SUPPLEMENTARY MATERIAL

Phenolic compounds, volatiles and antioxidant capacity of white myrtle berry liqueurs

Gabriele Serreli1, Igor Jerković2, Katarzyna Angelika Gil3, Zvonimir Marijanović4, Viviana Pacini5, Carlo Ignazio Giovanni Tuberoso3,*

1 Department of Biomedical Sciences, Unit of Experimental Pathology, University of Cagliari, Cittadella Universitaria SS 554, 09042 Monserrato, (CA), Italy

2 Department of Organic Chemistry, Faculty of Chemistry and Technology, University of Split, Ruđera Boškovića 35, 21000 Split, Croatia

3 Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy

4 Department of Food Technology, Marko Marulić Polytechnic in Knin, Petra Krešimira IV 30, 22300 Knin, Croatia

5 Distillerie Mario Pacini S.r.l, Via Cettolini 32, 09030 Elmas (CA), Italy

* Corresponding author. Tel.: +39 0706758644; fax: +39 0706758612.

E-mail address: (C.I.G. Tuberoso).

Material and Methods

Chemicals All the used chemicals were of analytical grade. Methanol, acetonitrile, pentane, diethyl ether, ferrous sulphate, phosphoric acid 85% w/w, Na2CO3, ammonium acetate, ferric chloride, CuSO4•5H2O, CuCl2·2H2O, anhydrous MgSO4, K2S2O8, DPPH●, Trolox, TPTZ, neocuproine (2,9-dimethyl-1,10-phenanthroline) hydrochloride, ABTS●+, Folin-Ciocalteu’s phenol reagent and gallic acid (≥ 98%) were obtained from Sigma-Aldrich (Milan, Italy). Ellagic acid (≥ 95%), malvidin-3-O-glucoside (≥ 95%), myricetin (≥ 99%), myricetin-3-O-galactoside (≥ 99%), and myricetin-3-O-rhamnoside (≥ 99%) were purchased from Extrasynthese (Genay Cedex, France). Ultrapure water (18 MΩ•cm) was obtained with a Milli-Q Advantage A10 System apparatus (Millipore, Milan, Italy).

Chromaticity coordinates The analysis of CIE L*, C*ab and h°ab chromaticity coordinates was performed by a Varian Cary 50 Scan spectrophotometer (Varian, Leini, Turin, Italy) and 10 mm quartz cuvettes. The colour analysis was done registering the transmittance in the visible spectrum (380-780 nm) using CIE 1964 (observer 10°) as an observation standard and CIE D65 as an illuminant. CIE L*C*abh°ab parameters were calculated using the Cary Win UV Color Application V. 2.00 software without transformations of the optical path.

Determination of total phenolic content (Folin-Ciocalteu’s assay), total reducing power (FRAP and CUPRAC assays) and free radical scavenging activity (DPPH● and ABTS●+ assays) All the assays were performed spectrophotometrically on the samples diluted with H2O (1:5-1:10, v/v for the liqueurs and macerates respectively). Total phenolic content was determined with a modified Folin-Ciocalteu’s method and the results were expressed as mg/kg of gallic acid equivalent (GAE) [1, 2]. The FRAP assay was assessed preparing a ferric complex of 2,4,6-tris(pyridin-2-yl)-1,3,5-triazine (TPTZ) and Fe3+ [2, 3], and CUPRAC assay was performed according to Bektaşǒglu et al. [4]. Both results were expressed as mmol/L of Fe2+ [1]. The whole procedure for the DPPH● assay was previously published [2]. The ABTS●+ assay was performed according to Re et al. [5] with some modifications [1]. DPPH● and ABTS●+ data were reported as Trolox equivalent antioxidant capacity (TEAC, mmol/L).

HPLC-DAD and LC-MS/MS Detection and quantitative analysis of phenolic compounds were carried out using an HPLC-DAD method as described by Tuberoso et al. [2]. The samples were diluted with ultrapure water (1:10 v/v), filtered through Econofilter RC membrane (0.45 μm, Ø 25 mm, Agilent Technologies, Milan, Italy) and injected in HPLC without any further purification. Hydroxybenzoic acids were detected and dosed at 280 nm, flavonols at 360 nm and anthocyanins at 520 nm. The method was validated in agreement with the ICH guidance note [6] by determining linearity, limits of detection (LOD), limits of quantification (LOQ), precision and recovery. The linearity was evaluated by preparing standard mixtures of all the commercial phenolic compounds at six different concentrations and analysing them by HPLC-DAD. Standard solutions were prepared in methanol, and the working standard solutions in ultrapure water. The calibration curves for commercial standards were plotted with the method of external standard, correlating the peak area with the concentration by means of the least-squares method. The correlation values were 0.9987–0.9999 in the range of 0.2–30 mg/L. The LODs and LOQs were calculated according to the equation LOD = 3.3 r/S and LOQ = 10 r/S, respectively (where r = standard deviation of the blank, and S = slope of the calibration curve). The precision of this method was evaluated testing intra- and interday repeatability. Six injections of the same standard containing all the phenolic compounds within one day and over three consecutive days, were performed. The relative standard deviation (RSD) for the area under the peak was determined as a measure of precision, and all RSDs were lower than 5%. Recovery rates were used to evaluate the accuracy of the method. Myrtle liqueur samples were spiked with two concentrations of gallic acid (50 and 200 mg/L), myricetin-3-O-rhamnoside (10 and 100 mg/L), and malvidin 3-O-glucoside (1 and 5 mg/L) and each spiked sample was analysed in triplicate. Recovery rates were between 98.2 and 101.5%. The matrix effect was evaluated comparing the response of a standards mix containing the previously listed compounds (each at 20 mg/L), prepared both in myrtle liqueur and in water. No statistical differences were observed (P < 0.05). LC-MS/MS method was used to confirm peaks’ attribution, and the system was optimised in positive mode for anthocyanins and in negative for other phenolics [2].

Headspace solid-phase microextraction (HS-SPME) and liquid-liquid extraction (LLE) HS-SPME was performed using a manual holder with polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibre from Supelco Co. (Bellefonte, PA, USA). The samples were placed in 15 mL glass vials and hermetically sealed with PTFE/silicone septa and placed in a water bath at 60°C during equilibration (15 min) and HS-SPME (45 min) under constant stirring (1000 rpm) with a magnetic stirrer. After sampling, the fibre was inserted into the injector (250 °C) of GC-FID and GC-MS for 6 min. For LLE, 2 mL of the samples were extracted three times with pentane : diethyl ether (1:2 v/v). The extracts were combined, dried over MgSO4, concentrated to 0.3 mL and 0.1 μL was analysed by GC-FID and GC-MS.

GC-FID and GC-MS Analyses GC-FID analyses were carried out with an Agilent Technologies (Palo Alto, CA, USA) gas chromatograph model 7890A equipped with a flame ionization detector (FID) and HP-5MS column (5% phenyl-methylpolysiloxane, Agilent J and W). The GC conditions were previously described by Jerković et al. [7]. GC-MS analyses were performed using an Agilent Technologies (Palo Alto, CA, USA) gas chromatograph model 7820A equipped with a mass selective detector model 5977E (Agilent Technologies) and a HP-5MS column, under the same conditions as previously described [7]. The volatiles identification was based on the comparison of their retention indices (RI) with those reported in the literature and their mass spectra with authentic compounds available in our laboratories or those listed in Wiley 9 (Wiley, New York, NY, USA) and NIST 14 (D-Gaithersburg) mass spectral libraries. The percentage composition was computed from the GC peak areas using the normalization method (without correction factors). The average component percentages in the tables were calculated from GC-FID and GC-MS analyses of triplicate extractions.

Statistical analyses The Graph Pad INSTAT software (GraphPad software, San Diego, CA, USA) was used to calculate the means and standard deviations of three independent experiments, involving triplicate or duplicate analyses for each sample/condition. Evaluation of statistical significance of differences was performed by one-way analysis of variance (One-way ANOVA). The statistical analysis was done using 2 - way analysis of variance, followed by Bonferroni post hoc test. All data are expressed as means ± standard error of the means. P < 0.05 was considered statistically significant.

References

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6.ICH Topic Q2 (R1) (2005). Validation of analytical procedures: Text and methodology. Accessed 25 October 2016

7.Jerković I, Prđun S, Marijanović Z, Zekić M, Bubalo D, Svečnjak L, Tuberoso CIG (2016) Traceability of Satsuma mandarin (Citrus unshiu Marc.) honey through nectar/honey-sac/honey pathways of the headspace, volatiles, and semi-volatiles: chemical markers. Molecules 21:1302

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