*2.4. Cytotoxicity of GrM<sup>1</sup>*

In order to verify the effects of the pure metabolite GrM1, 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]. **2019**, *24*, x 11 of 17

**Figure 10.** GrM1 cytotoxicity on SH-SY5Y cells and U-251 MG after 48 h exposure time. Mitochondrial redox activity (RA, %) was from MTT test data (**A**), whereas cell viability (CV, %) was from sulforhodamine B (SRB) test (**B**). Values are reported as mean ± SD of measurements carried out on 3 **Figure 10.** GrM<sup>1</sup> cytotoxicity on SH-SY5Y cells and U-251 MG after 48 h exposure time. Mitochondrial redox activity (RA, %) was from MTT test data (**A**), whereas cell viability (CV, %) was from sulforhodamine B (SRB) test (**B**). Values are reported as mean ± SD of measurements carried out on 3 samples (n = 3) analyzed 12 times.

samples (n = 3) analyzed 12 times. 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<sup>1</sup> 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<sup>2</sup> spectrum was super-imposable to that of GrM<sup>1</sup> (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.

*Molecules* **2019**, *24*, x; doi: www.mdpi.com/journal/molecules

**Figure 11.** Morphological analysis of cells treated with GrM1 pure compound after 48 h exposure time.

samples (n = 3) analyzed 12 times.

**Figure 10.** GrM1 cytotoxicity on SH-SY5Y cells and U-251 MG after 48 h exposure time. Mitochondrial redox activity (RA, %) was from MTT test data (**A**), whereas cell viability (CV, %) was from sulforhodamine B (SRB) test (**B**). Values are reported as mean ± SD of measurements carried out on 3

The SRB (sulforhodamine B) test confirmed the absence of cytotoxicity (Figure 10B) and the weak

**Figure 11.** Morphological analysis of cells treated with GrM1 pure compound after 48 h exposure **Figure 11.** Morphological analysis of cells treated with GrM<sup>1</sup> pure compound after 48 h exposure time. TOF-MS2 spectrum was super-imposable to that of GrM1 (Figure 12).

**Figure 12.** XIC (Extracted ion chromatogram) and TOF-MS2 spectrum of the ion at *m*/*z* 477.0696 from cell pellet extract**. Figure 12.** XIC (Extracted ion chromatogram) and TOF-MS<sup>2</sup> spectrum of the ion at *m*/*z* 477.0696 from cell pellet extract.

#### The data obtained were in agreement with the results previously reported in literature, **3. Materials and Methods**

HR-MS.

#### 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 *3.1. Plant Extraction and Fractionation*

ascribed to the most common flavonols and preservation of their structural entity in the intracellular environment. **3. Materials and Methods**  *3.1. 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 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 by flash column chromatography (FCC) on Merck Kieselgel 60 (40–63 µm) silica gel under pure N<sup>2</sup> pressure (h = 20 cm, Ø = 4.0 cm), eluting first with CHCl3, then with a CHCl3:EtOAc solution (1:1, *v*/*v*), subsequently with pure EtOAc, and finally with pure MeOH. The alcoholic fraction (20.63 g) was further chromatographed on Amberlite XAD-4 (h = 70 cm, Ø = 4.0 cm) eluting with water first (GrW) and then with MeOH (GrM). GrM fraction was analyzed by UHPLC-HR-MS.

removed using a rotary evaporator (Heidolph Hei-VAP Advantage, Schwabach,Germany). Ethanol extract was further fractionated by flash column chromatography (FCC) on Merck Kieselgel 60 (40– 63 μm) silica gel under pure N2 pressure (h = 20 cm, Ø = 4.0 cm), eluting first with CHCl3, then with a CHCl3:EtOAc solution (1:1, *v*/*v*), subsequently with pure EtOAc, and finally with pure MeOH. The An aliquot of GrM fraction (36 mg) was chromatographed by thin-layer chromatography (TLC) using a precoated silica gel 60 F254 (20 × 20 cm, 1 mm, Merck, Darmstadt, Germany) and eluting with EtOAc:MeOH:H2O:HCOOH (16:2:1:1) solution. GrM<sup>1</sup> (14.6 mg) was thus obtained and analyzed by UV–Vis and UHPLC-HR-MS. The fractionation scheme is depicted in Figure 13.

UV–Vis and UHPLC-HR-MS. The fractionation scheme is depicted in Figure 13.

An aliquot of GrM fraction (36 mg) was chromatographed by thin-layer chromatography (TLC) using a precoated silica gel 60 F254 (20 × 20 cm, 1 mm, Merck, Darmstadt, Germany) and eluting with EtOAc:MeOH:H2O:HCOOH (16:2:1:1) solution. GrM1 (14.6 mg) was thus obtained and analyzed by

alcoholic fraction (20.63 g) was further chromatographed on Amberlite XAD-4 (h = 70 cm, Ø = 4.0 cm)

*Molecules* **2019**, *24*, x; doi: www.mdpi.com/journal/molecules

*Molecules* **2019**, *24*, x 13 of 17

**Figure 13.** Extraction and fractionation scheme (FCC = flash column chromatography; TLC = thinlayer chromatography). **Figure 13.** Extraction and fractionation scheme (FCC = flash column chromatography; TLC = thin-layer chromatography).

#### *3.2. UHPLC-HR-MS and UV–Vis Analyses 3.2. 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 GrM and GrM<sup>1</sup> 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.

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 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).

#### spectrophotometer (Kyoto, Japan). *3.3. Cell Culture and Cytotoxicity Assessment*

containing 5% CO2.

*3.3. 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 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.

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, 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.

50.0 U/mL of penicillin and 100.0 μg/mL of streptomycin at 37 °C in a humidified atmosphere

3.3.1. MTT (3-(4, 5-Dimethylthiazolyl-2)-2,5-Diphenyltetrazolium Bromide) Cell Viability Test

### 3.3.1. MTT (3-(4, 5-Dimethylthiazolyl-2)-2,5-Diphenyltetrazolium Bromide) Cell Viability Test

Cells were seeded in 96-multiwell plates at a density of 1.0 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well. After 24 h, cells were treated with different doses of the GrM extract (15.625, 31.25, 62.5, 93.75, 125.0, 187.5 and 250.0 µg/mL) and pure GrM<sup>1</sup> metabolite (25, 50 and 100 µM) in a culture medium for 48 h. At the end of incubation, MTT (150 µL; 0.50 mg/mL in culture medium) was added. After 1 h at 37 ◦C in a 5% CO<sup>2</sup> humidified atmosphere, MTT solution was removed and formazan was dissolved in dimethyl sulfoxide (DMSO) (100 µL). The absorbance at 570 nm was determined using a Tecan Spectra Fluor fluorescence and absorbance reader. Mitochondrial redox activity inhibition (RAI, %) was calculated using the following formula: [(Absuntreated cells)−(Abstreated cells)/(Absuntreated cells)] × 100, where Abs stands for absorbance [26].

#### 3.3.2. SRB (Sulforhodamine B) Cell Viability Test

Cells were seeded in 96-multiwell plates at a density of 1.0 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well. After 24 h, cells were treated with pure GrM<sup>1</sup> metabolite (25, 50 and 100 µM) for 48 h. At the end of incubation, cells were fixed with ice-cold trichloroacetic acid (TCA) (10% *w*/*v*, 40 µL) for 1 h at 4 ◦C. The plates were washed five times in distilled water and allowed to dry. Then, sulforhodamine B (SBR; 50 µL, 0.4% *w*/*v* in 1% aqueous acetic acid) was added to each well and incubated at room temperature for 30 min. In order to remove unbound dye, the plates were quickly washed with 1% aqueous acetic acid and dried subsequently. The bound SRB was solubilized by adding 100 µL of 10 mM unbuffered Tris base (pH 10.5) to each well and shaking for 5 min on a shaker platform. Finally, the absorbance at 570 nm of each well was measured using a Tecan SpectraFluor fluorescence and absorbance reader. Cell viability inhibition (CVI, %) was determined using the following formula: [(Absuntreated cells)–(Abstreated cells)/(Absuntreated cells)]×100, where Abs stands for absorbance [26,30].

#### 3.3.3. Anti-Acetylcholinesterase (AChE) Activity Assay

SH-SY5Y cells were treated with GrM at a dose level equal to 25 µg/mL and then a colorimetric test was carried out using an acetylcholinesterase assay kit (Colorimetric) (Abcam, UK) in accordance with the manufacturer's instructions. The reaction was followed spectrophotometrically by the increase in absorbance at 412 nm. Donepezil (3 µM) was used as a positive standard [31].

#### 3.3.4. Cell Metabolomic Analysis

The SH-SY5Y and U-251 MG cells (2.5 <sup>×</sup> <sup>10</sup><sup>6</sup> cells in Petri dish) were treated with the metabolite GrM<sup>1</sup> for 48 h. At the end of the exposure period, the cells were first quenched in NaCl 0.9% in order to stop the metabolic activities. The cells were scraped and collected in Eppendorf tubes, centrifuged and extracted using 1.0 mL of a cold MeCN:H2O (1:1, *v:v*) solution. The extract was dried and reconstituted for UHPLC-HR-MS analysis [32].

#### **4. Conclusions**

In the present work the possibility of recovering bioactive components from the leaves of *Vitis vinifera* cv. Greco di Tufo was investigated. The employed extractive and fractionation procedures favored the preparation of an extract enriched in flavonol hexuronides variously oxygenated at the B-ring level.

The UHPLC-HR-MS/MS characterization of these molecules showed that the analytical protocol can be used for their rapid identification. The absence of cytotoxicity of the GrM and GrM<sup>1</sup> extracts opens up new investigations at the cellular level aimed at ascertaining their neuroprotective potential for the functional recovery of a precious waste made in the Campania Region (Italy).

**Author Contributions:** Conceptualization, M.P. and S.P. (Severina Pacifico); methodology, S.P. (Severina Pacifico), S.P. (Simona Piccolella), and G.C.; formal analysis, S.P. (Simona Piccolella) and G.C.; data curation, S.P. (Simona Piccolella) and S.P. (Severina Pacifico); writing—original draft preparation, M.G.V., M.P., S.P. (Simona Piccolella), and S.P. (Severina Pacifico); writing—review and editing, S.P. (Severina Pacifico) and S.P. (Simona Piccolella); supervision, S.P. (Severina Pacifico).

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflicts of interest.
