Effect of domestic cooking on human bioavailability of naringenin, chlorogenic acid, lycopene and beta-carotene in cherry tomatoes.

BACKGROUND: Epidemiological data showed that tomato and tomato product (sauce, paste) consumption is associated with a protective effect against the development of some chronic-degenerative diseases. Tomato antioxidant bioactive molecules such as carotenoids and polyphenols could be responsible, at least in part, for the healthy effect observed. The bioavailability of these compounds is an essential requirement to sustain their in vivo role. While it is well known that many factors can influence the bioaccessibility of carotenoids from the food matrix, there is little information about the factors affecting phenolic compounds' bioaccessibility. AIM OF THE STUDY: This investigation was carried out to evaluate the effect of domestic cooking on the bioavailability in humans of antioxidant molecules after the administration of a test meal containing cherry tomatoes. METHODS: A cross-over design was conducted. Subjects (3 females and 2 males) consumed experimental meals containing fresh and cooked cherry tomatoes. Blood collection was performed at different time intervals (0, 2, 4, 6, 8 and 24 h). RESULTS: Carotenoid and phenol plasma concentrations were measured. Plasma levels of lycopene and beta-carotene were not significantly different with respect to the baseline after ingestion of both the test meals, while plasma concentrations of naringenin and chlorogenic acid increased significantly with respect to the baseline (P<0.05) after administration of cooked cherry tomatoes, but not after administration of fresh cherry tomatoes. CONCLUSIONS: The present study indicated that domestically cooked tomatoes significantly increase naringenin and chlorogenic acid plasma levels. Considering that both naringenin and chlorogenic acid are widely studied for their potential healthy properties, evidence of their bioavailability and of the factors influencing their bioaccessibility is an important tool to sustain the possibility that these polyphenols play a biological role in human physiology.

Flavonoids uptake and their effect on cell cycle of human colon adenocarcinoma cells (Caco2).

Green tea, mainly through its constituents epigallocatechin gallate, epigallocatechin, epicatechin gallate and epicatechin, has demonstrated anticarcinogenic activity in several animal models, including those for skin, lung and gastro-intestinal tract cancer, although less is known about colorectal cancer. Quercetin, the major flavonoid present in vegetables and fruit, exerts potential anticarcinogenic effects in animal models and cell cultures, but less is known about quercetin glucosides. The objectives of this study were to investigate (i) the antioxidant activity of the phenolic compounds epicatechin, epigallocatechin gallate, gallic acid and quercetin-3-glucoside; (ii) the cytotoxicity of different concentrations of epicatechin, epigallocatechin gallate, and gallic acid; (iii) the cellular uptake of epicatechin, epigallocatechin gallate, gallic acid and quercetin-3-glucoside and (iv) their effect on the cell cycle. Human colon adenocarcinoma cells were used as experimental model. The results of this study indicate that all dietary flavonoids studied (epicatechin, epigallocatechin gallate, gallic acid and quercetin-3-glucoside) show a significant antioxidant effect in a chemical model system, but only epigallocatechin gallate or gallic acid are able to interfere with the cell cycle in Caco2 cell lines. These data suggest that the antioxidant activity of flavonoids is not related to the inhibition of cellular growth. From a structural point of view, the galloyl moiety appears to be required for both the antioxidant and the antiproliferative effects.

Tryptic digestion of soybean lipoxygenase-1 generates a 60 kDa fragment with improved activity and membrane binding ability.

Lipoxygenases are key enzymes in the metabolism of unsaturated fatty acids. Soybean lipoxygenase-1 (LOX-1), a paradigm for lipoxygenases isolated from different sources, is composed of two domains: a approximately 30 kDa N-terminal domain and a approximately 60 kDa C-terminal domain. We used limited proteolysis and gel-filtration chromatography to generate and isolate a approximately 60 kDa fragment of LOX-1 ("mini-LOX"), produced by trypsin cleavage between lysine 277 and serine 278. Mini-LOX was subjected to N-terminal sequencing and to electrophoretic, chromatographic, and spectroscopic analysis. Mini-LOX was found to be more acidic and more hydrophobic than LOX-1, and with a higher content of alpha-helix. Kinetic analysis showed that mini-LOX dioxygenates linoleic acid with a catalytic efficiency approximately 3-fold higher than that of LOX-1 (33.3 x 10(6) and 10.9 x 10(6) M(-1) x s(-1), respectively), the activation energy of the reaction being 4.5 +/- 0.5 and 8.3 +/- 0.9 kJ x mol(-1) for mini-LOX and LOX-1, respectively. Substrate preference, tested with linoleic, alpha-linolenic, and arachidonic acids, and with linoleate methyl ester, was the same for LOX-1 and mini-LOX, and also identical was the regio- and stereospecificity of the products generated thereof, analyzed by reversed-phase and chiral high-performance liquid chromatography, and by gas chromatography/mass spectrometry. Mini-LOX was able to bind artificial vesicles with higher affinity than LOX-1, but the binding was less affected by calcium ions than was that of LOX-1. Taken together, these results suggest that the N-terminal domain of soybean lipoxygenase-1 might be a built-in inhibitor of catalytic activity and membrane binding ability of the enzyme, with a possible role in physio(patho)logical conditions.

Activation of different lipoxygenase isozymes induces apoptosis in human erythroleukemia and neuroblastoma cells.

We investigated the ability of different hydroperoxides generated by lipoxygenase isozymes to induce programmed cell death (PCD) in human cells. Erythroleukemia K562 and neuroblastoma CHP100 cells were used, because they showed high basal activity of lipoxygenase. The hydroperoxides generated by 5-, 12-, or 15-lipoxygenases from linoleate, linolenate, or arachidonate, and the corresponding hydroxides, were able to induce PCD in both cell types, in a concentration- and time-dependent manner. After 24 h, K562 and CHP100 cells showed 2.5- to 3.5-fold more apoptotic bodies than the untreated controls. PCD elicited by lipoxygenase products was independent of intracellular glutathione concentration, and did not require mRNA transcription or protein synthesis. On the other hand, lipoxygenase products evoked an immediate and sustained rise in cytoplasmic calcium (within seconds), followed by mitochondrial uncoupling (within hours). Unlike the hydro(pero)xides, the terminal products of the arachidonate cascade (i.e., leukotrienes, prostaglandins and thromboxane) were not cytotoxic.

The early phase of apoptosis in human neuroblastoma CHP100 cells is characterized by lipoxygenase-dependent ultraweak light emission.

Human neuroblastoma CHP100 cells were forced into apoptosis (programmed cell death, PCD) or necrosis by treatment with calcium chloride or sodium nitroprusside (a nitric oxide donor), respectively. Cellular luminescence, a marker of membrane lipid peroxidation, was increased by calcium but not by nitroprusside, and reached a maximum of 4-fold the control value 2 hours after treatment. The increase in luminescence was paralleled by increased 5-lipoxygenase (up to 250% of the control value) and decreased catalase (down to 50%) activity within the same time window. Consistently, incubation of CHP100 cells with inhibitors of 5-lipoxygenase (5,8,11,14-eicosatetraynoic acid and MK886) reduced light emission and PCD, whereas inhibition of catalase by 3-amino-1, 2,4-triazole enhanced both processes. Treatment of CHP100 cells with retinoic acid or cisplatin, unrelated PCD inducers reported to activate the lipoxygenase pathway, also gave enhanced light emission parallel to PCD increase. Altogether, these results suggest that cellular luminescence is an early marker of apoptotic, but not necrotic, program(s) involving generation of hydrogen peroxide and activation of 5-lipoxygenase.

Effect of tunicamycin on the activity and immunoreactivity of ascorbate oxidase (Cucurbita pepo medullosa) expressed in cultured green zucchini cells.

Ascorbate oxidase activity and immunoreactivity were evaluated in crude tissue extracts obtained from callus cell cultures induced by green zucchini sarcocarp and grown in the presence of tunicamycin, a powerful N-glycosylation inhibitor. Tunicamycin at 2 or 4 microg ml(-1) blocked cell growth within a couple of weeks, although a sustained cell viability was observed in the same period. A significant inhibition of total protein synthesis was observed at 10 and 15 days of culture time, with a decrease of 30% and 43% respectively when cells were grown in the presence of 2 microg ml(-1) tunicamycin, and of 48% and 57% respectively when the tunicamycin concentration was 4 microg ml(-1). After the same culture times ascorbate oxidase specific activity assayed in crude tissue extracts showed increases of about 1.9-fold and 3.5-fold (10 days) and 1.7-fold and 3.1-fold (15 days) at 2 and 4 microg ml(-1) tunicamycin, respectively. Ascorbate oxidase mRNA levels, however, did not appreciably differ between control and treated samples, measured at the same growing times. Lectin-blot, based on the use of concanavalin A, indicated a marked decrease of glycosylated proteins in tunicamycin-treated cultures. As judged by immunoblot, anti-native ascorbate oxidase antibodies scarcely recognized the enzyme expressed in tunicamycin-treated cells; on the contrary, anti-deglycosylated ascorbate oxidase antibodies were more reactive to the enzyme expressed in tunicamycin-treated cultures.

Plasma (carotenoids, retinol, alpha-tocopherol) and tissue (carotenoids) levels after supplementation with beta-carotene in subjects with precancerous and cancerous lesions of sigmoid colon.

OBJECTIVES: (1) To compare tissue and plasma carotenoids status of healthy subjects and subjects with pre-cancer and cancer lesions; (2) to evaluate the effect of beta-carotene supplementation on the concentrations of other carotenoids in tissue (luteine + zeaxanthin, cryptoxanthin, lycopene, alpha-carotene) and in plasma and also retinol and alpha-tocopherol levels. DESIGN: Eighteen subjects were divided into three groups on the basis of colonoscopy and histological analytical findings: four healthy subjects (control group A); seven subjects affected by adenomatous polyps (group B with pre-cancer lesions); seven subjects suffering from colonic cancer (group C). Blood and colonic biopsy samples were taken (of colon and rectal mucosa) before and after beta-carotene supplementation in all subjects. Groups A and B received a daily dose of beta-carotene (30 mg/die) for 43 d. Group C's supplementation was terminated at the time which was performed, usually within 15 d. The tissue and plasma concentration of carotenoids, retinol and alpha-tocopherol were determined by high-performance liquid chromatography. RESULTS: The tissue concentrations of each carotenoid were similar in all the intestinal sites examined as regards groups A and B, although there was a high degree of intra individual variability within each group. Only beta-carotene made significant increases (P < 0.001) after supplementation. The subjects with cancer show tissue levels for each carotenoid lower than those of healthy subjects or subjects with polypous. The plasma levels of alpha-tocopherol did not change after supplementation while significant increases were noted of retinol, alpha-carotene (P < 0.01) and of beta-carotene (P < 0.001). CONCLUSIONS: The patients with colonic cancer seemed to undergo a significant reduction in their antioxidant reserves with respect to the normal subjects and or polyps. We can confirm that oral B-carotene supplementation induces also an increase in plasma alpha-carotene in all groups.

Application of a new high-performance liquid chromatographic method for measuring selected polyphenols in human plasma.

We developed a method to measure plasma levels of selected polyphenols before and after ingestion of green tea. Blood samples were obtained from four healthy women before and 30 and 50 min after the ingestion of 300 ml of green tea infusion. A 1-ml volume of plasma was hydrolysed with 0.5 M HCl-methanol (1:1, v/v) for 30 min at room temperature, extracted with ethyl acetate and separated by reversed-phase chromatography. Polyphenols were identified on the basis of their retention times and by spectrum analysis. Green tea caffeine has the same retention times as caffeic acid. Consumption of green tea produces a notable increase in the plasma levels of caffeine plus caffeic acid and the appearance of measurable levels of epigallocatechingallate. In conclusion, the method was found to have the requisite features of specificity and sensitivity for monitoring plasma levels of selected tea polyphenols.

Binding and iron delivering of ovotransferrin to cholesterol-depleted chick-embryo red blood cells.

Binding and iron delivering of ovotransferrin (OTf) were evaluated using 14-day old chick-embryo red blood cells (CERBC) and cholesterol-depleted by treatment with chicken egg phosphatidyl choline (E-PC) liposomes. Liposome-treated CERBC assayed for their cholesterol content showed a cholesterol depletion depending on the incubation time, being 25% (w/w) of the maximum cellular removal of cholesterol seen after 22 h incubation at 37 degrees C. Total phosphorus content did not change either for the various samples or during the different incubation times, indicating that specific cholesterol removal occurred, as confirmed also by the increased membrane fluidity revealed through fluorescence anisotropy measurements. The apparent dissociation constant (Kd) of control and treated CERBC was almost of the same value at the same incubation time, ranging from 0.30 microM after 0.25 h incubation to 0.19 microM after 14 or 22 h incubation. In all experiments, the maximum value of bound OTf molecules per cell (Bmax) notably decreased as incubation time increased. But, in cholesterol partly depleted CERBC, the decrease of the Bmax values was less pronounced as the incubation time increased. As far as binding experiments were concerned, iron uptake studies showed that uptaking capacities decreased as incubation time increased. Considering both binding and iron uptake, at the same incubation time, liposome-treated CERBC were slightly more efficient with respect to untreated samples. In any case a passive iron delivering could be evidenced after 22 h incubation. It is suggested that cholesterol may tune binding and iron uptake by either regulating or affecting the expression or mobility of the OTf receptor.

Primary structure of the major glycan from human seminal transferrin.

Human seminal transferrin (HSmT) is an iron-containing glycoprotein whose structural properties have not been adequately investigated. The carbohydrate content of the purified glycoprotein amount to 6.1%, and monosaccharide analysis revealed the major oligosaccharide moiety to be of the N-glycoside type. The carbohydrate chains were released from the iron-free form by digestion with peptide N-glycosidase F (PNGase F) in the presence of detergents such as SDS and beta-octylglucoside. After ethanol precipitation and fractionation on Bio-Gel P-6 and Bio-Gel P-2, the oligosaccharide was further purified on Mono-Q and desalted on Bio-Gel P-2. By 600-MHz 1H-NMR spectroscopy, the primary structure of the major N-linked oligosaccharide component was established to be: [formula: see text]

Exhaustive removal of N-glycans from ascorbate oxidase: effect on the enzymatic activity and immunoreactivity.

Purified ascorbate oxidase from Cucurbita pepo medullosa has been subjected to enzymatic deglycosylation using peptide N-glycosidase F. Experimental conditions were chosen to obtain efficiently deglycosylated and active ascorbate oxidase: in particular, three different detergent solutions were added separately to the incubation mixtures prior to the peptide N-glycosidase F. The detergent solution made of 0.1% (w/v) sodium dodecyl sulphate + 0.5% (v/v) Nonidet P-40 proved to be the only one effective for our purpose. Our results indicate that: (i) the presence of detergents did not affect the enzymatic activity; (ii) fully deglycosylated enzyme retained its activity compared with the native form. Moreover, anti-native ascorbate oxidase antibodies scarcely recognized deglycosylated protein.

Structural analysis of seminal and serum human transferrin by second derivative spectrometry and fluorescence measurements.

Denaturation of human seminal transferrin (HSmT) compared with human serum transferrin (HSrT) was followed to check structural differences between these two proteins. Second derivative UV spectroscopy indicated that treatment with 6 M guanidine hydrochloride (Gnd.HCl) induced greater structural changes in HSrT than in HSmT and, in particular; (i) the exposure value of tyrosinyl residues was almost 2.5-fold higher in native HSmT than in native HSrT; and (ii) a much more pronounced movement of tryptophanyl residues toward a higher polar environment could be noticed in HSrT after incubation with denaturing agent. Fluorescence measurements showed that: (i) a shift of the maximum emission wavelength of HSmT occurred (maximum emission was centered at 333 nm instead of 323 nm as for HSrT; excitation = 280 nm); (ii) the intrinsic tryptophan fluorescence intensity of HSmT increased after 36 hr in the range of 1.5-4.0 M of denaturant, whereas an opposite behavior was found for HSrT in the range 0.0-2.0 M; and (iii) the wavelength maximum of fluorescence emission changed in a biphasic manner for HSrT and, conversely, under the same experimental conditions, HSmT gave a linear and parallel increase of fluorescence emission after 1 and 36 hr. We can conclude that this different behavior of HSmT with respect to HSrT might be due mainly to the fact that both the number and the exposure of tyrosinyl and tryptophanyl residues are different. Lately, these effects are discussed in relationship with the fact that HSmT contains less than half disulphide bridges than HSrT.