Source: https://pubs.rsc.org/en/content/articlehtml/2019/fo/c8fo02175a
Timestamp: 2019-04-22 14:41:08+00:00

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Several studies have shown that fresh grape and its derivatives contain phenolic compounds exhibiting antioxidant and health promoting effects, particularly in relation to the cardiovascular system. In this study, two methods were developed to characterize sixteen varieties of table and wine grapes: (1) a LC-MS method to identify major and minor phenolic compounds; and (2) a HPLC-DAD method to quantify the most representative compounds. Sixty-seven molecules belonging to different classes of phenolic compounds were identified: anthocyanins, flavan-3-ols, flavonols, stilbenes and organic acids. In parallel, the free radical scavenging activity and anti-inflammatory activities of the 16 grape varieties were evaluated. The results showed a good correlation between the total phenolic content and the biological activity. Extracts from Exalta and Albarossa grape varieties were the most active in reducing IL-8 release by gastric epithelial cells (IC50 = 8.48 μg mL−1 and 6.68 μg mL−1, respectively), a biomarker of inflammatory processes. The observed biological activities were mainly associated with skin and seed extracts/portions. The interest in studying table grapes and their non-fermented derivatives as sources of healthy compounds has increased in the last few years and our findings suggest that table grapes and their fresh derivatives, in addition to wine, could be involved in the health promoting effects of the Mediterranean diet.
Oxidative stress and inflammation are closely related factors for the induction of pathophysiological processes, one of which can be modulated by the other. As a consequence, these two conditions can be found at the same time in many pathological conditions, including metabolic syndrome and neurodegenerative disorders.
Several studies have been performed to evaluate the modulatory properties of flavonoids on those inflammatory factors which are responsible for different chronic diseases; however, only few studies have been focused on the effects of grapes and their derivatives on gastric inflammation.6,7 Gastritis is one of the most common gastrointestinal inflammatory disorders, frequently associated with the bacterium Helicobacter pylori. During H. pylori infection, gastric epithelial cells show higher levels of cytokines including IL-1β, TNF-α, and IL-8, a potent chemokine playing a key role in gastric diseases. This response is highly dependent on NF-κB activation, leading to the transcription of several pro-inflammatory mediators and to the worsening of the inflammatory conditions. The search for new compounds capable of modulating positively these mechanisms could prevent a prolonged inflammation state with positive effects on human health.
Polyphenols could represent good candidates for this purpose, but the effect on gastric inflammation of polyphenols from fresh grapes has not been investigated so far, even though data on grape seeds or skin extracts have indicated promising applications.
On these bases, the objective of the present study was the chemical characterization by HPLC-DAD-ESI-MSn of sixteen different grape varieties with the evaluation of their antioxidant potential and gastric anti-inflammatory activity. The results obtained with the whole fruit will be compared to those derived from isolated skin and pulp to define the relative contribution to the biological properties and to identify the most promising varieties.
Acetonitrile, methanol, formic acid and water (LC-MS grade) were purchased from VWR International (Fontenay-sous-Bois, France). Folin-Ciocalteu's reagent, sodium carbonate and reagents used for biological assays were from Sigma Aldrich (Germany). The commercial standards at disposal were: flavonols (quercetin, quercetin-3-O-rutinoside, quercetin-3-O-galactoside, quercetin-3-O-glucoside, quercetin-3-O-glucuronide, kaempferol and kaempferol-3-O-glucoside), flavan-3-ols (epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, and procyanidin B2), stilbene resveratrol and trans-piceid; these standards and the reactive 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH) were from Sigma-Aldrich (Steinem, Germany). Organic acids (caftaric acid, ferulic acid, chlorogenic acid and caffeic acid) and anthocyanins (delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, peonidin-3-O-glucoside and malvidin-3-O-glucoside) were from Extrasynthèse (Lyon, France). All standards were stored according to the supplier's instructions, then solubilized at a concentration of 1 mg mL−1 in a water : methanol mixture (1 : 1, v/v), diluted to a final concentration of 10 μg mL−1 and injected into a chromatographic system.
Thirteen table grapes (two red and four white seed-containing varieties, three red and three white seedless varieties), harvested in 2015, were included in this study. Three wine varieties were studied in parallel for comparison. Grape varieties and their origin are reported in Table 1.
a A specimen voucher of each grape variety was labeled and stored at the Department of Pharmacological and Biomolecular Sciences (Università degli Studi di Milano), Lab. Food Chemistry and Toxicology.
Aliquots of 50 g of berries from each type of grape were weighed, homogenized and freeze-dried. All samples were maintained at −20 °C till the analysis.
For spectrophotometric and chromatographic analyses, 0.4 g of each homogenate grape sample were mixed with 3 mL of a methanol : water (1 : 1, v/v) mixture, and sonicated for 15 minutes by using an ultrasonic bath; red and white grape samples were centrifuged for 15 minutes at 3000 and 8000 r.c.f. (relative centrifugal force), respectively. The supernatant was collected and filtered on 0.45 μm filters (Millipore, Billerica, MA, USA). Grape precipitates were extracted again with 2 mL of a methanol : water mixture, and the supernatants were combined and brought to a volume of 5 mL with the same extraction mixture.
The total polyphenol content was determined according to the Folin–Ciocalteu's method, as reported by Singleton and Rossi.8 Aliquots of 300 μL prepared as described above were solubilized in 1 mL of a 50 : 50 (v/v) water : methanol solution, mixed in test tubes with 1.5 mL of Folin–Ciocalteu's reagent diluted 10 times, and 1.2 mL of 7.5% (w/v) sodium carbonate. After 30 min, the absorbances were measured at 765 nm in a UV-visible spectrophotometer (Varian Cary 50 SCAN, Palo Alto, CA, USA). The polyphenol content was calculated using a standard curve of gallic acid, and results were expressed as equivalents of gallic acid in mg g−1.
A HPLC method combined with electrospray ionization mass spectrometry (ESI-MS) and use of a Diode Array Detector (DAD) has been set up. The analytic platform was composed of a Surveyor LC system, which was connected to an LCQ Advantage mass spectrometer through a Finnigan IonMax electrospray ionization (ESI) source assembled with a high flow stainless steel emitter (Thermo Fisher Scientific, Rodano, MI, Italy). Full instrument control and data analysis were realized using the Xcalibur software (version 2.0.7, Thermo Fisher Scientific, Rodano, MI, Italy). The chromatographic column was a Synergi 4u MAX-RP 80A (250 × 2.0 mm 4 μm) (Phenomenex, Torrance, CA, USA).
The analysis was performed using a linear gradient elution at a flow rate of 0.3 mL min−1, where the elution phases had the following composition: (A) water : acetonitrile : formic acid 96.99 : 3 : 0.01 (v/v/v); and (B) acetonitrile : water : formic acid 50 : 49.99 : 0.01 (v/v/v). The gradient was programmed as follows: 0–15 min: 94–70% A, 15–30 min: 70–50% A, 30–35 min: 50–10% A, 35–38 min: 10% A isocratic, 38–48 min: 10–94% A.
The mass spectrometer operated in electrospray for negative ions (ESI−) using nitrogen as a sheath gas. The capillary temperature was 275 °C, source voltage was 3.50 kV, sheath gas flow was 35 arb and sweep gas flow was 10 arbs. The collision energy for MS2 scans was 60%.
Quantitative HPLC-DAD analysis. A HPLC method combined with the use of a diode array detector (DAD) has been set up and validated;6 the method was applied to grape samples to quantify anthocyanins (delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, peonidin-3-O-glucoside, malvidin-3-O-glucoside) and other flavonoids (caftaric acid, rutin, hyperoside, quercetin-3-O-glucoside, quercetin-3-O-glucuronide, kaempferol-3-O-glucoside). In addition, the recovery and stability of phenolic compounds were evaluated. The recovery of phenolic compounds was evaluated by adding the different classes of phenolic compounds at three different concentrations (low, medium, and high) to the matrix.12 Short and long term stability of polyphenols was assessed at room temperature and at −20 °C for 30 days.
Stock solutions of anthocyanins were diluted in methanol : water (50 : 50, v/v) at a final concentration of 100 μg mL−1, and then diluted with 0.1 N HCl to obtain the working solutions at concentrations ranging from 0.1 to 12.5 (μg mL−1).
The anthocyanin separation was performed using a HPLC equipment Thermo (San Josè, CA, USA). The instrument consisted of a pump (P2000, Thermo Separation products, San Josè, CA, USA), an interface (SN4000, Thermo Separation products, San Josè, CA, USA), a UV detector (975-UV), a diode array detector (6000 LP, Thermo Separation products, San Josè, CA, USA), and an injection valve (Rheodyne, Cotati, CA, USA) with a 20 μL loop. The chromatographic column was a Synergi 4u MAX-RP 80A (250 × 4.60 mm 4 μm) (Phenomenex, Torrance, CA, USA). Iso-Disc Filters PTFE 0.45 μm were from Supelco Analytical (Bellefonte, PA, USA).
The analysis was performed using a gradient elution at a flow rate of 0.8 mL min−1, where (A) water : acetonitrile : formic acid 87 : 3 : 10 (v/v/v); and (B) acetonitrile : water : formic acid 50 : 40 : 10 (v/v/v) were used. The gradient was set as follows: 0–15 min: 94–70%, A, 15–30 min: 70–50% A, 30–35 min: 50–10% A, 35–38 min: 10% A isocratic, 38–48 min: 10–94% A. The detection was performed at 520 nm.
Stock solutions of caftaric acid, rutin monohydrate, hyperoside, quercetin-3-O-glucoside, quercetin-3-O-glucuronide, kaempferol-3-O-glucoside, epicatechin (EC) and epicatechin-gallate (ECG) were prepared in methanol : water (50 : 50, v/v) at a final concentration of 200 μg mL−1. Solutions were diluted with 0.1 N HCl in order to obtain working solutions with concentrations ranging from 0.1 to 50 (μg mL−1). Finally, they were stored at −20 °C, till the analysis.
The analysis of this group of flavonoids was performed by using the same instrumentation described above but with a different chromatographic column (Synergi 4u MAX-RP 80A – 250 × 2.0 mm 4 μm-Phenomenex, Torrance, CA, USA).
The analysis was performed using the gradient elution described above, but detection was performed at 360 nm, apart from that of EC and ECG, performed at 280 nm.
DPPH assay. The radical scavenging activity or antioxidant activity (AOA) was assessed using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radicals.13,14 Aliquots of 1 mL of DPPH (Sigma Aldrich, Germany) solubilized in methanol (5 mg per 100 mL) were mixed with 0.5 mL of each sample prepared as described above. The absorbance was measured after 30 minutes at 517 nm. The results were expressed as mg g−1 Trolox equivalents (TE).
Oxygen radical absorbance capacity (ORAC) assay. The assay was performed according to the method proposed by Ou et al. and modified by Dávalos et al.15,16 It was carried out using a Victor X3 plate reader (PerkinElmer, USA) equipped with a fluorescence detector set at excitation and emission wavelengths of 484 and 528, respectively. Analyses were performed in phosphate buffer (pH 7.4, 75 mM). Peroxyl radicals were generated using AAPH (12 mM) and fluorescein (70 nM) was used as the substrate. Trolox (6.25–50 μM) or samples (400 μg mL−1) were added and, after shaking, values were registered every minute for 120 min at 37 °C. The area under the curve (AUC) was calculated by integrating the relative fluorescence curve. The net AUC was calculated by subtracting the AUC of the blank. The final ORAC values were determined from the linear regression equation of Trolox concentrations and expressed as μM Trolox per mL.
Preparation of water extracts from grapes. Aliquots of 2 g of freeze-dried fruits (see methods) were extracted for 4 hours with 20 mL of deionized water at room temperature, under stirring and dark conditions. Then, the mixture was filtered, and the recovered material was subjected to further extraction for 16 hours with the same volume of deionized water under the same conditions. Then, the aqueous extracts were combined, frozen and immediately freeze-dried. The extracts were stored in aliquots at −20 °C until the biological assays.
Skin, pulp and seeds from Exalta and Albarossa varieties were manually separated and extracted following the method previously described.
Cell culture. Human epithelial gastric cells (AGS) were grown in a 75 cm2 flask (Euroclone S.p.A, Pero, Italy) using DMEM F12 medium supplemented with 100 U mL−1 penicillin, 100 mg mL−1 streptomycin, 2 mM L-glutamine, and 10% heat-inactivated FBS (Euroclone S.p.A, Pero, Italy). AGS cells were incubated under a humidified atmosphere containing 5% CO2.
Cytotoxicity assay. AGS cells were seeded in 24-well plates (30 000 cells per well), and after 72 hours they were co-treated with increasing concentrations of the extract and the stimulus TNFα (10 ng mL−1) for 6 hours. The integrity of the cell morphology was analyzed before and after the treatment through microscopic inspection. Cell viability was measured at the end of the treatment by the MTT method. The culture medium was removed from the plate and 200 μL of MTT solution was added into each well for 45–60 minutes. The absorbance was read at 550 nm using a microplate reader (iMarkTM Microplate Absorbance Reader, Bio-Rad). No evidence of cytotoxicity was observed in AGS cells treated for 6 hours with the extracts at the concentrations used for testing biological activity.
IL-8 release measurement. AGS cells were seeded in 24-well plates (30 000 cells per well); after 72 hours they were co-treated with the extract (5–100 μg mL−1) and the stimulus TNFα (10 ng mL−1) for 6 hours. IL-8 released in the culture medium was measured through an ELISA Development Kit (Peprotech Inc., London, UK). Corning 96 well EIA/RIA plates from Sigma-Aldrich (Milan, Italy) were coated with the antibody provided in the ELISA Kit and incubated overnight at room temperature. After blocking the reaction with 1% albumin solution, each sample (200 μL) was added into wells for 2 hours at room temperature. The amount of IL-8 was detected by spectrophotometry (λ: 450 nm, 0.1 s) using biotinylated and streptavidin–HRP conjugate antibodies, and evaluating the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate reaction. Quantification of IL-8 contained in each sample was performed using an optimized standard curve supplied in the ELISA Kit (8.0–1000.0 pg mL−1). EGCG 20 μM was used as a reference inhibitor of IL-8 release.
Biological assays were performed in triplicate.
Statistical analysis. All data are expressed as mean ± SD of at least three independent assays performed in duplicate; data were analyzed by unpaired one-way analysis of variance (ANOVA) followed by Bonferroni's post-hoc test. Statistical analysis was done using the GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA). *p < 0.05 was considered statistically significant. IC50 was calculated using the GraphPad Prism 6.00 software.
The total phenolic content (TPC) in red and white grape samples ranged, respectively, between 0.44 ± 0.02 and 7.94 ± 0.19 mg gallic acid equivalents per g of fresh weight (Fig. 1). These values are in agreement with data from the scientific literature, where a TPC content between 0.70 and 5.7 mg gallic acid equivalents per g is reported.17–19 As expected, the TPC of red wine varieties was higher than that of red table grapes: Albarossa showed the highest polyphenol content (7.94 ± 0.19 mg g−1) followed by Barbera and Nebbiolo (4.08 ± 0.25 and 3.85 ± 0.07, respectively).
Fig. 1 TPC (mg g−1 of gallic acid equivalents) in grape samples.
Grapes are characterized with a complex polyphenol profile. Due to the similar spectral characteristics of individual compounds belonging to the same subclass and the limited commercial availability of reference compounds, HPLC coupled to mass spectrometry was a strategic tool for the complete characterization of samples.
The phenolic profiles of sixteen varieties of grapes (including red and white varieties) was investigated by HPLC-DAD-ESI-MSn. This method allowed the identification of 25 anthocyanins, 18 flavan-3-ols, 18 flavonols, 3 stilbenes and 3 organic acids. Table 2 shows the distribution of polyphenol classes and their relative abundance in the individual varieties included in the study.
PS: Pasiga; RF: Red Flame; RG: Red Globe; Bes: Beauty seedless; KRs: King's Ruby seedless; NE: Nerona; NB: Nebbiolo; BR: Barbera; AL: Albarossa; KH: King's Husainy; SC: Sugraone (from Conegliano); EX: Exalta; SL: Sultanina; CAs: Canner seedless; CEs: Centennial seedless; SG: Sugraone (from Beja). +++ = high relative abundance; ++ = medium relative abundance; + = low relative abundance.
The specific LC-MS data of the identified compounds are reported in the ESI (Tables 1S, 2S, 3S, and 4S†). In addition, HPLC-DAD and MS/MS spectra of one red (Albarossa) and one white (Exalta) grape samples are reported in Fig. 1S and 2S,† respectively.
Considering anthocyanins, 25 compounds were separated by LC-MS: five of them were identified as the 3-O-monoglucosides of delphinidin, cyanidin, petunidin, peonidin, and malvidin. Additionally, 20 further compounds were detected and identified as acylated (including acetyl, coumaroyl and caffeoyl esters) or di-glucoside anthocyanins, in agreement with previous studies.20 Generally, wine varieties showed the highest relative abundance of anthocyanin compounds. In all grapes analyzed (table and wine varieties), the most representative anthocyanins were peonidin-glucoside and malvidin-3-O-glucoside; the former was detected in all red varieties, whereas malvidin-3-O-glucoside was present in all samples except for Red Globe. Flavan-3-ol dimers and trimers were quite abundant and equally distributed in all grape samples: flavan-3-ol monomers showed less presence; only catechin, epicatechin and epigallocatechin monomers were identified. Proanthocyanidin dimers showed a relatively high abundance in all grape samples, while proanthocyanidin trimers were significant in all grape varieties except for Sugraone (from Portugal). Apart from proanthocyanidin trimers, a similar flavan-3-ol pattern was observed in Sugraone grapes coming from two different countries (Italy and Portugal).
In red table grapes the most common flavonols were quercetin galactoside, quercetin glucoside, quercetin glucuronide and quercetin rutinoside as reported previously.17,21 A similar pattern was observed in the red wine grapes, which were rich in flavonol compounds. Generally speaking, the white varieties showed the lowest relative abundance in flavonols and the distribution of these phenolics was quite homogeneous, with quercetin-3-O-glucoside, quercetin-3-O-glucuronide and trihydroxyflavone-riboside being the most abundant.
Finally, caftaric acid (an organic acid) was present in all samples, as reported previously.17Trans-coutaric acid and cis-coutaric acid were detected only in wine grapes, apart from Nebbiolo.
DP: Delphindin-3-O-glucoside; CY: cyanidin-3-O-glucoside; PT: petunidin-3-O-glucoside; PO: peonidin-3-O-glucoside; MV: malvidin-3-O-glucoside.a Red wine grapes.
a Total content of phenolic compounds measured by HPLC; ^<LOD (43.5 ng mL−1); §<LOD (4.3 ng mL−1); °<LOD (3.3 ng mL−1); #<LOD (4.9 ng mL−1); *<LOD (4.0 ng mL−1); **<LOD 0.012 ng mL−1; ***<LOD 0.009 ng mL−1; N.D. = Not Detectable.
In addition, the recovery and stability of the phenolic compounds were assessed according to FDA recommendations.12 Recovery was always >90%, and the percentage of variation after short- and long-term stability evaluation was always within ±15%. A significant difference between wine and table grape varieties was found in terms of the delphinidin-3-O-glucoside content, which was particularly abundant only in wine grapes. Malvidin-3-O-glucoside was the most abundant in the table grape varieties Pasiga (41.87 ± 1.25 μg g−1), King's Ruby (24.00 ± 0.49 μg g−1), Nerona (71.70 ± 2.71 μg g−1), Beauty Seedless (141.68 ± 5.63 μg g−1) and in wine cultivar Albarossa (1067.40 ± 34.66 μg g−1) and Barbera (653.78 ± 12.55 μg g−1).
Cyanidin-3-O-glucoside and petunidin-3-O-glucoside were the less abundant anthocyanins both in table and wine grape varieties. Compared to other table grapes, Red Flame was the variety showing a lower anthocyanin content.
Excluding anthocyanins, the phenolic compound content ranged between 5.94 (King's Ruby seedless) and 151.38 μg g−1 (Canner seedless) (Table 4). The total phenolic content measured by HPLC was lower than the TPC measured by Folin-Ciocalteu's assay. This difference can be due to: (1) the non-specific nature of Folin-Ciocalteu's assay, affected by some interfering molecules such as sugars, ascorbic acid, and aromatic amines, leading to a possible overestimation of the total phenolic content; and (2) other compounds could contribute to the total phenolic content, as in the case of procyanidin dimers and trimers detected by LC-MS analysis, which were relatively abundant in some white varieties (e.g. Exalta and Canner seedless, see Table 2). Unfortunately, pure compounds to quantify these molecules are not commercially available.
Fig. 2 presents HPLC-DAD and MS/MS spectra of the four most representative phenolic compounds: (A) epicatechin (B) procyanidin B2; (C) malvidin-3-O-glucoside; and (D) quercetin. Generally speaking, flavonols and flavan-3-ols were the most abundant compounds. Quercetin-3-glucuronide and epicatechin were the most frequently detected molecules, which were present mainly in white table grapes and, in particular, in the seedless varieties Canner and Centennial.
Kaempferol-3-O-glucoside was equally distributed in red and white table grapes (0.14–4.21 μg g−1 and 0.17–1.22 μg g−1, respectively). The only exception was the seedless variety Canner, where the concentration was significantly higher (21.94 ± 0.30 μg g−1).
A significant amount of ECG was present only in two white table grape samples, Sultanina and Exalta (53.87 μg g−1 and 34.50 μg g−1, respectively), while in the other varieties this compound was not detectable (<LOD 0.012 and 0.009 μg mL−1 for EC and ECG, respectively). In red varieties flavan-3-ol monomers were not quantified due to interference with anthocyanins.
DPPH assay (Trolox equivalents). Fig. 3 shows the free radical scavenging capacity measured in red and white grape samples (fresh fruit). The red wine varieties showed the highest free radical scavenging activity (10.92 ± 0.08; 7.45 ± 0.08; and 4.88 ± 0.18 mg g−1 for Albarossa, Barbera and Nerona, respectively), followed by the white grape Exalta (3.41 ± 0.04 mg g−1). Among white varieties, even Centennial seedless and King's Husainy showed a significant antioxidant activity (1.51 ± 0.01 mg g−1 and 1.24 ± 0.01 mg g−1, respectively), higher than some red varieties. The lowest content was found in the white table grape Sultanina (0.31 ± 0.002 mg g−1) and in the red table grape King's Ruby seedless (0.31 ± 0.001 mg g−1). Samples with the highest content of phenolic compounds showed the highest antioxidant activity; these parameters were highly correlated (R2 > 0.95) (not shown).
Fig. 3 DPPH radical scavenging activity (AOA, mg g−1 Trolox equivalents) and ORAC values (AUC) of grape samples obtained by analysing grapes at 400 μg mL−1.
ORAC assay. The results obtained by ORAC assay are reported in Fig. 3, where the AUC obtained by analyzing grape samples (400 μg mL−1) are compared with that of the positive control (Trolox 24 μM).
Effect on the TNFα-induced IL-8 release in human epithelial gastric cells. The sixteen grape water extracts were tested for their ability to inhibit TNFα-induced IL-8 release, a chemokine widely involved in gastric inflammation. Screening was performed in AGS cells at a concentration of 50 μg mL−1. Among white grapes included in the study, Exalta showed the highest inhibitory activity whereas Albarossa was the most active among red grapes. The extracts (50 μg mL−1) were able to decrease the IL-8 release of about 85% and 75%, respectively (Fig. 4).
Fig. 4 A: Effect of sixteen grape water extracts on the TNFα-induced IL-8 release. AGS cells were treated for 6 h with the stimulus TNFα (10 ng mL−1) and the extract at a concentration of 50 μg mL−1. IL-8 release in the culture medium was measured with an ELISA assay. The graphs show the means ± s.d. of at least 3 experiments performed in duplicate. *p < 0.05, ***p < 0.001 vs. TNFα alone. According to the literature, EGCG 20 μM was used as the reference inhibitor of IL-8 release. Effects of Exalta (B) and Albarossa (C) grape extracts (whole fruit) on the TNFα-induced IL-8 release. AGS cells were treated for 6 h with the stimulus TNFα (10 ng mL−1) and the extract at different concentrations (5–100 μg mL−1). IL-8 released in the culture medium was evaluated with an ELISA assay. The graphs show the means ± s.d. of at least 3 experiments performed in duplicate. **p < 0.01, ***p < 0.001 vs. TNFα alone. According to the literature, EGCG 20 μM was used as the reference inhibitor of IL-8 release.
On the basis of these findings, AL and EX water extracts were selected for further studies. In order to study more deeply the inhibitory activity on IL-8 release, assays were performed using AGS cells with increasing concentrations of the extracts (5–100 μg mL−1) in the presence of TNFα for 6 hours. Biological assays did not show significant variability between experiments. As shown in Fig. 4, both Exalta and Albarossa showed a great inhibitory effect on the TNFα-induced IL-8 release, with low IC50s (8.48 μg mL−1 and 6.68 μg mL−1 for Exalta and Albarossa, respectively).
Contribution of each part of the fruit to the IL-8 inhibitory effect in AGS cells. The next step of the study was to investigate the contribution of the different parts of Exalta and Albarossa grapes (skin, pulp and seeds) to the inhibitory activity observed with the whole fruit. Exalta and Albarossa from skin, pulp and seeds were prepared as reported in the Materials and methods section.
As shown in Fig. 5, the extract from skin and seeds of Exalta and Albarossa caused a dose dependent inhibition of TNFα-induced IL-8 secretion. The extract from Exalta skin inhibited the IL-8 release with an IC50 about 5-fold lower than that from Albarossa skin (IC50: 9.77 μg mL−1 and 51.47 μg mL−1, respectively), while the IC50 of the extract from Exalta seeds was about 2-fold that from Albarossa seeds (IC50: 8.31 μg mL−1 and 4.58 μg mL−1, respectively). These results could be partially explained by the high amounts of anthocyanins in Albarossa skin (Table 3) and flavonols in Exalta, seeds (Tables 2 and 4). Both anthocyanins and flavonols (mainly procyanidins) showed anti-inflammatory activity in previous studies, with the inhibition of the NF-κB pathway in a concentration-dependent pattern.24,25 The relative abundance of procyanidins and flavan-3-ol dimers and trimers showed by LC-MS analysis supports the hypothesis of a role of these compounds in the observed biological activity. Even though Exalta is a seedless variety, it contained little undeveloped seeds, probably derived from the incomplete parthenocarpic process. This characteristic could contribute to the particular abundance of flavonols in Exalta grapes.
Fig. 5 Effects of skin, pulp and seed extracts from Exalta (A–C) and Albarossa (D–F) cultivar on the TNFα-induced IL-8 release. AGS cells were treated for 6 h with the stimulus TNFα (10 ng mL−1) and increasing concentrations of each extract (5–100 μg mL−1). IL-8 released in the culture medium was evaluated with an ELISA assay. The graphs show the means ± s.d. of at least 3 experiments performed in duplicate. **p < 0.01, ***p < 0.001 vs. TNFα alone. According to the literature, EGCG 20 μM was used as the reference inhibitor of IL-8 release.
No effect on TNFα-induced IL-8 release was observed with extracts from Exalta and Albarossa pulp, even at the highest concentration tested (100 μg mL−1).
The aim of the study was the chemical characterization of different Vitis vinifera L. (grape) varieties and the measurement of the relative free radical scavenging activity and anti-inflammatory activities.
The LC-MS analysis allowed a preliminary characterization of samples included in the study, with the identification of 67 phenolic compounds including anthocyanins, flavan-3-ols, flavonols, stilbenes and organic acids. After the screening analysis, the most abundant compounds were quantified by the HPLC-DAD method.
Among anthocyanins, malvidin-3-O-glucoside and peonidin-3-O-glucoside were the most abundant compounds in red grape samples. Proanthocyanidins were identified by MSn fragmentation; among them, dimers and trimers of flavan-3-ols were the most abundant in table grape varieties containing seeds. With regard to flavonols, quercetin and kaempferol derivatives were widely distributed in all varieties analysed. Among the water extracts tested, those from the wine variety Albarossa and the table grape Exalta inhibited significantly TNFα-induced IL-8 secretion and expression in human gastric epithelial cells. In agreement with these data, Albarossa and Exalta were particularly rich in polyphenols (7.94 ± 0.19 and 2.86 ± 0.045 mg g−1, respectively). Taking into consideration the results obtained from the chromatographic analysis, a significant contribution to the high polyphenol content of Exalta could be due to the presence of high amounts of procyanidin dimers and trimers (as shown in Table 2). The most active grapes were also among the varieties with the highest antioxidant potential (measured by DPPH assay). As for the anti-inflammatory activity, grape varieties can be classified from the most active to the least active in the following way: Albarossa > Exalta > Red Globe > Nebbiolo > King's Husainy. For the free radical scavenging activity the rank was: Albarossa > Barbera > Nebbiolo > Exalta > Red Globe. This correlation between the two biological assays suggests that the active components in grapes could counteract the oxidative stress occurring during the inflammatory process in vivo. Furthermore, the free radical scavenging capacity could be part of the mode of action of the observed anti-inflammatory activity. However, further studies are needed to investigate this aspect in more detail.
Considering the mean serving of grapes in Mediterranean countries corresponding to 200 g, it is possible to calculate that the active dose observed in the in vitro test can be easily reached at the gastric level, also taking into consideration the dilution performed by gastric fluids (about 20 mL). The effect at the gastric level observed in the in vitro test can be reasonably extrapolated at the in vivo situation, since no metabolic changes of active molecules are done at this level.
The results described in this paper show that, when consumed regularly, some varieties of table grapes could contribute significantly to the intake of active molecules and in particular of phenolic derivatives, which could counteract oxidative stress and modulate inflammatory processes in the stomach, a district so frequently involved in inflammation.
This research was supported by grants from MIUR Progetto Eccellenza.
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