Separation of five oligostilbenes from Vitis amurensis by flow‐rate gradient high‐performance counter‐current chromatography

A rapid and efficient high‐performance counter‐current chromatography (HPCCC) method was developed to separate five oligostilbenes from the roots of Vitis amurensis. An n‐hexane/ethyl acetate/methanol/water system (4:8:4:10, v/v/v/v) was selected as an optimal two‐phase solvent system of which the upper phase was used as the stationary phase and the lower phase was used as the mobile one. Partition coefficient values for the target compounds under these optimized conditions were 0.28 ( 1 , ampleosin A), 7.12 ( 2 , (+)‐g‐viniferin), 2.26 ( 3 , vitisin A), 5.38 ( 4 , wilsonol C), and 11.23 ( 5 , vitisin B). Flow‐rate gradient HPCCC (4 mL/min in 0–70 min, 8 mL/min in 70–250 min) was applied to isolate the target compounds in as high purity as possible within the shortest possible run time. Under these conditions, ampelopsin A (12.1 mg), (+)‐g‐viniferin (10.4 mg), vitisin A (2.8 mg), wilsonol C (3.2 mg), and vitisin B (37 mg) were isolated with >95% purity from 150 mg of enriched oligostilbene extract. Although the K_D of the last eluted compound, vitisin B (K_D = 11.23), was relatively large, it was eluted in 115–145 min using the two‐phase solvent system. This study shows that HPCCC is an efficient tool for the isolation and purification of natural products.     

Development of hybrid elution systems for efficient purification of stilbenoids using centrifugal partition chromatography coupled to mass spectrometry

The phytochemical study of the root extract of the stilbenoid-rich Vitis riparia × Vitis berlandieri grapevine was carried out by centrifugal partition chromatography (CPC). For this reason, we developed a new elution mode we named back-step, which allowed us to obtain cleaner fractions and a more efficient separation process when used in conjunction with a classical elution approach. Three hydroxystilbenes: (E)-resveratrol, (E)-?-viniferin and (E)-vitisin C, with greater than 90% purity were thus obtained through such process, with minimal sample handling and purification steps. Online coupling of CPC to ESI mass spectrometry was used for optimization of the separation parameters and to facilitate the characterization of the stilbenoids. This study details the first phytochemical investigation of stilbenoids from the hybrid species together with a new elution mode able to widen the range of ARIZONA biphasic systems.     

Quantification of abscisic acid in grapevine leaf (Vitis vinifera) by isotope-dilution liquid chromatography–mass spectrometry

A specific, sensitive, precise, and accurate method for the determination of abscisic acid (ABA) in grapevine leaf tissues is described. The method employs high-performance liquid chromatography and electrospray ionization–mass spectrometry (LC-ESI-MS) in selected ion monitoring mode (SIM) to analyze ABA using a stable isotope-labeled ABA as an internal standard. Absolute recoveries ranged from 72% to 79% using methanol/water pH 5.5 (50:50 v/v) as an extraction solvent. The best efficiency was obtained when the chromatographic separation was carried out by using a porous graphitic carbon (PGC) column. The statistical evaluation of the method was satisfactory in the work range. A relative standard deviation (RDS) of < 5.5% and < 6.0% was obtained for intra-batch and inter-batch comparisons, respectively. As for accuracy, the relative error (%Er) was between −2.7 and 4.3%, and the relative recovery ranged from 95% to 107%.     

The effect of methyl jasmonate and light irradiation treatments on the stilbenoid biosynthetic pathway in Vitis vinifera cell suspension cultures

Grape stilbenes are a well-known family of plant polyphenolics that have been confirmed to have many biological activities in relation to health benefits. In the present study, we investigated the effect of methyl jasmonate (MeJA) elicitor at four different concentrations (25, 50, 100 and 200 μM) in combination or not with high-level light irradiation (10,000 LUX) on a cell line obtained from the pulp of Vitis vinifera cv. Shahani. Our results showed that the stilbene synthesis pathway is inhibited by high-light conditions. A concentration of 50 μM MeJA was optimum for efficient production and high accumulation of total phenolics and total flavonoids as well as total stilbenoids. Furthermore, we showed that there is a significant negative correlation between the production of these metabolites and cell growth. These data provide valuable information for the future scale-up of cell cultures for the production of these very high value compounds in bioreactor system.     

Effect of Extraction Solvent and Temperature on Polyphenol Profiles,Antioxidant and Anti-Inflammatory Effects of Red Grape Skin By-Product

A fully-detailed LC-MS qualitative profiling of red grape skin, extracted with a mixture of ethanol and water (70:30 v:v) has permitted the identification of 65 compounds which can be classified into the following chemical classes: organic and phenolic acids (14 compounds), stilbenoids (1 compound), flavanols (21 compounds), flavonols (15 compounds) and anthocyanins (14 compounds). The extraction yield obtained with water at different temperatures (100 °C, 70 °C, room temperature) was then evaluated and the overall polyphenol content indicates that EtOH:H₂O solvent is the most efficient and selective for polyphenol extraction. However, by analyzing the recovery yield of each single polyphenol, we found that water extraction under heating conditions is effective (extraction yield similar or even better in respect to the binary solvent) for some polyphenolic classes, such as hydrophilic procyanidins, phenolic acids, flavonol glucosides and stilbenoids. However, according to their lipophilic character, a poor yield was found for the most lipophilic components, such as flavonol aglycones, and in general for anthocyanins. The radical scavenging activity was in accordance with the polyphenol content, and hence, much higher for the extract obtained with the binary solvent in respect to water extraction. All the tested extracts were found to have an anti-inflammatory activity in the R3/1 cell line with NF-kb reporter challenged with 0.01 µg/mL of IL-1α, in a 1 to 250 µg/mL concentration range. An intriguing result was that the EtOH:H₂O extract was found to be superimposable with that obtained using water at 100 °C despite the lower polyphenol content. Taken together, the results show the bioactive potentialities of grape skin extracts and the possibility to exploit this rich industrial waste. Water extraction carried out by heating is an easy, low-cost and environmentally friendly extraction method for some polyphenol classes and may have great potential for extracts with anti-inflammatory activities.     

Potential use of secondary products of the agri-food industry for topical formulations and comparative analysis of antioxidant activity of grape leaf polyphenols

The aim of the present study was to develop a phytocosmetic using Vitis waste by-products, for use as a topical formulation for skin protection against ultraviolet radiation damage. The study also evaluates the free radical scavenger activity of the crude extracts of dried leaves of Vitis vinifera and Vitis labrusca, as well as the anthocyanins, flavonoid fraction and isolated compounds. Next, release and permeation studies of hydrogels were performed using Franz-type diffusion cells. Flavonoid acted more intensively in TRAP and conjugated dienes antioxidant assays, whereas anthocyanins had higher antioxidant activity in hydroxyl and nitric oxide assay. Only quercetin-3-O-glucuronide (5) was released from hydrogels, and the flavonoid retention in porcine ear skin after eight hours of permeation was below of limit of quantification for this compound. The polyphenols present in Vitis are capable of absorbing UV and visible light, justifying their potential as sunscreens for the development of a phytocosmetic.     

Preparative isolation and purification of antioxidative stilbene oligomers from Vitis chunganeniss using high‐speed counter‐current chromatography in stepwise elution mode

Preparative high‐speed counter‐current chromatography (HSCCC) was successfully applied to the isolation and purification of three stilbene oligomers from Vitis chunganeniss using stepwise elution with a pair of two‐phase solvent systems composed of n‐hexane–ethyl acetate–methanol–water at (2:5:2:5, v/v) and (1:2:1:2, v/v). The preparative HSCCC separation was performed on 800 mg of crude sample yielding hopeaphenol (21.1 mg), amurensin G (37.2 mg) and vitisin A (95.6 mg) in a one‐step separation, with purities over 95% as determined by HPLC. The structures of these three compounds were identified by MS, ¹H NMR and ¹³C NMR. In addition, their antioxidant activities were screened by DPPH assay, where vitisin A showed strong antioxidant activity. Further EPR experiments with spin‐trapping technique demonstrated that vitisin A is a potent and selective singlet oxygen quencher, which may be used in singlet oxygen‐mediated diseases as a pharmacological agent.     

Ecophysiological and phytochemical response to ozone of wine grape cultivars of Vitis vinifera L.

Vitis vinifera sensitivity to tropospheric ozone (O₃) has been evidenced in several studies. In this work, physiological and metabolic effects of O₃ on two wine cultivars of V. vinifera (i.e. Maturano and San Giuseppe) have been studied. Moreover, chlorogenic acid (CGA) production, in consideration of its importance in the biosynthetic pathway of polyphenols and as antioxidant, has been investigated. Maturano cultivar resulted more sensitive to O₃, as evidenced by the gas exchange reduction at the early stage of treatment, and by the increase in Ci/Ca and the decoupling of net photosynthesis and the stomatal conductance at the end of the treatment. Unexpectedly, O₃ did not activate stilbene production. Ozone induced an early CGA decrease, significantly more consistent in cv. Maturano, and an increase after 8 days, more consistent in cv. S. Giuseppe. These results suggest that CGA could be considered a biochemical marker of O₃-induced stress in V. vinifera.     

Investigation of common fluorophores for the detection of nitrated explosives by fluorescence quenching

This study assessed the impact of fruit temperature on the phenolic metabolism of grape berries (Vitis vinifera L. cv. Merlot) grown under field conditions with controlled exposure to sunlight. Individual cluster temperatures were manipulated in situ. Diurnal temperature fluctuation was damped by daytime cooling and nighttime heating of clusters. Daytime-only and nighttime-only temperature controls were applied for comparison. Berry temperatures were recorded continuously to compare the chemical data. Samples collected at véraison indicated that damping the diurnal temperature fluctuation advanced the onset of ripening. Those berries were larger (double-damped: 0.753 ± 0.015 g berry⁻¹ vs control: 0.512 ± 0.034 g berry⁻¹) and more colored than all others. Development of phenolic metabolites was followed by two reversed-phase high performance liquid chromatography methods and gel permeation chromatography. These methods provided information on anthocyanins, proanthocyanidins, flavonols, flavan-3-ol monomers, and polymeric material. Damping the diurnal temperature fluctuation reduced proanthocyanidin mean degree of polymerization (double-damped: 21.8 ± 1.0 vs control: 28.0 ± 1.7). Proanthocyanidin accumulation at véraison was linearly related to heat summation over the developmental period with nighttime heating yielding the highest concentration and daytime cooling yielding the lowest (night-heat: 1.46 ± 0.13 mg berry⁻¹ vs day-cool: 0.97 ± 0.09 mg berry⁻¹). Damping the diurnal temperature fluctuation had a marked effect on the rate of fruit development whereas total heat summation had more of an effect on phenolic metabolism alone. The results provide insight on the direct effect of temperature on phenolic metabolism.     

A White Grape Juice Extract Reduces Fat Accumulation through the Modulation of Ghrelin and Leptin Expression in an In Vivo Model of Overfed Zebrafish

A caloric surplus and a sedentary lifestyle are undoubtedly known to be the leading causes of obesity. Natural products represent valuable allies to face this problematic issue. This study was planned to assess the effect of a white grape (Vitis vinifera) juice extract (WGJe) in diet-induced obese zebrafish (Danio rerio). Fish were divided into four different diet groups: (i) normally fed (NF); (ii) overfed (OF); (iii) WGJe-supplemented NF (5 mL/L in fish water); (iv) WGJe-supplemented OF. Body mass index (BMI) was extrapolated each week. After the fourth week, euthanized zebrafish were processed for both microscopic evaluations and gene expression analyses. OF zebrafish showed higher BMI values with respect to NF counterparts, an effect that was hindered by WGJe treatment. Moreover, histological analyses showed that the area of the adipose tissue, as well as the number, size, and density of adipocytes was significantly higher in OF fish. On the other hand, WGJe was able to avoid these outcomes both at the subcutaneous and visceral levels, albeit to different extents. At the gene level, WGJe restored the altered levels of ghrelin and leptin of OF fish both in gut and brain. Overall, our results support the anti-obesity property of WGJe, suggesting its potential role in weight management.