritis (OA) is a highly prevalent disease that affects the quality of life of its victims. Interleukin-1 β (IL-1β) plays a major role in the pathogenesis of OA and is present in the arthritic joints at high levels. IL-1β has been shown to cause increased production of catabolic enzymes such as MMPs, and inflammatory mediators such as prostaglandins and nitric oxide through the activation of several signaling pathways including NF-κB. Novel approaches using plant derived polyphenols to inhibit signal transduction pathways have shown promise and could overcome some of the toxicity issues related to current therapies. One key signaling mechanism implicated in OA that could be a target of orally bioavailable inhibitors is the NF-κB pathway. Delphinidin (2-(3,4,5-trihydroxyphenyl)chromenylium-3,5,7-triol) is an anthocyanidin that is widely distributed in pigmented fruits and flowers and has been shown to possess anti-inflammatory activity and block the activation of NF-κB in cancer cells. No such study with human chondrocytes has been reported. In the present study using primary human chondrocytes we determined (a) the chondroprotective effect of Delphinidin; and (b) mechanism of NF-κB inhibition in IL-1β-stimulated human chondrocytes. Methods: Chondrocytes were derived by enzymatic digestion of human cartilage (OA chondrocytes) from patients undergoing total knee arthroplasty. OA chondrocytes were pretreated with Delphinidin for different time points and were then stimulated with IL-1β (10ng/ml) in vitro and total RNA or cell lysate was prepared following our standard protocols. To determine the gene expression, single stranded cDNA was synthesized and the expression of target mRNAs (COX-2, MMP-13, iNOS) was quantified using TaqMan Assays. HeLa cells were transfected with promoter reporter vectors and negative control vectors using the Fugene-6 reagent and the effect on promoter activity was verified by Luciferase assay. Activation of NF-κB p65 was determined by specific ELISA-based DNA binding assay. Total protein levels were determined by detergent compatible Biorad Protein Assay. Nitric oxide production was determined by Griess reaction. Protein expression and the phosphorylated forms of different kinases were determined by Western immunoblotting. Data were analyzed using Origin 6.1 software package and p<0.05 was considered significant. Results: Pretreatment of chondrocytes with Delphinidin (50 μg/ml) for 2 hrs significantly inhibited the IL-1β induced expression of COX-2, iNOS and MMP-13 and the production of nitric oxide. Delphinidin also inhibited the phosphorylation of IKKβ and was also a potent suppressor of IKKβ gene and protein expression in OA chondrocytes. IKK promoter had a strong activity in HeLa cells and this activity and production of the Luciferase reporter enzyme was blocked by pre-treatment with Delphinidin. Functional pathway analyses showed that IL-1β-induced phosphorylation of NF-κB-inducing Kinase (NIK), which interacts with and activates IKKα and IKKβ which then phosphorylate IκB, was inhibited by pretreatment of OA chondrocytes with Delphinidin. In agreement with these findings, pre-treatment with Delphinidin inhibited the phosphorylation and degradation of IκB, as determined by Western immunoblotting, and the activation and the DNA binding activity of NF-κB p65 determined by a highly specific ELISA based assay. Conclusions: Taken together, the data presented here identifies a novel mechanism of NF-κB inhibition by Delphinidin by inhibiting NIK, a kinase upstream of IKK complex in the NF-κB activation pathway. Given the important role played by IL-1β and NF-κB in OA, these results may provide important clues to develop useful pharmacological inhibitors of NF-κB for the prevention and/or treatment of OA.