Polyphenol pattern and antioxidant activity of different tomato lines and cultivars.

BACKGROUND/AIMS: Besides antioxidant vitamins and minerals, fruits and vegetables contain flavonoids and related phenolics. The biological activities of these polyphenols have become well known in recent years evidencing their beneficial effects on human health. In this context, the characterization of the flavonoids present in tomatoes is of great interest. Thus the polyphenol pattern (including flavonols, flavanones and cinnamate derivatives), lycopene and beta-carotene concentrations and the total antioxidant activity (TAA) of the phenolic fraction from different tomato lines and cultivars have been determined. METHODS: The characterization was obtained by means of spectrophotometry and HPLC analyses. RESULTS: Mean values for single flavonoids were 0.68 +/- 0.16 for naringenin, 0.74 +/- 0.12 for rutin and 0.32 +/- 0.06 for a rutin-pentoside. Mean total polyphenol content was 13.15 +/- 1.15 mg/100 g and mean TAA value was 1.3 +/- 0.10 mmol/g. The obtained TAA values resulted in good accordance with the total polyphenol content (R(2) = 0.7928). The main phenolic acids were chlorogenic (mean +/- SE 0.20 +/- 0.03) and caffeic acid (mean +/- SE 0.03 +/- 0.01). Mean levels of lycopene and beta-carotene were 5.38 +/- 0.90 and 1.18 +/- 0.40 mg/100 g, respectively. CONCLUSIONS: Almost all the lines characterised by low carotenoid content produce high levels of polyphenols, and consequently have the most powerful antioxidant potential.

Analytical methods for quality control of propolis.

Propolis is a resinous substance collected by honeybees from leaf buds and cracks in the bark of various plants, and it is composed of 50% resin (composed of flavonoids and related phenolic acids), 30% wax, 10% essential oils, 5% pollen and 5% various organic compounds. Propolis cannot be used as raw material, and it must be purified by extraction with solvents. This process should remove the inert material and preserve the polyphenolic fraction, which is considered to contribute more to the observed healing effects than the other propolis constituents. Therefore, the assay of propolis polyphenols is of interest, and this paper describes the results obtained in the analysis of propolis by means of a gradient HPLC or mass spectrometry. HPLC in the gradient mode and coupled with photodiode array detection remains the method of choice for the assay of most relevant components of propolis. Direct analysis by APCI-IT-MS represents a valuable alternative to obtain typical fingerprints of propolis and a reliable identification of a large number of propolis components.

Plasma levels of caffeic acid and antioxidant status after red wine intake.

The aim of this study was to evaluate the bioavailability of caffeic acid and the modification of plasma antioxidant status following red wine intake. Five healthy male volunteers consumed 100, 200, and 300 mL of red wine corresponding to approximately 0.9, 1.8, and 2.7 mg of caffeic acid, respectively. Plasma samples collected at different times (0-300 min) were evaluated for their content of caffeic acid and their total antioxidant status. Both these parameters, i.e., plasma concentration of caffeic acid and antioxidant potential, were dose-dependent and the C(max) was reached at about 60 min after red wine intake. The results indicate that caffeic acid is bioavailable and it may be correlated with the antioxidant potential of plasma.

Polyphenol content and total antioxidant activity of vini novelli (young red wines).

Eight commercial Italian vini novelli (red wines prepared by carbonic maceration and supposed to be consumed within three months from their wine-making) were evaluated for their total antioxidant activity. The wines had an average total phenol content (1605.4 +/- 337.4 mg/L gallic acid equivalents) lower than that of wines prepared by traditional maceration and consumable after aging (2057. 3 +/- 524.0 mg/L gallic acid equivalents). The average flavanol content (424.7 +/- 121.3 mg/L catechin equivalents) and the total antioxidant activity (16.8 +/- 3.8 mmol/L Trolox equivalents) of vini novelli were higher than the corresponding values (382.7 +/- 174.5 mg/L catechin equivalents and 12.3 +/- 3.3 mmol/L Trolox equivalents) found for aged wines. Three couples of experimental wines were prepared from the same grapes by traditional or carbonic maceration. These wines showed a different phenolic pattern, anthocyanins being more abundant in vini novelli. However, the average total antioxidant activities of the wines were similar, suggesting that aging (and not the wine-making technique) is the main factor influencing the antioxidant activity of red wines.

Electrospray ionization mass spectrometry of synthetic oligonucleotides using 2-propanol and spermidine

Oligonucleotides have become widely used tools in molecular biology and molecular diagnostics. Their parallel synthesis in large numbers and the increasing interest in microarray technology has raised the requirement for fast and informative analytical tools for their quality control. A direct injection electrospray ionization mass spectrometry (ESI-MS) technique based on the use of aqueous 2-propanol as running eluent, and spermidine (or triethylamine) as DNA modifiers, has been applied to analyze a large set of samples (about 200 synthetic oligonucleotides) ranging from 5 to 15 kDa (17-51mers) with good results in terms of sensitivity, suppression of sodium adduct formation, and speed of analysis. Copyright 2000 John Wiley & Sons, Ltd.

Relationship between rate and extent of catechin absorption and plasma antioxidant status.

Flavonoids are described to exert a large array of biological activities, which are mostly ascribed to their radical-scavenging, metal chelating and enzyme modulation ability. Most of these evidences have been obtained by in vitro studies on individual compounds and at doses largely exceeding those dietary. Little is known about a possible relationship between rate and extent of the absorption and modifications of plasma antioxidants. To elucidate this aspect, human volunteers were supplemented with single doses of green tea catechins in free (Greenselect) or phospholipid complex form (Greenselect Phytosome) equivalent to 400 mg epigallocatechingallate (EGCg). EGCg was chosen as biomarker for green tea catechin absorption, and its time course plasma concentration was correlated to the subsequent percent variations of plasma ascorbate, total glutathione, alpha-tocopherol, beta-carotene and Total Radical Antioxidant Parameter (TRAP). Green tea catechins were absorbed more extensively when administered as phospholipid complex rather than as free catechins. Single dose intake of both forms of catechins produced a transient decrease (10-20%) of plasma ascorbate and total glutathione and an increase of plasma TRAP (16-19%). These variations were consistent with the plasmatic levels of EGCg, ascorbate and total glutathione.

Analytical strategies in the structural characterization of elcatonin.

Elcatonin is a synthetic peptide of 32 amino acid residues, that differs from natural peptide hormone (eel calcitonin) in that the 1 and 7 cystine residues are replaced with alpha-amino suberic acid (Asu). Elcatonin is pharmacologically important, since it inhibits osteoclastic bone reserption and induces calcium uptake from body fluids. It is also used for the treatment of Page's disease and hypercalcemic conditions. Until now the structural characterization of elcatonin has been obtained by proteolytic digestion followed by high performance liquid chromatographic (HPLC) analysis of the peptide fragments. Capillary electrophoresis and fast-atom bombardment have also been employed. This work describes the results obtained when a liquid chromatograph, coupled to mass spectrometer using electrospray ionization (LC/ESI-MS) was applied to elcatonin analysis. After digestion with trypsin, the resulting peptides were separated by HPLC with 'on-line' UV detection, and directly injected into the ESI source. The molecular weights of all the fragments were detected, and the sequences of two of them were determined by collisionally induced dissociation in the ESI source. To confirm these 'on-line' results, the 'off-line' approach was also applied. In this case, the fragments from tryptic digestion were isolated by preparative HPLC, concentrated and analyzed by direct infusion into the ESI-MS system. Then, different elcatonin digests obtained using other proteases, e.g. protease V8 and clostripain, were analyzed by direct infusion, and these results combined with those achieved by the 'on-line' analysis allowed us to obtain the entire mapping of elcatonin.

Echinacoside and caffeoyl conjugates protect collagen from free radical-induced degradation: a potential use of Echinacea extracts in the prevention of skin photodamage.

The protective effect of caffeoyl derivatives (echinacoside, chlorogenic acid, chicoric acid, cynarine, and caffeic acid, typical constituents of Echinacea species) on the free radical-induced degradation of Type III collagen has been investigated. The macromolecule was exposed to a flux of oxygen radicals (superoxide anion and hydroxyl radical) generated by the xanthine/xanthine oxidase/Fe2+/EDTA system and its degradation assessed qualitatively by SDS-PAGE and quantitatively as the amount of soluble peptides (according to the 4-hydroxyproline method) released from native collagen after oxidative stress. The SDS-PAGE pattern of native collagen is markedly modified by free radical attack, with formation of a great number of peptide fragments with molecular masses below 97 kDa: in the presence of microM concentrations of echinacoside, there is a complete recovery of the native profile. Collagen degradation was, in fact, dose-dependently inhibited by all the compounds, with the following order of potency: echinacoside approximately chicoric acid > cynarine approximately caffeic acid > chlorogenic acid, with IC50 ranging from 15 to 90 microM. These results indicate that this representative class of polyphenols of Echinacea species protects collagen from free radical damage through a scavenging effect on reactive oxygen species and/or C-, N-, S-centered secondary radicals, and provide an indication for the topical use of extracts from Echinacea species for the prevention/treatment of photodamage of the skin by UVA/UVB radiation, in which oxidative stress plays a crucial role.

High-performance liquid chromatography determination of enzyme activities in the presence of small amounts of product.

Enzymes can be assayed by HPLC by calculating the amount of substrate(s) left over, or product formed, through the peak area ratios with a suitable internal standard. However, sometimes the substrates used are contaminated with small amounts of products and this can lead to errors in the determination of the enzyme activity. A method for a HPLC test of such enzymes, which prevents eventual errors, uses the ratio substrate/product at time zero as internal standard and the kinetics can be followed with the aid of a simple mathematical equation. This approach was applied to the determination of the activities of papain, urokinase, NAD glycohydrolase, and pyruvate kinase samples and it was compared with the data obtained by the internal standard method, giving reproducible results in all cases.

High-performance liquid chromatographic assay of serotonin in human plasma.

A sensitive and reproducible reversed-phase high-performance liquid chromatography with electrochemical detection for determination of serotonin in human plasma is described. The method has an average coefficient of variation of 3.5%; the recovery of serotonin and 5-hydroxy-N-methyltryptamine (internal standard) accounts for 95 +/- 2%. Average serotonin concentrations in platelet-free plasma and in platelet-rich plasma from 21 normal subjects 22-63-year-old) ranged from 0.6 to 4.9 ng/ml (mean 2.8 +/- 1.3 SD) and from 38.3 to 106.5 ng/10(8) platelets (mean 66.8 +/- 18.9 SD), respectively. The release of endogenous serotonin from human aggregating platelets challenged by collagen or adenosine diphosphate has been quantitated.