*2.2. GrM<sup>1</sup> 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<sup>1</sup> (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 (λmax) 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 λmax 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<sup>1</sup> 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.

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

(MOR) flavonol isomers**.** 

superimposable.

reported.

*2.2. GrM1 Purification* 

band II of 2 nm according to the quercetin glucuronidate in C-3.

e− 2CO − H]– (*m*/*z* 245.0450), all with an intensity lower than 10%.

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 GrM1 (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 (λmax) 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 λmax 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]. GrM1 spectrum showed, compared to the standard quercetin, a blue shift of the band I of 16 nm and a red shift of

**Figure 5.** Comparison of the UV–Vis spectra of the metabolite GrM1 and of quercetin (QUE) and morin **Figure 5.** Comparison of the UV–Vis spectra of the metabolite GrM<sup>1</sup> and of quercetin (QUE) and morin (MOR) flavonol isomers.

The TOF mass spectrum of the molecule is completely superimposable to that recorded for peak **13** of the mixture (Figure 6) with the deprotonated molecular ion at *m*/*z* 477.0685, the ion [2M − H]– at *m*/*z* 955.1420 and, again, the ion [aglycone − H]– at *m*/*z* 301.0355. The TOF-MS2 spectrum of the ion at *m*/*z* 477.0685 provided the ion [aglycone − H]– as base peak and the ions [aglycone – H2O – H]– (*m*/*z* 283.0244), [aglycone − CO − H]– (*m*/*z* 273.0399), [aglycone – CO − H2O − H]– (*m*/*z* 255.0293) and [aglycon The TOF mass spectrum of the molecule is completely superimposable to that recorded for peak **<sup>13</sup>** of the mixture (Figure 6) with the deprotonated molecular ion at *<sup>m</sup>*/*<sup>z</sup>* 477.0685, the ion [2M <sup>−</sup> H]– at *<sup>m</sup>*/*<sup>z</sup>* 955.1420 and, again, the ion [aglycone <sup>−</sup> H]– at *<sup>m</sup>*/*<sup>z</sup>* 301.0355. The TOF-MS<sup>2</sup> spectrum of the ion at *<sup>m</sup>*/*<sup>z</sup>* 477.0685 provided the ion [aglycone <sup>−</sup> H]– as base peak and the ions [aglycone – H2O – H]– (*m*/*<sup>z</sup>* 283.0244), [aglycone <sup>−</sup> CO <sup>−</sup> H]– (*m*/*<sup>z</sup>* 273.0399), [aglycone – CO <sup>−</sup> <sup>H</sup>2<sup>O</sup> <sup>−</sup> H]– (*m*/*z* 255.0293) and [aglycon e<sup>−</sup> 2CO <sup>−</sup> H]– *Molecules*  (*m*/*z* 245.0450), all with an intensity lower than 10%. **2019**, *24*, x 8 of 17

**Figure 6. (A**) TOF-MS and (**B**) TOF-MS2 spectra of the quercetin-3-*O*-glucuronide. (**C**) Quercetin-3-*O*glucoside TOF-MS2 spectrum. **Figure 6. (A**) TOF-MS and (**B**) TOF-MS<sup>2</sup> spectra of the quercetin-3-*O*-glucuronide. (**C**) Quercetin-3-*O*glucoside TOF-MS<sup>2</sup> spectrum.

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 1,2A– and 1,2B– (Figure 7). The loss of a CO2 unit from the ion at *m*/*z* 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 1,2A– and 1,2B – (Figure 7). The loss of a CO<sup>2</sup> unit from the ion at *m*/*z* 151.0039 provided

OH O

*m/ z* 301.0354



HO O OH

1 2

OH

OH


retrocyclization

HO O

OH

*m/ z* 151.0037

HO

OH

*m/ z* 107.0139

O

O





*m/ z* 178.9986

OH O

HO <sup>O</sup>

O

OH

+ O

OH OH OH

176.0321 Da

<sup>+</sup> OH


*m/ z* 121.0295

**1,2A- 1,2B-**

O

O

OH OH OH

OH

OH

*m/ z* 229.0506

HO

OH


OH


OH O


OH

HO O OH

*m/ z* 273.0405

HO O OH

O

*m/ z* 477.0675

OH O

OH O

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

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

**Figure 7.** Proposed fragmentation pathway of GrM1 [aglycone – H]– ion. Theoretical *m*/*z* values are

*2.3. Cytotoxicity of GrM* 

9).

glucoside TOF-MS2 spectrum.

the ion at *m*/*z* 107.0134 (calcd. 107.0139). Comparing the spectrum of the standard quercetin with that obtained by GrM<sup>1</sup> deprotonated aglycone dissociation, the two realities were fully superimposable. quercetin with that obtained by GrM1 deprotonated aglycone dissociation, the two realities were fully superimposable.

151.0039 provided the ion at *m*/*z* 107.0134 (calcd. 107.0139). Comparing the spectrum of the standard

**Figure 6. (A**) TOF-MS and (**B**) TOF-MS2 spectra of the quercetin-3-*O*-glucuronide. (**C**) Quercetin-3-*O*-

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

*Molecules* **2019**, *24*, x 8 of 17

**Figure 7.** Proposed fragmentation pathway of GrM1 [aglycone – H]– ion. Theoretical *m*/*z* values are reported. **Figure 7.** Proposed fragmentation pathway of GrM<sup>1</sup> [aglycone – H]– ion. Theoretical *m*/*z* values are reported. *Molecules* **2019**, *24*, x 9 of 17

*Molecules* **2019**, *24*, x; doi: www.mdpi.com/journal/molecules The [aglycone−H2O−H]– ion (*m*/*z* 283.0244), detected in GrM<sup>1</sup> TOF-MS<sup>2</sup> 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. The [aglycone−H2O−H]– ion (*m*/*z* 283.0244), detected in GrM1 TOF-MS2 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.

**Figure 8.** Proposed mechanism for the formation of the ion at *m*/*z* 283.0249. The calculated *m*/*z* value is reported (error < 5 ppm). **Figure 8.** Proposed mechanism for the formation of the ion at *m*/*z* 283.0249. The calculated *m*/*z* value is reported (error < 5 ppm).

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

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

primary cells is much more challenging, especially for neuronal cells [26].

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

#### *2.3. 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-40 -*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). *Molecules* **2019**, *24*, x 10 of 17

**Figure 9.** Redox mitochondrial inhibition (RAI%) of GrM extract towards SH-SY5Y cell line at 48 h exposure time. Values are reported as mean ± SD of measurements carried out on 3 samples (n = 3) **Figure 9.** Redox mitochondrial inhibition (RAI%) of GrM extract towards SH-SY5Y cell line at 48 h exposure time. Values are reported as mean ± SD of measurements carried out on 3 samples (n = 3) analyzed 12 times.

analyzed 12 times. 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 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].

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

inhibition of cell proliferation of about 30% in SH-SY5Y cells [27].

*2.4. Cytotoxicity of GrM1* 

for neurons [28].

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

*Molecules* 
