HRMS Profile of a Hazelnut Skin Proanthocyanidin-rich Fraction with

Article pubs.acs.org/JAFC

HRMS Profile of a Hazelnut Skin Proanthocyanidin-rich Fraction with Antioxidant and Anti-Candida albicans Activities Anna Lisa Piccinelli,† Imma Pagano,†,‡ Tiziana Esposito,†,‡ Teresa Mencherini,*,† Amalia Porta,† Anna Maria Petrone,†,‡ Patrizia Gazzerro,† Patrizia Picerno,† Francesca Sansone,† Luca Rastrelli,† and Rita Patrizia Aquino† †

Department of Pharmacy and ‡Ph.D. Program in Drug Discovery and Development, University of Salerno, Via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy S Supporting Information *

ABSTRACT: Roasted hazelnut skins (RHS) represent a byproduct of kernel industrial processing. In this research, a RHS extract (RHS-M) and its fraction RHS-M-F3 enriched in proanthocyanidins (PAs), with antioxidant activity, were characterized in terms of total phenolic compound and PA contents. RHS-M and RHS-M-F3 showed antifungal properties against Candida albicans SC5314 (MIC2 = 3.00 and 0.10 μg/mL and MIC0 = 5.00 and 0.50 μg/mL, respectively), determined by the microbroth dilution method and Candida albicans morphological analysis. No cytotoxic effect on HEKa and HDFa cell lines was exhibited by RHS-M and RHS-M-F3. The metabolite profiling of RHS-M and RHS-M-F3 was performed by thiolysis followed by HPLC-UVHRMS analysis and a combination of HRMS-FIA and HPLC-HRMSn. Extract and fraction contain oligomeric PAs (mDP of 7.3 and 6.0, respectively, and DP up to 10) mainly constituted by B-type oligomers of (epi)-catechin. Also, (epi)-gallocatechin and gallate derivatives were identified as monomer units, and A-type PAs were detected as minor compounds. KEYWORDS: hazelnut skins, industry byproducts, proanthocyanidins, Candida albicans, HRMS flow injection analysis, HPLC-HRMSn analysis

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additional bond between C2−O7 (A-type PAs, less frequently identified in foods). PAs of hazelnut skins are mainly composed of B-type PCs with an average degree of polymerization (DP) of 7−11.6−8 Also, PC−PD heteropolymers are distinctive of this matrix as well as galloylated PCs.7,8 Recently, the occurrence of A-type PAs, as minor constituents, in hazelnut skin has been reported.2,8,10 PAs showed protective effects against cardiovascular diseases, atherosclerosis, cancer, and urinary tract infections. All of these activities might be related to their strong antioxidant properties.11 Moreover, recent studies have shown the efficacy of cranberry-derived A-type PAs as well as prodelphinidins from Stryphnodendron adstringens against Candida albicans biofilm formation.12,13 The ability to form biofilms on biotic and abiotic surfaces and the hyphal form of growth play key roles during Candida albicans human pathogenesis and fungal drug resistance. In addition to health consequences, the yeast is also involved in food or cosmetics contamination, which is evident as appearance or flavor modifications or nutritional value reduction of the products, as well as allergies or intoxications for the final consumer. Therefore, the food and cosmetic industries commonly use preservatives to prevent microbial growth. A recent trend is the development of safe natural antimicrobial ingredients from food industry byproducts, both to minimize the use of synthetic ones

INTRODUCTION Hazelnut (Corylus avellana L., Betulaceae family) is one of the most cultivated tree nuts worldwide, and Italy is the second largest hazelnut-producing area, behind Turkey. The hazelnut edible kernel is consumed whole in its raw (with skin) and peeled (without skin) forms. However, the most common use of the peeled hazelnut is as an ingredient in processed foods, such as bakery, candy, and chocolate products. The skin, which represents about 2.5% of the total hazelnut kernel weight, is usually removed by blanching or roasting to improve the kernel flavor, color, and crunch and for the use of the kernel in the bakery and confectionery industry.1 Roasted hazelnut skins (RHS) are a byproduct of the industrial roasting process,2 and the disposal of this biodegradable waste material represents a serious environmental and economic problem for the hazelnut industry. Nowadays, RHS are used without treatment for animal feed, but several studies have demonstrated the possibility to upgrade the hazelnut skins as an inexpensive source of bioactive compounds for nutraceutical, pharmaceutical, or cosmetic applications.3,4 Indeed, the hazelnut phenolic compounds, with human health beneficial effects over and above their basic nutritional value, are mainly located in the skin.1,5 In particular, RHS are a rich source of proanthocyanidins (PAs).1,3,6−9 PAs are oligomers or polymers, also known as condensed tannins, classified in procyanidins (PCs), propelargonidins (PPs), or prodelphinidins (PDs) on the basis of the flavan-3-ol unit (epi)catechin (eC), (epi)afzelechin (eA), or (epi)gallocatechin (eG), respectively.8 The monomers can be either linked at the C4−C8 or C4−C6 position (B-type PAs, more common in food products) or doubly linked through an © XXXX American Chemical Society

Received: November 12, 2015 Revised: January 4, 2016 Accepted: January 7, 2016

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DOI: 10.1021/acs.jafc.5b05404 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 1. Total Phenol and Procyanidin Contents and Free Radical Scavenging Activity of RHS Extracts and Fractions total phenol contenta (μmol GAE/mg)b RHS-M80 RHS-E80 RHS-A80 RHS-M RHS-M-F2 RHS-M-F3 α-tocopherolf

0.98 0.84 1.02 1.27

± ± ± ±

0.01 0.01 0.04 0.01

5.11 ± 0.11

total phenol contenta (μmol CE/mg)c 1.01 0.86 1.05 1.33

± ± ± ±

total procyanidin content (g CE/100 g)c

0.01 0.01 0.04 0.01

4.7 4.7 4.1 5.4

5.44 ± 0.12

± ± ± ±

1.1 1.8 0.4 8.3

19.6 ± 0.5

free radical scavenging activityd (EC50, μg/mL) 58.9 58.7 58.5 10.6

± ± ± ±

1.8e 0.9e 2.0e 0.7

7.8 ± 0.3 10.1 ± 1.2

a

Evaluated by Folin−Ciocalteu method. bExpressed as gallic acid equivalents (GAE). cExpressed as catechin equivalents (CE). dDetermined by DPPH test. Range of tested concentrations (5.0−200 μg/mL). ep < 0.05. fPositive control. extractions (n = 3) were conducted under stirring for 12 h at a solid to solvent ratio of 1:10 (w/v). Another RHS aliquot (340 g), preliminarily defatted with n-hexane, was extracted successively by exhaustive maceration (3 × 1 L for 24 h) with chloroform and methanol (RHS-M).17 After the removal of the organic solvent under vacuum at 40 °C in a rotary evaporator (Rotavapor R-200, Buchi Italia s.r.l, Cornaredo, Italy), the extracts were lyophilized to give the solid residues M80 (yield = 15.8 ± 1.4%, w/w), E80 (yield = 10.3 ± 0.9%, w/w), A80 (yield = 16.5 ± 1.3%, w/w), and M (yield = 7.8 ± 0.8%, w/ w). Column Chromatography of Hazelnut Skin Methanol Extract (RHS-M). Separation of RHS-M was carried out according to the method previously described3 with slight modifications. Three grams of RHS-M was dissolved in 20 mL of 96% (v/v) ethanol and loaded on a chromatographic column (1.0 m × 3.0 cm i.d., GE Healthcare) packed with Sephadex-LH 20. Fractions RHS-M-F1 and RHS-M-F2 were eluted from the column using 2 L of 96% (v/v) ethanol. To obtain fraction 3 (RHS-M-F3), rich in condensed tannins, the column was washed with 2 L of 50% (v/v) acetone. RHS-M-F3 was lyophilized after the removal of the organic solvent in a rotary evaporator. Determination of DPPH Radical Scavenging Activity. The antiradical activities of RHS extracts (RHS-M80, RHS-E80, RHS-A80, and RHS-M), fraction RHS-M-F3, and α-tocopherol (positive control) were determined using the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) and the procedure previously described.20 An aliquot (37.5 μL) of the methanol solution containing different amounts of each extract, fraction, or compound was added to 1.5 mL of daily prepared DPPH• solution (0.025 g/L in MeOH); the maximum concentration employed was 200 μg/mL. An equal volume (37.5 μL) of the vehicle alone was added to the control tubes. Absorbance at 517 nm was measured on a UV−visible spectrophotometer (Evolution 201, Thermo Fisher Scientific, Milan, Italy) 10 min after starting the reaction. The DPPH• concentration in the reaction medium was calculated from a calibration curve (range = 5−36 μg/mL) analyzed by linear regression (y = 0.0228x − 0.0350, R2= 0.9999). All experiments were carried out in triplicate; the mean effective scavenging concentrations (EC50, μg/mL) were calculated, and the results are reported in Table 1. Determination of Total Phenol Content (Folin−Ciocalteu Method). RHS extracts (RHS-M80, RHS-E80, RHS-A80, and RHSM) and RHS-M-F3 were analyzed for their total phenolic compound content according to the Folin−Ciocalteu (FC) colorimetric method.21 Briefly, 50 μL of extract or fraction (200 μg/mL in methanol) was added to 25 μL of undiluted FC reagent and 150 μL of Na2CO3 (20%, w/v) in a 1 mL volumetric flask made up to final volume with distilled water. A control without FC reagent and a blank with methanol instead of sample were included in the assay. Tube contents were vortexed, and the reaction mixture absorbance was read at 725 nm after 45 min by a microplate spectrophotometer reader Multiskan Go (ThermoFisher Scientific, Milan, Italy). The total polyphenol content was expressed as gallic acid or catechin equivalents (GAE μmol/mg extract or CE μmol/mg extract, means ± SD of three determinations) calculated by calibration curves (y = 0.577x − 0.0053,

with potential adverse health effects and to overcome the drug resistance problems of microorganisms.14−16 On the basis of all of this evidence and keeping our research of botanicals as bioactive ingredients of functional foods and human health products useful to prevent fungal infections and spoilage of products themselves,17,18 a RHS extract (RHS-M) with powerful antioxidant activity and high polyphenol content, mainly PAs, and a PA-rich fraction (RHS-M-F3) were produced. The activity of RHS-M and its subfractions against Candida albicans SC5314 was determined by the microbroth dilution method and Candida albicans morphological analysis. Moreover, the effect of RHS-M and PA-rich fraction (RHS-MF3) on primary human epidermal keratinocyte (HEKa) and dermal fibroblast (HDFa) cell lines viability was evaluated by MTT test.19 Finally, a comprehensive chemical characterization of RHS-M and RHS-M-F3 was performed using thiolysis coupled to HPLC-UV analysis and a combination of highresolution mass spectrometry (HRMS) techniques. A direct HRMS flow injection analysis (HRMS-FIA), by the use of ESIFT-MS instrumentation, was performed to establish the metabolomic profile of the active fraction and to determine the PA types and mass distributions. Then, tandem MS coupled to liquid chromatography (HPLC-HRMSn) was applied to elucidate the detailed structures of PA oligomers.

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MATERIALS AND METHODS

Materials. Gallic acid (GA), α-tocopherol, Folin−Ciocalteu (FC) phenol reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 4(dimethylamino)cinnamaldehyde (DMAC) ≥98% HPLC grade, benzyl mercaptan, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), (+)-catechin hydrate (C) ≥98% HPLC grade, procyanidin B2, analytical grade acetone, ethanol, methanol, and n-hexane were obtained from Sigma Chemical Co. (Milan, Italy). Sephadex LH-20 was supplied by GE Healthcare, Uppsala, Sweden. Ultrapure water (18 MΩ) was prepared by a Milli-Q purification system (Millipore, Bedford, MA, USA). MS grade methanol and water were supplied by Romil (Cambridge, UK), and MS grade ammonium formate and formic acid were provided by Sigma-Aldrich (Milan, Italy). Hazelnut Skin Samples. RHS samples were kindly supplied by an Italian hazelnut processing industry (Hazelnuts South Italy Manufacturing S.r.l., Baiano, Avellino, Italy). RHS represented the waste of daily industrial processing, carried out on Campania hazelnut varieties (90% Mortarella and 10% Lunga San Giovanni). RHS material was finely blended using a knife mill Grindomix GM 200 (Retsch, Haan, Germany). Extraction Procedures. Three portions of RHS (50 g each) were defatted with n-hexane for 8 h, and the residues were air-dried. The defatted samples were extracted in dark flasks as previously reported9 with slight modifications. Three different solvents, methanol/water (80:20, v/v, RHS-M80), ethanol/water (80:20, v/v, RHS-E80), and, acetone/water (80:20, v/v, RHS-A80) were separately employed. The B


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Journal of Agricultural and Food Chemistry R2 = 0.999; y = 0.5335x − 0.0006, R2 = 0.998 for GA and C, respectively). Determination of Total Procyanidin Content (DMAC Assay). To estimate the total procyanidin (PA) content, the DMAC method was used.22 The DMAC solution (0.1% DMAC reagent, w/v, in ethanol/water/HCl 75:12.5:12.5, v/v/v) was prepared immediately before use. In brief, 70 μL of extracts or fraction (200 μg/mL in methanol) was mixed with 210 μL of DMAC solution in a well of a 96well microplate (ThermoFisher Scientific, Milan, Italy). After 15 min at room temperature, absorbance was read at 640 nm using a microplate spectrophotometer reader Multiskan Go (ThermoFisher Scientific, Milan, Italy). A control without DMAC reagent and a blank with methanol instead of sample were included in the assay. The concentration of total PAs was estimated from a calibration curve using catechin (range = 1−50 μg/mL), and the data were expressed as catechin equivalents (CE μg/mg extract, means ± SD of three determinations). In Vitro Antifungal Activity of RHS-M and RHS-M-F3. Susceptibility Testing. The in vitro minimal inhibitory concentrations (MICs) of RHS-M and fractions RHS-M-F2 and RHS-M-F3 were determined against Candida albicans SC5314 (kindly provided by Professor W. A. Fonzi, Georgetown University, Washington, DC, USA) by the microbroth dilution method as previousily reported.17 Briefly, microtiter plates (96-well microtiter microplates, ThermoFisher Scientific, Milan, Italy) containing 100 μL of 2-fold serial dilutions of extract or fraction in RPMI 1640 medium (with Lglutamine, without glucose and NaHCO3, buffered to pH 7.0 with 0.165 M 4-morpholinepropanesulfonic acid (MOPS) buffer) were inoculated with 100 μL of 2.5 × 103 yeast/mL and incubated at 35 °C for 24 h. The MIC2 of the extract or fractions was read as the lowest concentration that produced an inhibition of growth ≥50%, and MIC0 was the lowest concentration that produced an absence of growth (optically clear) compared with control cells containing only medium and vehicle. Candida albicans Morphological Analysis. Hyphal growth of Candida albicans-treated cells was induced using RPMI 1640 medium, supplemented with 2.5% fetal calf serum, 20 mM HEPES, 2 mM Lglutamine, and 16 mM sodium hydrogen carbonate (pH 7.0, GibcoBRL). Stationary yeast cells were inoculated into a fresh prewarmed medium at a density of 6 × 104 cells/mL in a flat-bottom 96-well microtiter plate. Different concentrations of extract or fraction (ranging from 0.3 to 5.0 μg/mL) were added to each well. After incubation at 37 °C for 24 h, each microtiter plate was examined using an inverted microscope (Zeiss Axiovert 200M, Carl Zeiss Light Microscopy, Göttingen, Germany) to monitor phenotypic modification and hyphae formation. Images were acquired using a Micron (EVOS) Digital Image software (ThermoFisher Scientific Inc., USA) and processed using Adobe Photoshop 5.0. In Vitro Cytotoxic Activity of RHS-M and RHS-M-F3. Primary human dermal fibroblasts (HDFa) and primary human epidermal keratinocytes (HEKa), isolated from adult skin, were obtained from Gibco, Life Technology Corp. HDFa cells were routinely grown in M106 medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, fetal bovine serum (2% v/v), hydrocortisone (1 μg/ mL), human epidermal growth factor (10 ng/mL), and basic fibroblast growth factor/heparin (10 μg/mL) in monolayer cultures at 37 °C in a humidified atmosphere containing 5% CO2 in air. HEKa cells were routinely grown in M154 basal medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, bovine pituitary extract (0.2% v/ v), bovine insulin (5 μg/mL), hydrocortisone (0.18 μg/mL), bovine transferrin (5 μg/mL), and human epidermal growth factor (0.2 ng/ mL) in monolayer cultures at 37 °C in a humidified atmosphere containing 5% CO2 in air. All reagents and supplements for cell cultures were purchased from Life Technologies. HDFa (4 × 103 cells/ well) and HEKa (6 × 103 cells/well) were cultured in triplicate in 96well plates in a final volume of 100 μL of M106 complete medium and in M154 complete medium, respectively. All experiments were performed in cell lines grown for not more 5 passages. DMSOdissolved RHS-M and water-dissolved RHS-M-F3 were added to the cells to achieve final concentrations ranging from 7.5 to 150 μg/mL.

After 20 or 44 h of growth at 37 °C, MTT was added to each well (0.5 mg/mL, final concentration), and plates were incubated for 4 h at 37 °C. Formazan product was solubilized with 100 μL of lysis buffer (10% SDS-0.01 M HCl), and the absorbance of each well was measured at 570 nm using a microplate reading spectrophotometer.23,24 Thiolysis. Thiolysis of RHS-M and RHS-M-F3 was performed according to a procedure previously reported,25 with some modifications. Briefly, 50 μL of methanol acidified with concentrated HCl (3.3%, v/v) and 100 μL of benzyl mercaptan solution (5%, v/v, in methanol) were added to 50 μL of the sample (4 mg/mL in methanol). The mixture was vortexed, and the vial was sealed. The reaction was carried out for 30 min at 40 °C and was stopped by placing the vial in an ice bath. Immediately after, 200 μL of water was added to the samples and 10 μL of the reaction mixture was injected directly for HPLC analysis. Also, pure catechin (1 mg/mL) and procyanidin B2 (0.5 mg/mL) were thiolyzed. Triplicate experiments for sample were performed. HPLC-UV analyses were carried out with a Dionex Ultimate 3000 UHPLC system (ThermoFisher Scientific, Milan, Italy) using a Hibar Purospher STAR RP-18 end-capped column (3 μm, 150 × 3 mm) (Merck, Darmstadt, Germany). A linear gradient (5−75% B in 45 min) of water (A) and methanol (B), both with 2 mM ammonium formate and 0.1% formic acid, was applied to separate the thiolytically degraded PAs mixtures. The flow rate was 300 μL min−1 and the injection volume 5 μL. The column was thermostated at 25 °C. The detection was performed at the wavelength of 280 nm. Peaks were identified by HPLC-HRMS method reported in a following section and literature data.6,25 The proportions of constituent flavan-3-ol and mDP were calculated according to a published method.25 HRMS-FIA. HRMS-FIA experiments were performed with a linear ion trap−Orbitrap hybrid mass spectrometer (LTQ OrbiTrap XL, ThermoFisher Scientific, Milan, Italy) using electrospray ionization (ESI) in negative ion mode. RHS-M and RHS-M-F3 (50 and 100 μg/ mL in methanol/water 1:1, v/v) were pumped at 5 μL/min, and the MS data were acquired in two ranges (normal mass range, m/z 500− 2000; and high mass range, m/z 1000−3000) with a resolution of 60000 and maximum ion injection time of 100 ms. Under optimal analysis conditions the following conditions were used: sheath and auxiliary gas, 30 and 5 (arbitrary units), respectively; spray voltage, 4.0 kV; capillary temperature, 280 °C; capillary voltage, − 49 V; tube lens, 146.5 V. Xcalibur 2.2 software (ThermoFisher Scientific, Milan, Italy) was used to interpret the data obtained. HPLC-HRMSn Analysis. Analyses were performed with LTQ OrbiTrap XL connected to an Accela system (ThermoFisher Scientific, Milan, Italy). The mass spectrometer, equipped with ESI source, was operated in negative mode. The chromatographic conditions were the same as those used for UHPLC-UV analysis of thiolysis reaction. Instrumental parameters were as follows: source voltage, 4.0 kV; capillary voltage, −49 V; tube lens voltage, −146.5 V; capillary temperature, 300 °C; sheath and auxiliary gas flow (N2), 35 and 10 (arbitrary units), respectively. MS spectra were acquired by full range acquisition covering m/z 250−2000. For fragmentation study, a data-dependent scan was performed by deploying the collisioninduced dissociation (CID). The normalized collision energy of the CID cell was set at 30 eV. PAs were identified according to the corresponding spectral characteristics: mass spectra, accurate mass, characteristic fragmentation, and retention time. Xcalibur 2.2 software was used for instrument control, data acquisition, and data analysis. Statistical Analysis. Data were expressed as the mean ± standard deviation of triplicate measurements. The data were statistically analyzed using statistical software, Statgraphic Centurion XVI, version 16.1, from Statistical Graphics (Rockville, MD, USA). Pearson correlation coefficients were conducted, and a significant difference was defined at p < 0.05.

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RESULTS AND DISCUSSION Total Phenolic Compound and PA Contents and Antioxidant Activity of RHS Extracts and Fractions. The polyphenol-rich extracts from defatted RHS were produced by C


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In Vitro Activity against Candida albicans SC 5314 and Cytotoxic Effect on Human Skin Cell Lines of RHS-M and Fractions. According to the aims of this research and on the basis of the recently reported antifungal activity for cranberry A-type proanthocyanidins and S. adstringens prodelphinidins, the activity against Candida albicans SC5314 of the PAs-richer RHS extract (RHS-M) and its purified fractions (RHS-M-F2 and RHS-M-F3) was evaluated. Candida albicans is an opportunistic fungal pathogen that causes a wide range of human diseases from oral thrush and vaginitis, affecting the superficial mucosa, to candidaemia and systemic candidiasis in patients with compromised immunity. Nowadays, there is great interest in researching new natural derivatives active against Candida albicans to reduce the side effects of synthetic ones and to overcome the emergence of drugresistant strains. The effectiveness of RHS-M, RHS-M-F2, and RHS-M-F3 against Candida albicans SC5314, determined by the in vitro microbroth dilution method,17 was expressed as minimal inhibitory concentration (MIC). RHS-M exhibited antimycotic activity with a MIC2 of 3.00 ± 0.36 μg/mL and MIC0 of 5.00 ± 0.60 μg/mL at 48 h (Table 2). The RHS-M

maceration at room temperature under stirring, using 80% of aqueous methanol (RHS-M80), ethanol (RHS-E80), and acetone (RHS-A80) as solvent systems, according to literature data.9 Moreover, defatted RHS were also processed by exhaustive maceration at room temperature using solvents with increasing polarity to produce the dried extract RHS-M.17 The efficiency in extracting phenolic compounds between the different solvents and procedures was evaluated in terms of extraction yield, as well as total phenolic compound and proanthocyanidin (PA) contents, determined by Folin− Ciocalteu (FC) and DMAC assay, respectively. The extraction yields of RHS extracts (ranging from 7.8 to 16.5%, w/w) resulted in lower values than those previously reported (ranging from 27.8 to 32.6%, w/w).9 This difference might be ascribed to different cultivars and geographic origins of the employed skin samples. As is well-known, these parameters are strictly related to the biosynthesis of secondary metabolites, such as phenolic compounds.26 In our conditions, the exhaustive maceration using solvents with increasing polarity appeared as the most suitable method to recover the RHS phenolic compounds (Table 1). Indeed, RHS-M showed a higher total phenolic compound (1.27 μmol GAE/mg) and PA content (5.4 mg CE/100g) with respect to RHS-M80, RHS-E80, and RHS-A80 (1.3−1.5- and 1.1−1.3-fold highest, respectively). The antioxidant activity of nut byproduct derivatives, such as hazelnut skin extracts, has been extensively studied using several methods.1,8,27 Among the different assays, the DPPH test seems to be the most sensitive method to evaluate the antiradical activity of hazelnut skins.26 For such reasons, the produced RHS extracts were screened for their ability to scavenge the radical DPPH. As reported in Table 1, all of the extracts exhibited a significant and concentration-dependent free radical scavenging activity expressed as EC50, μg/mL of antioxidant required to decrease the initial DPPH• concentration by 50%. Our findings revealed that the antiradical activities of RHS-M80, RHS-E80, and RHS-A80 were much alike (EC50 = 58.9, 58.7, and 58.5 μg/mL, respectively). Meanwhile, the ability of RHS-M to scavenge the DPPH radical (EC50 = 10.6 μg/mL) appeared >5-fold higher than that of other extracts and comparable to that of α-tocopherol (EC50 = 10.1 μg/mL) used as positive control. The strong correlation found between the antioxidant potency of RHS extracts and both total phenolic compound and PA contents (Pearson correlation, r −0.90668 and −0.8838, respectively) could explain the superimposable activity of RHS-M80, RHS-E80, and RHS-A80, showing also a similar total phenolic compound and PA content, with respect to the richest in active components, RHS-M. On the basis of these preliminary results, RHS-M was subjected to a fractionation on a Sephadex-LH 20 column obtaining a fraction enriched in condensed tannin, according to the procedure previously reported.3 Fractions RHS-M-F1 and RHS-M-F2 were collected using ethanol 96% as elution solvent, and RHS-M-F3 was eluted with aqueous acetone (50%, v/v). RHS-M-F1 was not subjected to further analysis due to its insolubility in organic and aqueous solvents, whereas the free radical scavenging activities of RHS-M-F2 and RHS-M-F3 were determined. RHS-M-F3 showed a powerful antioxidant activity (EC50 = 7.8 μg/mL) correlated to its very high polyphenol (5.11 μmol GAE/mg and 5.44 μmol CE/mg) and PA (19.6 g CE/100g) contents (Table 1). On the contrary, no significant antioxidant activity was observed for RHS-M-F2 up to 500.0 μg/mL. RHS-M-F2 total phenol compound and PA contents were not detectable by FC and DMAC methods.

Table 2. Anti-Candida albicans Activity of RHS-M Extract and Fractions against C. albicans Evaluated by Broth Microdilution Methoda RHS-M RHS-M-F2 RHS-M-F3 a

MIC2b (μg/mL)

MIC0b (μg/mL)

3.00 ± 0.36 >50.00 0.10 ± 0.01

5.00 ± 0.60 >100.00 0.50 ± 0.06

RPMI pH 7.0. bMean ± SD of three determination.

activity seems to be correlated to its PAs content. In fact, the purified PAs-rich RHS-M-F3 showed a higher activity (MIC2 0.10 ± 0.01 μg/mL and MIC0 0.50 ± 0.06 μg/mL), and the fraction RHS-M-F2, no containing PAs, was not active up to 50.00 μg/mL (MIC2) and 100.00 μg/mL (MIC0) (Table 2). The ability to produce the filamentous hyphae is a key component of the invasive Candida albicans growth, tissue penetration, and biofilm development. It has also been reported that the fungal hyphae generate significant amounts of reactive oxygen species (ROS) during their germinal phase.28 Because one important factor underlying the pathogenicity and virulence of Candida albicans is its morphological transition from yeast to filamentous form, the RHS-M, RHS-M-F2, and RHS-M-F3 (Figure 1) interference with hyphae formation was investigated by inverted fluorescence microscopy. Our results showed that the treatment of Candida albicans with RHS-M, at concentrations corresponding to MIC2 and MIC0 (Figure 1b,c), not only reduced the number of fungal cells but also inhibited germination and the generation of true hyphae compared to the control (Figure 1a). As presumed, among the RHS-M fractions, RHS-M-F3, at concentrations from 0.30 to 3.00 μg/mL (Figure 1g−i) impairs the yeast-to-hyphae transition in a concentration-dependent manner. In particular, using RHS-M-F3 from 0.30 to 1.00 μg/mL (Figure 1g,h), hyphal branching was reduced in number and length (Candida albicans yeast form is labeled with arrows in Figure 1h), whereas from 1.00 to 3.00 mg/mL (Figure 1h,i) RHS-M-F3 determined a total inhibition of germination. Because RHS-M and RHS-M-F3 have also a strong antioxidant activity, they can perform the doubly useful action of inhibiting the hyphae formation of Candida albicans and the simultaneous ROS D


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Figure 1. Effect of RHS-M, at concentrations corresponding to MIC2 and MIC0 (3.0 (b) and 5.0 (c) μg/mL, respectively), on Candida albicans growth and germ tube formation compared to control (a) (cells treated with only vehicle). Effect of RHS-M-F2 (d−f) and RHS-M-F3 (g−i), at different concentrations (0.3 (d, g), 1.0 (e, h), and 3.0 (f, i) μg/mL, respectively) on Candida albicans hyphal growth compared to control (a) (cells treated with only vehicle). The arrows (panel h) label C. albicans yeast form.

F3 on normal human skin cells. However, the effectiveness of RHS as dietary supplements must be proved considering the PA metabolites derived from the action of the microbiota, as well as from phase I and phase II metabolism.29 RHS Metabolite Profiling by Mass Spectrometry. To advance understanding of RHS-M and RHS-M-F3 activity, it is important to thoroughly characterize their putatively active components. The identification of PAs by HPLC does not allow a complete chromatographic resolution of PAs in complex samples containing a great PA diversity or polymers (DP > 6).30 Acidic depolymerization of PAs with nucleophiles (thiolysis or phloroglucinolysis) is currently the method most used to determine mDP value and the average flavonol composition of complex PA mixtures.30 Also, mass spectrometry, alone or coupled to HPLC, has been successfully applied to analyze complex PA mixtures from various plant sources.30 In particular, flow injection ESI-MS31−34 and MALDI-TOFMS35 have been used for direct analyses of PAs, and they provide a full picture of PA mass distributions. HPLC-MS/MS

release. On the other hand, RHS-M-F2 had no effect on growth, morphological transition, and germ tube formation up to 3.00 μg/mL (Figure 1d−f). Owing to the powerful antimicrobial activity against Candida albicans, RHS-M or its active purified fraction RHS-M-F3 could be used as biopreservatives and/or active ingredients in foods or topical formulations. In this context, the investigation of their cytotoxic effects in different kinds of primary skin cells is necessary. For this purpose, human epidermal keratinocytes (HEKa) and dermal fibroblasts (HDFa) viability was evaluated by MTT assay. The unmetabolized phenolic compounds of RHS-M and RHS-M-F3, directly in touch with cells, did not affect the growth of both HEKa and HDFa cells up to 150.0 μg/mL, after 24 h of treatment. A decrease in viability of HEKa cells (about 60%) was observed only at the highest RHS-M-F3 tested concentration (150.0 μg/mL) after 48 h. However, this cytotoxic concentration is about 300-fold greater than the antimycotic active dose (MIC0). Globally, these results supported the safe use of the functional RHS-M and RHS-ME


Article


Table 3. High-Resolution Masses, Molecular Formulas, and Compositions of PAs Found in HRMS-FIA Spectra of RHS-M and RHS-M-F3 [M − H]− m/z (ppm) 575.1197 577.1356 591.1147 593.1301 607.1096 609.1251 727.1316 729.1468 743.1262 745.1425 863.1841 865.1995 877.1631 879.1786 881.1960 893.1577 895.1741 897.1903 1017.2100 1151.2477 1153.2633 1167.2424 1169.2579 1181.2203 1183.2374 1185.2480 1199.2330 1201.2504 1305.2787 1439.3124 1441.3260 1453.2927 1455.3044 1457.3156 − 1471.3015 1485.2782 1487.2963 1489.3111 − 1729.3919 1743.3720 1745.3856 − 1759.3662 1761.3816 1175.3529 1777.3685 − − − − − − − − − − − −

(2.2) (2.7) (2.3) (2.0) (2.3) (1.9) (3.1) (2.4) (2.6) (3.5) (2.7) (2.4) (2.3) (2.1) (4.1) (1.9) (2.7) (3.4) (1.6) (2.2) (2.1) (2.0) (1.9) (0.8) (2.0) (−2.2) (2.6) (4.0) (5.3) (2.6) (1.3) (3.4) (0.7) (−3.6) (3.5) (0.4) (2.0) (1.4) (2.5) (2.9) (1.8) (2.5) (2.3) (−2.1) (−2.1)

[M2 − H]2− m/z (ppm) − − − − − − − − − − − − − − − − − − − − − − − − − − − − − 719.1515 720.1596 726.1412 727.1491 728.1572 734.1392 735.1470 742.1348 − − 863.1841 864.1883 871.1812 872.1849 878.1680 879.1786 880.1823 887.1761 888.1789 1008.2227 1015.2142 1022.2021 1023.2073 1024.2172 1152.2526 1159.2437 1160.2518 1167.2424 1296.2918 1304.2794 1312.2851

(1.9) (2.3) (2.0) (2.1) (2.4) (2.7) (2.7) (0.3)

(2.6) (−1.5) (2.2) (−2.4) (−1.0) (2.1) (−2.5) (2.2) (−3.5) (1.4) (3.2) (1.5) (−1.0) (0.9) (−0.4) (0.9) (1.2) (2.0) (5.5) (−2.1) (4.1)

mol formula

DPa

A-bond

eCb

eGc

gd

C30H24O12 C30H26O12 C30H24O13 C30H26O13 C30H24O14 C30H26O14 C37H28O16 C37H30O16 C37H28O17 C37H30O17 C45H36O18 C45H38O18 C45H34O19 C45H36O19 C45H38O19 C45H34O20 C45H36O20 C45H38O20 C52H42O22 C60H48O24 C60H50O24 C60H48O25 C60H50O25 C60H46O26 C60H48O26 C60H50O26 C60H48O27 C60H50O27 C67H54O28 C75H60O30 C75H62O30 C75H58O31 C75H60O31 C75H62O31 C75H58O32 C75H60O32 C75H58O33 C75H60O33 C75H62O33 C90H72O36 C90H74O36 C90H72O37 C90H74O37 C90H70O38 C90H72O38 C90H74O38 C90H72O39 C90H74O39 C105H86O42 C105H84O43 C105H82O44 C105H84O44 C105H86O44 C120H98O48 C120H96O49 C120H98O49 C120H96O50 C135H110O54 C135H110O55 C135H110O56

2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 7 7 7 7 7 8 8 8 8 9 9 9

1 − 1 − 1 − 1 − 1 − 1 − 2 1 − 2 1 − − 1 − 1 − 2 1 − 1 − − 1 − 2 1 − 2 1 2 1 − 1 − 1 0 2 1 0 1 0 − 1 2 1 − − 1 − 1 − − −

2 2 1 1 − − 2 2 1 1 3 3 2 2 2 1 1 1 3 4 4 3 3 2 2 2 1 1 4 5 5 4 4 4 3 3 2 2 2 6 6 5 5 4 4 4 3 3 7 6 5 5 5 8 7 7 6 9 8 7

− − 1 1 2 2 − − 1 1 − − 1 1 1 2 2 2 − − − 1 1 2 2 2 3 3 − − − 1 1 1 2 2 3 3 3 − − 1 1 2 2 2 3 3 − 1 2 2 2 − 1 1 2 − 1 2

− − − − − − 1 1 1 1 − − − − − − − − 1 − − − − − − − − − 1 − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − −

F


Article

Journal of Agricultural and Food Chemistry Table 3. continued [M − H]− m/z (ppm) − − a

[M2 − H]2− m/z (ppm) 1440.3177 (0.9) 1448.3144 (0.4)

mol formula

DPa

A-bond

eCb

eGc

gd

C150H122O60 C150H122O61

10 10

− −

10 9

− 1

− −

Degree of polymerization. b(epi)catechin. c(epi)gallocatechin. dgalloyl.

linkages (Table 3). Also, with the use of ESI-FT-MS instrumentation it was possible to determine the charge state of the molecular species by the analysis of the isotopic distribution of the mass signals.38 Figure 2 shows the HRMS profiles of RHS-M and RHS-M-F3, and Table 3 summarizes their PA composition. Both extract (RHS-M) and its bioactive fraction (RHS-M-F3) exhibited the same PA mass distribution (Figure 2). The main peaks corresponded to [M − H]− ions of B-type PCs up to DP 6: a series of signals with a peak occurring every 288.06 mass units starting at m/z 577.1356 (2). [M2 − H]2− ions allowed the identification of PAs with a higher DP, up to DP 10 (Figure 2 and Table 3), whereas [M3 − H]3− ions were not observed. The HRMS spectrum revealed also peaks of PAs with one (Figure 2 and Table 3) or more eG units (Table 3) and gallate derivatives up to DP 4 (Figure 2 and Table 3). Moreover, the signals 2.02 or 4.03 Da lower in mass than the Btype indicated the presence of A-type PAs with one and two extra interflavanic linkages (Figure 2 and Table 3). HRMS profile of RHS-M-F3 revealed a PA composition similar to the related extract (RHS-M) and that reported for hazelnut skins,2,7,8,10 suggesting that it is a fraction of hazelnut skinenriched PAs. HPLC-HRMSn Analysis. Finally, HPLC-HRMSn analysis of RHS-M-F3 was performed to elucidate the detailed structures of di-, tri-, and tetraoligomer isomers and galloylated derivatives. HPLC-MS/MS is a valid tool to obtain important information concerning the sequence of flavanol units, the type and position of linkages, the hydroxylation scheme, and the galloylation sites of oligomeric PAs (DP < 5). On the basis of the proposed fragmentation patterns,6,36−38 information about the hydroxylation pattern and type of interflavan bond is obtained by the fragment ions derived from retro-Diels−Alder (RDA) reaction (loss of 152, 136, and 168 Da for eC, eA, and eG, respectively) and from heterocyclic ring fission (HRF) of the extension unit (loss of 126 Da for eC, eA, or eG). Quinone methide (QM) cleavage of interflavan bonds produces diagnostic fragment ions ([Mt − H]− and [MEX − 3H]− ions for B-type PAs and [MT − 5H]− ions for A-type PAs, where EX = extension unit and T = terminal unit) useful to identify the connection sequence of the oligomers. Using the information from HPLC-HRMS and MS/MS experiments, 7 monomers were identified and a total of 59 PA oligomers (14 isomeric groups) were structurally sequenced from RHS-M-F3. Table 4 lists the retention times, [M − H]− ions, molecular formulas, and diagnostic MS2 product ions of the identified monomers and oligomeric PAs. Figures S2 and S3 show the extracted ion chromatograms and representative MS2 spectra of RHS-M-F3, respectively. The natural occurrence of identified PA oligomers in RHS-M-F3 was confirmed by HPLC-HRMSn analysis of the filtrate obtained after precipitation of polymers,2 enabling the exclusion of the origin of [M − H]− ions from the in-source fragmentation of higher PAs. Among the identified PAs, numerous isomers of B-type PC dimers (7 C30H25O12), trimers (11 C45H37O18), and tetramers (6 C60H49O24) were successfully identified (Table 4). Also, the

has been instead employed for the structural characterization of PA oligomers (DP ≤ 6) by the study of fragmentation patterns.6,36−38 In this study, RHS-M and RHS-M-F3 chemical characterizations were performed by thiolysis followed by HPLC-UV-HRMS analysis and a combination of HRMS techniques, HRMS-FIA and HPLC-HRMSn, to establish the nature and the proportion of the flavanol units, the mDP and PA mass distributions, the type of linkages, the presence of derivatives, and the sequence of flavanol units in PA oligomers. Thiolysis. Initially, the acid-catalyzed depolymerization in the presence of benzyl mercaptan as nucleophilic reagent (thiolysis), coupled to HPLC-UV-HRMS analysis, was applied to characterize PAs of RHS-M and RHS-M-F3. Thiolysis is a useful method in the characterization of PAs, as it distinguishes between extension and terminal units (released as flavanols). This method allows the determination of the nature and the proportion of the constitutive flavanol units and mDP of PA mixtures.25 The flavanol molar proportions of thiolytically degraded PAs in RHS-M and RHS-M-F3 are listed in Table S1, and a representative chromatogram of thiolytically degraded PAs (RHS-M-F3) is shown in Figure S1. Thiolysis of RHS-M and RHS-M-F3 revealed mDP values of 7.3 ± 0.07 and 6.0 ± 0.01, respectively. Flavonol units of RHS-M and RHS-M-F3 consisted of mostly epicatechin (RHS-M, 51.2 ± 0.1%; RHSM-F3, 47.8 ± 0.2%) and catechin (RHS-M, 38.6 ± 0.2%; RHSM-F3, 39.6 ± 0.2%). Catechin-3-O-gallate (RHS-M, 6.4 ± 0.3; RHS-M-F3, 7.2 ± 1.0%), epigallocatechin (RHS-M, 2.4 ± 0.01; RHS-M-F3, 2.2 ± 0.04%), gallocatechin (RHS-M, 1.1 ± 0.01; RHS-M-F3,1.8 ± 0.1%), and gallocatechin-3-O-gallate (RHSM, 0.4 ± 0.01; RHS-M-F3,1.4 ± 0.1%) were also found as constitutive flavanols of RHS-M and RHS-M-F3. Catechin and epicatechin occurred as both terminal and extension units, and overall catechin was the main terminal unit (about 70%, Table S1) and epicatechin was the major extension unit found (55− 57%, Table S1). The data showed that the mDP and the nature and the proportion of the flavanol units of RHS-M and RHSM-F3 are closely similar. According to the results of biological studies (antioxidant and antifungal activities), we can conclude that RHS-M-F3 is an enriched-PA fraction of whole extract (RHS-M). HRMS-FIA. Subsequently, HRMS-FIA, with ESI source and the negative ion mode, was applied to investigate the metabolite profiling of the active fraction of RHS-M and RHS-M-F3. ESI-MS-FIA data together with the complementary information on thiolysis provide a detailed picture of the composition of PAs because the latter supplies the mDP value, whereas ESI-MS is expected to give an indication of the distribution of DPs around the mean.31 HRMS-FIA rapidly enabled the detection of signals corresponding to monocharged ions ([M − H]− PAs, DP up to 5−6) and to doubly charged ions ([M2 − H]2− for DP > 4). The m/z values allowed the high-resolution molecular weight and exact molecular formula to be established as well as the determination of the type of flavanol units, the degree of polymerization (DP), the presence of galloyl or glycosyl groups, and the number of A- or B-type G


Article


Figure 2. FIA-HRMS profiles (negative ion mode) of RHS-M and RHS-M-F3. Abbreviations: eC and eG, (epi)catechin and (epi)gallocatechin, respectively; g, galloyl.

flavanol sequence of B-type dimers (5 eG-eC, 2 eC-eG and 2 eG-eG) and trimers containing eG (3 eG-eC-eC, 1 eC-eG-eC and 1 eG-eG-eC) was well established by HRMS and MS/MS data (Table 4). With regard to A-type PAs, both dimers and

trimers were detected as minor compounds (Table 4). A full characterization of A-type dimer isomers (3 eC-A-eC, 6 eG-AeC) was obtained (Table 4), whereas in the case of dimers eGA-eG and A-type trimers their low abundance in RHS-M-F3 H


Article


Table 4. Retention Times, [M − H]− Ions, Molecular Formulas, Sequences, and Diagnostic MS2 Product Ions of the Identified Monomers and Oligomeric PAs in RHS-M-F3 tR (min)

[M − H]− (m/z)

ppm

21.3 29.7 12.5 20.8 35.1 36.8 28.0 29.7 34.1 36.6 15.5 16.5 18.5 19.6 22.1 24.0 28.3 22.0 23.3 25.2 27.2 28.6 29.9 10.1 10.4 11.4 14.7 14.9 12.0 12.4 12.7 19.6 26.3 8.1 9.2 24.5 27.5 26.1

289.0713 289.0716 305.0664 305.0668 441.0820 441.0823 457.0773 575.1189 575.1189 575.1191 577.1348 577.1346 577.1347 577.1349 577.1350 577.1350 577.1353 591.1141 591.1139 591.1140 591.1137 591.1143 591.1139 593.1290 593.1298 593.1296 593.1296 593.1296 593.1298 593.1236 607.1088 607.1088 607.1088 609.1241 609.1243 729.1458 729.1460 729.1460

22.9

mol formula

compounda−f

2.3 3.3 2.8 3.9 0.3 1.5 1.7 0.9 0.9 1.2 1.2 0.9 1.1 1.5 1.6 1.7 2.1 1.4 0.9 1.2 0.6 1.7 1.0 0.2 1.4 1.5 1.1 1.1 1.5 1.4 1.0 0.9 1.0 0.3 0.7 1.1 1.4 1.4

C15H13O6 C15H13O6 C15H13O7 C15H13O7 C22H17O10 C22H17O10 C22H17O11 C30H23O12 C30H23O12 C30H23O12 C30H25O12 C30H25O12 C30H25O12 C30H25O12 C30H25O12 C30H25O12 C30H25O12 C30H23O13 C30H23O13 C30H23O13 C30H23O13 C30H23O13 C30H23O13 C30H25O13 C30H25O13 C30H25O13 C30H25O13 C30H25O13 C30H25O13 C30H25O13 C30H23O14 C30H23O14 C30H23O14 C30H25O14 C30H25O14 C37H29O16 C37H29O16 C37H29O16

catechin epicatechin gallocatechin epigallocatechin catechin-3-g epicatechin-3-g eG-3-g eC-A-eC eC-A-eC (A2) eC-A-eC eC-eC eC-eC eC-eC eC-eC eC-eC eC-eC (B2) eC-eC eG-A-eC eG-A-eC eG-A-eC eG-A-eC eG-A-eC eG-A-eC eG-eC eG-eC eG-eC eG-eC eG-eC eC-eG eC-eG eG-A-eG eG-A-eG eG-A-eG eG-eG eG-eG eC-eCg eC-eCg eCgal-eC

745.1406

1.0

C37H29O17

eC-eGg

23.8

745.1402

0.3

C37H29O17

eG-eCg

9.4 8.7 15.4 17.0 17.8 18.6 19.0 20.0 24.6 25.9 27.7 29.1 7.8 12.0 13.9 12.8 14.5

863.1827 865.1976 865.1978 865.1978 865.1981 865.1978 865.1978 865.1975 865.1977 865.1978 865.1980 865.1979 881.1922 881.1921 881.1932 881.1928 881.1932

1.1 0.2 0.4 0.4 0.8 0.4 0.4 0.1 0.3 0.4 0.6 0.6 −0.1 −0.3 1.0 0.5 1.0

C45H35O18 C45H37O18 C45H37O18 C45H37O18 C45H37O18 C45H37O18 C45H37O18 C45H37O18 C45H37O18 C45H37O18 C45H37O18 C45H37O18 C45H37O19 C45H37O19 C45H37O19 C45H37O19 C45H37O19

eC-eC-eC eC-eC-eC eC-eC-eC eC-eC-eC eC-eC-eC eC-eC-eC eC-eC-eC eC-eC-eC eC-eC-eC eC-eC-eC eC-eC-eC eG-eC-eC eG-eC-eC eG-eC-eC mixture mixture

diagnostic MS2 ionsg (m/z) 175, 179, 205, 231, 245 165, 175, 179, 219, 221, 247, 261, 287 169, 193, 271, 289, 331 169, 193, 225, 305, 331, 385, 389, 429 285 (QMEX), 289 (QMT), 407 (RDA − H2O), 423 (RDA) 449 (HRF)

287 (QMEX), 289 (QMT), 299 (RDA + HRF), 407 (RDA − H2O), 425 (RDA), 451 (HRF)

301 (QMEX), 289 (QMT), 407 (RDA − H2O), 439 (RDAT), 465 (HRF)

303 (QMEX), 289 (QMT), 407 (RDAEX − H2O), 425 (RDAEX), 441 (RDAT), 467 (HRF)

305 (QMEX), 287 (QMT), 423 (RDAEX − H2O), 425 (RDAT), 441 (RDAEX), 467 (HRF) not recorded

305 (QMT), 423 (RDA − H2O), 441 (RDA), 483 (HRF) 287 (QMEX), 441 (QMT), 289 (QMT − gal), 407 (RDA − H2O − gal), 451 (HRF − gallate), 559 (RDA − H2O), 577 (RDA/− gal), 603 (HRF) 439 (QMEX), 289 (QMT), 287 (QMEX − gal), 407 (RDA − H2O − gal), 425 (RDA − gal), 451 (HRF − gal), 559 (RDA − H2O), 577 (RDA/− gal), 603 (HRF) 457 (QMT), 305 (QMT − gal), 423 (RDAEX − H2O − gal), 467 (HRF − gal), 575 (RDAEX − H2O), 593 (RDAEX/− gal), 619 (HRF) 303 (QMEX), 441 (QMT), 289 (QMT − gal), 407 (RDAEX − H2O − gal), 467 (HRF − gal), 559 (RDAEX − H2O), 577 (RDAEX), 593 (− gal), 619 (HRF) not recorded 287 (QMEX(CD)), 577 (QMT(CD)), 575 (QMEX(FG)), 289 (QMT(FG)), 407 (QMT(CD) + RDA − H2O), 425 (QMT(CD) + RDA), 449 (QMEX(FG) + HRF), 451 (QMT(CD) + HRF), 695 (RDA − H2O), 713 (RDA), 739 (HRF)

303 (QMEX(CD)), 577 (QMT(CD)), 591 (QMEX(FG)), 289 (QMT(FG)), 695 (RDAeG − H2O), 711 (RDAeC − H2O), 713 (RDAeG), 729 (RDAeC), 755 (HRF)

I


Article

Journal of Agricultural and Food Chemistry Table 4. continued tR (min)

[M − H]− (m/z)

ppm

8.3

881.1925

0.2

C45H37O19

eC-eG-eC

10.4

897.1872

−0.1

C45H37O20

eG-eG-eC

32.8

1017.2092

0.8

C52H41O22

eC-eC-eCg

13.1 16.4 17.1 22.4 21.5 23.0

1153.2619 1153.2621 1153.2616 1153.2618 1153.2614 1153.2618

0.9 1.1 0.7 0.8 0.5 0.8

C60H49O24 C60H49O24 C60H49O24 C60H49O24 C60H49O24 C60H49O24

eC-eC-eC-eC eC-eC-eC-eC eC-eC-eC-eC eC-eC-eC-eC eC-eC-eC-eC eC-eC-eC-eC

mol formula

compounda−f

diagnostic MS2 ionsg (m/z) 287 (QMEX(CD)), 593 (QMT(CD)), 591 (QMEX(FG)), 289 (QMT(FG)) 407 (QMT(CD) + RDAeG − H2O), 425 (QMT(CD) + RDAeG), 695 (RDAeG − H2O), 711 (RDAeC − H2O), 713 (RDAeG), 729 (RDAeC), 755 (HRF) 303 (QMEX(CD)), 593 (QMT(CD)), 607 (QMEX(FG)), 543 (2RDAeG − H2O), 603 (RDAeG + HRF), 711 (RDAeG − H2O), 729 (RDAeG), 771 (HRF) 729 (QMT(CD)), 575 (QMEX(FG)), 441 (QMT(FG)), 577 (QMT(CD) + RDA/− gal), 603 (QMT(CD) + HRF), 847 (RDA − H2O), 865 (RDA/− gal), 891 (HRF) 865 (QMT(CD)), 575 (QMEX(FG)), 577 (QMT(FG)), 863 (QMEX(IL)), 739 (QMT(CD) + HRF), 983 (RDA − H2O), 1001 (RDA), 1027 (HRF)

a

eG, (epi). beC, (epi)catechin. cA2, procyanidin A-type dimer A2; dB2, procyanidin B-type dimer B2; eeCg, (epi)catechin-3-O-gallate; feGg, (epi)gallocatechin-3-O-gallate; gEX, extension unit; T, terminal unit; CD, FG and IL, nomenclature of flavanol ring by Li and Deinzer, 2007.

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prevented their characterization by MS/MS experiments. Also, the galloylated PC dimers (2 eC-eCg and 1 eCg-eC) and trimer (eC-eC-eC-g) and galloylated dimers containing eG (eC-eGg and eG-eCg) were structurally sequenced through the diagnostic QM, RDA, and HRF ions. In particular, the galloylation positions in the isomers eC-eCg and eCg-eC were established on the basis of the product ions corresponding to QMEX (m/z at 287 and 439, respectively) and QMT (m/z at 441 and 289, respectively). The extension units of isomers eCeGg and eG-eCg were identified by the diagnostic product ions of RDAEX (m/z at 593 and 577, respectively) and RDAEX − H2O (m/z at 575 and 559, respectively) and corroborated from QMT ion at m/z 457 and 441, respectively (Table 4). HRMSFIA and HPLC-HRMSn data were fully consistent with thiolysis results. The results showed that condensed tannins present in RHS-M-F3 consist of PCs, PC−PD heteroligomers, and galloyl derivatives. The sequence of flavanol units was also elucidated to some extend by means of tandem mass spectrometry. In conclusion, the results obtained in this research highlighted that roasted hazelnut skins, recovered as a byproduct of edible kernel industrial processing, can be considered as a newsworthy source of bioactive PAs. The condensed tannins present in whole extract (RHS-M) and its active fraction (RHSM-F3) consist of PCs, PC−PD heteroligomers, and galloyl derivatives. The flavanol sequence of the main PA oligomers was elucidated by extended tandem mass spectrometry analysis. Bioactivity data indicated that RHS-M and RHS-M-F3 could be used as functional, safe ingredients of functional foods or human health products based on their powerful antioxidant and antifungal activities against Candida albicans.

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AUTHOR INFORMATION

Corresponding Author

*(T.M.) E-mail: [email protected]. Phone: +39 (0)89 968294. Notes

The authors declare no competing financial interest.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05404. Table S1. Flavanol molar proportions, average DP, and galloylation degree of PAs in RHS-M and RHS-M-F3. Figure S1. HPLC-UV chromatogram of thiolytically degraded PAs in RHS-M-F3. Figure S2. Extracted ion chromatograms of RHS-M-F3. Figure S3. Representative MS2 spectra of PA oligomers (DP 2−4) detected in RHS-M-F3 (PDF) J


Article

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HRMS Profile of a Hazelnut Skin Proanthocyanidin-rich Fraction with

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