Characterization of phenolic compounds and antioxidant and anti-inflammatory properties of red cabbage and purple carrot extracts

Journal of Functional Foods 21 (2016) 133–146

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Characterization of phenolic compounds and antioxidant and anti-inflammatory properties of red cabbage and purple carrot extracts Paulina Mizgier a, Alicja Z. Kucharska a,*, Anna Sokół-Łe˛towska a, Joanna Kolniak-Ostek a, Marcin Kidon´ b, Izabela Fecka c a

Department of Fruit, Vegetable and Cereals Technology, Wrocław University of Environmental and Life Sciences, Chełmon´skiego 37, 51-630 Wrocław, Poland b Department of Fruit and Vegetable Technology, Poznan´ University of Life Sciences, Wojska Polskiego 31, 60624 Poznan´, Poland c Department of Pharmacognosy, Wrocław Medical University, Borowska 211, 50-556 Wrocław, Poland

A R T I C L E

I N F O

A B S T R A C T

Article history:

The qualitative and quantitative evaluation of phenolic compounds contained in extracts

Received 1 September 2015

derived from red cabbage and purple carrot were performed. Their antioxidant and anti-

Received in revised form 22

inflammatory properties were determined. In purple carrot and red cabbage extracts, 7 and

November 2015

21 anthocyanins were identified, respectively, of which 83 and 88% were acylated. The total

Accepted 3 December 2015

anthocyanin content of purple carrot and red cabbage extracts was 154.0 mg cyanidin 3-O-

Available online

glucoside equivalents (Cy 3-glcE) per g dry matter (DM) and 175.1 mg Cy 3-glcE/g DM, respectively. The content of phenolic acids in the purple carrot extract was 133.7 mg 5-O-

Keywords:

caffeoylquinic acid equivalents per g DM. In red cabbage extract, 21 hydroxycinnamic acid

Red cabbage

derivatives (HCAs) were identified for the first time. These compounds mainly include resi-

Purple carrot

dues of p-coumaric, ferulic and sinapic acids or their hydrated forms. Purple carrot extract

Anti-inflammatory activity

showed a superior ability to inhibit COX-2 (44%) compared to red cabbage (24%).

Anthocyanins

© 2015 Elsevier Ltd. All rights reserved.

Phenolic compounds

1.

Introduction

Anthocyanins are natural pigments that belong to the group of flavonoids and are responsible for the attractive colors of fruits, vegetables, and flowers. Anthocyanins are compounds having multidirectional biological activity. Numerous scientific studies have confirmed the antioxidant, anti-inflammatory and antitumor activities (Hou, Fujii, Terahara, & Yoshimoto, 2004; Wang & Stoner, 2008). The antioxidant properties of an-

thocyanins are related to their ability to reduce or prevent the harmful effects of free radicals on the human body, and their protective role in the inflammatory process consists in reducing the pro-inflammatory enzymes, e.g. COX-2, and activating the synthesis of prostacyclin (PGI2) (Bowen-Forbes, Zhang, & Nair, 2010). During inflammation, there occurs formation of large amounts of reactive oxygen species (ROS), e.g. superoxide (O2●−) and hydroperoxyl (HO2●), as well as reactive nitrogen species (RNS) consisting of nitrogen dioxide (NO2), nitric oxide (●NO) and peroxynitrite (ONO 2 − ), which leads to a distortion of

* Corresponding author. Department of Fruit, Vegetable and Cereals Technology, Wrocław University of Environmental and Life Sciences, Chełmon´skiego 37, 51-630 Wrocław, Poland. Tel.: +48 713207712; fax: +48713207707. E-mail addresses: [email protected], (A.Z. Kucharska). http://dx.doi.org/10.1016/j.jff.2015.12.004 1756-4646/© 2015 Elsevier Ltd. All rights reserved.

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Journal of Functional Foods 21 (2016) 133–146

cellular redox balance. This results in increased activity of proinflammatory agents such as inducible nitric oxide synthase (iNOS), inducible cyclooxygenase-2 (COX2), as well as cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα). As demonstrated, anthocyanins have the ability to inhibit the inducible form of nitric oxide synthase (iNOS), thereby reducing the synthesis and release of the active, in the processes of oxidation, nitric oxide ( ● NO) (Joseph, Edirisinghe, & Burton-Freeman, 2014; Lau, Joseph, McDonald, & Kalt, 2009). Therefore, the addition of anthocyanin to products makes it possible to not only improve the color but also to enrich them in bioactive compounds. Acylated anthocyanins have the greatest potential to be used as natural dyes, because they exhibit greater stability at higher pH values under the action of heat and radiation as compared to non-acylated anthocyanins. They can, therefore, be used for coloring products of neutral or slightly alkaline pH, such as powder desserts, dairy products, and also for dyeing batches of fruit or vegetable beverages (Ba˛kowska-Barczak, 2005; de Pascual-Teresa & Sánchez-Ballesta, 2008; Giusti, Rodríguez-Saona, & Wrolstad, 1999; Giusti & Wrolstad, 2003). A good source of acylated anthocyanins is red vegetables. High concentrations of anthocyanins and availability of raw materials makes red cabbage and purple carrot interesting in terms of obtaining the dye extracts. Red cabbage (Brassica oleracea L. var. capitata L. f. rubra) is one of the most important vegetables grown around the world for consumption. For many years, it has also been used for therapeutic purposes (Wiczkowski, Szawara-Nowak, & Topolska, 2013). Recent studies by Zielin´ska et al. (2015) show the possibility of applying red cabbage extract as a dietary supplement in the therapy of inflammatory bowel disease. In addition to the acylated anthocyanins, also glucosinolates, carotenoids and tocopherols are responsible for its health benefits (Podse˛dek, 2007; Volden et al., 2008). Many authors have noted (Ahmadiani, Robbins, Collins, & Giusti, 2014; Podse˛dek, 2007) that, due to the prevalence, intense coloration and high content of anthocyanins (40–188 mg Cy 3-glcE/100 g FW), red cabbage is an excellent vegetable suitable for obtaining natural dyes for food products. Purple carrot (Daucus carota subsp. sativus var. atrorubens Alef) is grown in Middle Asia, Far East and in Europe (Kammerer, Carle, & Schieber, 2004a, 2004b; Türkyılmaz, Yemis¸, & Özkan, 2012). Acylated anthocyanins contained therein are an excellent source of natural dyes and are used among other things for coloring nectars and soft drinks, jellies and sugar confectionery (Türkyılmaz et al., 2012). This material, in addition to anthocyanins (1.5–126.4 mg Cy 3-glcE/100 g FW) (Algarra et al., 2014; Montilla, Arzaba, Hillebrand, & Winterhalter, 2011) also contains phenolic acids and their derivatives in the amount of 74.64 mg in 100 g FW (Alasalvar, Grigor, Zhang, Quantick, & Shahidi, 2001). As seen from the short outline above, both vegetables are rich sources of acylated anthocyanins, but only extracts from purple carrot are commonly used in food industry as colorants. Thus, the question arises, whether extracts from red cabbage could find similar use. Therefore, in this paper, we decided to perform comparison of extracts from the two vegetables. Red vegetables, besides anthocyanins, also contain other phenolic compounds. They are present in significantly smaller

amounts than in fruits, but during preparation of the red dyes, after removal of sugars, aliphatic carboxylic acids and other ingredients, their concentration in the extracts is increased. The presence of these compounds in the anthocyanin extracts can favorably influence the color (due to intermolecular co-pigmentation effect) and enhance their biological value. In purple carrot extracts, phenolic compounds have been identified (Alasalvar et al., 2001; Kammerer et al., 2004a), but there is no information on the content of phenolic compounds, other than anthocyanins, in red cabbage. There is also no information on the anti-inflammatory activity of extracts from red cabbage and purple carrot. Therefore, the aim of this study was to perform qualitative and quantitative comparison of phenolic compounds, contained in red cabbage and purple carrot extracts, and to determine their antioxidant and antiinflammatory activity.

2.

Materials and methods

2.1.

Chemicals

The following were acquired from Sigma-Aldrich (Steinheim, Germany): 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azinobis(3ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tri(2-pyridyl)- S-triazine (TPTZ), dimethyl sulphoxide (DMSO), FeCl 3 , acetonitrile, formic acid, TMPD (N,N,N′,N′tetramethyl-p-phenylenediamine), cyclooxygenase 1 from sheep (C0733-5000UN), cyclooxygenase 2 human (C0858-1000UN), arachidonic acid from porcine liver and hematin porcine. Tris(hydroxymethyl)-aminomethane (TRIS), acetic acid, acetone and NaHSO3 were obtained from Chempur (Piekary S´la˛skie, Poland). Acetonitrile for LC–MS was purchased from POCh (Gliwice, Poland). Cyanidin 3-O-glucoside (C 3-glc) and 5-Ocaffeoylquinic acid (5-CQA), 3-O-caffeoylquinic acid (3-CQA), 4-O-caffeoylquinic acid (4-CQA), rosmarinic acid (RA), o-, m- and p-coumaric (o-, m-, p-CuA) acid, caffeic acid (CA), ferulic acid (FA), isoferulic acid (iFA), sinapic acid (SA), protocatechuic acid (PA) and syringic acid (SgA) were purchased from Extrasynthese (Genay, France). Cyanidin 3-O-coumaroyl-sambubioside-5-Oglucoside (Cy 3-coumsamb-5-glc) was purchased from Polyphenols (Sandnes, Norway). All reagents were of analytical grade.

2.2.

Plant material

Research material consisted of commercially available heads of red cabbage (Brassica oleracea L. var. capitata L. f. rubra) of the ‘Garance’ variety. The roots of the purple carrot variety ‘Deep Purple’ (Daucus carota subsp. sativus var. atrorubens Alef) came from a private farm. Seeds were from the company Bejo Zaden (Oz˙arów Mazowiecki, Poland).

2.3.

Extraction procedure

Heads of red cabbage were shredded in Thermomix (Wuppertal, Vorwerk, Germany). The resulting material (1.0 kg) was extracted with acetone with the addition of acetic acid (2.0 L)

Journal of Functional Foods 21 (2016) 133–146

(5 mL of acetic acid per 1 L acetone). Extraction was conducted in an ultrasonic bath (Sonic 6D, Polsonic, Warsaw, Poland) for 25 min, and then the material was molded in a hydraulic press (Rawtech, De˛bno, Poland). Blanched roots of purple carrot were ground on a Bücher-Guyer grinder (Niederweningen, Switzerland), and then molded in an Arauner-Kitzinger laboratory hydraulic press (Kitzinger, Germany). The obtained extracts were concentrated by a Rotavapor rotary evaporator (Büchi, Flawil, Switzerland) in a water bath at 40 °C. The concentrated acetone extract of red cabbage and purple carrot juice was passed through a column with Amberlite XAD-16 resin (Rohm and Haas, Chauny Cedex, France). The column was washed with distilled water to rinse the organic acids, sugars and other compounds. Anthocyanins were eluted with 80% ethanol with the addition of glacial acetic acid (0.5 mL of glacial acetic acid/L alcohol). The collected fractions were concentrated by a Rotavapor rotary evaporator (Büchi, Flawil, Switzerland) in a water bath at 40 °C, and then dried in an SPT-200 vacuum oven (ZUT Colector, Kraków, Poland) (40 °C, 0.094 MPa).

2.4. Identification of phenolic compounds by UPLC-qTOF-MS/MS The method was previously described by Kucharska, Szumny, Sokół-Łe˛towska, Piórecki, and Klymenko (2015). Identification of phenolic compounds was performed on the Acquity ultra-performance liquid chromatography (UPLC) system, coupled with a quadruple time-of-flight (Q-TOF) MS instrument (UPLC/Synapt Q-TOF MS, Waters Corp., Milford, MA, USA), with an electrospray ionization (ESI) source. Separation was achieved on the Acquity BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm; Waters). The mobile phase was a mixture of 4.5% (v/v) aq. formic acid (A) and acetonitrile (B). The gradient program was as follows: initial conditions – 1% B in A, 12 min – 25% B in A, 12.5 min – 100% B, 13.5 min – 1% B in A. The flow rate was 0.45 mL/min and the injection volume was 5 µL. The column was operated at 30 °C. UV–vis absorption spectra were recorded on-line during UPLC analysis, and the spectral measurements were made in the wavelength range of 200–600 nm, in steps of 2 nm. The major operating parameters for the Q-TOF MS were set as follows: capillary voltage 2.0 kV, cone voltage 40 V, cone gas flow of 11 L/h, collision energy 28–30 eV, source temperature 100 °C, desolvation temperature 250 °C, collision gas, argon; desolvation gas (nitrogen) flow rate, 600 L/h; data acquisition range, m/z 100–2000 Da; ionization mode, negative and positive. The data were collected with MassLynx v. 4.1 software. The runs were monitored at the following wavelengths: anthocyanins at 520 nm, phenolic acids and their derivatives at 320 and 280 nm.

2.5. Quantitative determination of phenolic compounds by HPLC-DAD The analysis was previously described by Sokół-Łe˛towska et al. (2014). The HPLC analysis was performed using a Dionex (Germering, Germany) system equipped with the diode array detector model Ultimate 3000, quaternary pump LPG-3400A, autosampler EWPS-3000SI, thermostated column compartment TCC-3000SD, and controlled by Chromeleon v.6.8 software. Cadenza Imtakt column C5-C18 (75 × 4.6 mm, 5 µm) was used.

135

The mobile phase was composed of solvent A (4.5% aq. formic acid, v/v) and solvent B (100% acetonitrile). The elution system was as follows: 0–1 min 5% B in A, 20 min 25% B in A, 21 min 100% B, 26 min 100% B, 27 min 5% B in A. The flow rate of the mobile phase was 1.0 mL/min, and the injection volume was 20 µL. The column was operated at 30 °C. Hydroxycinnamic (HCA) and hydroxybenzoic (HBA) acid derivatives and depsides were detected at 320 nm and 280 nm, and anthocyanins at 520 nm. Caffeoylquinic acids were expressed as mg of 5-Ocaffeoylquinic acid equivalents (5-CQAE) per g of dry matter (DM), other HCA derivatives as sinapic acid equivalents (SAE) per g DM, and anthocyanins as cyanidin 3-O-glucoside equivalents (Cy 3-glcE) per g DM.

2.6.

Acid and alkaline hydrolysis

Red cabbage extract was subjected to both alkaline and acid hydrolysis, in order to identify HCA derivatives. 1 mg of extract was dissolved in 5 mL of methanol and hydrolyzed for 60 min in conditions of a water bath in the presence of 1 M NaOH or 1 M HCl. After the process, the alkaline hydrolysate was adjusted to pH ~ 3 with 2 M HCl. Next, the hydrolysates were filtered through a hydrophilic PTFE 0.20 µm membrane (Millex Samplicity Filter, Merck) and analyzed by UPLC-qTOF-MS/MS.

2.7.

Antioxidant capacity

The total antioxidant potential of samples was determined using a ferric reducing antioxidant power ability of plasma (FRAP) assay by Benzie and Strain (1996) as a measure of antioxidant power. The DPPH radical scavenging activity of samples was determined according to the method of Yen and Chen (1995). The antiradical activity against ABTS•+ of samples was determined according to the method of Re, Pellegrini, Proteggente, Pannala, and Yang (1999). For all analyses, a standard curve was prepared using different concentrations of Trolox. All determinations were performed in triplicate using a Shimadzu UV-2401 PC spectrophotometer (Kyoto, Japan). The results were corrected for dilution and expressed in mmol Trolox equivalents (TE) per g of extract.

2.8.

Anti-inflammatory activity

Anti-inflammatory activity was determined by a modified method described by Seeram, Momin, Nair, and Bourquin (2001) and Strugała, Gładkowski, Kucharska, Sokół-Łe˛towska, and Gabrielska (2015), consisting of spectrophotometric measurement of the inhibition of cyclooxygenase (COX-1, COX-2) at a wavelength of 611 nm. To the cuvette containing TRIS-HCl buffer (pH 8.0), the test enzyme inhibitor (solution of anthocyanin formulations at a concentration of 8 µg/mL or standard: cy 3-coumsamb-5-glc (0.6 µg/mL), hematin (0.1026 mmol), cyclooxygenase (COX-1/COX-2) (1 mg/mL)) was added. After mixing and incubation (approx. 10 minutes at 37 °C), TMPD (24.35 mmol) and arachidonic acid (35 mmol) were added. The final volume of the sample was 1 mL. Measurements were recorded on a Shimadzu UV-1800 spectrophotometer (Kyoto, Japan) over 3 minutes. The percentage of inhibition of cyclooxygenase activity was calculated by the following formula:

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Journal of Functional Foods 21 (2016) 133–146

% Inhibition =

ΔAcontrol − ΔAsample ΔAcontrol

⋅ 100%

where: ΔAcontrol and ΔAsample denote the increase of absorbance 3 min after substrate addition to the probe without or with the extract tested, respectively.

2.9.

Statistical analysis

Results are presented as the mean ± standard deviation of three technical replications. All statistical analyses were performed with Statistica version 9.0 (StatSoft, Tulsa, OK, USA). One-way analysis of variance (ANOVA) by Duncan’s test was used to compare the mean values. Differences were considered to be significant at α = 0.05.

3.

Results and discussion

3.1. Qualitative and quantitative identification of anthocyanins The results of identification of anthocyanins and other phenolic compounds from red cabbage and purple carrot extracts are shown in Tables 1 and 2 and Fig. S1–S5. The compounds were identified based on the analysis and comparison of retention times and spectra of the individual peaks of the anthocyanin extract of red cabbage and purple carrot, on the basis of spectral data (UPLC-qTOF-MS/MS) and by comparison with literature data (Algarra et al., 2014; Charron, Clevidence, Britz, & Novotny, 2007; Montilla et al., 2011; Wiczkowski et al., 2013). In the red cabbage extract, 21 anthocyanin compounds including 19 acylated were identified (Table 1, Fig. S1). Other authors, in extracts from this material, detected from 9 (Pliszka, Huszcza-Ciołkowska, Mieleszko, & Czaplicki, 2009) to 24 (Arapitsas, Sjöberg, & Turner, 2008), and even to 36 (Charron et al., 2007) anthocyanins. Such varied results may be affected by many factors, such as variety of raw materials, growing conditions, climate and the extraction process itself (parameters, the type of solvent) and the method of marking and identification (Wiczkowski et al., 2013). After fragmentation of all compounds in cabbage extract, ion at m/z 287, characteristic for cyanidin, was obtained, which is consistent with the studies of Arapitsas et al. (2008) and Wiczkowski et al. (2013). McDougall, Fyffe, Dobson, and Stewart (2007) identified in red cabbage, in addition to cyanidin, pelargonidin, and Charron et al. (2007) identified peonidin, but in our study, those aglycones were not detected. Identified anthocyanins were acylated singly (12 compounds) and doubly (7 compounds) mainly with p-coumaric, ferulic and sinapic acids. As in previous studies by other authors (Charron et al., 2007; Giusti et al., 1999), there was no cleavage of ester linkages between glucosides and acyl groups. Acyl groups were assigned by own calculation of the neutral loss and compared with calculations of other authors (Charron et al., 2007). P-coumaric, ferulic and sinapic acid were attached to the tetraglucosides (9RC, 11RC, 13RC, 15RC), triglucosides (8RC, 24– 26RC), (sinapoyl)tetraglucosides (20RC, 21RC, 23RC), and

(sinapoyl)triglucosides (29RC, 31–32RC) of cyanidin. In addition, combinations of caffeic and sinapic acids with triglucoside and diglucoside of cyanidin, respectively, were determined. Peak 11RC was identified as cyanidin 3-p-coumaroyl-triglucoside5-O-glucoside (MS1 [M + H]+ m/z 1081.2990; MS2 m/z 919 [M162 + H] + , m/z 449 [M-162-470 + H] + ). The loss of 470 Da corresponds to the loss of diglucose and p-coumaroyl. Other authors (Charron et al., 2007; Wiczkowski et al., 2013) in a similar red cabbage anthocyanin extract reported a loss of 470 Da corresponding to the loss of glucose, and two acyl residues derived from of p-coumaric acid and caffeic acid. Compound 20RC exhibited a pseudomolecular ion at m/z 1287.3749 ([M + H]+), which corresponded to the molecule of the cyanidin 3-p-coumaroylsinapoyl-triglucoside-5-O-glucoside. It is consistent with studies of Charron et al. (2007), while Wiczkowski et al. (2013) identified in red cabbage cyanidin 3-diferuloyl-triglucoside-5-Oglucoside ([M + H]+ m/z 1287). Compound 30RC was identified as cyanidin 3-OH-feruloyl-sinapoyl-triglucoside-5-O-glucoside (MS1 [M + H]+ m/z 1171.3279; MS2 m/z 1009 [M-162 + H]+, m/z 449 [M-162-560 + H]+). The loss of 560 Da corresponds to the loss of glucose and two acyls of sinapic acid and hydroxyferulic acid (monodesmethylsinapic acid). Charron et al. (2007) in red cabbage identified compound with [M + H]+ at m/z 1171 as cyanidin 3-O(p-hydroxybenzoyl)(malonoyl)-triglucoside-5-O-glucoside. Similar to previous studies by other authors, in a cabbage extract, four isomers of cyanidin 3-O-sinapoly-diglucoside-5-O-glucoside (8RC, 14RC, 16RC, 26RC) were identified. In purple-carrot extracts, 7 anthocyanins including 2 nonacylated (7PC and 8PC) and 5 singly acylated: with sinapic acid (10PC), ferulic acid (12PC, 14PC and 15PC) and p-coumaric acid (compound 13PC) were identified (Table 2, Fig. S2). After fragmentation, ions with [M + H]+ at m/z 287 (7PC, 8PC, 10PC, 12PC and 13PC), m/z 271 (14PC), and m/z 301 (15 PC), characteristic for cyanidin, pelargonidin and peonidin, respectively, were obtained, which is consistent with previous studies (Algarra et al., 2014; Türkyılmaz et al., 2012). Compounds 10PC, 12PC, 13PC had [M + H]+ at m/z 949.2607, 919.2571, 889.2410 (the difference of 30 or 60 Da), and they were identified as acylated glycosides of cyanidin: 3-O-xylosyl-sinapoylglucosylgalactoside, 3-O-xylosyl-feruloylglucosyl-galactoside, 3-Oxylosyl-p-cumaroylglucosyl-galactoside. Compounds 14PC and 15PC had pseudomolecular ions at m/z 903.2582 and m/z 933.2641, respectively. The fragment ions at m/z 271 (14PC) and m/z 301 (15PC) correspond to a similar loss of xylosyl(feruloylglucosyl)galactoside (632 Da). According to other authors (Algarra et al., 2014; Kammerer et al., 2004b; Montilla et al., 2011) in purple carrot also peonidin 3-O-xylosyl(glucosyl)galactoside and cyanidin 3-Oxylosyl(glucosyl)galactoside esterified with caffeic and sinapic acids may occur. However, in our studies, these compounds were not detected. The contents of anthocyanin compounds in the tested extracts are shown in Tables 1 and 2. The total content of anthocyanins present in the extract obtained from red cabbage was 175.0 mg Cy 3-glcE/g DM, whereas in an extract of purple carrot it was 154.4 mg Cy 3-glcE/g DM. Higher concentration of anthocyanins in the extract of cabbage than in the extract of carrot may be due to different levels of pigments in the raw material, and from various extraction conditions. Among anthocyanins identified in red cabbage extract, monoacylated and

Table 1 – Content (mg/g DM) and characterization of anthocyanins and hydroxycinnamic acids derivatives (HCAs) of extract from red cabbage (RC) determined using their spectral characteristics in positive and negative ions in LC–ESI/MS. Peaka

tr (min)

UV λmax (nm)

[M + H]+/ [M-H]− (m/z)

MS/MS (m/z)

Exact mass

Measured mass

Δm (ppm)

Tentative identification

Content (mg/g)b

Anthocyanins [M + H]+ 3 RC

3.70

513

773.2146

611 [M-162 + H]+; 449 [M-162-162 + H]+; 287 [M-162-162 162 + H]+

772.6660

772.2067

0.459

Cy 3-diglc-5-glc

4 RC

3.96

512

611.1614

449 [M-162 + H]+; 287 [M-162-162 + H]+

610.1612

610.1535

0.007

Cy 3,5-diglc

2.97 ± 0.14

8 RC

4.95

528

979.2661

817 [M-162 + H]+; 449 [M-162-368(162 + 206) + H]+; 287 [M-162-368-162 + H]+

978.2640

978.2582

0.014

Cy 3-sin-diglc-5-glc

9.29 ± 0.69

9 RC

4.95

526

1141.3282

979 [M-162 + H]+; 449 [M-162-530(162 + 162 + 206) + H]+; 287 [M-162-530-162 + H]+

1140.9069

1140.3203

0.587

Cy 3-sin-triglc-5-glc

17.82 ± 1.14

6.18

520

1081.2990

919 [M-162 + H]+; 449 [M-162-470(162 + 162 + 146) + H]+; 287 [M-162-470-162 + H]+

1081.2824

1080.2911

0.991

Cy 3-p-coum-triglc-5-glc

6.45 ± 0.16

6.36

521

1111.3154

949 [M-162 + H]+; 787 [M-162-162 + H]+; 449 [M-162-162-338(162 + 176) + H]+; 287 [M-162-162-338-162 + H]+

1110.3089

1110.3075

0.001

Cy 3-fer-triglc5-glc

4.94 ± 0.47

978.2719

978.2646

0.008

Cy 3-sin-diglc-5-glc

1140.9069

1140.3134

0.593

Cy 3-sin-triglc-5-glc

14 RC

6.36

526

979.2725

15 RC

6.51

526

1141.3213

817 [M-162 + H]+; 449 [M-162-368(162 + 206) + H]+; 287 [M-162-368-162 + H]+ 949 [M-162 + H]+; 817 [ M-162-162 + H]+; 449 [M-162-162-368(162 + 206) + H]+; 287 [M-162-162368-162 + H]+ 817 [M-162 + H]+; 449 [M-162-368(162 + 206) + H]+; 287 [M-162-368-162 + H]+

3.04 ± 0.69

16 RC

6.66

526

979.2661

978.2640

978.2582

0.006

Cy 3-sin-diglc-5-glc

20 RC

7.11

535

1287.3749

1125 [M-162 + H]+; 449 [M-162-676(162 + 162 + 176 + 176) + H]+; 287 [M-162-676-162 + H]+

1286.4415

1286.3670

0.074

Cy 3-p-coum-sin-triglc-5-glc

21 RC

7.33

536

1317.3746

1155 [M-162 + H]+; 449 [M-162-706(162 + 162 + 206 + 176) + H]+; 287 [M-162-706-162 + H]+

1316.3745

1316.3667

0.008

Cy 3-fer-sin-triglc-5-glc

5.46 ± 0.35

22 RC

7.47

537

935.2587

934.2770

934.2508

0.026

Cy 3-caff-diglc-5-glc

2.68 ± 0.10

23 RC

7.47

536

1347.3896

24 RC

8.27

522

919.2509

773 [M-162 + H]+; 449 [M-162-324(162 + 162) + H]+; 287 [M-162-324-162 + H]+ 1023 [M-324 + H]+; 449 [M-324-574(162 + 206 + 206) + H]+; 287 [M-324-574-162 + H]+

4.60 ± 0.49

1.17 ± 0.16

1346.4162

1346.3817

0.034

Cy 3-sin-sin-triglc-5-glc

757 [M-162 + H]+; 449 [M-162-308(162 + 146) + H]+; 287 [M-162-308-162 + H]+

918.2435

918.2430

0.0005

Cy 3-p-coum-diglc-5-glc

15.0 ± 0.84 42.50 ± 3.04

25 RC

8.47

523

949.2607

787 [M-162 + H]+; 449 [M-162-338(162 + 179) + H]+; 287 [M-162-338-162 + H]+

948.2534

948.2528

0.0006

Cy 3-fer-diglc-5-glc

26 RC

8.47

526

979.2725

817 [M-162 + H]+; 449 [M-162-368(162 + 206) + H]+; 287 [M-162-368-162 + H]+

978.2640

978.2646

0.008

Cy 3-sin-diglc-5-glc

28 RC

8.89

520

817.2166

655 [M-162 + H]+; 449 [M-162-206 + H]+; 287 [M-162-206-162 + H]+

816.6694

816.2087

0.460

Cy 3-sin-glc-5-glc

29 RC

9.08

534

1125.3179

963 [M-162 + H]+; 449 [M-162-514(162 + 146 + 206) + H]+; 287 [M-162-514-162 + H]+

1124.3207

1124.3100

0.011

Cy 3-p-coum-sin-diglc-5-glc

30 RC

9.19

535

1171.3279

1009 [M-162 + H]+; 449 [M-162-560(162 + 206 + 192) + H]+; 287 [M-162-560-162 + H]+

1170.8487

1170.320

0.529

Cy 3-OHfer-sin-triglc-5-glc

31 RC

9.33

535

1155.3163

993 [M-162 + H]+; 449 [M-162-544(162 + 176 + 206) + H]+; 287 [M-162-544-162 + H]+

1154.3112

1154.3084

0.003

Cy 3-fer-sin-diglc-5-glc

32 RC

9.41

536

1185.3218

1023 [M-162 + H]+; 449 [M-162-574(162 + 206 + 206) + H]+; 287 [M-162-574-162 + H]+

1184.3218

1184.3139

0.008

Cy 3-sin-sin-diglc-5-glc

5.48 ± 0,34

Total

10.07 ± 0.36 2.03 ± 0.15 10.44 ± 0.40 31.06 ± 1.13 175.0

HCAs derivatives [M-H]− 1RC

2.52

325

353.0879

191 [M-162(caff) – H]−; 179 [CA – H]−; 135 [CA-44 – H]−

354.3087

354.0958

0.213

3-CQA

1.02 ± 0.19

2RC

2.87

329

789.2097

627 [M-162 – H]−; 609 [M-162-18 – H]−; 517 [M – 272 – H]−; 491 [M-298 (272 + 26) – H]−; 447 [609162 – H]−; 285; 267 [285-18 – H]−; 241 [OH-diH-SA – H]−; 223 [SA – H]−; 205 [sin f.– H]−; 145; 137

790.5084

790.2176

0.291

di-(OHsin-glc) or glc-OHsin-OHsin-glc

1.26 ± 0.28

5RC

4.45

306

1097.2990

935 [M-162 – H]−; 917 [M-162-18 – H]−; 609 [M-488 – H]−; 447 [935-488(609-162) – H]−; 285; 223 [SA – H]−; 205 [sin f.– H]−; 145; 137

1098.6926

1098.3069

0.385

di-glc-OHp-coum→sin-OHsin-glc

0.27 ± 0.05

6RC

4.78

322

1127.2983

965 [M-162 – H]−; 947 [M-162-18 – H]−; 609 [M-518 – H]−; 590 [di-sin-glc – H]−; 447 [965-518 (609162) – H]−; 285; 205 [sin f. – H]−; 145; 137

1128.7142

1128.3080

0.406

di-glc-OHfer→sin-OHsin-glc

0.20 ± 0.0.4

7RC

4.91

326

1157.3125

995 [M-162 – H]−; 977 [M-162-18 – H]−; 833 [M-162-162(995-162) – H]−; 815 [833-18 – H]-; 609 [M548 – H]−; 447 [609-162 – H]−; 223 [SA– H]−; 205 [sin f. – H]−; 190 [sin f.-15 – H]•−; 175.00 [ sin f.15-15 – H ]•−; 145; 137

1158.5478

1158.3204

0.228

di-glc-OHsin→sin-OHsin-glc

0.57 ± 0.12

10RC

5.07

328

385.1113

386.3505

386.1192

0.231

1-O-sinapoyl-glucose

1.82 ± 0.34

223 [M-162 – H]−; 2O5 [M-180(223-18) – H]−; 190 [sin f.-15 – H]•−; 175 [ sin f.-15-15 – H]•−; 164

Journal of Functional Foods 21 (2016) 133–146

11 RC 13 RC

(continued on next page)

137

138

Table 1 – (continued) Peaka

tr (min)

UV λmax (nm)

[M + H]+/ [M-H]− (m/z)

MS/MS (m/z)

Exact mass

Measured mass

Δm (ppm)

Tentative identification

Content (mg/g)b

6.21

313

935.2443

917 [M-18 – H]−; 773 [M-162 – H]−; 755 [M-162-18 – H]−; 663 [M-272 – H]−; 637 [M-298(272 + 26) – H]−; 447 [M-488 – H]−; 325 [glc-p-CuA – H]−; 285; 267 [285-18 – H]−; 223 [SA – H]−; 205 [sin f. – H]−; 190 [sin r.-15 – H ]•−; 179 [SA-44(canolol) – H]−; 145; 137

936.6394

936.2522

0.387

di-glc-OHp-coum→sin-OHSA

6.30 ± 0.45

17RC

6.67

327

965.2625

803 [M-162 – H]−; 785 [M-162-18 – H]−; 693 [M-272 – H]−; 667 [M-298(272 + 26) – H]−; 447 [M-518 – H]−; 285; 267 [285-18 – H]−; 205 [sin f. – H]−; 145; 137

966.6224

966.2704

0.352

di-glc-OHfer→sin-OHSA

5.81 ± 0.32

18RC

6.79

328

995.2705

833 [M-162 – H]−; 815 [M-162-18 – H]−; 723 [M-272 – H]−; 697 [M-298(272 + 26) – H]−; 447 [M-548 – H]−; 285 ; 267 [285-18 – H]−; 223 [SA – H]−; 205 [sin f. – H]−; 145; 137

996.6054

996.2784

0.327

di-glc-OHsin→sin-OHSA

8.63 ± 0.55

19RC

7.02

321

949.2606

787 [M-162 – H]−; 431 [M-518 – H]−; 387 [431-44 – H]−; 223 [SA – H]−; 205 [sin f. – H]−; 193 [FA – H and SA-15-15 – H]−; 164 [SA-15-44 – H]−; 145; 137

di-glc-OHfer→ sin-diH-SA

1.04 ± 0.20

27RC

8.85

325

1201.3308

1039 [M-162 – H]-; 815 [M-162-224(1039-224) – H]−; 653 [815-162 – H]−; 447 [M-754 – H]−; 285; 267 [285-18 – H]−; 223 [SA – H]−; 205 [sin f. – H]−; 193 [SA-15-15-H]•−; 145; 137

di-glc-sin-OHsin→sin-OHSA

0.45 ± 0.17

33RC

9.96

324

945.2669

721 [M-224 – H]−; 529 [M-416(224 + 192) – H]−; 223 [SA – H]−; 205 [sin f. – H]−; 145; 137

di-glc-sin→(OH)fer-SA

0.30 ± 0,07

34RC

10.04

326

753.2221

529 [M-224 – H]−; 223 [SA – H]−; 205 [sin f. – H]−; 179 [SA-44(canolol) – H]; 145; 137

35RC

10.08

327

1185.3309

36RC

10.18

326

37RC

10.55

38RC 39RC

di-glc-sin-SA

1.39 ± 0.03

753 [M-432 – H]−; 529 [753-224 – H]−; 223 [SA – H]−; 205 [sin f. – H]−; 145; 137

OHsin-diH-sin→sin-sin-di-glc

1.27 ± 0.21

1241.3562

1079 [M-162 – H]−; 753 [M-488 – H]−; 285; 223 [SA – H]−; 205 [sin f. – H]−; 145; 137

di-glc-OHp-coum→sin-sin-di-glc isomer

0.42 ± 0.10

312

1181.3390

1019 [M-162 – H]−; 993 [1019-26 – H]−; 975 [993-18 – H]−; 693 [M-488 – H]−; 667 [693-26 – H]−; 487 [ M-693(693-205) – H]−; 325 [487-162 – H]−; 223 [SA – H]−; 205 [sin f. – H]−; 145; 137

di-glc-OHp-coum→p-coum-sin-diglc

1.41 ± 0.29

10.72

327

1121.3416

897 [M-224 – H]−; 223 [SA – H]−; 145

tri-glc-di-sin-SA

0.50 ± 0.04

10.92

324

1211.3469

1049 [M-162 – H]−; 1006 [M-205 – H]−; 723 [M-488 – H]−; 487 [M-723 – H]−; 325 [487-162 – H]; 223 [SA – H]−; 205 [sin f. – H]−; 145; 137

di-glc-OHp-coum→fer-sin-di-glc

0.56 ± 0.06

40RC

11.02

327

1241.3562

1079 [M-162 – H]−; 1035 [M-206 – H]−; 753 [M-488 – H]−; 487 [M-753 – H]−; 325 [glc-p-CuA – H]−; 223 [SA – H]−; 205 [sin f. – H]−; 145; 137

di-glc-OHp-coum→sin-sin-di-glc isomer

0.90 ± 0.20

41RC

11.28

325

959.2817

735 [M-224 – H]−; 529 [M-430(735-206) – H]−; 511 [M-448(430 + 18) – H or 735-224 – H]−; 223 [SA – H]−; 205 [sin f. – H]−; 145; 137

di-glc-di-sin-SA

6.39 ± 0.44

42RC

11.45

327

1241.3562

1079 [M-162 – H]−; 1035 [M-206 – H]−; 1017 [M-224 – H]−; 873.25 [M-162-206(1079-206) – H]−; 223 [SA – H]−; 205 [sin f. – H]−; 145; 137

di-glc-OHp-coum→sin-sin-di-glc isomer

0.50 ± 0.08

754.6001

960.7944

754.2300

960.2896

Total

0.370

0.505

41.03

Data are expressed as mean ± SD, n = 3. a

Peaks are shown in Figs. S1 and S4.

b

Anthocyanins expressed as cyanidin 3-O-glucoside; 3-O-caffeoylquinic acid expressed as 5-O-caffeoylquinic acid, other HCAs derivatives expressed as sinapic acid.

Cy 3-diglc-5-glc, cyanidin 3-diglucoside-5-glucoside; Cy 3,5-diglc, cyanidin 3,5-diglucoside; Cy 3-sin-diglc-5-glc, cyanidin 3-(sinapoyl)-diglucoside-5-glucoside; Cy 3-sin-triglc-5-glc, cyanidin 3-(sinapoyl)-triglucoside-5-glucoside; Cy 3-p-coum-triglc-5-glc, cyanidin 3-(p-cumaroyl)-triglucoside-5-glucoside; Cy 3-fer-triglc5-glc, cyanidin 3-(feruloyl)-triglucoside-5-glucoside; Cy 3-sin-diglc-5-glc, cyanidin 3-(sinapoyl)-diglucoside-5-glucoside; Cy 3-sin-triglc-5-glc, cyanidin 3-(sinapoyl)-triglucoside-5-glucoside; Cy 3-sin-diglc-5-glc, cyanidin 3-(sinapoyl)-diglucoside-5-glucoside; Cy 3-p-coum-sin-triglc-5-glc, cyanidin 3-(p-cumaroyl)(sinapoyl)-diglucoside5-glucoside; Cy 3-fer-sin-triglc-5-glc, cyanidin 3-(feruloyl)(sinapoyl)-triglucoside-5-glucoside; Cy 3-caff-diglc-5-glc, cyanidin 3-(caffeoyl)-diglucoside-5-glucoside; Cy 3-sin-sin-triglc-5-glc, cyanidin 3-(sinapoyl)(sinapoyl)-triglucoside-5-glucoside; Cy 3-p-coum-diglc-5-glc, cyanidin 3-(p-cumaroyl)-diglucoside-5-glucoside; Cy 3-fer-diglc-5-glc, cyanidin 3-(feruloyl)-diglucoside-5-glucoside; Cy 3-sin-diglc-5-glc, cyanidin 3-(sinapoyl)-diglucoside-5-glucoside, Cy 3-sin-glc-5-glc, cyanidin 3-(sinapoyl)-glucoside-5glucoside; Cy 3-p-coum-sin-diglc-5-glc, cyanidin 3-(p-cumaroyl)(sinapoyl)-diglucoside5-glucoside; Cy 3-OHfer-sin-triglc-5-glc, cyanidin 3-(α-OH-dihydro-feruloyl)(sinapoyl)-diglucoside-5-glucoside, Cy 3-fer-sin-diglc-5-glc, cyanidin 3-(feruloyl)(sinapoyl)-diglucoside-5-glucoside; Cy 3-sin-sin-diglc-5-glc, cyanidin 3-(sinapoyl)(sinapoyl)-diglucoside-5-glucoside → ester bond between HCA fragments X and Y; 3-CQA, 3-O-caffeoylquinic acid CA, caffeic acid; caff, caffeoyl; diH, dihydro; FA, ferulic acid; fer, feruloyl; glc, glucoside; ma, malonoyl; (OH)fer, α-OH-feruloyl (monodesmethylsinapoyl); OHfer, α-OH-dihydroferuloyl; OHp-coum, α-OH-dihydro-p-coumaroyl; OHsin, α-OHdihydrosinapoyl; p-coum, p-coumaroyl; p-CuA, p-coumaric acid; p-OHB, p-hydroxybenzoyl; OHSA, α-OH-dihydro-sinapic acid; SA, sinapic acid; sin, sinapoyl; sin f.–, stabile form of sinapoyl.

Journal of Functional Foods 21 (2016) 133–146

12RC

Table 2 – Content (mg/g) and characterization of anthocyanins and hydroxycinnamic acids derivatives (HCAs) of extract from purple carrot (PC) determined using their spectral characteristics in positive and negative ions in LC–ESI/MS. Peaka

tr (min)

UV λmax (nm)

5 PC 6 PC

3.72 3.92

321 326

9 PC 11 PC 16 PC

4.55 5.44 6.52

324 324 328

18 PC

9.17

327

Total

MS/MS (m/z)

Exact mass

Measured mass

Δm (ppm)

Compound

Content (mg/g)b

743.2062 581.1523 949.2607 919.2571 889.2410 903.2582 933.2642

449 [M-294(132 + 162 + H]+; 287 [M-294-162 + H]+ 449 [M-132 + H]+; 287 [M-132-162 + H]+ 449 [M-500(132 + 162 + 206) + H]+; 287 [M-500-162 + H]+ 449 [M-470(132 + 162 + 176) + H]+; 287 [M-470-162 + H]+ 449 [M-440(132 + 162 + 146) + H]+; 287 [M-440-162 + H]+ 271 [M-632(132 + 162 + 176 + 162) + H]+ 301 [M-632(132 + 162 + 176 + 162) + H]+

742.1995 580.1467 948.2534 918.2502 888.2338 902.2579 932.7826

742.1983 580.1456 948.2528 918.2492 888.2331 902.2503 932.2563

0.001 0.001 0.001 0.001 0.0007 0.329 0.526

Cy 3-xyl-glc-gal Cy 3-xyl-gal Cy 3-xyl-sin-glc-gal Cy 3-xyl-fer-glc-gal Cy 3-xyl-p-coum-glc-gal Pg 3-xyl-fer-glc-gal Pn 3-xyl-fer-glc-gal

8.77 ± 0.40 17.43 ± 0.64 8.63 ± 0.15 97.62 ± 0.90 19.63 ± 0.53 1.98 ± 0.14 0.37 ± 0.08 154.44

353.0840 365.0521 341.0849 353.0879, 707 [2M – H] 179.0321 353.0879, 707 [2M – H] 355.1022 517.1562 365.0521, 731 [2M – H] 527.0800

191 [M-162 – H]−, 179 [M-174 – H]−, 135 [M-174-44 – H]− 203 [M-162 – H]−, 179 [M-162-24 – H]−, 135 [M-162-24-44 – H]− 179 [M-162-24 – H]−, 135 [M-162-24-44 – H]− 191 [M-174 – H]−

354.3087 366.3187 340.2920 354.3087

354.0919 366.0600 340.0770 354.0958

0.217 0.259 0.215 0.212

3-CQA CAD hex-CA 5-CQA

5.67 ± 0.19 0.53 ± 0.12 0.41 ± 0.02 80.17 ± 1.37

135 [M-44 – H]− 191 [M-174 – H]−, 179 [M-174 – H]−, 135 [M-174-44 – H]−

178.2332 354.3087

178.0242 354.0958

0.209 0.213

CA 4-CQA

0.76 ± 0.07 19.86 ± 0.77

193 [M-162 – H]−, 175 [M-162-18 – H]− 355 [M-162 – H]−, 193 [M-162-162 – H]−, 175 [M-162-162-18 – H]− 203 [ M-162 – H]−, 179 [M-174 – H]−, 135 [M-174-44 – H]−

356.3238 518.4640 366.3187

356.1101 518.1641 366.0600

0.214 0.299 0.259

hex-FA dihex-FA CAD

5.51 ± 0.45 5.25 ± 0.39 12.64 ± 0.54

365 [M-162 – H]−, 203 [M-162-162 – H]−, 179 [M-174 – H]−, 135 [M-174-44 – H]−

528.4606

528.0879

0.373

di-CAD

2.92 ± 0.19 133.72

Journal of Functional Foods 21 (2016) 133–146

Anthocyanins [M + H]+ 7 PC 4.03 516 8 PC 4.39 517 10 PC 5.30 531 12 PC 5.51 528 13 PC 5.64 526 14 PC 6.08 511 15 PC 6.16 529 Total HCAs derivatives [M-H]− 1 PC 2.37 326 2 PC 2.62 327 3 PC 3.02 324 4 PC 3.52 326

[M + H]+/ [M-H]− (m/z)

Data are expressed as mean ± SD, n = 3. Cy 3-xyl-glc-gal, cyanidin 3-xylosyl-glucosyl-galactoside; Cy 3-xyl-gal, cyanidin 3-xylosyl-galactoside; Cy 3-xyl-sin-glc-gal, cyanidin 3-xylosyl(sinapoylglucosyl)galactoside; Cy 3-xyl-fer-glc-gal, cyanidin 3-xylosyl(feruloylglucosyl)galactoside; Cy 3-xyl-p-coum-glc-gal, cyanidin 3-xylosyl(coumaroylglucosyl)galactoside; Pg 3-xyl-fer-glc-gal, pelargonidin 3-xylosyl(feruloylglucosyl)galactoside; Pn 3-xyl-fer-glc-gal, peonidin 3-xylosyl(feruloylglucosyl)galactoside; 3-CQA, 3-caffeoylquinic acid; 5-CQA, 5 caffeoylquinic acid; 4-CQA, 4-caffeoylquinic acid; CAD, caffeic acid derivative; hex-CA, caffeic acid hexoside; CA, caffeic acid; hex-FA, ferulic acid hexoside; dihex-FA, ferulic acid dihexoside; di-CAD, di-caffeic acid derivative. a Peaks are shown in Figs. S2 and S5. b Anthocyanins expressed as cyanidin 3-O-glucoside, caffeoylquinic acids and other HCAs derivatives expressed as 5-O-caffeoylquinic acid.

139

140

Journal of Functional Foods 21 (2016) 133–146

Fig. 1 – Structures of basic hydroxycinnamic acids and their derivatives (A) and mechanism of the ion (m/z 205) formation (B).

diacylated anthocyanins were dominant, accounting for 51 and 37.1% of total anthocyanins, respectively (Table 1). There was only 11.9% of non-acylated anthocyanins (3RC and 4RC). According to Wu and Prior (2005), red cabbage is a great source of acylated anthocyanins, which may constitute up to 85% of all anthocyanins of this raw material, which also was confirmed in our study. Of the seven anthocyanins identified in the purple carrot extracts, cyanidin 3-O-xylosyl (feruoylglucosyl)galactoside (12PC) was the dominant compound. It constituted 63.4% of the total anthocyanins. As in the case of red cabbage extract, in the extracts of purple carrot acylated anthocyanins prevailed (83.0% of total anthocyanins). In purple carrot roots examined by Algarra et al. (2014) and Kammerer et al. (2004b), cyanidin 3-O-xylosyl (feruoylglucosyl)galactoside was the predominant compound, which, depending on the variety, accounted for 40– 84% of the total anthocyanin content. According to Kammerer et al. (2004b), the proportion of acylated anthocyanins in the purple carrot is 80%, which is consistent with the results of our research.

3.2. Qualitative and quantitative identification of hydroxycinnamic acid derivatives In both analyzed extracts, numerous derivatives of HCAs were identified, present as esters, glycosides and glycoside-ester

forms. HCA derivatives in their structure contain residues of p-coumaric, caffeic, ferulic, monodesmethylferulic and sinapic acids or their hydrated forms, such as α-OH-dihydro-pcoumaric, α-OH-dihydroferulic and α-OH-dihydrosinapic acid (Fig. 1A). In order to confirm the presence of elementary HCAs, red cabbage extract was subjected to alkaline (1 M NaOH, 60 min, water bath) and acid (1 M HCl, 60 min, water bath) hydrolysis, and analyzed by UPLC-qTOF-MS/MS in positive and negative ionization. The use of two kinds of hydrolysis enabled the identification of the types of bonds present in the structures of HCAs, namely ester and/or glycosidic (stable under alkaline conditions) linkage. In addition, under similar LCMS conditions, fragmentation of reference HCAs (o-, m- and p-coumaric, caffeic, ferulic, isoferulic and sinapic acid), their derivatives (monocaffeoylquinic acids and rosmarinic acid) and the corresponding HBAs (protocatechuic and syringic acid) was analyzed. In the case of phenolic acids and their derivatives, better results were obtained in the negative ionization. Free phenolic acids from both chemical groups were subject to decarboxylation [M-44(CO2)–H]−, and in the case of methoxy derivatives, also gradual demethylation with release of mono[M-15(CH3)–H]•− and didesmethyl [M-30(2 × CH3)–H]•− (Table 3). Ferulic and isoferulic acid had an identical fragment ion composition but differed mainly in signal intensity of m/z 178 [M-15(CH3)–H]•− – ferulic 30% and isoferulic 60%. Coumaric acid positional isomers (o-, m-, p-) showed the same

Table 3 – Characterization of standard phenolic acids and products of alkaline hydrolysis of red cabbage HCAs (HRC) using their spectral characteristic in negative ions in LC–ESI/MS. tr (min)

UV λmax (nm)

[M-H]− (m/z)

MS/MS (m/z)

Measured mass

Δm (ppm)

Compound

154.1201

154.0271

0.093

PA

354.3087

354.0958

0.213

3-CQA

354.3087

354.0958

0.213

5-CQA

180.1574

180.0428

0.115

CA

354.3087

354.0958

0.213

4-CQA

164.1580

164.0481

0.109

p-CuA

194.1840

194.0563

0.128

FA

164.1580

164.0481

0.109

m-CuA

194.1840

194.0563

0.128

iFA

198.1727

198.0531

0.119

SgA

164.1580

164.0481

0.109

o-CuA

224.2099

224.0671

0.143

SA

360.2080

360.0950

0.113

RA

518.2490 518.2490 326.2986 356.1969 386.3505

518.1340 518.1340 326.1024 356.1121 386.1192

0.115 0.115 0.196 0.085 0.231

cis/trans isomers of di-glc-FA glc-p-CuA glc-FA glc-SA

Journal of Functional Foods 21 (2016) 133–146

Standards of phenolic acids and depsides 109 [M-44 – H]−; 107 1.68 258; 293 153.0192; 307.0470 [2M–H]− 2.52 323 353.0879; 191 (M-162 – H]−; 179 [M-12 – H]−; 135 [M-44 – H]− 707.1797 [2M–H]− 3.90 324 353.0879; 191 (M-162 – H]−; 137 [M-54 – H]− 707.1797 [2M–H]− 4.09 322 179.0349; 135 [M-44 – H]− 359.0781 [2M–H]− 4.38 325 353.0879; 191 (M-162 – H]−; 179 [M-12 – H]−; 173 [M-6 – H]−; 135 [M-38 – H]− 707.1852 [2M–H]− 5.96 310 163.0402; 119 [M-44 – H]−; 117 [M-46 – H]−; 104 [M-44-15 – H]− 327.0868 [2M–H]− 6.66 321 193.0484; 178 [M-15 – H]•−; 149 [M-44 – H]•−; 134 [M-15-44 – H]•−; 117; 109 387.1068 [2M–H]−; 581.1669 [3M–H]− 7.05 277 163.0402; 119 [M-44 – H]−; 117 [M-46 – H]−; 104 [M-44-15 – H]− 327.0868 [2M–H]−; 491.1345 [3M–H]− 7.06 322 193.0484; 178 [M-15 – H]•−; 149 [M-44 – H]•−; 134 [M-15-44 – H]•−; 117 387.1028 [2M–H]−; 581.1718 [3M–H]− 182 [M-15 – H]•−; 167 [M-15-15 – H]•−; 153 [M-44 – H]−; 7.49 290 197.0452; 395.1000 [2M–H]− 138 [M-15-44 – H]•−; 123 [M-15-15-44 – H]•−; 121 7.98 277;323 163.0402; 119 [M-44 – H]− 327.0868 [2M–H]−; 491.1345 [3M–H]− 9.31 310 223.0592; 208 [M-15 – H]•−; 193 [M-15-15 – H]•−; 179 [M-44(canolol) – H]−; 447.1306 [2M–H]− 164 [M-15-44 – H]•−; 149 [M-15-15-44 – H]•−; 121 11.94 328 359.0871; 197 ([M-162(OHCA) – H]−; 179 [M-180(CA) – H]−; 161 [M-198 – H]−; 719.1661 [2M–H]− 135 [CA-44 – H]−; 133 [M-198-28 – H]; 123 Products of alkaline hydrolysis of extract from red cabbage HCAs (HRC)a 2.36 238, 326 517.1261 355 [M-180 – H]−; 193 [FA – H]−; 165; 137 2.54 237; 325 517.1261 355 [M-180 – H]−; 193 [FA – H]−; 165; 137 2.96 236, 294 325.0945 163 [M-162 (p-CuA) – H]−; 119 [p-CuA-44 – H]− 3.52 238; 289; 313 355.1042 193 [M-162(FA) – H]−; 149 [FA-44 – H]− 223 [M-162(SA) – H]−; 179 [SA-44(canolol) – H]− 3.97 237; 298 385.1079; 771.2384 [2M–H]−

Exact mass

Main signals are underlined. PA, protocatechuic acid; 3-CQA, neochlorogenic acid; 5-CQA, chlorogenic acid; CA, caffeic acid; 4-CQA, cryptochlorogenic acid; p-CuA, p-coumaric acid; FA, ferulic acid; m-CuA, m-coumaric acid; iFA, isoferulic acid; SgA, syringic acid; o-CuA, o-coumaric acid; SA, sinapic acid; RA, rosmarinic acid; glc, glucose; OHCA, α-hydroxydihydrocaffeic acid; diH, dihydro. a Peaks are shown in Fig. S3.

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fragmentation but differed in intensity of fragment ion 119 [M44(CO2)–H]•− – 100% ortho, 30% meta and 90% para. Depsides, as a result of fragmentation, release the residue of phenolic acids and other components as quinic acid, and released phenolic acids were subject to, among other things, decarboxylation. Among analyzed products of the alkaline hydrolysis of cabbage dye, large amounts of unknown compounds were present (5 peaks: 5.25, 5.99, 6.14, 9.45, 9.91 min) (Fig. S3), which were poorly ionized, in both negative and positive modes. They are probably stable forms of HCAs or anthocyanidin fragments. Free phenolic acids were not detected. In alkaline conditions, the ester bond underwent hydrolytic degradation, while from the α-OH-dihydro derivatives, a water molecule was released. As a result of these processes, three monoglucosides of p-coumaric (2.96 min, m/z 325.0945), ferulic (3.52 min, m/z 355.1042) and sinapic (3.97 min, m/z 385.1159) acid, and two isomers of ferulic acid diglucoside, e.g. gentiobioside (2.36 and 2.54 min, m/z 517.1215), were formed (Table 3). The obtained products of hydrolysis confirmed the complex glycoside-ester structure of identified polyphenols. As a result of their partial degradation, at the site of the ester bond of HCA derivatives molecules, the specific products X and Y were obtained, which come from two fragments creating the pseudomolecular ions [M(X→Y) – H]−: 1) An even fragment X = [MX-17(OH) – H]− derived from a structure formed from parts of HCA with a carbonyl moiety of an ester bond (acyl), which combines (→) with an odd fragment; 2) An odd fragment Y = [MY – H]− derived from part of HCA with -OH in a benzene ring or side chain, to which an even fragment (acyl) is connected. In the red cabbage extract, a total of 21 compounds from the HCA derivatives were identified (Table 1, Fig. S4). Compound 1RC with m/z at 353.0879, as a result of fragmentation, released ions characteristic for the 3-O-caffeoylquinic acid standard, and was identified as neochlorogenic acid. Compound 2RC with m/z at 789.2097 fragmented at 627 corresponding to the loss of glucose [M-162 – H]−, 609 corresponding to the loss of glucose, and H2O [M-162-18 – H]− and 447 corresponding to the loss of another glucose [M-162-18-162 – H]−. In 2RC, also signals from α-OH-dihydrosinapic acid (m/z 241), sinapic acid (m/z 223) and the stable form of sinapoyl as an ion of 3-(4-hydroxy-3,5dimethoxy-phenyl)prop-2-ynal (m/z 205; measured 205.0516, exact mass: 205.0506) (Fig. 1B) were detected. This compound is a diglucoside derivative of α-OH-dihydrosinapic acid dimer, where glucose may be attached to a carbonyl group of the first acid (an ester bond like 1-O-sinapoyl-glucose) and to the phenolic group of the second acid (a glycosidic bond at C-4 of benzene). It can be schematically shown as: glc-di-α-OHdihydrosinapoyl-glc or di-(α-OH-dihydrosinapoyl-glc). Compounds 5–7RC, with pseudomolecular ions at m/z 1097.2990, 1127.2983 and 1157.3125, respectively, differing from each other by approx. 30 or 60 Da (likewise as acylated anthocyanins, e.g. 11RC, 13RC and 15RC), are characterized by similar fragmentation, which corresponds to the loss of the glucose (ions 935, 965 and 995, respectively), and glucose and H2O (ions 917, 947, and 977, respectively). Also, less intensive ions with m/z at 447 and 609 were observed after fragmentation, derived from

sinapoyl-α-OH-dihydrosinapic acid or α-OHdihydrosinapoylsinapic acid and corresponding glucoside (447 = 609 − 162 Da) (glc is attached at the phenolic group of the benzene ring, optionally at α-OH of the side chain) or ester with glucose (glc is attached through the carboxylic function), as well as sinapic acid and the stable form of sinapoyl (m/z 223 and 205). An odd ion Y (609 Da) is formed by cleavage of the pseudomolecular ion [M(X+609) – H]− into two fragments. An even fragment X in the case of 5 RC comes from the structure with mass of 488 Da, in 6 RC from 518 Da, and in 7 RC from 548 Da. As in the case of pseudomolecular ions, they differ by 30 or 60 Da. Nevertheless, fragment ions corresponding to these structures were not found. Most likely, the HCA X fragment is a combination of a suitable α-OH-dihydroxycinnamic acid, and two glucose residues, which in the form of a disaccharide (e.g. gentiobiose) can be attached to the phenolic group, to form a linear glycoside, or separately to the phenolic and alcoholic group of the side chain of α-OH-HCAs, forming a branched glycoside. 5RC, 6RC and 7RC structures can be schematically represented as X→Y: di-glc-α-OH-dihydrocoumaroyl→sinapoylα-OH-dihydrosinapoyl-glc, di-glc-α-OH-dihydroferuloyl→ sinapoyl-α-OH-dihydrosinapoyl-glc and di-glc-α-OHdihydrosinapoyl→ sinapoyl-α-OH-dihydrosinapoyl-glc. Compounds 12RC, 17RC and 18RC with m/z at 935.2443, 965.2625 and 995.2705 have analogic structures. Among the fragment ions of these structures, intensive signals with a mass reduced by glucose (−162) – 773, 803, 833 – and glucose and H2O (−180) – 755, 785, 815 – were present. There was no signal at m/z 609, while ions with 447, 223 and 205 and a very weak 241 occurred. Even fragments X of these compounds are the structures described above, i.e. 488, 518 and 548 Da, which are also not recorded in the current fragmentation ions. In the case of 12RC an additional signal with m/z at 325 corresponding to the glucose derivative of coumaric acid was present. Compounds 12RC, 17RC and 18RC can be schematically represented as X→Y: di-glc-α-OH-dihydrocoumaroyl→sinapoyl-α-OHdihydrosinapic acid, di-glc-α-OH-dihydroferuloyl→sinapoylα-OH-dihydrosinapic acid (Fig. 2) and di-glc-α-OHdihydrosinapoyl→sinapoyl-α-OH-dihydrosinapic acid; compared to 5RC, 6RC and 7RC, they are deprived of glucose in the odd fragment Y (which confirms the lack of signal m/z 609). Compounds 37RC, 39RC and 40RC with m/z 1181.3390, 1211.3469 and 1241.3562, by breaking the molecule, provide ions without glucose [M-162 – H]−, 1019, 1049 and 1079 Da, respectively, odd Y ions [M-488 – H]−, 693, 723 and 753 Da, respectively, and ion at m/z 487 derived from an even X ion with mass 488 Da, which is a diglucoside of α-OH-dihydro-p-coumaric acid (e.g. gentiobioside). The odd fragments from 37RC, 39RC and 40RC are structures differing by 30 or 60 Da, which can be described as diglucosides of sinapic acid esters with p-coumaric, ferulic or another sinapic acid (p-coumaroyl-sinapoyl-di-glc/ sinapoyl-p-coumaroyl-di-glc, feruloyl-sinapoyl-di-glc/sinapoylferuloyl-di-glc and di-sinapoyl-di-glc, respectively). Among fragmentation ions, also ions from p-coumaric acid glucoside (m/z 325), the rest of sinapic acid and a stable form of sinapoyl (m/z 223 and 205) were identified. Finally, compounds 37RC, 39RC and 40RC can be schematically represented as X→Y: di-glc-α-OH-dihydro-p-coumaroyl→p-coumaroyl-sinapoyl-diglc, di-glc-α-OH-dihydro-p-coumaroyl→feruloyl-sinapoyl-diglc and di-glc-α-OH-dihydro-p-coumaroyl→sinapoyl-sinapoyl-

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Fig. 2 – Structures of example hydroxycinnamic acid derivatives (10RC, 17RC) of extract from red cabbage.

di-glc. Compounds 36RC and 42RC are the isomers of 40RC (m/z 1241.3562). The first one has a similar composition of product ions and 42 RC different, which suggests significant differences in the combination of components of this glycosideester (Table 1). Peak 19RC corresponds to the pseudomolecular ion 949.2606, which is fragmented into 787 [M-162(glc) – H]−, 431 [M-518 – H]− and 387 [431.09-44(CO2) – H]−. Fragmentation also gave signals from sinapic and ferulic acids (m/z 223 and 193). An even fragment (acyl) of 19RC (518 Da) is formed from two molecules of glucose and α-OH-dihydroferuloyl, and the odd fragment (ion with m/z at 431) is a sinapic and dihydrosinapic acid depside, and it occurs primarily as a 387 ion formed by decarboxylation (−44) of the free carboxyl group. 19RC can be schematically presented as X→Y: di-glc-α-OHdihydroferuloyl→sinapoyl-dihydrosinapic acid or alternatively di-glc-α-OH-dihydroferuloyl→dihydrosinapoyl-sinapic acid. 27RC pseudomolecular ion with m/z at 1201.3308 fragmented at 1039 [M-162(glc) – H]−, 815 [M-162(glc)-224 – H]−, next 653 [815162(glc) – H]− and 447 [M-754 – H]−. Among fragmentation ions, also ions derived from sinapic and α-OH-dihydrosinapic acid (m/z 223 and 241), as well as from a stable form of sinapoyl (m/z 205), occurred. This compound is a diglucoside derivative of depside, formed of two sinapic and two α-OHdihydrosinapic acids, which can be represented as di-glcsinapoyl-α-OH-dihydrosinapic acid or di-glc-sinapoyl-α-OHdihydrosinapoyl→sinapoyl-α-OH-dihydrosinapic acid. Compound 35RC with m/z at 1185.3309 fragmented at 753 [M432 – H]− and 529 [753-224(SA) – H]− and m/z at 223 and 205, which correspond to sinapic acid and sinapoyl. An odd fragment Y of the compound is composed of two sinapoyl residues and two glucose molecules (m/z 753), while even fragment X is a depside of α-OH-dihydrosinapic and with dihydrosinapic acids (acyl: 449-17(-OH) = 432 Da). The structure of 35RC can be represented as α-OH-dihydrosinapoyl-dihydrosinapoyl→disinapoyl-di-glc. Among the daughter ion of 10RC, 34RC and 41RC compounds with m/z at 385.1113, 753.2221 and 959.2817, respectively, there was confirmed the presence of fragments

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derived from sinapic acid, i.e. an acid residue, a stable form of sinapoyl, its desmethyl radicals (m/z 223, 205, 190 and 175) and optionally canolol (m/z 179 produced by sinapic acid decarboxylation), which were released from glucoside bonds. 10RC was identified as a simple glucoside derivative of sinapic acid, probably 1-O-sinapoyl-glucose (main fragmentation ions at m/z 205 and 190 formed by cleavage of glucose and then methyl). 34RC is a diglucoside of sinapoyl-sinapic acid (main fragmentation ions at m/z 223 and 205, which are formed by release of two glucose residues and breaking of the ester bond of depside – dimer of sinapic acid, 529 Da, corresponds to a stable form of sinapoyl diglucoside/gentiobioside). Compound 41RC during fragmentation released successively sinapic and sinapoyl-sinapic acid (fragmentation ion at m/z 753 and 529), confirming that it is a diglucoside, e.g. gentiobioside of sinapic acid trimer (di-glc-sinapoyl-sinapoyl-SA). The total content of HCAs in the red cabbage extract was 41.03 mg/g (Table 1). Among these compounds, the most abundant compounds were 18RC, 41RC and 12RC, which constituted respectively 21, 16 and 15% of HCAs. Table 2 shows the results of the identification of phenolic compounds contained in the extract of purple carrot. Ten compounds belonging to the group of HCA derivatives were identified (Fig. S5). Compounds 1PC, 4PC and 6PC had m/z at 353.0840, and after fragmentation at m/z 191 and 179, which are characteristic for disintegration of monocaffeoylquinic acid isomers (neochlorogenic, chlorogenic and cryptochlorogenic acid). In purple carrot extract, also ferulic acid derivatives, with a characteristic ion at m/z 193 (9PC, 11PC), were identified. Fragmentation of 2PC, 16PC and 17PC compounds, with [M − H]− at m/z 365.0521 and 527.0800, gave an ion with m/z at 179, which corresponds to caffeic acid. Compounds 2PC and 16PC were identified as caffeic acid derivative isomers, while compound 17PC was identified as a derivative of dicaffeic acid. According to literature data, purple carrot phenolic acids include hydroxycinnamic acid derivatives (Alasalvar et al., 2001; Kammerer et al., 2004a), which was consistent with our research. The total content of phenolic acids in the extract of purple carrot was 133.7 mg 5-CQA/g DM (Table 2). Among those compounds, chlorogenic acid was dominant, and represented 57% of the total amount of phenolic acids. Alasalvar et al. (2001), studying the phenolic acids in orange, yellow, white, and purple carrot, demonstrated that chlorogenic acid in the largest amount is present in purple carrot, which contains from 6 to 12 times more chlorogenic acid than the other variety. According to Alasalvar et al. (2001), chlorogenic acid represents 72.5% of the total HCAs of purple carrots.

3.3.

Antioxidant capacity

Antioxidant properties of obtained extracts and anthocyanin standard (cy 3-coumsamb-5-glc) were measured as free radical scavenging activity (ABTS and DPPH methods) and ferricreducing capacity by the FRAP method (Table 4). Purple carrot extract showed higher DPPH and ABTS activity and FRAP reducing power than the extract of red cabbage. Previous literature results of test materials indicate, however, that red cabbage has higher antioxidant activity than purple carrot. Pliszka et al. (2009), examining three varieties of red cabbage, identified their

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Table 4 – Antioxidant activity (mmol TE/g) and antiinflammatory properties (%) of extract from red cabbage and purple carrot and standard of anthocyanin (cyanidin 3-O-coumaroyl-sambubioside-5-O-glucoside). Extract Red cabbage

Standard Purple carrot

Antioxidant activity 1.95 ± 0.42a DPPH 1.50 ± 0.18b FRAP 2.49 ± 0.35b 3.09 ± 0.21a ABTS 2.53 ± 0.49b 3.83 ± 0.07a Anti-inflammatory properties 31 ± 2a COX-1 38 ± 4a COX-2 24 ± 3b 44 ± 3a

Cy 3-coumsamb5-glc 1.54 ± 0.13c 0.42 ± 0.08c 1.76 ± 0.15c 25 ± 3b 43 ± 3a

Data are expressed as mean ± SD, n = 3. Cy-3-coumsamb-5-glc, Cyanidin 3-O-coumaroyl-sambubioside-5O-glucoside, TE, trolox equivalents; DPPH, 1,1-Diphenyl-2picrylhydrazyl radical; ABTS•+, 2,2′-azinobis(3-ethylbenzothiazoline6-sulphonic acid) radical cation; FRAP, ferric reducing antioxidant power; COX-1, cyclooxygenase 1; COX-2, cyclooxygenase 2. a,b,c Values with different letters are significantly different (p < 0.05).

activity as TEAC level 2.42–3.03 mmol TE/100 g FW, while Wiczkowski et al. (2013) reported activity of 8.65 mmol TE/ 100 g FW. On the other hand, FRAP and ORAC activities determined by Volden et al. (2008), were 2.94 mmol Fe2+/100 g FW and 3.10 mmol TE/100 g FW, respectively. Much lower content of anthocyanins in purple carrot, compared to red cabbage, was noted by Algarra et al. (2014). Purple carrot of ‘Antonia’ and ‘Purple Haze’ varieties was characterized by activity levels of 17.6 µmol TE/100 g FW and 240.0 µmol TE/ 100 g FW (DPPH), and 86.4 and 182.0 µmol TE/100 g FW (FRAP), respectively (Algarra et al., 2014). Another reason for the higher antioxidant activity of the purple carrot extract could be a higher content of phenolic acids and derivatives, including isomers of monocaffeoylquinic acid. According to the literature data, isomers of caffeoylquinic acid are characterized by higher antioxidant activity than other phenolic acids. This is related to the chemical structure of caffeoylquinic acid, which contains two -OH groups linked to the aromatic ring at meta and para sites (Karamac´, Kosin´ska, Estrella, Hernan´dez, & Duenˇas, 2012). Hotta et al. (2002) in their study demonstrated that the DPPH antioxidant activity of phenolic acids is as follows: chlorogenic > caffeic > sinapic > ferulic > p-coumaric acid. In the case of both red cabbage and purple carrot extracts, higher activity for the ABTS+• than for the DPPH• were observed. According to Floegel, Kim, Chung, Koo, and Chun (2011), the ABTS method better reflects the antioxidant properties of highly pigmented materials (i.e. red cabbage, black plum or spinach) than the DPPH method. In our study, similar relationships were observed. However, comparing the activity of extracts and the standard of acylated cyanidin, it can be seen that the extracts were characterized by values higher than or equal to those of cy 3-coumsamb-5-glc. The reason for this may be the additional presence of anthocyanins and other phenolic acids, mainly complex HCAs, in the extracts, and the synergy resulting from their presence. This favors, for coloring products, the use of extracts from raw materials which comprise a mixture of red dyes and phenolic compounds which enhance the antioxidant activity.

3.4.

Anti-inflammatory activity

Table 4 shows the results of anti-inflammatory activity (against COX-1 and COX-2) of red cabbage and purple carrot extracts, as well as the standard of cy 3-coumsamb-5-glc. The capacity of the compounds contained in red cabbage and purple carrot extracts and standard to inhibit COX-1 was at the level of 38, 31, and 25%, respectively. The ability of the compounds to inhibit the COX-2 enzyme was opposite. For this enzyme, purple carrot extract and standard were more effective, being characterized by almost twice the anti-inflammatory activity than the extract of red cabbage. Cyclooxygenase-1 and 2 are enzymes exhibiting similar catalytic properties, but differ in biological activity. COX-1 is a constitutive cyclooxygenase, which by regulating the synthesis of prostanoids is responsible for maintaining the normal function of internal organs, whereas the induced isoform COX-2 increases its expression under the influence of stress factors and inflammation. That is why compounds extracted from fruits or vegetables, which have the ability to inhibit COX-2, are considered to be anti-tumorigenic (Vareed, Reddy, Schutzki, & Nair, 2006). According to Zielin´ska et al. (2015), red cabbage extract showed anti-inflammatory properties when applied to the mouse model of Crohn’s disease. Our research indicates that purple carrot extract exhibits superior anti-inflammatory properties for COX-2 than red cabbage. Anti-inflammatory activity of the compounds depends on their molecular structure. In a study conducted by Seeram et al. (2001), cyanidin showed higher anti-inflammatory activity (COX1: 38.7%, COX-2: 46.8%) compared to cyanidin-rutinoside and cyanidin-glucosylrutinoside, while cyanidin-rutinoside exhibited higher anti-inflammatory activity than cyanidinglucosylrutinoside. As noted by Seeram et al. (2001), the higher the anti-inflammatory activity is, the lower is the number of sugar residues attached to the aglycone. Similar dependence occurred in our study for COX-2. Anti-inflammatory activity against this enzyme was higher for single acylated anthocyanins (extract from purple carrot and the standard) than for double acylated anthocyanins (extract from red cabbage). The ability of the extracts to inhibit COX-1 and COX-2 as a mixture of chemically different compounds also depends on the possible synergistic or antagonistic interactions between anthocyanins and phenolic acids. By comparing the percentages of inhibition of cy 3-coumsamb-5-glc (COX-1: 25% and COX2: 43%) and studied extracts (COX-1: 31–38% and COX-2: 24– 44%) the possibility of such effects in extracts can be seen. Similar effects were observed by Muntha, Alexander-Lindo, and Nair (2005). These authors, for a mixture of betanin and cyanidin 3-O-glucoside, observed much lower anti-inflammatory activity against COX-2 (59%) than for betanin alone (97%).

4.

Conclusions

The results indicate that both red cabbage and purple carrots are good materials to obtain acylated anthocyanin formulations. Despite a comparable total concentration of anthocyanin, there were differences in the content of phenolic acids, antioxidant and anti-inflammatory activity. Purple carrot extract showed a higher content of phenolic acids, higher antioxi-

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dant activity and almost 2-fold higher ability to inhibit COX2, compared to red cabbage extract. In red cabbage extract, 21 HCAs were identified for the first time. These compounds mainly include residues of p-coumaric, ferulic and sinapic acids or their hydrated forms. The obtained results, prevalence of red cabbage, and growing interest in purple carrot, give hope for the replacement of synthetic dyes with natural, biologically active anthocyanin pigments.

Acknowledgements The publication was supported by Wroclaw Centre of Biotechnology, under the program The Leading National Research Centre (KNOW) for the years 2014–2018. This work was supported by the National Science Centre, Grant N N312 279240. We would like to thank the company Bejo Zaden for providing the seeds of purple carrot.

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