Yerba mate tea (Ilex paraguariensis): Phenolics,antioxidant capacity and in vitro inhibition of colon cancer cell proliferation

Mate tea (MT) is a rich source of phenolic compounds that vary depending on geographical origin and mode of preparation. Total phenolic concentration and antioxidant capacity of Mate tea (Ilex paraguariensis) products were determined in this work. In addition, a representative MT was tested for in vitro inhibition of human colon carcinoma cell proliferation. Total polyphenol concentration, was measured using Folin–Ciocalteau method, ranged from 90 to 176 mg gallic acid eq (GAE)/g dry leaves (DL) in traditional MT and from 40 to 113 mg GAE/g DL in MT added with other flavouring ingredients. It was estimated that a cup of tea (250 ml) containing one teaspoon (5 g) of instant MT could provide an average intake of 1.5 g GAE. Fresh tea (FT) from Mate leaves displayed high antioxidant capacity (85 ± 1%) and preferentially inhibited 50% of net growth of human colorectal adenocarcinoma cells CaCo-2 (GI₅₀ = 1.0 ± 0.03 μg/ml) and HT-29 (GI₅₀ = 105.2 ± 15.2 μg/ml) when compared with the CCD-33Co normal colon fibroblast cell line (GI₅₀ > 300 μg/ml). MT inhibited in vitro colon cancer cell proliferation possibly mediated via pro-oxidant activities, therefore represents a potential source of chemopreventive agents that deserve further investigation.     

Evaluation of the potential of high pressure technology as an enological practice for red wines

The impact of high hydrostatic pressure (HHP) treatments on the phenolic compounds composition of a red wine was studied after storage in order to evaluate the potential of this technology as an enological practice. Red wines pressurized at 500 and 600 MPa at 20 °C for 5 and 20 min, respectively, showed a lower content of monomeric anthocyanins (13 to 14%), phenolic acids (8 to 11%), and flavonols (14 to 19%) after 5 months of storage, when compared to the unpressurized wine. These results, together with the different degree of tannin polymerization and flavan-3-ol content in the pressurized wines led to propose an effect of HHP in the increase of polymerization and cleavage reactions of proanthocyanidins. The sensorial analysis of pressurized wines showed lower astringency, a higher intensity of cooked fruit aroma and lower intensity of fruity notes, when compared with the unpressurized wine. These effects are associated to those observed during wine aging.Industrial relevanceDuring the last decade, the use of high hydrostatic pressure (HHP) as a non-thermal technology for food preservation and modification has increased substantially. Recently, pressure treatments have shown to influence long term red wine physicochemical and sensorial characteristics, leading to aged wine-like characteristics. Therefore, the use of HHP technology to modify wine composition could benefit the wine industry, especially to improve wines with low aging potential. This work shows that HHP can be potentially used as enological practice, modulating the organoleptic properties of wine by decreasing astringency and increasing pleasant aromas. It seems possible to exploit commercially the production of young red wines with distinct characteristics using this technology, addressing the market and consumer demand.     

Peptides derived from high oleic acid soybean meals inhibit colon,liver and lung cancer cell growth

Soybean meal, a co-product after oil extraction from seeds, is rich in protein. Our objective was to utilize this co-product, obtain gastrointestinal (GI) resistant peptides from the isolated protein, and test for bioactivity against colon, liver and lung cancer cell lines. N98-4445A, S03-543CR high oleic acid soybean lines, and R95-1705 high protein soybean line were used for this study. Protein isolates were prepared at alkaline pH and hydrolyzed using alcalase enzyme to generate peptide hydrolysates. After determining gastrointestinal resistance of the peptide hydrolysates they were fractionated into definite molecular sizes of < 5 kDa, 5–10 kDa, and 10–50 kDa and tested against human colon (HCT-116, Caco-2), liver (HepG-2) and lung (NCL-H1299) cancer cell lines. MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, cytotoxicity assay was performed to test in vitro cancer cell viability upon treatment with peptide fractions. The peptide fractions from N98-4445A and S03-543CR lines showed cell growth inhibition of 73% of colon cancer (HCT-116), 70% of liver cancer cells and 68% of lung cancer cells. Dose response showed that the peptides had significant inhibitory effect at higher concentrations (1000 μg/mL to 600 μg/mL) and gradually decreased with decreased dosage (500 μg/mL to 100 μg/mL). Reverse phase HPLC identified three single peptides from the 10–50 kDa fractions of N98-4445A soy line that have potential for enhanced activity. Soybean peptide fractions can thus be a source of bioactivity against colon, liver and lung cancer cell proliferation.     

Purification and characterization of angiotensin I-converting enzyme inhibitory peptides of small red bean (Phaseolus vulgaris) hydrolysates

Angiotensin I-converting enzyme (ACE) inhibitory activity was investigated for small red bean (Phaseolus vulgaris) protein hydrolysate produced by sequential digestion of Alcalase, papain followed by in vitro gastrointestinal simulation. The hydrolysate had ACE inhibitory activity with IC₅₀ of 67.2 ± 1.8 μg protein/mL. Peptides responsible for potent ACE inhibitory activity were isolated by a three-step purification process, including ultrafiltration, gel filtration and preparative reverse phase high performance chromatography (RP-HPLC). The fraction obtained after RP-HPLC fractionation with the highest activity yielded an IC₅₀ of 19.3 ± 1.4 μg protein/mL. Enzymatic kinetic studies using this fraction demonstrated competitive inhibition with K_i of 11.6 ± 1.7 μg protein/mL. Mass spectrometric characterization identified for the first time the octapeptide PVNNPQIH which demonstrated an IC₅₀ value of 206.7 ± 3.9 μM. The results expand the knowledge base of ACE inhibitory properties of small red bean protein hydrolysate and should be useful in further identification of specific ACE inhibitory peptides in beans.     

The chemopreventive effect of β-cryptoxanthin from mandarin on human stomach cells (BGC-823)

β-Cryptoxanthin, a provitaminic carotenoid, present in many fruits and vegetables, has been associated with decreased risk of chronic diseases, including cancer. The influence of β-cryptoxanthin derived from mandarin on the proliferation of the stomach tumor cell line BGC-823 was tested using MTT and cell count assay at 72 h and dose–response (from 0.01 to 20 μM). β-Cryptoxanthin suppressed the cell migration by the scratch assay. Furthermore, β-cryptoxanthin induced an accumulation of cells in the G1/G0 phase of the cell cycle (as detected by flow cytometry), which was in accordance with an increased expression of p21 and down regulations of cyclin D1 and cyclin E, detected by Western blot analysis, and β-cryptoxanthin increased the mRNA levels of retinoic acid receptor β (RARβ) with the treatment at 10 μM for 24 h. Collectively, the above findings suggest that β-cryptoxanthin could be therapeutic in the treatment of stomach cancer cell in vitro.     

Biological potential of extracts of the wild edible Basidiomycete mushroom Grifola frondosa

Partially purified polysaccharides (FP) and hot alkali extract (FNa) obtained from fruiting bodies of the wild basidiomycete Grifola frondosa were examined for their antimicrobial, antioxidant and cytotoxic activity. The structural properties of FP and FNa samples were investigated by FT-IR and high resolution 1H- and 13C-NMR spectroscopy. From a group of various G − and G + bacteria the antibacterial effects were highest against the G + B. cereus. FNa was the better antioxidant shown by the lower EC₅₀ values of DPPH scavenging ability, ferric-reducing antioxidant power and ferrous ion-chelating ability. Ferric-reducing antioxidant power and ferrous ion-chelating ability were mostly linked to total polysaccharides, total- and β-glucan content, as well as total protein content. Both extracts displayed a moderate dose dependent antiproliferative action towards malignant human breast cancer MDA-MB-453, cervical adenocarcinoma HeLa and myelogenous leukemia K562 cells not observed in the non cancer derived MRC-5 fibroblasts. The highest effect was found in HeLa cells for FP extract. The mean diameter of Ca-alginate bead loading FP was 960.7 μm while the mean diameter of beads encapsulating FNa extract was 1051.7 μm.     

On the staining of human tissue-cultured (Chang) conjunctival cells with rose bengal and lissamine green

PurposeTo assess the staining of Chang conjunctival epithelial cells with rose bengal and lissamine green.MethodsA human (Chang) conjunctival epithelial cell line was grown on plastic plates in 10% foetal calf serum-supplemented medium in 5% carbon dioxide–air at 37 °C. Cells were examined daily between days 2 and 10 of culture, reaching confluence after 7 days, for overall appearance before and after exposure for 3 min at 37 °C to solutions of either rose bengal or lissamine green (dissolved in saline, PBS or balanced salts solution). Measurements of nucleus and cell size were made on some confluent cultures.ResultsChang cells, regardless of the growth state or the vehicle used, stain very intensely with rose bengal, with the nucleus of the cells showing the most notable dye uptake. Cultures showing such intensive staining with rose bengal were consistently non-staining with lissamine green. Nucleus size, based on measurements of the long dimension (NUCLONG) were the same for rose bengal or lissamine green exposed cells (averaging 19.5 ± 2.4 μm and 18.8 ± 2.9 μm, respectively). Cell size at confluence, based on measurements of the long dimension of lissamine green-exposed cells (LONG), averaged 40.5 ± 7.2 μm. The cytoplasm-to-nucleus ratio (based on LONG–NUCLONG/NUCLONG) averaged 1.177 ± 0.406 (i.e. approximately a 1:1 ratio).ConclusionsCultured Chang conjunctival cells stain intensely with rose bengal, but the same is not seen with lissamine green. The cell morphology is indicative of flattened epithelial cells.     

Effect of germination and elicitation on phenolic composition and bioactivity of kidney beans

Ready-to-eat sprouts are becoming popular healthy fresh foods. Germination and elicitation may be used to improve phytochemical and functional quality of sprouts. The aim of this study was to unveil the efficacy of germination and the use of elicitors (500 μM ascorbic, 50 μM folic and 5 mM glutamic acid) to enhance the phenolic composition and antioxidant and angiotensin I converting enzyme (ACE) inhibitory activities of 8 day-old Phaseolus vulgaris L. var. Pinto sprouts. Sprouting produced a general decrease in flavan-3-ols and anthocyanins of sprouts that was compensated with a higher content of flavanones and flavonols. Although elicitor treatments reduced total phenolic content of sprouts, they promoted the synthesis of specific flavanones and flavonol O-glycosides, effect that was dependent on the type of elicitor. Antioxidant activity was not affected by ascorbic and folic acid treatments whereas it was slightly reduced in glutamic acid-treated sprouts. Folic acid was the only treatment that caused an outstanding increase in the ACE inhibitory activity of sprouts. In conclusion, elicitation may produce positive changes in the phenolic profile and improve health-promoting potential of sprouts, although selection of elicitor is crucial to deliver marketplace ready to eat sprouts enriched in specific bioactive phytochemicals.     

Adaptability of some French red grape varieties cultivated at two different Portuguese terroirs: Comparative analysis with two Portuguese red grape varieties using physicochemical and phenolic parameters

In the last year's, several French grape varieties have been introduced in different world wine regions, increasing their representation in the world. Consequently, it is important to analyze the adaptability of several red grape varieties from French origin to the other specific ‘terroirs’ and compare their characteristics with native grape varieties. Thus, the aim of this study was to evaluate the physicochemical and phenolic composition of five French red grape varieties and two Portuguese red grape varieties grown at two different Portuguese wine regions. In general, French (except ‘Alicante Bouschet’ grape variety) and Portuguese red grape varieties collected in the vineyard located in ‘Dão’ wine region had higher positive physicochemical characteristics, especially for estimated alcohol degree and titratable acidity. Regarding the phenolic composition, French grape varieties showed significantly higher values of total phenols and flavonoid compounds, especially for the grape samples collected in the vineyard located in ‘Dão’ region. Thus, for example ‘Alicante Bouschet’ grape variety showed the highest values for total phenolic (global average value from 0.636 to 0.894 mg g^− 1 of berry) and flavonoid compounds (global average value from 0.584 to 0.834 mg g^− 1 of berry). However, for total anthocyanins it was clear that the two Portuguese grape varieties studied (‘Touriga Nacional’ and ‘Tinta Roriz’) showed significantly highest values independently of the wine region considered (global average value ranged from 0.603 to 0.785 mg g^− 1 of berry). Similar tendency was also confirmed by significantly highest global average values for the sum of the glycosylated derivatives, where the values ranged from 0.389 to 0.539 mg g^− 1 of berry, for the two Portuguese grape varieties considered.     

A rational approach to prevent postprandial modification of LDL by dietary polyphenols

Food may undergo enhanced oxidation in the stomach leading to increases in the generation of deleterious lipid peroxidation products. Following meat consumption an escalation occurs in malondialdehyde (MDA) levels in human plasma. It was hypothesized that MDA could cause postprandial LDL modification in vivo, which can be abolished by the simultaneous addition of red wine polyphenols. Healthy volunteers consumed two test meals for four sequential days: meat cutlets (MC) and meat cutlets with red wine (MCRW). Postprandial plasma MDA levels after meal (MC) increased by 106 nmol/ml, and only by 57 nmol/ml after meal (MCRW). Following meal (MC) day 1 postprandial MDA–LDL levels increased by 27%. Following 4 days of repeated consumption of meal (MC), postprandial MDA–LDL levels increased by 96% (P = 0.021) and remained elevated after an overnight fast. Addition of red wine to the meal (MCRW) completely prevented postprandial MDA–LDL modification. It is concluded that the postprandial increase level of MDA in the plasma is partially responsible for LDL modification.     

Aging of red wines made from hybrid grape cv. BRS Violeta: Effects of accelerated aging conditions on phenolic composition,color and antioxidant activity

Aging is an enological technique usually employed with Vitis vinifera wines for improving and stabilizing wine sensory attributes like red wine color. However, red wines made from non-vinifera grape cultivars are considered not suitable for classic wine aging because they are prone to color loss during processing and storage, mainly due to the major occurrence of non-acylated anthocyanidin 3,5-diglucosides. The recently developed hybrid grape cv. BRS Violeta gives rise to deep red-purplish colored wines characterized by an important contribution of p-coumaroylated anthocyanidin 3,5-diglucosides. In this work, Violeta red wines were subjected to control (storage at 15 °C) and accelerated (storage at 25, 35 and 50 °C) aging for 120 days. Total phenolic content was only significantly decreased by 23% in accelerated aged wines at 50 °C, whereas antioxidant capacity was mainly reduced in the first 20 days of aging (around 35% for all the wines, except for 45% for wine aged at 50 °C) and then remained almost stable. In contrast, total anthocyanin content decreased following expected first-order kinetics and the variation of rate constants with regard to temperature fitted well with an Arrhenius-type equation. The calculated activation energy for disappearance of anthocyanins (47 kJ/mol) was close to the lower limit of the range of values reported for thermal degradation of anthocyanidin 3-glucosides. In addition, half-life values for p-coumaroylated anthocyanidin 3,5-glucosides of Violeta wine aged at 50 °C almost doubled those of their corresponding non-acylated derivatives. The latter result is suggested to be the main reason behind the relatively high resistance of Violeta wine towards the disappearance of anthocyanin under accelerated aging conditions. Finally, chromatic characteristics of Violeta wines did not significantly change their C* values during aging but L* and h* values increased as expected, the latter mainly due to higher increases of the yellow color component (b*) over lower decreases of the red color component (a*). These color changes were more drastic in the accelerated aged wines and the wine aged at 25 °C still maintained interesting color characteristics close to those of the control aged wine that did not change very much.     

Pomegranate husk extract,punicalagin and ellagic acid inhibit fatty acid synthase and adipogenesis of 3T3-L1 adipocyte

Fatty acid synthase (FAS) has been recognized as a potential therapeutic target for obesity. In this study, for the first time, the inhibitory effect of pomegranate husk extract, punicalagin and ellagic acid on FAS was investigated. We found them potently inhibiting the activity of FAS with half-inhibitory concentration values (IC₅₀) of 4.1 μg/ml (pomegranate husk extract), 4.2 μg/ml (4.50 μM, punicalagin) and 1.31 μg/ml (4.34 μM, ellagic acid), respectively. Moreover, they all exhibited time-dependent inactivation of FAS. Punicalagin and ellagic acid inhibited FAS with different mechanisms compared to previously reported inhibitors, through inactivating acetyl/malonyl transferase and β-ketoacyl synthase domains, respectively. Additionally, 100 μg/ml pomegranate husk extract, 5.24 μg/ml (5 μM) punicalagin and 4.5 μg/ml (15 μM) ellagic acid effectively reduced lipid accumulation inside FAS over-expressed 3T3-L1 adipocytes. Since FAS plays a key role in the biosynthesis pathway of fatty acid, these findings suggest that pomegranate husk extract, punicalagin and ellagic acid have potential in the prevention and treatment of obesity.     

HPLC-analysis of polyphenolic compounds in Spanish white wines and determination of their antioxidant activity by radical scavenging assay

A high-performance liquid chromatographic method coupled with UV and fluorescence detectors for the determination of polyphenols in white wines samples is reported. The proposed method was validated regarding linearity, repeatabilities within day and between days, recoveries and polyphenol stabilities.Eleven phenolic compounds including, flavanols and flavanols dimmers (procyanidins), hydroxycinnamates, flavonols and stilbene derivatives were identified and quantified. Samples were injected into the chromatograph directly without previous treatment.The in-house method validation provided good results with respect to repeatabilities (within day (RSD ? 4%) and between days (RSD ? 5%)) and recoveries (?97.3%).In the second part of the work the antioxidant activity of the wine samples was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. The correlation between specific polyphenols and the antioxidant activity in samples was also investigated.     

The use of active PET to package rosé wine: Changes of aromatic profile by chemical evolution and by transfers

Active Polyethylene Terephthalate (PET) bottles containing 1 or 3% of oxygen scavenger (named 1osPET and 3osPET) were used to pack rosé wine. Changes in the aromatic profiles were monitored during 12 months and compared to those of a wine packed in glass bottles. Wine in 1osPET bottles was differentiated from wine in glass or 3osPET bottles by ten aging markers such as cis-dioxane, ethyl pyruvate or furfural. Only trans-1,3-dioxolane allowed to discriminate wine in glass and in 3osPET bottles. Methionol, an oxygen sensitive aroma compound, was preserved in glass and 3osPET bottles but was slightly degraded (15%) in 1osPET bottles. Chemical reactions were the main cause of the aroma compound degradation. Indeed, the total amount of compounds sorbed only reached 160 μg considering the bottles and the joint of cap after 12 months of storage. The use of PET with 3% of oxygen scavenger is adapted to pack wine for at least 12 months.     

Fluorescein staining and physiological state of corneal epithelial cells

PurposeTo evaluate the physiological status of corneal epithelial cells exhibiting fluorescein staining.MethodsFluorescein staining properties of corneal epithelial cells under normal and stressed conditions were studied using cell-culture (human corneal limbal epithelial cells – HCLE) and organ-culture (rabbit) models. Stress stimuli comprised exposure to hypotonicity, hypertonicity, preservatives, scratch, and alkaline wounding. In addition to fluorescein, cells were stained with Hoechst-33342 (HO), Propidium-iodide (PI), and Annexin-V (AN-V) to identify live, dead and apoptotic cells. Clinical-slit-lamp and fluorescence confocal-microscopic (FCM) observations were performed. FCM images were quantified for fluorescence intensity using Image-J software.ResultsHealthy HCLE cells uniformly took up fluorescein to a moderate degree with a mean grey value of 62 ± 24 (mean ± SD) on a scale of 0–256 (no unit). Fluorescence levels similar to those observed prior to stress were associated with healthy cells. Apoptotic cells showed the highest fluorescence (138 ± 38). Dead cells showed minimal fluorescence (23 ± 7) that was similar to the background (20 ± 11, p > 0.05). Observations in whole rabbit eyes were in general agreement with these cell culture findings.ConclusionsThe clinical observation of corneal staining with fluorescein suggests the presence of epithelial cells that are undergoing apoptosis but does not indicate dead cells. Under in vitro or ex vivo conditions, healthy cells took up fluorescein at levels that were lower than those of apoptotic cells and thus, are not likely to be perceived as exhibiting staining during clinical observation. Sodium fluorescein may be considered as a probe for apoptotic epithelial cells.     

Effects of phenolic compounds in blueberries and muscadine grapes on HepG2 cell viability and apoptosis

Although blueberries and muscadine grapes have high contents of polyphenols, few studies have been conducted to assess their potential effects on cancer cells. The objective of this study was to systematically evaluate the effects of different fractions of phenolic compounds in blueberries and muscadine grapes on HepG2 liver cancer cell viability and apoptosis. Three cultivars of blueberries (‘Briteblue’, ‘Tifblue’ and ‘Powderblue’) and four cultivars of muscadine grapes (‘Carlos’, ‘Ison’, ‘Noble’, and ‘Supreme’) were assessed in this study. Polyphenols were extracted and further separated into phenolic acids, tannins, flavonols, and anthocyanins using a HLB cartridge and LH-20 column. The major compounds of different fractions were characterized. The phenolic acid fractions of muscadine grapes and blueberries showed a 50% inhibition of HepG2 cell population growth at the level of 1–2 mg/mL. The greatest inhibitory effects were observed from the anthocyanin fractions with 50% inhibitions of cancer cell population growth at concentrations of 70–150 and 100–300 μg/mL in blueberries and muscadine grapes, respectively. The flavonol and tannin fractions showed intermediate activities. In addition, DNA fragmentation was measured by using a Cell Death Detection ELISA kit to assess the induction of apoptosis. The anthocyanin fraction resulted in a two- to fourfold increase in DNA fragmentation compared to control in both muscadine grapes and blueberries. These findings of inhibition of cancer cell growth and induction of apoptosis suggest that blueberries and muscadine grapes may contribute to reduction in liver cancer risk.     

Anti-inflammatory and anti-oxidative effect of curcumin in connective tissue type mast cell

Curcumin in turmeric from rhizome of plant Curcuma longa was investigated on its anti-inflammatory effect with canine cutaneous mastocytoma mast cells. Though the degranulation induced by neuropeptide substance P, specific mast cell secregtagogue Compound 48/80 and immunoglobulin IgG were reached above 10%, curcumin reduced the release below 4.6% at 10 μM without cytotoxicity. Inhibitory effect of curcumin on [Ca²⁺]_i elevation was also confirmed. ROS induced by Compound 48/80 were evaluated with dichlorodihydrofluorescein diacetate (DCFH-DA), and were lowered by curcumin in dose dependent manner. Cell membrane lipid peroxidation was monitored with diphenyl-1-pyrenylphosphine (DPPP). Curcumin decreased cell membrane lipid peroxidation induced by arachidonic acid in the concentration above 0.01 μM. Curcumin also inhibited the protein tyrosine- and threonine-phosphorylations around 33 and 45 kDa, respectively. Taking these findings into consideration with the inhibitory effects on connective tissue type mast cell, there is a potential use to ameliorate inflammation-related chronic diseases.     

Cancer cell antiproliferation activity and metabolism of black carrot anthocyanins

The antiproliferation activity of a black carrot anthocyanin-rich extract (BC-ARE) on human cancer cells (HT-29 colorectal adenocarcinoma and HL-60 promyelocytic leukaemia) and metabolism of its characteristic anthocyanins (acylated and nonacylated forms) in humans were investigated. Cancer cells were exposed for 24 h to 0.0–2.0 mg/mL of BC-ARE. Anthocyanin-rich extract inhibited proliferation of both types of cancer cells in a dose-dependent manner. After ingestion of a black carrot concentrate, three acylated and two nonacylated anthocyanins were excreted in the volunteers (n = 4) urine (0.048% of the administered dose). The anthocyanins were characterized by HPLC-DAD-ESI-MS/MS. Urine recovery of nonacylated anthocyanins was 8-fold higher than that of acylated anthocyanins.Industrial relevanceNatural pigments have achieved commercial significance as food colorants due to the fact that consumers perceive them as safe additives. At present extracts rich in acylated anthocyanins, which are known for their outstanding stability, serve as a major source of natural food colours for the food colorant industry with purple sweetpotato, red cabbage and black carrot being the major sources. Beside aesthetic values, anthocyanins possess enhanced physiological activity due to their potent antioxidant properties and enhance the health-promoting qualities of foods. Our findings on cancer cell antiproliferation activity of an anthocyanin-rich black carrot extract as well as information about the bioavailability of black carrot anthocyanins could lead to an increased application of these natural food colorants by the food industry.