Availability of polyphenols in fruit beverages subjected to in vitro gastrointestinal digestion and their effects on proliferation,cell-cycle and apoptosis in human colon cancer Caco-2 cells

Three fruit beverages: Fb (grape–orange–apricot), FbFe (Fb + iron sulphate) and FbFeM (FbFe + skimmed milk) were subjected to in vitro gastrointestinal digestion. Addition of Fe and milk decreased the phenolic content of Fb by 13% and 55%, respectively. The digestion process decreased the phenolic content by 47%, 60%, and 70% in Fb, FbFe and FbFeM with respect to the initial, non-digested, Fb. Caco-2 cells were incubated for 4 h daily for four days or continuously for 24 h with bioaccessible fractions obtained after the digestion (digests). Polyphenols were not metabolised by Caco-2 cells. Fb digest (∼50 μM total phenolics) was the sample that led to the highest inhibition of cell proliferation under the two experimental conditions. Fb digest did not induce apoptosis but ceased cell-cycle in the S-phase which is associated to a decrease in B1 and D1 cyclin levels upon incubation for 24 h.     

Transepithelial transport of 6-O-caffeoylsophorose across Caco-2 cell monolayers

The aim of this study was to clarify the transport behaviour and mechanism of caffeic acid analogue bearing a sugar-moiety, 6-O-caffeoylsophorose (CS), in Caco-2 cells. The absorption of CS was investigated by its transport across Caco-2 cell monolayers using a high-performance liquid chromatography-time-of-flight-mass spectrometry (LC–TOF-MS). The permeation of CS was concentration-dependent and reached the plateau at >6 mM. The apparent permeability (P_app) of CS in the apical-to-basolateral direction was 5.4 × 10⁻⁷ cm/s, while in the reversed direction the P_app value was significantly reduced (1.9 × 10⁻⁷ cm/s). CS transport was competitively inhibited by phloretin, an inhibitor of monocarboxylic acid transporter (MCT). Benzoic acid, an MCT substrate, also reduced CS transport. A less significant change of CS transport was observed across Caco-2 cell monolayers pretreated with quercetin, a suppressor of tight-junction. These findings strongly indicate that CS, a caffeic acid analogue bearing sophorose moiety, can be transported across Caco-2 cell monolayers via the MCT pathway.     

Caseinophosphopeptides exert partial and site-specific cytoprotection against H2O2-induced oxidative stress in Caco-2 cells

Caseinophosphopeptides can sequester prooxidant metals and scavenge free radicals, and may thus be used as functional food ingredients. The total antioxidant capacity (TEAC and ORAC) of two pools of caseinophosphopeptides (1–3 mg/ml), obtained from casein subjected to simulated gastrointestinal digestion (at two different pH values) and selective precipitation, was evaluated to determine dose–response activity. Pool B (which showed the highest antioxidant capacity due to the presence of more antioxidant amino acids) was used to test its cytoprotective effect against H₂O₂-induced oxidative stress in Caco-2 cells. Caseinophosphopeptides protected the cells against oxidative damage by preserving cell viability, increasing GSH content, inducing catalase enzyme activity, diminishing lipid peroxidation and maintaining a correct cell cycle progression. However, they failed to exert protection at a mitochondrial level (ROS and mitochondrial membrane potential), implying a partial and site-specific effect. Thus, their mechanism of action is not only related to free radical scavenging activity, but also to metal chelation and the modulation of intracellular signaling cascades.     

Effects of high CO2 and low O2 atmospheres on the berry drop of ‘Kyoho’ grapes

To investigate the effects of low O₂ and high CO₂ atmospheres on the berry drop of ‘Kyoho’ grapes (Vitis vinifera X V. labrusca), changes of fruit detachment force (FDF) and berry abscission and enzyme activities in the abscission zone were examined during 60 days of storage in air (control), 4%O₂ + 9%CO₂ or 4%O₂ + 30%CO₂ at 0 °C and 95%relative humidity. There was a high negative correlation between FDF and berry drop. Cellulase activity increased over time and was closely correlated with berry abscission. Polygalacturonase (PG) activity rose markedly for the first 30 days and then remained relatively constant. Peroxidase (POD) activity dropped significantly for the first 15 days and subsequently rose sharply (P < 0.05). Cellulase, PG and POD activities were the lowest in fruits in 4%O₂ + 30%CO₂, followed by 4%O₂ + 9%CO₂ and air storage. Pectinesterase (PE) maintained a basal level of activity and there were no significant differences among three treatments (P < 0.05). Compared to air control, the combined effects of the lower level of O₂ and the higher level of CO₂ suppressed the activities of cellulase, PG and POD, maintained greater FDF, and reduced berry abscission during storage.     

Availability of iron from milk-based formulas and fruit juices containing milk and cereals estimated by in vitro methods (solubility,dialysability) and uptake and transport by Caco-2 cells

Iron solubility, dialysability and transport and uptake (retention + transport) by Caco-2 cells as indicators of iron availability have been estimated in the in vitro gastrointestinal digests of infant foods (adapted, follow-up and toddler milk-based formulas and fruit juices containing milk and cereals (FMC)). Low correlation coefficients (in all cases R-squared ? 37.1%) were obtained between iron solubility or dialysability versus transport or uptake efficiency – a fact emphasizing the importance of incorporating Caco-2 cell cultures to in vitro systems in order to adapt the conditions to those found in in vivo assays. The highest uptake efficiency corresponded to FMC (25.6–26.1%) and toddler formulas (32.1–41.9%), the samples with the highest ascorbic acid contents and ascorbic acid/iron molar ratios. In addition, the toddler formulas contained caseinphosphopeptides with the cluster sequence SpSpSpEE, representing the binding site for minerals. In adapted formulas, greater iron uptake efficiency was obtained for the formulation containing ferrous lactate (22.7%) versus ferrous sulfate (4.7%).     

Comparison of antioxidant capacities and cytotoxicities of certain fruit peels

This work was undertaken to explore the potential of fruit waste materials as sources of powerful natural antioxidants. The peels of eight kinds of fruits commonly consumed and grown in Thailand were used. The ethanolic fruit peel extracts were subjected to the scavenging tests of DPPH and ABTS radicals. Results from both assays were in good agreement that the top three markedly high free radical-scavenging power was from the peel extracts of Punica granatum (pomegranate), Nephelium lappaceum (rambutan), and Garcinia mangostana (mangosteen). The IC₅₀ values to quench the DPPH free radicals of these three extracts were 0.003, 0.006, and 0.023 mg/ml and the trolox equivalent antioxidant capacity (TEAC) values from ABTS assay were 4.066, 3.074, and 3.001 mM/mg, respectively. The extract of mangosteen peel showed moderate toxicity to Caco-2 cells and high toxicity to peripheral blood mononuclear cells (PBMC) with the IC₅₀ values of 32.0 and 4.9 μg/ml, respectively. Pomegranate peel extract stimulated Caco-2 cell and PBMC proliferation with the ED₅₀ of 4.7 and 44.4 μg/ml, respectively. Peel extract of rambutan exhibited extremely high value of IC₅₀ (>100 μg/ml) against both cell types indicating non-toxic activity to the cells. It was concluded that the peel of rambutan may be considered potentially useful as a source of natural antioxidants for food or drug product because of its high antioxidant activity and non-toxic property to normal cells.     

Phycobiliproteins or C-phycocyanin of Arthrospira (Spirulina) maxima protect against HgCl2-caused oxidative stress and renal damage

Our objective was to determine if the phycobiliproteins of Arthrospira (Spirulina) maxima protect renal cells against mercury-caused oxidative stress and cellular damage in the kidney. We used 40 male mice that were assigned into eight groups: (1) a control group that received 100 mM phosphate buffer (PB) ig and 0.9% saline ip, (2) PB + HgCl₂ (5 mg/kg ip), (3) PB plus phycobiliproteins (100 mg/kg ig), (4) PB plus C-phycocyanin (100 mg/kg ig), and four groups receiving HgCl₂ + phycobiliproteins or C-phycocyanin (50, and 100 mg/kg ig). The left kidneys were used to determine lipid peroxidation, quantification of reactive oxygen species, and reduced glutathione and oxidised content. The right kidneys were processed for histology. The HgCl₂ caused oxidative stress and cellular damage. All doses of phycobiliproteins or C-phycocyanin prevented enhancement of oxidative markers and they protected against HgCl₂-caused cellular damage.     

Combination of Pichia membranifaciens and ammonium molybdate for controlling blue mould caused by Penicillium expansum in peach fruit

The potential enhancement of Pichia membranifaciens by ammonium molybdate (NH₄Mo) to control blue mould caused by Penicillium expansum on peach fruit was investigated. Combining P. membranifaciens at 1 × 10⁸ cell/ml with 1 mM NH₄Mo provided a more effective control of blue mould rot than applying the yeast or NH₄Mo alone. Addition of 1 mM NH₄Mo significantly increased the growth of P. membranifaciens in peach wounds, but did not affect the population in nutrient yeast dextrose broth medium. The in vitro experiment showed that the combined treatment inhibited spore germination and germ tube elongation of P. expansum in comparison with the treatment of P. membranifaciens or NH₄Mo alone. Moreover, P. membranifaciens, NH₄Mo, and the combination of them did not impair the quality parameters including fruit firmness and content of total soluble solids, titratable acidity and vitamin C of peach fruit after 6 days of storage at 20 °C. These results suggested that the use of NH₄Mo is a useful approach to improve the efficacy of P. membranifaciens for postharvest disease control in peach fruit.     

Purification and characterization of olive (Olea europaea L.) peroxidase – Evidence for the occurrence of a pectin binding peroxidase

Peroxidase from olive fruit (Olea europaea L., cv Douro) in a black ripening stage was purified to electrophoretic homogeneity, resulting in four cationic and four anionic fractions. The anionic fractions accounted for 92% of recovered activity and showed molecular masses of 18–20 kDa. The anionic fraction PODa4, the predominant fraction that comprised about 70% of total recovered activity, showed an isoelectric point of 4.4 and optimum pH and temperature of, respectively, 7.0 and 34.7 °C, and apparent K_m values of 41.0 and 0.53 mM, for phenol and H₂O₂, respectively. From the activity-temperature profile, the denaturation temperature and the changes in enthalpy and heat capacity for unfolding of PODa4 were estimated as being, respectively, 36.5 °C, 411.2 and −13.6 kJ mol⁻¹ K⁻¹. The activation energy for phenol oxidation by PODa4 was 99.1 kJ mol⁻¹, corresponding to a calculated temperature coefficient (Q₁₀) of 4. The arabinose (39 mol%) and galacturonic acid (38 mol%) content of the carbohydrate moiety indicated the existence of pectic material in the purified PODa4 fraction. Co-migration of the carbohydrate with the protein band in the isoelectric focusing electrophoresis, points to PODa4 fraction as being a pectin type binding peroxidase.     

In vitro absorption and antiproliferative activities of monomeric and polymeric anthocyanin fractions from açai fruit (Euterpe oleracea Mart.)

Anthocyanins are among the most important and widely consumed natural pigments in foods, and have attracted increased attention as natural food colourants and potent bioactive agents. However, anthocyanins are generally unstable and may undergo chemical changes that include oxidative and polymerisation reactions during processing and storage. The role of anthocyanin polymerisation reactions on in vitro intestinal absorption and anti-cancer properties has not been assessed. This study investigated the chemical composition, antioxidant properties, antiproliferative activity, and in vitro absorption of monomeric and polymeric anthocyanin fractions from açai fruit (Euterpe oleracea Mart.). Cyanidin-3-rutinoside (58.5 ± 4.6%) and cyanidin-3-glucoside (41.5 ± 1.1%) were the predominant compounds found in monomeric fractions, while a mixture of anthocyanin adducts were found in polymeric fractions and characterised using HPLC–ESI-MSⁿ analyses. Monomeric fractions (0.5–100 μg cyanidin-3-glucoside equivalents/ml) inhibited HT-29 colon cancer cell proliferation by up to 95.2% while polymeric anthocyanin fractions (0.5–100 μg cyanidin-3-glucoside equivalents/ml) induced up to 92.3% inhibition. In vitro absorption trials using Caco-2 intestinal cell monolayers demonstrated that cyanidin-3-glucoside and cyanidin-3-rutinoside were similarly transported from the apical to the basolateral side of the cell monolayers (0.5–4.9% efficiency), while no polymeric anthocyanins were transported following incubation for up to 2 h. The addition of polymeric anthocyanin fractions also decreased monomeric anthocyanin transport by up to 40.3 ± 2.8%. Results from this study provide novel information regarding the relative size, absorption, and bioactive properties of anthocyanin monomers and polymer adducts.     

α-Glucosidase and α-amylase inhibitory activities of guava leaves

The 75% ethanol extract from guava (Psidium guajava Linn.) leaves was extracted further, in turn, with CH₂Cl₂, EtOAc and n-BuOH to afford four fractions, CH₂Cl₂-soluble, EtOAc-soluble, n-BuOH-soluble and residual extract fractions. Both the n-BuOH-soluble and EtOAc-soluble fractions showed high inhibitory activity against α-glucosidase and α-amylase. Seven pure flavonoid compounds, quercetin (1), kaempferol (2), guaijaverin (3), avicularin (4), myricetin (5), hyperin (6) and apigenin (7), were isolated (using enzyme assay-guide fractionation method) from the n-BuOH-soluble and EtOAc-soluble fractions. The structures of these pure compounds were determined on the basis of MS and NMR data and the activities of these compounds were evaluated. Compounds 1, 2 and 5 showed high inhibitory activities, with IC₅₀ values of 3.5 mM, 5.2 mM and 3.0 mM against sucrase, with IC₅₀ values of 4.8 mM, 5.6 mM and 4.1 mM against maltase and with IC₅₀ values of 4.8 mM, 5.3 mM and 4.3 mM against α-amylase, respectively. We found that myricetin showed the most powerful activity among these compounds with a 70% inhibition against sucrase at a concentration of 1.5 mg/ml. The hydroxyl group at the 3-position on the A-ring and a number of hydroxyl groups attached to the C-ring played important roles in the inhibition activity. There was an obvious synergistic effect (the mixing action of two compounds) against α-glucosidase, but against α-amylase this was not found. This is the first study of the active compositions of guava leaves and the biological activity of the active compositions against α-glucosidase and α-amylase.     

Purification and characterization of membrane-bound polyphenoloxidase (mPPO) from Snake fruit [Salacca zalacca (Gaertn.) Voss]

Membrane-bound polyphenoloxidase (mPPO) an oxidative enzyme which is responsible for the undesirable browning reaction in Snake fruit (Salacca zalacca (Gaertn.) Voss) was investigated. The enzyme was extracted using a non-ionic detergent (Triton X-114), followed by temperature-induced phase partitioning technique which resulted in two separate layers (detergent-poor phase at the upper layer and detergent-rich phase at the lower layer). The upper detergent-poor phase extract was subsequently fractionated by 40–80% ammonium sulfate and chromatographed on HiTrap Phenyl Sepharose and Superdex 200 HR 10/30. The mPPO was purified to 14.1 folds with a recovery of 12.35%. A single prominent protein band appeared on native-PAGE and SDS–PAGE implying that the mPPO is a monomeric protein with estimated molecular weight of 38 kDa. Characterization study showed that mPPO from Snake fruit was optimally active at pH 6.5, temperature 30 °C and active towards diphenols as substrates. The K_m and V_max values were calculated to be 5.46 mM and 0.98 U/ml/min, respectively, when catechol was used as substrate. Among the chemical inhibitors tested, l-cysteine showed the best inhibitory effect, with an IC₅₀ of 1.3 ± 0.002 mM followed by ascorbic acid (1.5 ± 0.06 mM), glutathione (1.5 ± 0.07 mM), EDTA (100 ± 0.02 mM) and citric acid (186 ± 0.16 mM).     

Production of enniatins A,A1, B,B1, B4, J1 by Fusarium tricinctum in solid corn culture: Structural analysis and effects on mitochondrial respiration

Enniatins (ENs) are secondary fungal metabolites with hexadepsipeptidic chemical structure and they possess a number of potent biological activities that can contaminate several kind of food and foodstuffs increasing the exposure risk for consumers. ENs are produced by several Fusariun strains including Fusarium subglutinans, Fusarium proliferatum and Fusarium tricinctum. Production of a mixture of ENs was performed by culturing F. tricinctum ITEM 9496 on white corn as substrate. The solid culture components were dried and subsequently extracted with water/methanol (50/50 v/v, 0.5% NaCl), homogenised, filtered, extracted by ethyl acetate and analysed by liquid chromatography with diode array detection (LC-DAD). The crude extract was first separated by low pressure liquid chromatography (LPLC) and then further purified by liquid chromatography (LC), resulting in six compounds with a purity higher than 95% as calculated by ¹H NMR, and with a yield of 30–300 mg per compound. The chemical structures of the ENs were determined by liquid chromatography coupled to mass spectrometry (LC–MS) and nuclear magnetic resonance (NMR). The biological activity of the resulting ENs was determined using a mitochondrial respiration test. We discovered that all the ENs studied induced an increase in the mitochondrial respiration resulting in uncoupling of the oxidative phosphorylation. This effect was most likely due to flux of K⁺ ions into the mitochondrial matrix. The order of potency of the ENs derivatives was: A₁ > B₁ > B > A > B₄ > J₁. These results suggest a correlation between the chemical structures and bioactivity and confirm the severe risks for human associated with consumption of enniatins.     

Characterization of polyphenoloxidase (PPO) and total phenolic contents in medlar (Mespilus germanica L.) fruit during ripening and over ripening

Characterization of polyphenoloxidase (PPO) enzyme and determination of total phenolic concentrations during fruit ripening and over ripening in medlar (Mespilus germanica L.) were determined. During ripening, PPO substrate specificity, optimum pH and temperature, optimum enzyme and substrate concentrations were determined. Among the five mono- and di-phenolic substrates examined ((p-hydroxyphenyl) propionic acid, l-3,4-dihydroxyphenylalanine, catechol, 4-methylcatechol and tyrosine), 4-methylcatechol was selected as the best substrate for all ripening stages. A range of pH 3.0–9.0 was also tested and the highest enzyme activity was at pH 7.0 throughout ripening. The optimum temperature for each ripening stage was determined by measuring the enzyme activity at various temperatures over the range of 10–70 °C with 10 °C increments. The optimum temperatures were found to be 30, 20 and 30 °C, respectively, for each ripening stage. Optimum enzyme and substrate concentrations were found to be 0.1 mg/ml and 40 mM, respectively. The V_max and K_m value of the reaction were determined during ripening and found to be 476 U/mg protein and 26 mM at 193 DAFB (days after full bloom) – stage 1, 256 U/mg protein and 12 mM at 207 DAFB – stage 2, 222 U/mg protein and 8 mM at 214 DAFB – stage 3. For all ripening stages sodium metabisulfite markedly inhibited PPO activity. For stage 1 of ripening, Cu²⁺, Hg²⁺ and Al³⁺, for stage 2, Cu²⁺ and Hg²⁺, and for stage 3, Cu²⁺, Hg²⁺, Al³⁺ and Ca²⁺ strongly inhibited diphenolase activity. Accordingly, it can be concluded that as medlar fruit ripen there is no significant changes in the optimum values of polyphenoloxidases, although their kinetic parametres change. As the fruit ripening progressed through ripe to over-ripe, in contrary to polyphenoloxidase activity, there was an apparent gradual decrease in total fruit phenolic concentrations, as determined by using the aqueous solvents and water extractions.     

Assessment of the ability of seaweed extracts to protect against hydrogen peroxide and tert-butyl hydroperoxide induced cellular damage in Caco-2 cells

The ability of brown seaweed extracts, Ascophyllum nodosum, Laminaria hyperborea, Pelvetia canaliculata, Fucus vesiculosus and Fucus serratus to protect against tert-butyl hydroperoxide (tert-BOOH) induced stress in Caco-2 cells was investigated. Oxidative stress was determined by measuring alteration in the enzymatic activity of catalase (CAT) and superoxide dismutases (SOD) and cellular levels of glutathione (GSH). L. hyperborea, P. canaliculata and F. serratus significantly protected against tert-BOOH induced SOD reduction but did not protect against the reduction in CAT activity or the increased cellular levels of GSH. The ability of F. serratus and F. vesiculosus to protect against H₂O₂ and tert-BOOH induced DNA damage was also assessed. The DNA protective effects of the two seaweed extracts was compared to those of three metal chelators; deferoxamine mesylate (DFO), 1,10-phenanthroline (o-phen) and 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (BAPTA-AM). F. serratus and F. vesiculosus significantly protected (P < 0.05) against H₂O₂ (50 μM) induced DNA damage but not tert-BOOH induced damage.     

The hydroxycinnamic acid content of barley and brewers’ spent grain (BSG) and the potential to incorporate phenolic extracts of BSG as antioxidants into fruit beverages

The hydroxycinnamic acid (HA) content of starting barley for brewers’ spent grains (BSG), whole BSG and phenolic extracts from BSG was measured using high performance liquid chromatography (HPLC) and correlated with antioxidant potential. The effect of BSG phenolic extracts on antioxidant activity of fruit beverages was also assessed (using the total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays). The concentration of HA present in barley extract and BSG was in the order of ferulic acid (FA), p-coumaric acid (p-CA) derivatives, FA derivatives, p-CA, caffeic acid (CA) and CA derivatives. Results suggested that brewing and roasting decreased the HA content. Antioxidant activity was significantly (P < 0.05) correlated with caffeic acid (R² = 0.8309) and total HA (R² = 0.3942) concentrations. Addition of extracts to fruit beverages resulted in a significant (P < 0.05) increase in antioxidant activity of cranberry juice, measured by the FRAP assay. In vitro digestion significantly (P < 0.05) reduced TPC, DPPH and FRAP activity of the fruit beverages.     

Estimation of oxygen uptake rate of tomato (Lycopersicon esculentum Mill.) fruits by artificial neural networks modelled using near-infrared spectral absorbance and fruit mass

The oxygen uptake rate of tomato fruits was estimated by an artificial neural network (ANN) model using near-infrared (NIR) spectral absorbance and fruit mass. The absorption peak apex from cytochrome c oxidase (COX) was confirmed at 841 nm for mitochondrial preparation and at 833 nm for intact fruits. The results of a proteome analysis that detected the putative COX subunit II PS17 from the mitochondrial preparation biochemically supported the presence of the absorption peak due to COX. An ANN model for estimating O₂ uptake rate was developed from the absorbance data at 11 wavelengths from 645 to 979 nm including 833 nm and fruit mass as input variables. The O₂ uptake rate was estimated by the proposed model with a correlation coefficient of 0.79 and a standard error of prediction of 0.091 mmol kg⁻¹ h⁻¹. This method may be effective for rapid estimation of shelf life and physiological activity of tomato fruits.     

An amperometric sensor based on Prussian blue and poly(o-phenylenediamine) modified glassy carbon electrode for the determination of hydrogen peroxide in beverages

An amperometric sensor based on Prussian blue (PB) and poly(o-phenylenediamine) (POPD) modified glassy carbon electrode (GCE) was developed for the determination of hydrogen peroxide (H₂O₂). The PB film was electrodeposited onto the GCE surface by amperometry, while the POPD film was formed on the top of PB layer by cyclic voltammetry. It was found that the POPD film remarkably improved the stability and selectivity of PB-based sensor. Under the optimised conditions, the developed electrode demonstrated a wide linear range from 0.1 μM to 0.12 mM with the detection limit of 0.05 μM. Furthermore, the developed electrode was applied for the amperometric determination of H₂O₂ in 10 different commercial beverages. The pretreatment of the beverage samples was the adjustment of pH value. Experimental results showed that the proposed electrode could be a useful tool to detect H₂O₂ in aseptically packaged beverages.