SUPPLEMENTARY MATERIAL
Phytochemical and antioxidant profiles of leaves from different Sorbus L. species
LinaRaudonėa*, RaimondasRaudonisa,b, Kristina Gaivelytėa, AudriusPukalskasc, PranasViškelisb,c, PetrasRimantasVenskutonisc and ValdimarasJanulisa
aDepartment of Pharmacognosy, Lithuanian University of Health Sciences, Eivenių str. 4, LT- 50161, Kaunas, Lithuania, e-mail:
bInstitute of Horticulture, Lithuanian Research Center for Agriculture and Forestry, Kauno str. 30, LT-54333, Babtai, Kaunas distr., Lithuania, e-mail:
cDepartment of Food Technology, Kaunas University of Technology, Radvilėnų pl. 19, LT-50254, Kaunas, Lithuania,e-mail:
*Corresponding author. Tel.: +370 682 41377; e-mail address: .
Abstract
Leaves of various Sorbus L. have been used in various traditional medicine systems. Pharmacological activities are attributed to a complex of phenolics. In this study phytochemical and antioxidant profiles of S. anglica, S. aria, S. arranensis, S. aucuparia, S. austriaca, S. caucasica, S. commixta, S. discolor, S. gracilis, S. hostii, S. semi-incisa, S. tianschanica were determined. Twenty four constituents were identified in Sorbus L. species using ultra high performance liquid chromatography coupled to quadruple and time-of-flight mass spectrometers (UPLC–QTOF–MS). Post-column FRAP assay identified compounds with reducing activity and revealed significantly greatest total antioxidant activity of 175.30 μmol TE/g DW, 169.20 μmol TE/g DW and 148.11 μmol TE/g DW in S. commixta, S. discolor and S. gracilis leaf samples, respectively with neochlorogenic and chlorogenic acids being most significant contributors. Characteristic fingerprints of phytochemical and antioxidant profiles could be applied for the quality evaluation of various raw materials of Sorbus L. species.
Keywords:Sorbus L., FRAP, chlorogenic acid, neochlorogenic acid, antioxidant profile, post-column assay.
1. Experimental
1.1 Chemicals
The chemicals used in this study were of analytical or chromatographic grade. Ultrapure water was prepared using a Millipore water purification system (Bedford, USA). Acetonitrile, formic acid and hydrochloric acid from Fluka Chemie (Buchs, Switzerland). Ethanol was provided by Stumbras (Kaunas, Lithuania). Trifluoroacetic acid, iron(III) chloride hexahydrate (FeCl3×6H2O), 2,4,6-tripyridyl-s-triazine (TPTZ), sodium acetate trihydrate (C2H3NaO2×3H2O) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) from Sigma-Aldrich Chemie (Steinheim, Germany). HPLC grade reference compounds: neochlorogenic acid ≥98.0%, chlorogenic acid ≥97.0%, rutin ≥94.0%, hyperoside ≥97.0%, isoquercitrin ≥90.0%, astragalin ≥95.0%, (-)-epicatechin ≥90.0%, procyanidin B2 ≥90.0% were purchased from Fluka (Buchs, Switzerland), Roth (Karlsruhe, Germany), Sigma-Aldrich (Buchs, Switzerland). The working FRAP solution comprised TPTZ (0.01 M dissolved in 0.04 M HCl), FeCl3×6H2O (0.02 M in water) and acetate buffer (0.3 M, pH 3.6) in the ratio of 1:1:25.
1.2 Plant material and preparation of extracts
The leaf samples of Sorbus L. species (S. anglica Hedl.; S. aria (L.) Crantz; S. arranensis Hedl.; S. aucupariaL.; S. austriaca (Beck) Hedl.; S. caucasica Zinserl.; S. commixta Hedl.; S. discolor Maxim.; S. gracilis K.Koch; S. hostii (J.Jacq.) K.Koch; S. semi-incisa (Borbas) Borbas; S. tianschanicaRupr.) were collected in Botanical Garden of Vilnius University (June 2011). Voucher specimens (No. 3640, 3641, 3642, 3651, 3654, 3652, 3653, 3658, 3659, 3662, 3663, 3664, respectively) were deposited in foreign department (WI) of herbarium of Vilnius University. Sorbus L. leaves were air dried in room temperature and milled up to the size of the particles passing through the 355 μm mesh. About 0.25 g (precise weight) of the milled leaves were extracted with 25 ml 50% v/v ethanol in ultrasound bath BioSonic UC100 (Coltčne/Whaledent, USA) for 20 min. The obtained leaf extracts were filtered through a 0.22 μm syringe filter (Carl Roth Gmbh, Germany) and subjected to analysis.
1.3 UPLC-QTOF-MS analysis
Extracts were separated using Waters Aquity UPLC system consisting of binary solvent manager, autosampler, column manager and PDA detector (Waters, Milford MA). Waters Aquity HSS T3 column was used for compound separation. Eluent A was 0.4% formic acid solution in water, and B – acetonitrile. The gradient was formed as follows: initially the separation was started with 88%A, it was kept at this concentration for 1 minute, and then in 8 minutes A was decreased to 70%, and from there in 1 minute to 90%, and was hold at 90% for 1 minute. After that the column was allowed to equilibrate for 2 minutes. Eluted compounds were analysed by MAXIS 4G QTOF mass spectrometer (BRUKER DALTONIK GmbH, Bremen, Germany) equipped with ESI ionization source. Capillary voltage was maintained at 4000 V. Nitrogen was used as nebulizing and drying gas, at 2.5 Bar pressure, and 10 l/min flow rate, respectively. Drying gas temperature was maintained 200 °C. MS spectra were recorded in negative mode, in the range from 80 to 1200 m/z. Spectra recording rate was 3 Hz.
1.4 HPLC analysis and FRAP post-column assay
The HPLC equipment system applied consisted of Waters 2695 Alliance solvent manager (Waters, Milford, MA) equipped with a Waters 2998 photodiode array detector. The chromatographic separation was carried out using a 3-μm ACE C18 analytical column (150 × 4.6 mm) with guard column 3-μm ACE C18 (20 × 4.0 mm) (Aberdeen, Scotland) thermostated at 25 °C. The mobile phase consisted of 0.05% (v/v) trifluoroacetic acid solution in water (solvent A) and acetonitrile (solvent B). The optimized linear gradient elution program was determined to be 12% solvent B at 0–5 min, 12–30% solvent B at 5–50 min, 30–90% solvent B at 50–51 min, 90% solvent B at 51–56 min, 12% solvent B at 57 min. The flow rate was set at 0.5 mL/min and the injection volume of all samples was 10 μl. The confirmation of the chromatographic peaks (neochlorogenic acid, chlorogenic acid, procyanidin B1, procyanidin B2, (-)-epicatechin, rutin, hyperoside, isoquercitrin, astragalin) identity was achieved by comparing the retention times and spectral characteristics (λ = 200–600 nm) of the eluting peaks with those of reference compounds. On-line post-column addition of working FRAP solution was performed by chromatograph Beckman programmable solvent module 126 (Fullerton, CA). The flow rate of the individual solution was set at 0.5 mL/min. The post-column reactor was made of 0.4 mL (PEEK, 0.25 mm i.d., 8 m) coil. The reactor temperature was set at 25 °C. The product chromatograms after FRAP post-column reaction were registered at 593 nm respectively, using Waters 2487 dual λ absorbance (UV/Vis) detector (Milford, MA).
The antioxidant activity of sample compounds was assessed by standard antioxidant Trolox. To generate calibration curve, ethanolic solutions of Trolox (5–100 μg/ml) were injected into the HPLC post-column system and analysed under isocratic conditions (50% solvent B). The antioxidant activity of phenolic compounds was expressed as μmol Trolox equivalent (TE) for 1 g of dry weight (DW) of leaves. TE was calculated by the following formula:
TE = c×V/m (μmol/g) (1)
c – the concentration of Trolox established from the calibration curve, μM; V – the volume of fruit extract, L; m – the weight (precise) of leaves, g.
Antioxidant profile enables the evaluation of contribution to antioxidant activity of individual compounds (CAA), because determined relevant peak areas correspond to relevant antioxidant activity of sample mixture (Beekwilder et al. 2005).
CAA = Area(x)/Area(total)×100 (%) (2)
Area(x) – the area of a peak of interest; Area(total) – the sum of the area of all peaks in antioxidant profile.
1.5 Statistical analysis
Statistical analysis was performed using SPSS version 15.0 (Chicago, USA) and Microsoft Excel. All determinations were done in triplicate, means and standard errors were calculated. Linear regression model was analysed. For the suitability of each regression model determination coefficient R2, Pearson’s correlation coefficient and p-value obtained by checking hypothesis on non-linear regression were used. The Mann-Whitney U test was performed for the hypothesis concerning equality of distributives. Level of significance α=0.05.
References
Beekwilder J, Jonker H, Meesters P, Hall RD, van der Meer IM, Ric de Vos CH. 2005. Antioxidants in raspberry: On-line analysis links antioxidant activity to a diversity of individual metabolites. J Agric Food Chem. 53:3313–3320.
Figure S1.Sorbus L. leavesphytochemical profiles: (a) S. anglica; (b) S. aria; (c) S. arranensis; (d) S. aucuparia; (e)S. austriaca; (f) S. caucasica; (g) S. commixta; (h) S. discolor; (i) S. gracilis; (j) S. hostii; (k)S. semi-incisa; (l) S. tianschanica. Peak number refer toTable S1.
Figure S2. Sorbus L. leavesantioxidant profiles in FRAP post-column assay: (a) S. anglica; (b) S. aria; (c) S. arranensis; (d) S. aucuparia; (e)S. austriaca; (f) S. caucasica; (g) S. commixta; (h) S. discolor; (i) S. gracilis; (j) S. hostii; (k)S. semi-incisa; (l) S. tianschanica. Peak number refer to Table 1.
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Table S1.UPLC/ESI-Q-TOF (negative ionization mode) phenolic compounds of Sorbus L. species leaves.
Peak No. / RT, min / Compounds / UPLC/ESI-Q-TOF / λmax,nm / Sorbus L. species
[M-H]- / Fragments / Formula [M-H]
1 / 3.87 / Quinic acid / 191 / C7H11O6 / a, b, c, d, e, f, g, h, i, j, k, l
2 / 6.65 / Neochlorogenic acid / 353 / 191 / C16H17O9 / 325.4 / a, b, c, d, e, f, g, h, i, j, k, l
3 / 7.01 / Procyanidin B1 / 577 / 289 / C30H25O12 / 278.9 / a, b, c, d, e, f, g, h, i, j, k, l
4 / 9.76 / Coumaroylquinic acid derivative / 337 / 163 191 / C16H17O8 / 309 / a, b, e, i, j, k
5 / 10.04 / Chlorogenic acid / 353 / 191 / C16H17O9 / 326.6 / a, b, c, d, e, f, g, h, i, j, k, l
6 / 11.25 / Coumaroylhexose / 325 / 145 / C15H17O8 / 315.8 / d, g, h, i
7 / 11.25 / Caffeoylquinic acid derivative / 353 / 191 / C16H17O9 / 326 / a, b, c, e, f, j, k, l
8 / 12.56 / Procyanidin dimer B2 / 577 / 289 / C30H25O12 / 278.9 / a, b, c, d, e, f, i, j, k, l
9 / 15.04 / Caffeoylquinic acid derivative / 353 / 135 191 297 / C16H17O9 / 325.4 / a, b, c, d, e, f, g, h, i, j, l
10 / 15.75 / (-)-Epicatechin / 289 / C15H13O6 / 286 / a, b, c, d, e, f, h, i, k, l
11 / 16.17 / Coumaroylquinic acid derivative / 337 / 163 191 / C16H17O8 / 311 / a, c, d, f, g, h, i
12 / 16.58 / Caffeoylshikimic acid / 335 / 135 161 179 / C16H15O8 / 327 / b, e, j, k, l
13 / 18.45 / Quercetin dihexoside / 609 / 301 447 / C27H29O16 / 256 355 / a, j, l
14 / 19.01 / Quercetin dihexoside / 609 / 301 447 / C27H29O16 / 255 350 / a, b, c, d, e, f, h, i, j
15 / 19.49 / Quercetin acetyl dihexoside / 651 / 301 / C29H31O17 / 254 355 / a, b, c, d, e, h, j
16 / 22.28 / Coumaroylquinic acid derivative / 337 / 163 191 / C16H17O8 / 311 / g, i
17 / 22.83 / Quercetin dihexoside / 609 / 301 / C27H29O16 / 255 354 / f, g, h, i
18 / 25.67 / Rutin / 609 / 301 / C27H29O16 / 255 354 / a, b, c, d, e, f, g, h, i, j, k, l
19 / 26.98 / Hyperoside / 463 / 301 / C21H19O12 / 255 354 / a, b, c, d, e, f, g, h, i, j, k, l
20 / 27.79 / Isoquercitrin / 463 / 301 / C21H19O12 / 255 354 / a, b, c, d, e, f, g, h, i, j, k, l
21 / 30.69 / Kaempferol coumaroyl glucoside / 593 / 285 / C27H29O15 / 264 346 / a, c, d, e, h, i, k, l
22 / 31.3 / Quercetin malonyl glucoside / 549 / 301 505 / C24H21O15 / 255 254 / a, b, c, d, e, f, h, j, k, l
23 / 32.88 / Astragalin / 447 / 285 / C21H19O11 / 265.8 / a, c, d, g, h, j, k, l
24 / 33.55 / Dicaffeoylquinic acid derivative / 515 / 191 353 / C25H23O12 / 326 / a, b, c, d, e, f, g, h, i, j, k, l
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