UHPLC-HR-MS/MS-Guided Recovery of Bioactive Flavonol Compounds from Greco di Tufo Vine Leaves

Leaves of Vitis vinifera cv. Greco di Tufo, a precious waste made in the Campania Region (Italy), after vintage harvest, underwent reduction, lyophilization, and ultrasound-assisted maceration in ethanol. The alcoholic extract, as evidenced by a preliminary UHPLC-HR-MS analysis, showed a high metabolic complexity. Thus, the extract was fractionated, obtaining, among others, a fraction enriched in flavonol glycosides and glycuronides. Myricetin, quercetin, kaempferol, and isorhamnetin derivatives were tentatively identified based on their relative retention time and TOF-MS2 data. As the localization of saccharidic moiety in glycuronide compounds proved to be difficult due to the lack of well-established fragmentation pattern and/or the absence of characteristic key fragments, to obtain useful MS information and to eliminate matrix effect redundancies, the isolation of the most abundant extract’s compound was achieved. HR-MS/MS spectra of the compound, quercetin-3-O-glucuronide, allowed us to thoroughly rationalize its fragmentation pattern, and to unravel the main differences between MS/MS behavior of flavonol glycosides and glycuronides. Furthermore, cytotoxicity assessment on the (poly)phenol rich fraction and the pure isolated compound was carried out using central nervous system cell lines. The chemoprotective effect of both the (poly)phenol fraction and quercetin-3-O-glucuronide was evaluated.


Introduction
Food by-products and waste exploitation practices are gaining a lot of attention as these materials are an untapped but rich source for the recovery of bioactive compounds, favorably relevant for other food and feed scopes [1][2][3][4]. In fact, a consistent and recent literature highlights that valorizing agrofood wastes is not only a considerable alternative to composting, but also, and above all, a highly sustainable opportunity for obtaining added-value molecules, which could be efficaciously exploited in the nutraceutical and/or cosmeceutical sector, through an integrated approach involving multiple actors for an ecofriendly industrial development [5]. Phenols, carotenoids, and some other beneficial phytochemicals, together with pectin, are just a few examples of bioactives in agrofood wastes [6]. In particular, phenols and polyphenols, commonly found in high amounts in fruit and vegetable waste products, are broadly hypothesized to be used as natural food and drink preservatives, thanks to their ability to extend the expiration date of a product, thus delaying its rancidity and/or avoiding alteration of taste or other organoleptic characteristics [7]. Moreover, the pectin advantageous recovery makes HR-MS/MS tools, and for its cytotoxic effects. The lack of toxic effects and its ability to inhibit acetylcholinesterase enzyme, together with the observation of its richness in glucuronidated flavonols, with dissimilar fragmentation pattern in respect to that of the most common glycosides, laid the foundation for the phytochemical investigation of the extract and the purification of the most abundant compound, quercetin-O-glucuronide (GrM1). The phytochemical approach represented a useful strategy to define the flavonol glucuronides' MS/MS chemical behavior for the rapid identification of these compounds.

GrM Chemical Composition
The alcoholic extract from the leaves of Vitis vinifera cv. Greco di Tufo, as evidenced by a preliminary UHPLC-HR-MS analysis, showed a high metabolic complexity. The extract was rich in (poly)phenol, alkylphenol, glycerolipid and glycerophospholipid components ( Figure 1A). The parental extract was further fractionated by normal-phase column chromatography, using three solvents with increasing polarity. Among the fractions obtained, the alcoholic one, named GrM, was peculiarly enriched in flavonol glycosides and glycuronides (Table 1; Figure 1B). Flavonol hexuronides, not massively produced in the plant environment, are commonly described as bioconversion products of the phytochemicals taken with the diet or introduced by supplementation with less toxicity [17]. Indeed, their presence is not negligible in plants with common analytical techniques. In fact, these compounds, whose chemical structure was deeply elucidated by NMR spectroscopy, were also isolated from Vitis × Labruscana cv. 'Isabella' leaf methanol crude extract [18]; recently, their presence was suggested as part of the minor components in hemp seed oil [19].
Based on the relative retention time and the TOF-MS 2 data, five derivatives of myricetin (4)(5)(6)(7)9), three derivatives of quercetin (12)(13)(14), two derivatives of kaempferol (15,16) and two derivatives of isorhamnetin (17,18) have been tentatively identified ( Table 1). The neutral loss of 162.05, 176.03 and 308.11 Da was in accordance with hexosyl, hexuronidyl and disaccharidic derivatives of the four flavonols. In particular, the neutral loss of 308.11 Da allowed us to hypothesize, for the metabolites 12, 15 and 17, a deoxyhexose and hexose moiety, which on the basis of the relative intensity of the radical aglycone ion ([aglycone-H] •-) and [aglycone-H] -, was linked to the -OH function in C-3 of the flavonolic nucleus in 12, and to the phenolic function in C-7 in 15 and 17 ( Figure 2). The identity The parental extract was further fractionated by normal-phase column chromatography, using three solvents with increasing polarity. Among the fractions obtained, the alcoholic one, named GrM, was peculiarly enriched in flavonol glycosides and glycuronides (Table 1; Figure 1B). Flavonol hexuronides, not massively produced in the plant environment, are commonly described as bioconversion products of the phytochemicals taken with the diet or introduced by supplementation with less toxicity [17]. Indeed, their presence is not negligible in plants with common analytical techniques. In fact, these compounds, whose chemical structure was deeply elucidated by NMR spectroscopy, were also isolated from Vitis × Labruscanacv. 'Isabella' leaf methanol crude extract [18]; recently, their presence was suggested as part of the minor components in hemp seed oil [19].
Based on the relative retention time and the TOF-MS 2 data, five derivatives of myricetin (4)(5)(6)(7)9), three derivatives of quercetin (12)(13)(14), two derivatives of kaempferol (15,16) and two derivatives of isorhamnetin (17,18) have been tentatively identified ( Table 1). The neutral loss of 162.05, 176.03 and 308.11 Da was in accordance with hexosyl, hexuronidyl and disaccharidic derivatives of the four flavonols. In particular, the neutral loss of 308.11 Da allowed us to hypothesize, for the metabolites 12, 15 and 17, a deoxyhexose and hexose moiety, which on the basis of the relative intensity of the radical aglycone ion ([aglycone-H] •-) and [aglycone-H] -, was linked to the -OH function in C-3 of the flavonolic nucleus in 12, and to the phenolic function in C-7 in 15 and 17 ( Figure 2). The identity of the flavonol glycoside 12 as rutin (rutinosyl derivative of quercetin) was further estimated by comparing the retention time and fragmentation pattern with that of the reference pure compound. of the flavonol glycoside 12 as rutin (rutinosyl derivative of quercetin) was further estimated by comparing the retention time and fragmentation pattern with that of the reference pure compound. The neutral loss of 176.03 Da, corresponding to a dehydrated hexuronic acid, characterized the MS/MS spectra of metabolites 6, 13, and 16, whose deprotonated molecular ion dissociated providing the product ion [aglycone-H] -as base peak; the only exception was represented by compound 18, for which the most favourable CH3 • loss gave an abundant ion at m/z 300.0250 ( Figure 3). The neutral loss of 176.03 Da, corresponding to a dehydrated hexuronic acid, characterized the MS/MS spectra of metabolites 6, 13, and 16, whose deprotonated molecular ion dissociated providing the product ion [aglycone-H]as base peak; the only exception was represented by compound 18, for which the most favourable CH 3 • loss gave an abundant ion at m/z 300.0250 ( Figure 3).  The lack of well-established fragmentation pattern and/or the absence of characteristic key fragments make difficult the localization of the hexuronyl moiety. In fact, the main fragment detected, for example, for the abundant metabolite 13 was that corresponding to the deprotonated aglycone  The lack of well-established fragmentation pattern and/or the absence of characteristic key fragments make difficult the localization of the hexuronyl moiety. In fact, the main fragment detected, for example, for the abundant metabolite 13 was that corresponding to the deprotonated aglycone quercetin, as well as other characteristic ions of the flavonol such as those at m/z 273.0399, due to CO loss ([aglycone-28] -), and at m/z 255.0293, which showed a relative intensity of only 5.1% and 8.5%, respectively. Other characteristic fragments of quercetin were identified in the ions at m/z 151.0029 and 178.9979 corresponding, respectively, to the deprotonated A ring, released by a retro-Diels Alder mechanism, and to the product of retrocyclization on bonds 1 and 2 [20]. A similar behavior was evident for the other hexuronyl derivatives and in particular, for those of myricetin. In Figure 4, the TOF-MS 2 spectra of myricetin derivatives 5, 6 and 9 are reported; they were tentatively identified as myricetin hexoside, hexuronide and hexosyl hexuronide, respectively. It is evident that the presence of an hexuronyl moiety massively influences the fragmentation of the aglycone, impoverishing in intensity its characteristic ions.

GrM 1 Purification
To obtain useful MS information and to eliminate matrix effect redundancies, a GrM fraction aliquot underwent thin-layer chromatography, which yielded, among others, the metabolite GrM 1 (the compound 13 of the GrM mixture). The UV-Vis spectrum of the molecule confirmed the presence of a flavonol skeleton molecule ( Figure 5). In fact, flavonols, like flavones, present two major absorption peaks (λ max ) in the region between 240-280 nm (commonly referred to as band II) and between 300-380 nm (band I). This latter band favors the distinction of the two classes of flavonoids since the flavone λ max is between 304-350 nm, while that of the flavonols is between 352-385 nm. Band I is associated with the absorption of the cinnamoyl system and band II with that of the benzoyl system (ring A). Literature evidence suggests that when glucuronidation occurs at the C-3 position, a hypsochromic shift of band I of about 14-29 nm is observed, whereas the glucuronidation at the phenolic function in C-7 does not lead to variations if not minimal or void [21]. GrM 1 spectrum showed, compared to the standard quercetin, a blue shift of the band I of 16 nm and a red shift of band II of 2 nm according to the quercetin glucuronidate in C-3. www.mdpi.com/journal/molecules a hypsochromic shift of band I of about 14-29 nm is observed, whereas the glucuronidation at the phenolic function in C-7 does not lead to variations if not minimal or void [21]. GrM1 spectrum showed, compared to the standard quercetin, a blue shift of the band I of 16 nm and a red shift of band II of 2 nm according to the quercetin glucuronidate in C-3. The TOF mass spectrum of the molecule is completely superimposable to that recorded for peak 13 of the mixture (  When [aglycone -H] -ion dissociated (spectrum not shown), it provided, with relative abundance of 20%, the ion at m/z 151.0039 (calcd. 151.0337). The latter is the result of a loss of CO from the ion at m/z 178.9988 (calcd. 178.9986), whose presence, together with that of the ion at m/z 121.0300 (calcd. 121.0295), confirmed the presence of the flavonol quercetin. In fact, the two ions were attributable to the retrocyclization that is realized between the bonds 1 and 2 of the flavonol nucleus with formation of the fragments 1,2 Aand 1,2 B - (Figure 7). The loss of a CO2 unit from the ion at m/z 151.0039 provided the ion at m/z 107.0134 (calcd. 107.0139). Comparing the spectrum of the standard quercetin with that obtained by GrM1 deprotonated aglycone dissociation, the two realities were fully superimposable.  When [aglycone -H]ion dissociated (spectrum not shown), it provided, with relative abundance of 20%, the ion at m/z 151.0039 (calcd. 151.0337). The latter is the result of a loss of CO from the ion at m/z 178.9988 (calcd. 178.9986), whose presence, together with that of the ion at m/z 121.0300 (calcd. 121.0295), confirmed the presence of the flavonol quercetin. In fact, the two ions were attributable to the retrocyclization that is realized between the bonds 1 and 2 of the flavonol nucleus with formation of the fragments 1,2 Aand 1,2 B - (Figure 7). The loss of a CO 2 unit from the ion at m/z 151.0039 provided the ion at m/z 107.0134 (calcd. 107.0139). Comparing the spectrum of the standard quercetin with that obtained by GrM 1 deprotonated aglycone dissociation, the two realities were fully superimposable.
Molecules 2019, 24, x; doi: www.mdpi.com/journal/molecules abundance of 20%, the ion at m/z 151.0039 (calcd. 151.0337). The latter is the result of a loss of CO from the ion at m/z 178.9988 (calcd. 178.9986), whose presence, together with that of the ion at m/z 121.0300 (calcd. 121.0295), confirmed the presence of the flavonol quercetin. In fact, the two ions were attributable to the retrocyclization that is realized between the bonds 1 and 2 of the flavonol nucleus with formation of the fragments 1,2 Aand 1,2 B - (Figure 7).   The [aglycone−H2O−H] -ion (m/z 283.0244), detected in GrM1 TOF-MS 2 spectrum, could also represent a characteristic fragment defining the localization of the hexuronic acid in C-3 of the aglycone (Figure 8). The electronic delocalization on oxygen in C-4 could favor the formation of an enolic function, the proton abstraction with the formation of a good leaving group, whose detachment defines the formation of an anion in which there is a charge separation.

Cytotoxicity of GrM
Glucuronidated flavonoids display important health properties [22,23]. Baicalein 7-O-β-glucuronide was observed to promote wound healing and to exert antitumor activity [24,25]; quercetin 3-O-β-glucuronide is anti-inflammatory and neuroprotective, whereas 3-methoxyflavonol-4 -O-glucuronide is anti-allergenic and epicatechin glucuronide promotes vascular function. Glucuronidation greatly affects flavonoids' physiological properties, frequently their solubility and thus bioavailability. Bioactivity is differently influenced by glucuronidation and glucuronate moiety localization as it could be increased or decreased, whereas intra-and extra-cellular transport, understood as excretion, commonly increases [16]. In particular, it was reported that the oral administration of a blend of vine supplements is effective in protecting against neuropathologies and cognitive impairment that occurs with aging. Based on this evidence, the cytotoxicity of the GrM extract on the SH-SY5Y cell line was preliminarly evaluated. The choice of this cell line is based on its common use in in-vitro studies related to neurotoxicity and neurodegenerative diseases. Indeed, it is evident that primary cultures would be the best choice for this kind of investigation, as they are able to mimic the properties of neuronal cells in vivo. However, the preparation and culture of primary cells is much more challenging, especially for neuronal cells [26].
The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) test was used for this purpose. It is able to measure the capacity of the mitochondrial dehydrogenases to reduce the tetrazolium ring of MTT, yellow colored, generating a chromogenic compound, the purple formazan. Obviously, this conversion is possible only in metabolically active cells. The results of the MTT assay suggested that the extract at doses ranging from 15.6 to 62.5 µg/mL did not massively influence the activity of mitochondrial dehydrogenases and a weak inhibitory effect on mitochondrial redox activity, equal to 31% and 38%, is recorded at the doses of 93.75 and 125 µg/mL, respectively (Figure 9). The inhibition resulted was dose and time dependent and reached 62.1% of redox activity inhibition (RAI) for exposure to the highest tested dose. The lack or weak toxicity of the extract, especially at low doses, together with the chemical constitution that sees the co-presence of notoriously antioxidant molecules, led us to undertake studies evaluating the inhibitory properties of acetylcholinesterase. Acetylcholinesterase (AChE) plays an important biological role in the termination of the nerve impulse at the level of cholinergic synapses by rapid hydrolysis of its substrate, acetylcholine. Our data, although preliminary, show that the phytocomplex at 25 μg/mL inhibits the activity of the enzyme (16.8 ± 3.2%) similar to donepezil (DP; 21.7 ± 1.8), one of the drugs most commonly used to increase memory function in patients with Alzheimer's disease. The drug was tested, based on literature data, at the concentration of 3 μM, and was shown to exert an inhibition of cell proliferation of about 30% in SH-SY5Y cells [27].

Cytotoxicity of GrM1
In order to verify the effects of the pure metabolite GrM1, SH-SY5Y cells were exposed to the The inhibition resulted was dose and time dependent and reached 62.1% of redox activity inhibition (RAI) for exposure to the highest tested dose. The lack or weak toxicity of the extract, especially at low doses, together with the chemical constitution that sees the co-presence of notoriously antioxidant molecules, led us to undertake studies evaluating the inhibitory properties of acetylcholinesterase. Acetylcholinesterase (AChE) plays an important biological role in the termination of the nerve impulse at the level of cholinergic synapses by rapid hydrolysis of its substrate, acetylcholine. Our data, although preliminary, show that the phytocomplex at 25 µg/mL inhibits the activity of the enzyme (16.8 ± 3.2%) similar to donepezil (DP; 21.7 ± 1.8), one of the drugs most commonly used to increase memory function in patients with Alzheimer's disease. The drug was tested, based on literature data, at the concentration of 3 µM, and was shown to exert an inhibition of cell proliferation of about 30% in SH-SY5Y cells [27].

Cytotoxicity of GrM 1
In order to verify the effects of the pure metabolite GrM 1 , SH-SY5Y cells were exposed to the molecule and their cell viability was evaluated by the MTT test ( Figure 10A). The molecule, tested at 25, 50 and 100 µM doses, did not exert toxic effects on the activity of mitochondrial dehydrogenases. These cytoprotective effects were further confirmed in U-251 MG, one of the immortalized glial cell lines which could be used instead of primary culture systems as a model for neural cells, based on the assumption that they are more homogenous, thus providing more useful tools. Indeed, glial cells are known as supportive elements of the nervous system, providing an optimal microenvironment for neurons [28]. The SRB (sulforhodamine B) test confirmed the absence of cytotoxicity ( Figure 10B) and the weak proliferative effect was also verified by microscopic morphological change analysis ( Figure 11). The SRB (sulforhodamine B) test confirmed the absence of cytotoxicity ( Figure 10B) and the weak proliferative effect was also verified by microscopic morphological change analysis ( Figure 11).
Furthermore, after treating SH-SY5Y cells with GrM 1 at 50 µM concentration, in a preliminary cell metabolomics scenario, the extraction of the cell pellet with a solution of MeCN:H 2 O (1:1, v:v), after appropriate quenching and extraction operation, highlighted a peak at m/z 477.0696, whose TOF-MS 2 spectrum was super-imposable to that of GrM 1 (Figure 12).
The data obtained were in agreement with the results previously reported in literature, according to which the bioconversion of quercetin and rutin in the glucuronidate derivatives is accompanied, in the HL-60 leukemic cells [29], by a complete elimination of the toxic effect commonly ascribed to the most common flavonols and preservation of their structural entity in the intracellular environment. The SRB (sulforhodamine B) test confirmed the absence of cytotoxicity ( Figure 10B) and the weak proliferative effect was also verified by microscopic morphological change analysis (Figure 11).  Furthermore, after treating SH-SY5Y cells with GrM1 at 50 μM concentration, in a preliminary cell metabolomics scenario, the extraction of the cell pellet with a solution of MeCN:H2O (1:1, v:v), after appropriate quenching and extraction operation, highlighted a peak at m/z 477.0696, whose TOF-MS 2 spectrum was super-imposable to that of GrM1 ( Figure 12). The data obtained were in agreement with the results previously reported in literature, according to which the bioconversion of quercetin and rutin in the glucuronidate derivatives is accompanied, in the HL-60 leukemic cells [29], by a complete elimination of the toxic effect commonly ascribed to the most common flavonols and preservation of their structural entity in the intracellular environment.

Plant Extraction and Fractionation
Leaves of Vitis vinifera cv. Greco di Tufo were collected in Montefusco (Avellino, Italy) in October 2017; the leaves were freeze-dried for 3 days using the FTS-System Flex-dry™ instrument (SP Scientific, Stone Ridge, NY, USA). Cryo-dried leaves were pulverized, using a rotary knife homogenizer and a sample (386.8 g) underwent solid-liquid extraction by maceration using ethanol as extracting solvent. Three extraction cycles (24 h each) were performed at 4 °C in the complete absence of light in order to obtain complete recovery of the metabolic content from Vitis vinifera cv. Greco di Tufo leaves. At the end of each cycle, the sample was filtered and the extraction solvent was removed using a rotary evaporator (Heidolph Hei-VAP Advantage, Schwabach,Germany). Ethanol

Plant Extraction and Fractionation
Leaves of Vitis vinifera cv. Greco di Tufo were collected in Montefusco (Avellino, Italy) in October 2017; the leaves were freeze-dried for 3 days using the FTS-System Flex-dry™ instrument (SP Scientific, Stone Ridge, NY, USA). Cryo-dried leaves were pulverized, using a rotary knife homogenizer and a sample (386.8 g) underwent solid-liquid extraction by maceration using ethanol as extracting solvent. Three extraction cycles (24 h each) were performed at 4 • C in the complete absence of light in order to obtain complete recovery of the metabolic content from Vitis vinifera cv. Greco di Tufo leaves. At the end of each cycle, the sample was filtered and the extraction solvent was removed using a rotary evaporator (Heidolph Hei-VAP Advantage, Schwabach, Germany). Ethanol extract was further fractionated

UHPLC-HR-MS and UV-Vis Analyses
GrM and GrM1 fractions, placed in vials at a concentration of 10 mg/mL in pure methanol UHPLC grade, were analysed by the Shimadzu NEXERA UHPLC system and the Omega Luna C18 column (50 × 2.1 mm, 1.6 μm). The mobile phase consisted of a binary solution A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile. A linear gradient was used for the analysis: 0-5 min, 5 → 15% B; 5-10 min, 15% B; 10-12 min, 15 → 17.5% B; 12-15 min, 17.5 → 45% B; 15-16.50 min, 45% B; 16.50-16.51 min, 45 → 5% B; 16.51-18.00 min, 5% B. The injection volume was 2.0 μL and the flow was set at 0.5 mL/min. MS analysis was performed using the AB SCIEX TripleTOF 4600 (AB Sciex, Concord, ON, Canada) system with a DuoSpray ion source operating in negative electrospray ionization. The APCI (Atmospheric Pressure Chemical Ionization) probe of the source was used for fully automatic mass calibration using the calibrant delivery system (CDS). CDS injects a calibration solution matching polarity of ionization and calibrates the mass axis of the TripleTOF ® system in all the scan functions used (MS or MS/MS). Data were collected by information-dependent acquisition (IDA) using a TOF-MS survey scan of 100-1500 Da (250 ms accumulation time) and eight dependent TOF-MS/MS scans of 80-1300 Da (100 ms accumulation time), using a collision energy (CE) of 35 V with a collision energy spread (CES) of 25 V. The following parameter settings were also used: declustering potential (DP), 70 V; ion-spray voltage, -4500 V; ion source heater, 600 °C; curtain gas, 35 psi; ion source gas, 60 psi. Data processing was performed using the PeakView ® -Analyst ® TF 1.7 Software.
UV-Vis spectrum of GrM1, as well as those of the pure reference compounds quercetin and morin, were acquired in the range 200-600 nm by a Shimadzu UV-1700 double beam spectrophotometer (Kyoto, Japan).

Cell Culture and Cytotoxicity Assessment
Tests assessing cell viability and mitochondrial activity were performed to monitor the cytotoxic potential of the GrM fraction from Vitis vinifera cv. Greco di Tufo and of the purified GrM1. For this purpose, a stock solution of the two samples was prepared. Recorded activities were compared to an untreated blank arranged in parallel to the samples. Results are the mean ± SD values.
Human neuroblastoma cell line SH-SY5Y and glioma cell line U-251 MG were cultured in DMEM (Dulbecco's Modified Eagle Medium) medium supplemented with 10% fetal bovine serum, 50.0 U/mL of penicillin and 100.0 μg/mL of streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.

UHPLC-HR-MS and UV-Vis Analyses
GrM and GrM 1 fractions, placed in vials at a concentration of 10 mg/mL in pure methanol UHPLC grade, were analysed by the Shimadzu NEXERA UHPLC system and the Omega Luna C18 column (50 × 2.1 mm, 1.6 µm). The mobile phase consisted of a binary solution A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile. A linear gradient was used for the analysis: 0-5 min, 5 → 15% B; 5-10 min, 15% B; 10-12 min, 15 → 17.5% B; 12-15 min, 17.5 → 45% B; 15-16.50 min, 45% B; 16.50-16.51 min, 45 → 5% B; 16.51-18.00 min, 5% B. The injection volume was 2.0 µL and the flow was set at 0.5 mL/min. MS analysis was performed using the AB SCIEX TripleTOF 4600 (AB Sciex, Concord, ON, Canada) system with a DuoSpray ion source operating in negative electrospray ionization. The APCI (Atmospheric Pressure Chemical Ionization) probe of the source was used for fully automatic mass calibration using the calibrant delivery system (CDS). CDS injects a calibration solution matching polarity of ionization and calibrates the mass axis of the TripleTOF ® system in all the scan functions used (MS or MS/MS). Data were collected by information-dependent acquisition (IDA) using a TOF-MS survey scan of 100-1500 Da (250 ms accumulation time) and eight dependent TOF-MS/MS scans of 80-1300 Da (100 ms accumulation time), using a collision energy (CE) of 35 V with a collision energy spread (CES) of 25 V. The following parameter settings were also used: declustering potential (DP), 70 V; ion-spray voltage, -4500 V; ion source heater, 600 • C; curtain gas, 35 psi; ion source gas, 60 psi. Data processing was performed using the PeakView ® -Analyst ® TF 1.7 Software.
UV-Vis spectrum of GrM 1, as well as those of the pure reference compounds quercetin and morin, were acquired in the range 200-600 nm by a Shimadzu UV-1700 double beam spectrophotometer (Kyoto, Japan).

Cell Culture and Cytotoxicity Assessment
Tests assessing cell viability and mitochondrial activity were performed to monitor the cytotoxic potential of the GrM fraction from Vitis vinifera cv. Greco di Tufo and of the purified GrM 1 . For this purpose, a stock solution of the two samples was prepared. Recorded activities were compared to an untreated blank arranged in parallel to the samples. Results are the mean ± SD values.
Human neuroblastoma cell line SH-SY5Y and glioma cell line U-251 MG were cultured in DMEM (Dulbecco's Modified Eagle Medium) medium supplemented with 10% fetal bovine serum, 50.0 U/mL of penicillin and 100.0 µg/mL of streptomycin at 37 • C in a humidified atmosphere containing 5% CO 2 .