Paolo Simonetti

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 (Greense‐lect™) or phosphotipid complex form (Greenselect™ Phytosomer®) 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, α‐tocopherol, β‐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.

Evaluation of steviol and its glycosides in Stevia rebaudiana leaves and commercial sweetener by ultra-high-performance liquid chromatography-mass spectrometry

Stevia rebaudiana leaves contain non-cariogenic and non-caloric sweeteners (steviol-glycosides) whose consumption could exert beneficial effects on human health. Steviol-glycosides are considered safe; nonetheless, studies on animals highlighted adverse effects attributed to the aglycone steviol. The aim of the present study was to develop and validate two different ultra-high-performance liquid chromatography methods with electrospray ionization mass spectrometry (UHPLC-MS) to evaluate steviol-glycosides or steviol in Stevia leaves and commercial sweetener (Truvia). Steviol-glycosides identity was preliminarily established by UV spectra comparison, molecular ion and product ions evaluation, while routine analyses were carried out in single ion reaction (SIR) monitoring their negative chloride adducts. Samples were sequentially extracted by methanol, cleaned-up by SPE cartridge and the analytes separated by UHPLC HSS C18 column (150 mm x 2.1 mm I.D., 1.8 microm). The use of CH2Cl2 added to the mobile phase as source of Cl- enhance sensitivity. The LLOD for stevioside, rebaudioside A, steviolbioside and steviol was 15, 50, 10 and 1 ng ml(-1), respectively. Assay validation demonstrated good performances in terms of accuracy (89-103%), precision (<4.3%), repeatability (<5.7%) and linearity (40-180 mg/g). Stevioside (5.8+/-1.3%), rebaudioside A (1.8+/-1.2%) and rebaudioside C (1.3+/-1.4%) were the most abundant steviol-glycosides found in samples of Stevia (n=10) from southern Italy. Rebaudioside A was the main steviol-glycosides found in Truvia (0.84+/-0.03%). The amounts of steviol-glycosides obtained by the UHPLC-MS method matched those given by the traditional LC-NH2-UV method. Steviol was found in all the leaves extract (2.7-13.2 mg kg(-1)) but was not detected in Truvia (<1 microg kg(-1)). The proposed UHPLC-MS methods can be applied for the routine quality control of Stevia leaves and their commercial preparations.

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 β-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 (R2 = 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 β-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.

Melatonin, melatonin isomers and stilbenes in Italian traditional grape products and their antiradical capacity

Although polyphenols represent the paradigm of the health‐promoting effects ascribed to grape products, recently, attention has been paid to dietary melatonin, significantly present in Mediterranean foods. In this work, we measured melatonin, its isomers, stilbenes (trans‐ and cis‐resveratrol and their glucosides, piceids) and total polyphenols in some different grape products (red, white and dessert wines, grape juices and Modena balsamic vinegars) of distinct Italian areas. We also evaluated their antiradical activity by DPPH· and ABTS·+ assays. For indoleamine analysis, the separation was carried out on a 1.7‐μm C18 BEH column and the detection performed by means of mass spectrometry with electrospray ionization in positive ion mode with multiple reaction monitoring. The confirmation of the peak identity was accomplished by injection into the high‐resolution system (Orbitrap) using accurate mass measurements (error below 1.0 ppm). Mass spectrometry analyses revealed, for the first time, the presence of melatonin in dessert wines and balsamic vinegars, as well as the occurrence of three different melatonin isomers in grape products.

Toxicity and Antioxidant Activity in Vitro and in Vivo of Two Fucus vesiculosus Extracts

The consumption of seaweeds has increased in recent years. However, their adverse and beneficial effects have scarcely been studied. Two extracts from the brown seaweed Fucus vesiculosus containing 28.8% polyphenols or 18% polyphenols plus 0.0012% fucoxanthin have been obtained and studied to determine their toxicity in mice and rats and also their antioxidant activity. Both extracts were shown to lack any relevant toxic effects in an acute toxicity test following a 4 week daily treatment in rats. The extracts exhibited antioxidant activity in noncellular systems and in activated RAW 264.7 macrophages, as well as in ex vivo assays in plasma and erythrocytes, after the 4 week treatment in rats. Our ex vivo results indicated that compounds from extract 2 may be more easily absorbed and that the antioxidants in their parent or metabolized form are more active. These findings support the view that the daily consumption of F. vesiculosus extract 2 (Healsea) would have potential benefits to humans.

Liquid chromatography/electrospray ionization mass spectrometric characterization of flavonol glycosides in tomato extracts and human plasma.

Flavonoids continue to attract wide attention as possible very useful agents for combating free radical pathologies, i.e. the pathological states associated with free radical overproduction. Commonly used methods for the analysis of plant flavonoids include high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). On the other hand, the soft-ionization approach based on electrospray ionization (ESI-MS) permits highly selective analysis of complex matrices. In this work, we examined firstly the ESI-MS behaviour of representative aglycones and glycosides of flavonols, flavones and isoflavones with the aim of suggesting a possible relationship between structure and mass spectra. Using HPLC coupled to a diode array detector (DAD) for on-line UV spectra acquisition, and in parallel to ESI-MS for mass spectra (LC/DAD-ESI-MS), we have developed methodology to observe flavonols directly in tomato puree extract. In this way, it has been possible to detect intact flavonol glycosides in tomato extracts and to characterize a flavonol trisaccharide. For the first time, using LC/ESI-MS, it has been possible to detect intact flavonol glycosides in plasma of healthy volunteers and to provide further evidence on the absorption of flavonoid glycosides after consumption of common vegetables like tomatoes.

From vineyard to glass : agrochemicals enhance the melatonin and total polyphenol contents and antiradical activity of red wines

Abstract:  Resistance inducers are a class of agrochemicals, including benzothiadiazole and chitosan, which activate the plant own defence mechanisms. In this work, open‐field treatments with plant activators were performed on two red grape (Vitis vinifera L.) varieties cultivated in different localities, Groppello (Brescia, Lombardia, Italy) and Merlot (Treviso, Veneto, Italy). Treatments were carried out every 10 days until the véraison and, after harvesting, experimental wines (microvinificates) were prepared. In general, both melatonin and total polyphenol content, determined by mass spectrometry and Folin‐Ciocalteu assay, respectively, were higher in wines produced from grapes treated with resistance inducers than in those obtained from untreated control and conventional fungicide‐treated grapes. Accordingly, antiradical power of wines derived from plant activator‐treated grapes, measured by both DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) and the ABTS [(2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid)] radical–scavenging assay, was higher than in their counterparts. To the best of our knowledge, this is the first report on the effects of agrochemicals on the melatonin content of red wine.

The presence of melatonin in grapevine (Vitis vinifera L.) berry tissues

Abstract:  Melatonin has been reported in a variety of food plants and, consequently, in a number of plant‐derived foodstuffs. In grapevine (Vitis vinifera L.) products, it was found in berry exocarp (skin) of different cultivars and monovarietal wines. Herein, we assessed, by means of mass spectrometry, the occurrence of melatonin in all berry tissues (skin, flesh, and seed) at two different phenological stages, pre‐véraison and véraison. We detected the highest melatonin content in skin, at pre‐véraison, whereas, at véraison, the highest levels were reported in the seed. Furthermore, during ripening, melatonin decreased in skin, while increasing in both seed and flesh. The relative concentrations of melatonin in diverse berry tissues were somewhat different from those of total polyphenols (TP), the latter measured by the Folin‐Ciocalteau assay, and more abundant in seed at pre‐véraison and in exocarp at véraison. The highest antiradical activity, determined by both DPPH (2,2‐diphenyl‐1‐pycryl hydrazyl) and ABTS [(2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid)] radical‐scavenging assay, was reported at pre‐veráison in seed. To the best of our knowledge, we reported, for the first time, the occurrence of melatonin in grape seeds.

Flavanone plasma pharmacokinetics from blood orange juice in human subjects

Some blood orange juice (BOJ) flavanones may have antioxidant, anti-inflammatory, anti-allergic, hypolipidaemic, vasoprotective and anticarcinogenic properties. The aim of the present study was to evaluate the pharmacokinetics of hesperetin and naringenin in human subjects after BOJ intake. In a cross-over study, seven healthy female volunteers consumed 150 and 300 ml BOJ corresponding to about 51-102 mg hesperetin and to 6-12 mg naringenin, respectively. Plasma samples were collected before, each hour for 8 h and 24 h after BOJ administration and analysed for their content of hesperetin and naringenin by liquid chromatography-MS/MS. The plasma concentrations of these compounds were dose dependent and the peak concentration (Cmax) was reached in 5.1 (sd 0.6) h after BOJ intake. The Cmax of hesperetin was 43.4 (sd 32.4) and 79.8 (sd 60.1) ng/ml after 150 and 300 ml intake, respectively. For naringenin the plasma peak was 16.4 (sd 11.9) and 34.0 (sd 20.6) ng/ml. Moreover, the conjugated forms of these flavanones represent more than 95 % of the plasma concentration. The results indicate that both hesperetin and naringenin are bioavailable after BOJ intake; naringenin seemingly more so than hesperetin.

Nutritional Traits of Bean (Phaseolus vulgaris) Seeds from Plants Chronically Exposed to Ozone Pollution

The effect of chronic exposure to ozone pollution on nutritional traits of bean ( Phaseolus vulgaris L. cv. Borlotto Nano Lingua di Fuoco) seeds from plants grown in filtered and nonfiltered open-top chambers (OTCs) has been investigated. Results showed that, among seed macronutrients, ozone significantly raised total lipids, crude proteins, and dietary fiber and slightly decreased total free amino acid content, although with a significant reduction of asparagine, lysine, valine, methionine, and glycine, compensated by a conspicuous augmentation of ornithine and tryptophan. Phytosterol analysis showed a marked increase of beta-sitosterol, stigmasterol, and campesterol in seeds collected from nonfiltered OTCs. With regard to secondary metabolites, ozone exposure induced a slight increase of total polyphenol content, although causing a significant reduction of some flavonols (aglycone kaempferol and its 3-glucoside derivative) and hydroxycinnamates (caffeic, p-coumaric, and sinapic acids). Total anthocyanins decreased significantly, too. Nevertheless, ozone-exposed seeds showed higher antioxidant activity, with higher Trolox equivalent antioxidant capacity (TEAC) values than those measured in seeds collected from filtered air.