Characterization of Non-Anthocyanic Flavonoids in Some Hybrid Red Grape Extracts Potentially Interesting for Industrial Uses

Previous studies showed that hybrid grapes often have qualitatively and quantitatively higher polyphenolic contents than the common V. vinifera grape varieties. In general, these compounds are studied for grape chemotaxonomy and for nutraceutical purposes due to their relevant antioxidant activity. Non-anthocyanic flavonoid composition of five red hybrid grape varieties produced by crossing of V. vinifera, V. aestivalis, V. cinerea, V. berlandieri, V. labrusca, V. lincecumii, and V. rupestris were studied by liquid chromatography/high-resolution mass spectrometry. Thirty-one compounds were identified, including methylnaringenin, a tetrahydroxy-dimethoxyflavanone-hexoside, two flavonols (quercetin and a pentahydroxyflavone isomer), 20 glycoside flavonols (four quercetin, two myricetin, two kaempferol, three isorhamnetin, one laricitrin, two syringetin, one kaempferide and two dihydroflavonol derivatives; myricetin-glucoside-glucuronide; myricetin-diglucoside; syringetin-dihexoside), three flavan-3-ols (−)-epicatechin, (+)-catechin, (−)-epicatechin gallate) and four proantocyanidins (procyanidin B1, procyanidin B2, procyanidin B3 or B4/B5, procyanidin T2 or T3/T4/C1). Seibel 19881, Seyve Villard 12-347 and Seyve Villard 29-399 were particularly rich in polyphenols. These findings emphasize that these grapes are especially interesting for the production of antioxidant extracts for nutraceutical and pharmaceutical uses.


Introduction
The health benefits of grape and wine consumption are mainly associated with their polyphenolic content (the so-called French paradox [1]). Polyphenols are antioxidant compounds that act as reducing agents by donating hydrogen, acting as chelators, quenching singlet oxygen and by trapping free radicals [2]. Moreover, their activity in reducing coronary heart diseases, atherosclerosis [3,4], various types of cancer, and dermal disorders, was demonstrated [5,6].
Flavonols are located in the skin where are involved in the shielding from UV rays [13]. Principal are myricetin, quercetin and kaempferol mainly present as glucoside and glucuronide derivatives [14]. As well as glucoside, laricitrin, isorhamnetin and syringetin were found in grape in galactoside and glucuronide forms [15][16][17]. Flavonols are antioxidant and bioactive compounds present in dietary plants used for human nutrition [18]. For example, in vitro studies showed quercetin inhibits human platelet aggregation and acts as potential anticancer by inhibiting carcinogens and cancer cell growth in many experimental and human tumors [19,20].
Tannins are divided in condensed and hydrolysable. Condensed tannins (proanthocyanidins) are flavan-3-ol polymers mainly present in the solid parts of grape (skin and seeds) and, in minor amount, in the pulp [21]. In-vivo studies showed that grape seed procyanidin extract has an antioxidant activity as important as vitamin E, by preventing oxidative damage of tissues by reduction of the lipid oxidation and/or inhibition of free radicals production [22].
Traditional analytical methods for analysis of low molecular weight (MW) flavonoids are performed by reverse-phase high performance liquid chromatography (HPLC) coupled with UV-Vis spectrophotometry or mass spectrometry (MS) [14,23]. In general, liquid chromatography/mass spectrometry (LC/MS) and multiple mass spectrometry (MS/MS and MS n ) are effective for the structural characterization [24]. LC coupled to high-resolution mass spectrometry (HRMS) has proven to be very effective in grape metabolomics, in particular for characterization of stilbene derivatives, flavonols and anthocyanins [25]. Recently, a database for grape and wine metabolomics (GrapeMetabolomics), which currently contains around 1100 putative compounds identified by HRMS, was constructed [26].
In previous studies, some hybrid grape varieties were highlighted as particularly interesting for their qualitative and quantitative anthocyanin and flavonol profiles [27,28]. In the present work, the study of more promising varieties was extended to the other non-anthocyanic flavonoids to complete their evaluation as sources of bioactive and antioxidant compounds potentially useful for industrial purposes. Polyphenols of their seed extracts were recently investigated [29], as a consequence the study was focalized on the skin/pulp extract. Characterization of compounds by ultra-high performance-liquid chromatography/quadrupole-time of flight mass spectrometry (UHPLC/QTOF) analysis, was performed.

Results and Discussion
In high-resolution MS metabolomics, the identification of the molecular formula (MF) of compounds relies on the measurement of the mono-isotopic mass and isotopic pattern (relative abundances and m/z distances) followed by the raw data processing performed by using specific algorithms [30,31]. In this study, the first "targeted" identification of compounds was performed by comparing the theoretical isotopic pattern of the metabolites present in GrapeMetabolomics with the experimental ones calculated by the algorithms using the signals measured. Then, "untargeted" data processing of the remaining raw data was performed and provided the identification of possible other compounds not included in the database. Identification of compounds was based on the [M − H] − ion isotopic pattern and confirmed by MS/MS analysis. In the five grape samples, thirty-one non-anthocyanic flavonoids reported in Table 1 were identified including nineteen flavonols, two flavanonols, three flavan-3-ols, two flavanones, one flavone and four proanthocyanidins. Among them, methylnaringenin, pentahydroxy flavone quercetin isomer, syringetin-dihexoside, and tetrahydroxy-dimethoxyflavanone-hexoside were identified by the "untargeted" method. Almost all the compounds were identified with low mass error (maximum 2.7 ppm) and identification scores >90%. Just three of them-dihydrokaempferol-3-O-rhamnoside, kaempferide-pcoumaroylhexoside, and isorhamnetin-glucuronide-had a lower identification score (73%, 80% and 79%, respectively) probably due to their low signal intensity. For glucoside and glucuronide flavonols different characteristic fragmentations, were observed: glucoside compounds showed high signal of radical aglycone ion [Y0 − H] −• instead the glucuronide derivatives had higher signal of aglycone ion Y0 − . A putative quercetin isomer was also identified: main MS/MS fragments at m/z 149.0244, which corresponds to the formula C8H5O3, and at m/z 151.0035 of ion C7H3O4, were observed (Table 1). Potentially, they form by 1,3 B − fragmentation of flavones and flavonols [32] and the putative structure proposed for the compound is shown in Figure 1.
Currently, the grape varieties studied are not used for production and just five plants of each variety are present in the CREA-VIT vine collection. As a consequence, a quantitative study really representative of the compounds produced in the grape was not possible. Anyway, a semi-quantitative analysis of the compounds was performed in order to have main information on their presence in the grape. For some compounds, such as catechin, rutin, some quercetin derivatives, high variability was observed (±50%, Table 2), mainly due to the differences between two vintages. In general, flavonols were the most abundant non-anthocyanic flavonoids identified with a total content between 25 and 93 mg/kg calculated as sum of the single compounds in Table 2 (34%-73% of the total non-anthocyanic flavonoids identified). Total flavan-3-ol content was between 9 and 27 mg/kg (11%-36% of the total non-anthocyanic flavonoids) and proanthocyanidins between 5 and 20 mg/kg (5%-27% of the non-anthocyanic flavonoids). In a previous study, high contents of flavonols were found also in other hybrid red grape varieties, such as Seibel 9280 and Seibel 6339, and the white grape Seyve-Villard 12.303 [34]. In all samples, the flavonol profile was dominated by myricetin (myricetin-3-O-glucoside between 12.4 and 41.5 mg/kg) and the highest flavanol was procyanidin B1 (between 1.5 and 9.7 mg/kg). Similarly, high contents of procyanidin B1 and myricetin-3-O-glucoside were found in the V. labrusca grape variety Bordô and in skin of the table grape varieties BRS Clara and BRS Morena [35,36]. Myricetin-3-O-glucoside was the more abundant flavonol also in the hybrid grape cultivar BRS Violeta, but in this case the main flavanol was (+)-catechin in both skin and flesh [37].

LC/QTOF Mass Spectrometry
The analytical system used was Agilent UHPLC 1290 Infinity coupled to Agilent 1290 Infinity Autosampler (G4226A) and Agilent 6540 accurate-mass Q-TOF mass spectrometer (nominal resolution 40.000) with Jet Stream Ionization source (Agilent Technologies, Santa Clara, CA, USA). For each sample, two negative ionization mode analyses were performed with full scan acquisition. After each sample a blank was run. The data acquisition software was Agilent MassHunter version B.04.00 (B4033.2). Chromatography was performed using a Zorbax reverse-phase column (RRHD SB-C18 3 mm × 150 mm, 1.8 μm) (Agilent Technologies). The mobile phase was composed of (A) 0.1% v/v aqueous formic acid; and (B) 0.1% v/v formic acid in acetonitrile. Gradient elution program: 5% B isocratic for 8 min, from 5% to 45% B in 10 min, from 45% to 65% B in 5 min, from 65% to 90% in 4 min, and 90% B isocratic for 10 min. Flow rate was 0.4 mL/min; sample injection was 10 μL; column temperature was 35 °C. QTOF conditions: sheath gas nitrogen 10 L/min at 400 °C, drying gas 8 L/min at 350 °C, nebulizer pressure 60 psi, nozzle voltage 1 kV, and capillary voltage 3.5 kV. Signals in the m/z 100-1700 range were recorded. Negative mass calibration was performed with standard mix G1969-85000 (Supelco Inc., Bellefonte, PA, USA) and had residual error for the expected masses between ±0.2 ppm. Lock masses were: TFA anion at m/z 112.9856 and HP-0921(+formate) at m/z 966.0007 in negative-ion mode.
MS/MS conditions: collision energies between 20 and 60 eV were used to fragment ions in the m/z 100-1700 range. Acquisition rate was 2 spectra/s. Data processing was performed with Agilent Mass Hunter Qualitative Analysis software version B.05.00 (5.0.519.0) (Agilent Technologies). Confidence of the compound identification was based on accurate mass and isotope pattern and was expressed by an "overall identification score" computed as a weighted average of the compound isotopic pattern signals, such as exact masses, relative abundances, and m/z distances (spacing). Weight parameters were Wmass = 100, Wabundance = 60, and Wspacing = 50; mass expected data variation 2.0 mDa + 5.6 ppm, mass isotope abundance 7.5%, and mass isotope grouping peak spacing tolerance 0.0025 m/z + 7.0 ppm.

Conclusions
Among the samples studied, Seibel 19881 and the two Seyve Villard varieties are characterized by peculiar richness of non-anthocyanic flavonoids. In particular, Seyve Villard samples have high content of flavan-3-ols, procyanidin B1 and procyanidin B2. It is worth to highlight that these compounds have protective activity towards LDL oxidation greater than standard antioxidants like trolox and ascorbic acid [38]. Seibel 19881 is rich in antioxidant compounds, such as flavonols and flavanonols, and has relevant content of high isorhamnetin derivatives.
A previous study showed that these Seyve Villard grape varieties have low anthocyanin content [27]. On the other hand, these findings emphasize that they can be interesting for extraction of non-anthocyanic antioxidant compounds. Seibel 19881 confirmed its richness of non-anthocyanic polyphenols by showing that it is particularly interesting for production of extracts for nutraceutical and pharmaceutical uses. Despite in the previous study grape Seibel 8357 showed the highest anthocyanins content, this variety is not particularly rich in non-anthocyanic flavonoids, even if is characterized by relevant syringetin content. This step of the research was to investigate the presence in grape of non-anthocyanic flavonoids potentially interesting for nutraceutical purposes. If an industrial interest should arise, separated analysis of skins and pulps will have to be performed.

Author Contributions
M.G. and R.F. designed the research; M.D.R., A.P., A.D.V. and R.F. performed the research and analyzed the data; M.D.R., A.P. and R.F. wrote the paper. All authors read and approved the final manuscript.