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

**Simona Piccolella <sup>1</sup> , Giuseppina Crescente <sup>1</sup> , Maria Grazia Volpe <sup>2</sup> , Marina Paolucci 2,3 and Severina Pacifico 1,\***


Received: 20 September 2019; Accepted: 6 October 2019; Published: 8 October 2019

**Abstract:** 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-MS<sup>2</sup> 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.

**Keywords:** food waste recovery; grape leaves; UHPLC-HR-MS/MS analysis; flavonol glycuronides recovery

#### **1. 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 [1–4]. 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

the molecule largely exploitable as a gelling agent in pastry or as a fat replacement in meat products, or a binder in animal feed [8,9].

Considering that grape cultivation is one of the main agro-economic activities worldwide, with over 60 million tons produced globally every year [10], and that the entire wine production chain includes not only the production of grapes, their processing, and marketing, but also a large amount of wastes, it is reasonable to hypothesize fruitful recycle processes that go far beyond those already involved in the transformation of a grape waste part (such as that destined for distilleries) [11]. In fact, although during the wine-making process, different by-products are generated and found to be valuable for alternative use in a new production cycle (e.g., stems, grape-marc, and grapeseed) [12], waste and effluents, normally rich in sugars, proteins, fibres, and lipids as well as vitamins and other bioactive compounds, are also generated. Thus, they could represent an ideal source for obtaining chemicals and pharmaceuticals with high added value, as well as for creating biomaterials and substrates that can be used in different biological processes [13].

However, at the national and international level, an integrated approach, that allows a complete recovery of oenological wastes through the development of efficient and sustainable technologies from an economic and environmental point of view, leading to the final production of different products with standard features and demonstrating applications in specific sectors, is still lacking. Although marc and grapeseed already have an acclaimed use, even the leaves of the vine can be considered as the stalk, a curious waste to be recovered in the vine and wine industry [14]. Leaves, which unlike dregs, marc (skins and grape seeds), and stalks, are not included in the Italian Ministerial Decree from 27 November 2008 as renewable by-products, are waste material whose disposal during fruit harvesting is massive, not only in the early stages of winemaking, but mainly in destemming. Pre-bloom leaf removal, which consists of removing all or part of the leaves from the fruitful area in the period from spring to late season, is a practice commonly used with the aim of improving the quality of the harvest. On the other hand, countries such as Florida, Greece, and many Middle Eastern countries use the cultivation of vines also for the production of leaves used in kitchens for the preparation of typical dishes (e.g., the Arabian warak enab dish) in the knowledge that, like fruits, they contain numerous substances beneficial to humans such as organic acids, vitamins, and stilbenes [15]. The vine leaves are also rich in anthocyanins and tannins with vasoactive and vasoprotective properties and which have the ability to stimulate vascularization [16].

In this context and with the aim to exploit the recovery of bioactive molecules from wine production wastes, leaves of *Vitis vinifera* cv. Greco di Tufo, collected in the Campania Region (Italy), were considered. The production of this wine with the denomination of controlled and guaranteed origin represents a great resource of the territory. This ancient vine, whose name derives from the characteristic shape of the bunches, with grapes grouped in pairs, was introduced by the Greeks along the Tyrrhenian coasts. The geographical environment favors the production of this prized wine with a characteristic flavor.

Leaves were extracted through maceration and the alcoholic extract obtained was further fractionated. The phytoextract chemical composition was unravelled by UHPLC-HR-MS/MS analysis. The powerful analytical tool was thoroughly investigated for achieving suitable and valid information on the spectral behavior of Greco di Tufo leaf metabolites. The extract, and the most abundant compound isolated therefrom, were further evaluated for their potential cytotoxicity in SH-SY5Y neuroblastoma and U-251 MG glioblastoma cell lines.

#### **2. Results and Discussion**

The valorization of Greco di Tufo vine leaves, an unexplored source of bioactive molecules, took advantage of the design of a green and sustainable extraction method, pursued using ethanol, the most common biosolvent. The fraction GrM was broadly analyzed for its bioactives using UHPLC-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. 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.

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acetylcholinesterase enzyme, together with the observation of its richness in glucuronidated

#### *2.1. 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). *2.1. 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).

**Figure 1.** TIC (Total Ion Chromatogram) of (**A**) EtOH extract and (**B**) GrM fraction from Greco di Tufo vine leaves. **Figure 1.** TIC (Total Ion Chromatogram) of (**A**) EtOH extract and (**B**) GrM fraction from Greco di Tufo vine leaves.

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]. 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-MS2 data, five derivatives of myricetin (**4**–**7**, **9**), three derivatives of quercetin (**12**–**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 Based on the relative retention time and the TOF-MS<sup>2</sup> data, five derivatives of myricetin (**4**–**7**, **9**), three derivatives of quercetin (**12**–**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.

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134

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**Table 1.** *Cont*.

**Figure 2.** Flavonol rutinosyl derivatives; TOF-MS2 spectrum of metabolites (**A**) **12**; (**B**) **15**; and (**C**) **17. Figure 2.** Flavonol rutinosyl derivatives; TOF-MS<sup>2</sup> spectrum of metabolites (**A**) **12**; (**B**) **15**; and (**C**) **17.**

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<sup>3</sup> • *Molecules* **2019**, *24*, x 5 of 17 loss gave an abundant ion at *m*/*z* 300.0250 (Figure 3).

*Molecules* **2019**, *24*, x; doi: www.mdpi.com/journal/molecules . **Figure 3. (A**) Extracted ion chromatograms (XICs) of hexuronyl flavonols, whose structure is depicted **Figure 3.** (**A**) Extracted ion chromatograms (XICs) of hexuronyl flavonols, whose structure is depicted in the grey box; TOF-MS<sup>2</sup> spectra of (**B**) **16** and (**C**) **18**.

in the grey box; TOF-MS2 spectra of (**B**) **16** and (**C**) **18**.

intensity its characteristic ions.

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-MS2 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

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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<sup>2</sup> 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. *Molecules* **2019**, *24*, x 6 of 17

**Figure 4.** TOF-MS2 spectra of metabolites (**A**) **5**; (**B**) **6**; and (**C**) **9***.* **Figure 4.** TOF-MS<sup>2</sup> spectra of metabolites (**A**) **5**; (**B**) **6**; and (**C**) **9**.
