Two-photon absorption spectra of carotenoids compounds

Carotenoids are biosynthetic organic pigments that constitute an important class of one-dimensional pi-conjugated organic molecules with enormous potential for application in biophotonic devices. In this context, we studied the degenerate two-photon absorption (2PA) cross-section spectra of two carotenoid compounds (beta-carotene and beta-apo-8'-carotenal) employing the conventional and white-light-continuum Z-scan techniques and quantum chemistry calculations. Because carotenoids coexist at room temperature as a mixture of isomers, the 2PA spectra reported here are due to samples containing a distribution of isomers, presenting distinct conjugation length and conformation. We show that these compounds present a defined structure on the 2PA spectra, that peaks at 650 nm with an absorption cross-section of approximately 5000 GM, for both compounds. In addition, we observed a 2PA band at 990 nm for beta-apo-8'-carotenal, which was attributed to a overlapping of I(I)B(u) +-like and 2(I)Ag(-)-like states, which are strongly one- and two-photon allowed, respectively. Spectroscopic parameters of the electronic transitions to singlet-excited states, which are directly related to photophysical properties of these compounds, were obtained by fitting the 2PA spectra using the sum-over-states approach. The analysis and interpretations of the 2PA spectra of the investigated carotenoids were supported by theoretical predictions of one- and two-photon transitions carried out using the response functions formalism within the density functional theory framework, using the long-range corrected CAM-B3LYP functional. (C) 2011 American Institute of Physics. [doi:10.1063/1.3590157]

Influence of ethylene on carotenoid biosynthesis during papaya postharvesting ripening

The carotenoid composition was evaluated during ripening of papaya cv. `Golden` under untreated (control) conditions and treated with ethylene and 1-methylcyclopropene (1-MCP). At the end of the experiments, the total carotenoid content in the control group (2194.4 mu g/100 g) was twice as high as that found in ethylene (1018.1 mu g/100 g) and 1-MCP (654.5 mu g/100 g) gas-treated samples. Separation of 21 carotenoids by HPLC connected to photodiode array and mass spectrometry detectors showed that no minor carotenoids seemed to be particularly favoured by the treatments. Lycopene was the major carotenoid in all untreated and gas-treated samples, ranging from 461.5 to 1321.6 mu g/100 g at the end of the experiments. According to the proposed biosynthetic pathway, lycopene is the central compound, since it is the most abundant carotenoid indicating a high stimulation of its upstream steps during ripening, and it is the source for the synthesis of other derivative compounds, such as beta-cryptoxanthin. The influence of both gas treatments on the carotenoid biosynthetic pathway was considered. (C) 2011 Elsevier Inc. All rights reserved.

Cultivar influence on carotenoid composition of loquats from Brazil

Cultivar, growing conditions and geographical origin are factors that influence the carotenoid composition in fruits. Because the loquat cultivars evaluated in this study, CentenAria, Mizauto, Mizuho, Mizumo and Nectar de Cristal, have not previously been investigated, the present work was carried out to determine and compare the carotenoid composition of these five loquat cultivars, by applying high-performance liquid chromatography connected to a photodiode array and mass spectrometry detectors (HPLC-PDA-MS/MS). Twenty-five carotenoids were separated on a C(30) column, and 23 of them were identified. All-trans-beta-carotene (19-55%), all-trans-beta-cryptoxanthin (18-28%), 5,6:5`,6`-diepoxy-beta-cryptoxantilin (9-18%) and 5,6-epoxy-beta-cryptoxanthin (7-10%) were the main carotenoids. The total carotenoid content ranged from 196 mu g/100 g (cv. Nectar de Cristal) to 3020 mu g/100 g (CV. Mizumo). The carotenoid profile of cv. Nectar de Cristal was different from the other cultivars, which was in agreement with its cream pulp colour, in contrast to the other four cultivars with orange pulp colour. Cultivars Mizauto, Mizuho, Mizumo and CentenAria showed provitamin A values between 89 and 162 mu g RAE/100 g, and can be considered good source of this provitamin. (C) 2009 Elsevier Inc. All rights reserved.

Supercritical CO(2) extraction of carotenoids from pitanga fruits (Eugenia uniflora L.)

Supercritical carbon dioxide (SC-CO(2)) extraction was employed to extract carotenoids from the freeze-dried pulp of pitanga fruits (Eugenia uniflora L.), an exotic fruit, rich in carotenoids and still little explored commercially. The SC-CO(2) extraction was carried out at two temperatures, 40 and 60 degrees C, and seven pressures, 100, 150, 200, 250, 300, 350 and 400 bar. The carotenoids were determined by high-performance liquid chromatography connected to photodiode array and mass spectrometry detectors. Lycopene, rubixanthin and P-cryptoxanthin were the main carotenoids present in the freeze-dried pitanga pulp, whereas beta-cryptoxanthin concentration was negligible in the SC-CO(2) extracts, for all the investigated state conditions. The maximum recovery of carotenoids was obtained at 60 degrees C and 250 bar, extracting 55% of the total carotenoid content, 74% of the rubixanthin and 78% of the lycopene from the pulp. Under these state conditions, the total carotenoid concentration in the extract was 5474 mu g/g, represented by 66% lycopene and 32% rubixanthin. The experimental state conditions produced different SC-CO(2) extracts with respect to the extraction yield and concentration of different carotenoids, indicating that the supercritical carbon dioxide was selective in the extraction of the pitanga carotenoids as a function of temperature and pressure. (C) 2008 Elsevier B.V. All rights reserved.

Validation of a food frequency questionnaire to assess the consumption of carotenoids, fruits and vegetables among adolescents: the method of triads

The aim of this study was to validate the intake of carotenoids, fruits and vegetables estimated by the Food Frequency Questionnaire for Adolescents (FFQA) using the method of triads. Blood samples were collected from 80 elementary school adolescents to assess serum levels of β-carotene. Partial correlation coefficients (r) were calculated between an estimated intake of carotenoids, fruits and vegetables and the serum levels of β-carotene. Validity coefficients were calculated using the method of triads. With the exception of carotenoids, partial r from the food frequency questionnaire (FFQ) were greater than those of the 24-hour recall (24hR). The fruit/vegetable group showed the highest partial r for the FFQ (r = 0.235) and the 24hR (r = 0.137). The highest validity coefficient was obtained for the vegetable group, as assessed by the FFQ (r = 0.873). On average, the validity coefficient values for the FFQ were greater than those obtained for the 24hR or the β-carotene serum levels. The FFQA is an accurate tool for estimating the intake of carotenoids, fruits and vegetables in this population group.

Aqueous Extract of Brazilian Green Propolis: Primary Components, Evaluation of Inflammation and Wound Healing by Using Subcutaneous Implanted Sponges

Propolis is a chemically complex resinous bee product which has gained worldwide popularity as a means to improve health condition and prevent diseases. The main constituents of an aqueous extract of a sample of green propolis from Southeast Brazil were shown by high performance liquid chromatography/mass spectroscopy/mass spectroscopy to be mono- and di-O-caffeoylquinic acids; phenylpropanoids known as important constituents of alcohol extracts of green propolis, such as artepillin C and drupanin were also detected in low amounts in the aqueous extract. The anti-inflammatory activity of this extract was evaluated by determination of wound healing parameters. Female Swiss mice were implanted subcutaneously with polyesther-polyurethane sponge discs to induce wound healing responses, and administered orally with green propolis (500mg kg(-1)). At 4, 7 and 14 days post-implantation, the fibrovascular stroma and deposition of extracellular matrix were evaluated by histopathologic and morphometric analyses. In the propolis-treated group at Days 4 and 7 the inflammatory process in the sponge was reduced in comparison with control. A progressive increase in cell influx and collagen deposition was observed in control and propolis-treated groups during the whole period. However, these effects were attenuated in the propolis-treated group at Days 4 and 7, indicating that key factors of the wound healing process are modulated by propolis constituents.

Induction of Heme Oxygenase-1 Can Halt and Even Reverse Renal Tubule-Interstitial Fibrosis

Background: The tubule-interstitial fibrosis is the hallmark of progressive renal disease and is strongly associated with inflammation of this compartment. Heme-oxygenase-1 (HO-1) is a cytoprotective molecule that has been shown to be beneficial in various models of renal injury. However, the role of HO-1 in reversing an established renal scar has not yet been addressed. Aim: We explored the ability of HO-1 to halt and reverse the establishment of fibrosis in an experimental model of chronic renal disease. Methods: Sprague-Dawley male rats were subjected to unilateral ureteral obstruction (UUO) and divided into two groups: non-treated and Hemin-treated. To study the prevention of fibrosis, animals were pre-treated with Hemin at days -2 and -1 prior to UUO. To investigate whether HO-1 could reverse established fibrosis, Hemin therapy was given at days 6 and 7 post-surgery. After 7 and/or 14 days, animals were sacrificed and blood, urine and kidney tissue samples were collected for analyses. Renal function was determined by assessing the serum creatinine, inulin clearance, proteinuria/creatininuria ratio and extent of albuminuria. Arterial blood pressure was measured and fibrosis was quantified by Picrosirius staining. Gene and protein expression of pro-inflammatory and pro-fibrotic molecules, as well as HO-1 were performed. Results: Pre-treatment with Hemin upregulated HO-1 expression and significantly reduced proteinuria, albuminuria, inflammation and pro-fibrotic protein and gene expressions in animals subjected to UUO. Interestingly, the delayed treatment with Hemin was also able to reduce renal dysfunction and to decrease the expression of pro-inflammatory molecules, all in association with significantly reduced levels of fibrosis-related molecules and collagen deposition. Finally, TGF-beta protein production was significantly lower in Hemin-treated animals. Conclusion: Treatment with Hemin was able both to prevent the progression of fibrosis and to reverse an established renal scar. Modulation of inflammation appears to be the major mechanism behind HO-1 cytoprotection.

Ethanol-induced vasoconstriction is mediated via redox-sensitive cyclo-oxygenase-dependent mechanisms

The present study investigated the role of ROS (reactive oxygen species) and COX (cyclooxygenase) in ethanol-induced contraction and elevation of [Ca(2+)](i) (intracellular [Ca(2+)]). Vascular reactivity experiments, using standard muscle bath procedures, showed that ethanol (1-800 mmol/l) induced contraction in endothelium-intact (EC(50): 306 +/- 34 mmol/l) and endothelium-denuded (EC(50): 180 +/- 40 mmol/l) rat aortic rings. Endothelial removal enhanced ethanol-induced contraction. Preincubation of intact rings with L-NAME [N(G)-nitro-L-arginine methyl ester; non-selective NOS (NO synthase) inhibitor, 100 mu mol/l], 7-nitroindazole [selective nNOS (neuronal NOS) inhibitor, 100 mu mol/l], oxyhaemoglobin (NO scavenger, 10 mu mol/l) and ODQ (selective inhibitor of guanylate cyclase enzyme, 1 mu mol/l) increased ethanol-induced contraction. Tiron [O(2)(-) (superoxide anion) scavenger, 1 mmol/l] and catalase (H(2)O(2) scavenger, 300 units/ml) reduced ethanol-induced contraction to a similar extent in both endothelium-intact and denuded rings. Similarly, indomethacin (non-selective COX inhibitor, 10 mu mol/l), SC560 (selective COX- I inhibitor, 1 mu mol/l), AH6809 [PGF(2 alpha) (prostaglandin F(2 alpha))] receptor antagonist, 10 mu mol/l] or SQ29584 [PGH(2)(prostaglandin H(2))/TXA(2) (thromboxane A(2)) receptor antagonist, 3 mu mol/l] inhibited ethanol-induced contraction in aortic rings with and without intact endothelium. In cultured aortic VSMCs (vascular smooth muscle cells), ethanol stimulated generation of O(2)(-) and H(2)O(2). Ethanol induced a transient increase in [Ca(2+)](i), which was significantly inhibited in VSMCs pre-exposed to tiron or indomethacin. Our data suggest that ethanol induces vasoconstriction via redox-sensitive and COX-dependent pathways, probably through direct effects on ROS production and Ca(2+) signalling. These findings identify putative molecular mechanisms whereby ethanol, at high concentrations, influences vascular reactivity. Whether similar phenomena occur in vivo at lower concentrations of ethanol remains unclear.

Carotenoids production from a newly isolated Sporidiobolus pararoseus strain by submerged fermentation

This work aimed at evaluating the total carotenoids production by a newly isolated Sporidiobolus pararoseus. Bioproduction was carried out in an orbital shaker, using 10% (w/v) of inoculum (25 A degrees C, 180 rpm for 35 h), incubated for 120 h in a dark room. Liquid N(2) and dimethylsulphoxide (DMSO) were used for cell rupture, and carotenoids were extracted with a solution of acetone/methanol (7:3, v/v). Optimization of carotenoids bioproduction was achieved by experimental design technique. Initially, a Plackett-Burman design was used for the screening of the most important factors, after the statistical analysis, a complete second-order design was carried out to optimize the concentration of total carotenoids in a conventional medium. Maximum concentration of 856 mu g/L of total carotenoids was obtained in a medium containing 60 g/L of glucose, 15 g/L of peptone, and 15 g/L of malt extract, 25 A degrees C, initial pH 4.0 and 180 rpm. Fermentation kinetics showed that the maximum concentration of total carotenoids was reached after 102 h of fermentation and that carotenoids bioproduction was associated with cell growth.

Antigenotoxicity and antimutagenicity of lycopene in HepG2 cell line evaluated by the comet assay and micronucleus test

Epidemiological studies have provided evidence that high consumption of tomatoes effectively reduces the risk of reactive oxygen species (ROS)-mediated diseases such as cancer. Tomatoes are rich sources of lycopene, a potent singlet oxygen-quenching carotenoid. In addition to its antioxidant properties, lycopene shows an array of biological effects including antimutagenic and anticarcinogenic activities. In the present study, the chemopreventive action of lycopene was examined on DNA damage and clastogenic or aneugenic effects of H2O2 and n-nitrosodiethylamine (DEN) in the metabolically competent human hepatoma cell line (HepG2 cells). Lycopene at concentrations of 10. 25, and 50 mu M, was tested under three protocols: before, simultaneously, and after treatment with the mutagen, using the comet and micronucleus assays. Lycopene significantly reduced the genotoxicity and mutagenicity of H2O2 in all of the conditions tested. For DEN, significant reductions of primary DNA damage (comet assay) were detected when the carotenoid (all of the doses) was added in the cell culture medium before or simultaneously with the mutagen. In the micronucleus test, the protective effect of lycopene was observed only when added prior to DEN treatment. In conclusion, our results suggest that lycopene is a suitable agent for preventing chemically-induced DNA and chromosome damage. (C) 2007 Elsevier Ltd. All rights reserved.

DIVERGENT ROLE OF HEME OXYGENASE INHIBITION IN THE PATHOGENESIS OF SEPSIS

The reduction of neutrophil migration to an infectious focus is associated with a high mortality in severe sepsis. Previously, we showed that heme oxygenase (HO) products downregulate neutrophil recruitment in a noninfectious inflammatory model. The present study was designed to determine the role of HO in sepsis induced by cecal ligation and puncture (CLP) model. We demonstrated that pretreatment, but not the combination of pretreatment plus posttreatment with zinc protoporphyrin IX (ZnPP IX), an HO inhibitor, prevented the reduction of CXCR2 on circulating neutrophils and the failure of intraperitoneal neutrophil migration to the site of infection. Consequently, bacterial dissemination, systemic inflammatory response, and organ injury were prevented. In addition, pretreatment with the HO inhibitor avoided hypotension and consequently increased survival. Moreover, in mice subjected to severe CLP, the pretreatment, but not the combination of pretreatment plus posttreatment with ZnPP IX, prevented the increase of plasmatic free heme observed in nontreated severe CLP. The administration of exogenous hemin to mice subjected to moderate sepsis consistently increased the mortality rate. Furthermore, hemin resulted in a reduction of neutrophil migration both in vivo and in vitro. Altogether, our results demonstrated that pretreatment with the HO inhibitor prevents the pathological findings in severe CLP. However, the combination of pretreatment plus posttreatment with ZnPP IX enhances sepsis severity because of an increase in circulating levels of heme, which is deleterious to the host tissues and also inhibits neutrophil migration.

Gastroprotective effect of heme-oxygenase 1/biliverdin/CO pathway in ethanol-induced gastric damage in mice

Our objective was to evaluate the role of heme-oxygenase 1 (HO-1)/biliverdin/CO pathway in gastric defense against ethanol-induced gastric damage in mice. Mice were pre-treated with saline, hemin (HO-1 inducer), biliverdin (HO-1 product), dimanganese decacarbonyl (DMDC, CO donor) or zinc protoporphyrin IX (ZnPP IX, HO-1 antagonist). Another group received soluble guanylate cyclase (sGC) inhibitor (ODQ) 30 min before hemin, biliverdin or DMDC. After 30 min, gastric damage was induced by ethanol. After one hour, rats were sacrificed. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malonylaldehyde (MDA), glutathione (GSH) or bilirubin. HO-1 expression was determined after saline or ethanol administration by polymerase chain reaction (PCR) or immunohistochemistry. Ethanol (25% or 50%) induced gastric damage, increased MDA levels and reduced GSH in the gastric tissue. Ethanol 50% increased HO-1 mRNA transcripts, HO-1 immunoreactivity, and bilirubin concentration in gastric mucosa. Pre-treatment with hemin reduced gastric damage and MDA formation and increased GSH concentration in the gastric mucosa. ZnPP IX amplified the ethanol-induced gastric lesion, increased MDA formation and decreased GSH concentration in gastric mucosa. Biliverdin and DMDC reduced gastric damage and MDA formation and increased GSH concentration in the gastric tissue. ODQ completely abolished the DMDC protective gastric effect However, effects of hemin or biliverdin did not change with ODQ treatment. Our results suggest that HO-1/biliverdin/CO pathway plays a protective role against ethanol-induced gastric damage through mechanisms that can be dependent (CO) or independent (biliverdin) of sGC activation. (C) 2010 Elsevier B.V. All rights reserved.

Human salivary gland morphogenesis: myoepithelial cell maturation assessed by immunohistochemical markers

Aims: Myoepithelial cells are important components of salivary gland structure, aiding the expulsion of saliva from acinar lobules. The aim was to evaluate the expression of smooth muscle actin (SMA), calponin, caldesmon, CD10, CD29, S100 protein, glial fibrillary acidic protein (GFAP) and p63 in myoepithelial cells during salivary gland morphogenesis to understand the maturation process of these cells and their possible use in the diagnosis of salivary gland lesions. Methods and results: Major and minor human salivary glands at various stages of development, derived from fetuses at 8-26 weeks of gestation, were studied immunohistochemically. Fully developed salivary glands were used as controls. The protein p63 was present in all stages of salivary gland morphogenesis from initial bud to terminal bud stage. CD29, S100 and calponin were detected increasingly as salivary gland structure matured and in fully developed salivary gland. Proteins GFAP, CD10 and caldesmon were not observed in myoepithelial cells of salivary glands. Conclusions: The proteins SMA, calponin, CD29, S100 and p63, which are present from the earliest stages of salivary gland maturation, are valuable myoepithelial markers but, although very specific, are not exclusive markers for this cell type.

Development of human minor salivary glands: expression of mucins according to stage of morphogenesis

The formation of salivary glands entails the proliferation of epithelial cells from the stomatodeum into the underlying ectomesenchyme, culminating in a complex network of ducts and acinar bulbs. The extent to which mucins regulate this process is unknown, but they appear to mediate luminal space formation and maturation. Our aim was to examine mucin expression patterns during the morphogenesis of human salivary glands. Mucin expression - MUC1, 2, 3, 4, 5AC, 5B, 6, and 16 - was analyzed in specimens of developing human salivary glands, obtained from fetuses at 4-24 weeks` gestation, and fully developed salivary glands by immunohistochemistry. Expression patterns were analyzed qualitatively according to the development stage of the salivary glands. Mucins 1, 3, 4, 5B, and 16 were expressed during salivary gland development - being stronger in all ductal segments by the final phases of branching morphogenesis and in mature glands. Acinar cells were negative for most mucins, including MUC1 in mature salivary glands. Mucins 2, 5AC, and 6 were not expressed. Mucins MUC1, 3, 4, 5B, and 16 are expressed in developing human salivary glands and in mature glands, suggesting important roles in the maturation and maintenance of the ductal network.

Role of the spinal cord heme oxygenase-carbon monoxide-cGMP pathway in the nociceptive response of rats

The aim of the present study was to investigate the role of the spinal cord heme oxygenase (HO)-carbon monoxide (CO)-soluble guanylate cyclase (sGC)-cGMP pathway in nociceptive response of rats to the formalin experimental nociceptive model. Animals were handled and adapted to the experimental environment for a few days before the formalin test was applied. For the formalin test 50 mu l of a 1% formalin solution was injected subcutaneously in the dorsal surface of the right hind paw. Following injections, animals were observed for I h and flinching behavior was measured as the nociceptive response. Thirty min before the test, rats were pretreated with intrathecal injections with the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is known to induce the HO pathway. Control animals were treated with vehicles. We observed a significant increase in nociceptive response of rats treated with ZnDPBG, and a drastic reduction of flinching nociceptive behavioral response in the heme-lysinate treated animals. Furthermore, the HO pathway seems to act via cGMP, since methylene blue (a sGC inhibitor) prevented the reduction of flinching nociceptive behavioral response caused by heme-lysinate. These findings strongly indicate that the HO pathway plays a spinal antinociceptive role during the formalin test, acting via cGMP. (C) 2007 Elsevier B.V. All rights reserved.