Optimized ultrasonic-assisted extraction of phenolic antioxidants from grape (Vitis vinifera L.) skin using response surface methodology.

Currently, the analysis of bioactive compounds is easily carried out by modern chromatographic and spectrometric techniques, but the success of the procedure still depends on the efficiency of the extraction methods. This work aimed at finding the optimal conditions for ultrasonic-assisted extraction (UAE) of phenolic antioxidants from grape skin using single-factor experimentation combined with response-surface methodology. Our extraction parameters were ethanol concentration, time and temperature. Total phenolic content (TPC and TPC-280), 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity and oxygen radical absorbance capacity (ORAC) were dependent variables. A second-order polynomial model satisfactorily described the experimental data. Optimized extraction conditions for UAE from grape skin were: 59.5% ethanol, 113.6 min and 66.8 °C for TPC extraction; 61.1% ethanol, 99.4 min and 66.1 °C for TPC-280 extraction; 57.1% ethanol, 99.5 min and 66.8 °C for DPPH radical-scavenging activity; 60.2% ethanol, 82.4 min and 66.8 °C for ORAC value. Our work provides detailed and useful information to optimize the extraction of grape skin phenols, which are widely used as dietary supplements and nutraceuticals.

Ampelometric Leaf Trait and SSR Loci Selection for a Multivariate Statistical Approach in Vitis vinifera L. Biodiversity Management.

High estimated heritability values were recently revealed for mature leaf traits in grape (Vitis vinifera L.), thus redeeming ampelography in the era of molecular markers. The "Organisation Internationale de la Vigne et du Vin (OIV)" set a list of hundreds of descriptors for grapevine in order to standardize ampelographic and ampelometric scores. Therefore, the selection and reduction of the number of OIV codes can represent a major goal for leaner biodiversity assessment studies. The identification of ampelometric traits associated with grape diversity allows to construct Classification Trees with chi squared automatic interaction detection (CHAID) algorithm, a stepwise model-fitting method that produces a tree diagram in which at each step the sample pool is splitted based on the independent variables statistically different for the dependent variable. A collection of 100 table and wine grapevines (Vitis vinifera L.) was characterized and evaluated by means of six microsatellites and twenty-two ampelometric traits on mature leaves. Nine ampelometric traits were selected by principal component analysis and employed to build the classification trees based on CHAID algorithm. The strategy can represent an effective tool for grape biodiversity management, right allocations, and identification of new grape genotypes, implemented by a further microsatellite investigation only when unsolved cases occur, allowing faster and cheaper results.

In-time and in-space tandem mass spectrometry to determine the metabolic profiling of flavonoids in a typical sweet cherry (Prunus avium L.) cultivar from Southern Italy.

This paper presents a comprehensive analytical methodology, based on 'in-time' and 'in-space' tandem mass spectrometry (MS) techniques, to identify and quantify flavonoid compounds in a typical Italian sweet cherry cultivar (cv. Ferrovia). Five anthocyanins, four flavan-3-ols and nine flavonols were determined by means of hyphenated high-performance liquid chromatography - multi-stage MS (HPLC-MS(n)) analyses (MS(n) up to MS(4)), among which quercetin-3-O-rutinoside-7-O-glucoside, kaempferol-3-O-rutinoside-7-O-glucoside, quercetin-3-O-galactosyl-rhamnoside and quercetin-3-O-coumaroylglucoside were tentatively identified in sweet cherries for the first time. Ultrafast HPLC and tandem MS (UHPLC-MS/MS) analyses through multiple reaction monitoring experiments showed that cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside were the main anthocyanins of cv. Ferrovia at maturity. Moreover, consistent levels of catechin and epicatechin as well as quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside were also found. Because flavonoids have been ascribed as potential health-promoting compounds, gathered findings provide new insight into the knowledge of the quali-quantitative profile of these phytochemicals into a widespread fruit such as sweet cherry.

Morphological variability in leaves and molecular characterization of novel table grape candidate cultivars (Vitis vinifera L.).

The present work report the characterization of twenty-one table grapes candidate cultivars plus five registered ones included as reference, by means of 47 ampelographic traits, 23 ampelometric measurements and six microsatellite loci. The final goal of the research was to analyse the possibility of reducing the number of morphological and molecular tools required for a precise and effective description of a grape genotype or cultivar. This would be of great help for future biodiversity description on a larger sample of more than 300 table grapes accessions today grown at the 'Consiglio per la Ricerca e la sperimentazione in Agricoltura (C.R.A.)-Unità di ricerca per l'uva da tavola e la vitivinicoltura in ambiente mediterraneo (Bari-Italy)'. OIV ampelographic traits showed a clear distinction among all twenty-six genotypes analysed, suggesting the relevant morphological variability investigated. Principal component analysis based on ampelometric traits revealed main veins ON(3), ON(4) and O(3)N(4); ratios between main veins; angles between main veins and of petiolar sinus, to be the most effective records in differentiating cultivars, for a total variation of 69.9 % described by the first three components. Molecular analysis based on six microsatellite loci was performed on all genotypes, providing a detailed molecular profile and a dendrogram of genetic similarity, in which all genotypes were clearly distinguishable. Finally, with the goal of using the minimum possible number of markers to differentiate genotypes, microsatellites VVMD5 and VVMD27 were selected to be sufficient to distinguish among all the candidate cultivars included in the analysis, representing a possible 'step by step' approach when a molecular characterization has to be undertaken on a large number of genotypes, by first testing few markers and increasing their number only if necessary.

Validation assay of p3_VvAGL11 marker in a wide range of genetic background for early selection of stenospermocarpy in Vitis vinifera L.

DNA markers technology, derived from research in molecular biology and genomics, offers great promise for plant breeding, allowing the "molecular breeding" via marker-assisted selection. Grapevine genomic resources allowed, in recent years, the characterization at molecular level of genes involved in interesting phenotypes such as stenospermocarpic seedlessness, a trait really appreciated by consumers. Recent studies in table grapes revealed that the VvAGL11 gene, member of the D-lineage MADS-box family, controls the ovule identity, and thus potentially playing an important role in stenospermocarpy. Intragenic markers of VvAGL11 have been found and tested for breeding purposes. In the present paper, we describe an in deep assay on a total of 475 genotypes derived by our own grape germplasm and seeded × seedless crosses F1 offspring, to evaluate and verify the "diagnostic" power of VvAGL11 in marker-assisted selection. We found only 8/475 that were seeded and carried the seedless-associated allele in the STS p3_VvAGL11. However, and most importantly, there were no seedless varieties without such allele. We validated the marker as a 100 % effective tool for early negative selection of stenospermocarpy in Vitis vinifera L. crosses.

HPLC-DAD-ESI-MS analysis of flavonoid compounds in 5 seedless table grapes grown in Apulian Region.

UNLABELLED: Flavonoids present in skin extracts of red seedless table grape varieties Summer Royal, Autumn Royal, and Crimson, and white seedless varieties Carati and Thompson were analyzed by HPLC-DAD-MS, in 3 y of study (2006 to 2008). The anthocyanins, delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside (with their corresponding p-coumaroyl derivatives), peonidin-3-O-glucoside, and malvidin-3-O-glucoside (with their corresponding acetyl, caffeoyl, and p-coumaroyl derivatives) were found. In addition the flavonols quercetin-3-O-glucuronide, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, kaempferol-3-O-galactoside, and the flavan-3-ols procyanidin B1, procyanidin B2, and catechin were also detected. Anthocyanins were the main flavonoids in red grapes ranging from 24 (Crimson) to 500 (Summer Royal) mg/kg fresh weight of grapes; consistent levels of flavonols and flavan-3-ols were also quantified in all varieties. To determine the effective climatic influence on flavonoids content in field conditions, viticultural practices have been developed, that could exclude the effects of direct solar radiation from confounding the assessment of those related to thermal conditions alone. A strong positive correlation was determined between flavonoids and temperature data that seem to be responsible for the difference of these metabolites along the years; furthermore, it has been possible to define a linear relationship (R(2) = 0.6871, P = 0.0057) between thermal amplitude and total flavonoids values in the red grapes. PRACTICAL APPLICATION: Grapes are economically the most important fruit species in the world and approximately 30% of its production is used as fresh fruit. Because of the very important role of flavonoids in food quality as well as their health-promoting properties, and considering that our experiments were performed along 3 consecutive years, gathered results in this research are quite promising to give a useful information on the flavonoid contents and their evolution in 5 seedless table grapes that are widespread in Mediterranean regions but also in California and South America, and are grown in a viticultural climate (Apulia, South Italy) very close to some regions of Spain, Turkey, Tunisia, and Israel.