Article pubs.acs.org/JAFC

Characterization of Phenolic Compounds and Antioxidant Activity of Solanum scabrum and Solanum burbankii Berries J. Oszmiański, J. Kolniak-Ostek, and A. Wojdyło* Department of Fruit, Vegetable and Grain Technology, Wroclaw University of Environmental and Life Sciences, 37/41 Chełmońskiego Street, 51 630 Wroclaw, Poland ABSTRACT: The purpose of this research was to quantify and characterize phenolic compounds and to measure the antioxidant activity of Solanum scabrum and Solanum burbankii berries. The antioxidant activity of Solanum berry extracts was assayed by electrochemical and spectrophotometric methods, whereas liquid chromatography (LC)/quadrupole time-of-flight mass spectrometry and ultra-performance LC-photodiode detector were used for identification and quantification of their polyphenols. Eighteen phenolic compounds were identified in these fruits. The presence of seven phenolic acid derivatives and two flavonols was reported for the first time. In both cultivars, the major compound was found to be anthocyanin petunidin-3-(pcoumaroyl-rutinoside)-5-O-glucoside. Additional anthocyanins in S. burbankii and S. scrabum berries were characterized as petunidin, delphinidin, and malvidin with the same glycosidic substitution pattern and acylation with p-coumaric and ferulic acids. S. scabrum was richer in phenolic compounds, especially anthocyanins, and was characterized by more powerful antioxidant activity than S. burbankii. KEYWORDS: UPLC-PDA−Q/TOF-MS, identification, Solanaceae, phenolic acid, flavonols, acylated antocyanins, antioxidant activity, electrochemical detector

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INTRODUCTION

The second investigated berry is S. burbankii, being an interspecies nightshade hybrid developed by Luther Burbank. Its berries are tiny, smaller (approximately 0.5 cm in diameter) than those of S. scabrum, with a different flavor and sweet taste. The low-growing plant hides its berries well under the foliage and is a bit more difficult to harvest than other edible nightshades. They are good fresh but even better when sweetened and cooked. Short plants produce good yields rather quickly. Similarly to S. scabrum, S. burbankii is very rich in anthocyanin. Its fruits are widely used for the preparation of pies, jams, and sauces, with an intense purple color.4,5 Some of the anthocyanin composition of the S. scabrum and S. burbankii berries has already been established;2 however, these authors did not identify other phenolic compounds presented in these fruits, i.e., quercetin and hydroxycinnamic acid derivatives. The major pigments in the berries of this species were anthocyanidins, especially petunidin-3-(p-coumaroyl-rutinoside)-5-O-glucoside (93%). Seven other pigments included delphinidin-3-O-rutinoside-5-O-glucosides acylated with p-coumaric, ferulic, and an unidentified acid; malvidin-3O-rutinoside-5-O-glucosides acylated with p-coumaric and ferulic acid; and petunidin-3-O-rutinoside-5-O-glucoside acylated with two p-coumaric acid moieties. Over 97% of the anthocyanins present in S. scabrum are acylated with cinnamic acids. In other fruits belonging to the family of Solanum, i.e., in Solanum quineese, Francis et al.1 identified also petunidin-3-(pcoumaroyl-rutinoside)-5-O-glucoside, as the major pigment, and 3-O-rutinoside-5-O-glucosides of petunidin or malvidin.

Solanaceae are a family that includes a number of toxic and edible plants. The name of the family originates from the Latin Solanum, “the nightshade plant”.1 Most likely, the name comes from the perceived resemblance that some of the flowers bear to the sun and its rays, and in fact, a species of Solanum (Solanum nigrum) is known as the “sunberry”. Solanaceae species are often rich in alkaloids the toxicity of which to humans and animals ranges from mildly irritating to fatal in small quantities.1 In most parts of the world, particularly in Europe and North America, these species are considered to be troublesome weeds of agriculture, but in many developing countries, they constitute a minor food crop, with the shoots and berries not only being used as vegetables and fruits, but also for various medicinal and local uses. Some of species, however, are often considered to be edible forms, and consumption of their leaves and fruits in man’s diet is widespread, particularly in Africa and SE Asia.3 In Poland and other countries of Eastern Europe two Solanum species are well-known and cultivated, namely, Solanum scabrum and Solanum burbankii, which are consumed fresh and as homemade products in the form of juices, sauces, and jams. S. scabrum is relatively easy recognizable by its large-sized leaves, strong stem with distinct dented wings, and manyseeded fruits 15−17 mm in diameter that do not drop at maturity. It is additionally characterized by vigorous growth and easy cultivation.1,2 The berry is green when immature and purple to purplish black when ripe, with a high concentration of anthocyanins. Therefore, these anthocyanin pigments are used as colorants in fruit juices and apple sauce. Additionally, S. scabrum is a widely cultivated species and is used as a leafy vegetable, as a medicinal plant, and as a source of ink dye.3 © 2014 American Chemical Society

Received: Revised: Accepted: Published: 1512

July 18, 2013 January 27, 2014 January 29, 2014 February 7, 2014 dx.doi.org/10.1021/jf4045233 | J. Agric. Food Chem. 2014, 62, 1512−1519

Journal of Agricultural and Food Chemistry

Article

Plant Material. S. scabrum and S. burbankii berries were collected in 2011 from the Garden of Medicinal Plants herbarium at the Medical University in Wroclaw, Poland, by cultivation in the University’s experimental field. The fruits were harvest successively during 10 days and after harvest, the berries (100 g per day, finally 1 kg) were immediately frozen and stored at −20 °C until used for analysis. Extraction Procedure. The extraction procedure of polyphenols was carried out as described previously by Oszmiański et al.18 The frozen berries were quick thawed in a microwave oven and heated only to 20 °C and then directly crushed in a laboratory mill (IKA A11). This extraction procedure was applied because use of the microwave oven was previously found to be the best extraction method when berries were exposed for 1 min. Polyphenols were isolated from berries by maceration with water containing 200 mg/L SO2 and subsequently extracted in a ultrasonic bath at 20 °C for 15 min. The ratio of this solvent to berries was 10:1 (v/v). The homogenized mixture was then centrifuged for 5 min (4000 rpm; MPW 360R; Warsaw, Poland), and the extraction was repeated twice. The experiment were performed in triplicate. Four milliliters of the extract was passed through a C18 Sep-Pak cartridge (360 mg/0.7 mL; Waters) preconditioned with 5 mL of ethyl acetate, 5 mL of methanol, and 5 mL of 0.01 N HCl. The loaded cartridge was washed with 3 mL of 0.01% HCl and then the cartridge was dried with a flow of nitrogen gas for 3 min. Phenolic compounds other than anthocyanins were eluted with 20 mL of ethyl acetate. The solvent was removed using a rotary vacuum evaporator at 20 °C, and the phenolic residue was dissolved in 20% acetonitrile for UPLCPDA−MS analysis. Anthocyanins adsorbed in the Sep-Pak C18 were eluted with methanol containing 0.5% HCOOH, and the anthocyanin fraction was analyzed by the UPLC-PDA−MS method. Absorption Spectra of Berry Extracts. Berry extract was used for measurement of absorption spectra, which were recorded in the visible wavelength range from 450 to 600 nm. These spectra were measured using a PC 2401 UV−vis spectrophotometer (Shimadzu; Tokyo, Japan) in a 10-mm path length cell, with buffer solutions as a reference. Buffer solutions of two different pH values (pH 2.5 and 7.5) were prepared by mixing 50 mM ammonium formate with acetonitrile (50/50; v/v). pH was adjusted with ammonium hydroxide. All experiments were repeated in triplicate. Identification of Polyphenols by the UPLC-PDA−Q/TOF-MS Method. Identification of polyphenol of S. scabrum and S. burbankii berry extracts was carried out using an ACQUITY ultra-performance LC system (UPLC) with binary solvent manager (Waters Corp.; Milford, MA) and a Micromass Q-Tof Micro mass spectrometer (Waters; Manchester, UK) equipped with an electrospray ionization (ESI) source operating in negative and positive mode. Separations of individual polyphenols were carried out using a UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm, Waters Corp.; Milford, MA) at 30 °C. Samples (10 μL) were injected, and elution was completed in 15 min with a sequence of linear gradients and isocratic flow rates of 0.45 mL min−1. The mobile phase was composed of solvent A (4.5% formic acid, v/v) and solvent B (100% acetonitrile). The program began with isocratic elution with 99% A (0−1 min), and then a linear gradient was used until 12 min, lowering A to 0%; from 12.5 to 13.5 min, the initial composition (99% A) was used and then held constant to reequilibrate the column. Analysis was carried out using full scan, datadependent MS scanning from m/z 100 to 1500. Leucine enkephalin was used as the internal reference compound during ESI-MS accurate mass experiments and was permanently introduced via the LockSpray channel using a Hamilton pump. The lock mass correction was ±1.000 for the mass window. All quadrupole time-of-flight mass spectrometry (Q/TOF-MS) chromatograms are displayed as base peak intensity (BPI) chromatograms and scaled to 12 400 counts per second (cps) (=100%). The effluent was led directly to an electrospray source with a source block temperature of 130 °C, desolvation temperature of 350 °C, capillary voltage of 2.5 kV, and cone voltage of 30 V. Nitrogen was used as desolvation gas at flow rate 300 L h−1. The characterization of the single components was carried out via the retention time and the accurate molecular masses. Each compound was optimized to its estimated molecular mass [M − H]− in the

Spectral inspection indicates that there is a considerable number of interfering compounds that absorb in the UV−vis region. These compounds make it difficult to confirm the identical nature of the individual pigment peaks. Anthocyanins are able to capture free radicals by donation of phenolic hydrogen atoms.10,11 Acylation of the anthocyanin molecule improves their stability through intramolecular and/ or intermolecular copigmentation and self-association reactions. Therefore, sources of acylated anthocyanins of the S. scabrum and S. burbankii berries may ensure the desirable stability for food applications. The anthocyanins with acylating substituents are more stable during processing and storage than the unacylated pigments. The antioxidative activity of phenolic compounds, especially anthocyanin, was studied by different chemical methods. The Folin−Ciocalteu method, the oxygen radical absorbance capacity (ORAC) assay, and the Trolox equivalent antioxidant capacity (TEAC) assay are the most considered methods. Kilmartin et al.13 developed an electrochemical method for the characterization of the antioxidant properties of wine and wine phenolics. Recently, Janeiro et al.14 studied the redox behavior of anthocyanins by voltammetric techniques and found that all the phenolic hydroxyl groups can be oxidized electrochemically. Also differences in electroactive substituents on analogous structures can lead to characteristic differences in their voltammetric behavior.15 Pereira at al.16 studied the effects of substitution for the hydroxyl group at position C4′ in the B-ring of the flavylium cation, which indicated that the para-position of the ring B is important for the increase in the delocalization of π-electrons in the chromophore. Electrochemical studies reveal general trends in the electron-donating abilities of flavonoids. Cren-Olivé at al.17 shown the importance the catechol group in the B-ring, which is more easily oxidizable than the resorcinol group in the A-ring. They indicated that the 4′-OH is the group that deprotonates preferentially in the flavylium cation of cyanidin and cyanidin-3-O-glucoside.17 The purpose of this research was to tentatively identify and quantify phenolic compounds, main flavonoids, phenolic acids, and anthocyanins for better characterization of S. scabrum and S. burbankii berries. Additionally, the study was aimed at monitoring the changes in the antioxidant activity by spectrometric methods (ABTS and FRAP assay) and an electrochemical technique, cyclic voltammetry. These rapid analytical techniques are very useful in determining the antioxidant activity, which is a consequence of the ability of an antioxidant to donate electrons around the potential of the anodic wave.

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MATERIALS AND METHODS

Reagents and Standard. Acetonitrile and methanol for UPLC and LC/MS purity, formic acid, leucine enkephalin, 2,2-azinobis(3ethylbenzothiazoline-6-sulfonic acid (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), ammonium formate, and methanol were purchased from Sigma-Aldrich (Steinheim, Germany). Quercetin-3-O-glucoside and -3-O-rutinoside; peonidin, delphinidin and petunidin aglycon; pcoumaric acid; and sinapic acid were purchased from Extrasynthese (Lyon, France). Chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid were purchased from TRANS MIT GmbH (Giessen, Germany). UPLC-grade water, prepared by using an HLP SMART 1000s system (Hydrolab, Gdańsk, Poland), was additionally filtered through a 0.22 μm membrane filter immediately before use. 1513

dx.doi.org/10.1021/jf4045233 | J. Agric. Food Chem. 2014, 62, 1512−1519

a

malvidin-3-O-rutinoside-5-O-glucoside

malonyl-caffeoylquinic acid

acetylo-p-coumaroylquinic acid

quercetin-3-O-rutinoside

7

8

9

10

1514

petunidin-3-O-p-coumaroyl-hexoside-5-O-hexose

petunidin-3-O-feruoyl-hexoside-5-O-hexose

malvidin-3-O-p-coumaroyl-hexoside-5-O-hexose

malvidin-3-O-feruoyl-hexoside-5-O-hexose

15

16

17

18

10.75

10.28

9.82

9.30

9.10

8.89

8.44

8.37

8.30

6.24

5.76

5.53

4.66

4.43

4.16

3.61

3.33

2.27

tR (min)

532

532

532

531

328

529

351

528

352

310

325

526

325

529

324

324

525

323

λmax (nm) [M − H] 353.0870 (100%) 191.0555 (100%)/173.0446 (43%)/135.0452 (62%) [M + H]+ 773.2146 (100%) 611.1609 (26%)/465.1017 (21%)/303.0496 (100%) [M − H]− 707.1823 (100%) 353.0875 (88%)/191.0558 (100%) [M − H]− 353.0879 (100%) 191.0555 (84%)/173.0449 (100%) [M + H]+ 787.2300 (100%) 625.1765 (35%)/479.1188 (25%)/317.0671 (100%) [M − H]− 439.0872 (100%) 353.0879 (20%)/233.0660 (100%)/191.0555 (32%)/173.0449 (20%) [M + H]+ 801.2440 (100%) 639.1934 (42%)/493.1325 (30%)/331.0814 (100%) [M − H]− 439.0872 (100%) 395.0979 (29%)/353.0879 (20%)/233.0665 (97%)/191.0555 (25%)/173.0453 (100%) [M − H]− 379.1031 (100%) 319.0818 (31%)/163.0386 (245%)/145.0296 (46%) [M − H]− 609.1432 (100%) 301.0277 (86%) [M + H]+ 919.2509 (100%) 757.1982 (70%)/465.1034 (46%)/303.0496 (100%) [M − H]− 463.0887 (100%) 301.0277 (100%)/173.0447 (30%) [M + H]+ 949.2607 (100%) 787.2077 (85%)/465.1032 (55%)/303.0496 (100%) [M − H]− 339.0716 (100%) 223.0606 (100%)/149.0241 (57%) [M + H]+ 933.2642 (100%) 771.2128 (99%)/479.1187 (59%)/317.0671 (100%) [M + H]+ 963.2780 (100%) 801.2242 (100%)/479.1100 (87%)/317.0671 (86%) [M + H]+ 947.2829 (100%) 785.2296 (99)/493.1341 (55%)/331.0814 (100%) [M + H]+ 977.2892 (100%) 815.2397 (100%)/613.3407 (86%)/493.1344 (54%)/331.0814 (97%)

−

[M + H]+/[M − H]− (m/z) and MS/MS fragments in ESI-MS-Q-TOF

Results represent mean values of triplicated determinations ± standard deviation (p < 0.001). bnd: not detected.

total phenolics (mg/100 g)

sinapoyl malic acid

14

delphinidin-3-O-feruoyl-rutinoside-5-O-glucoside

malonyl-caffeoylquinic acid

6

13

petunidin-3-O-rutinoside-5-O-glucoside

5

quercetin-3-O-glucoside

4-caffeoylquinic acid

4

12

5-caffeoylquinic acid

3

delphinidin-3-O-p-coumaroyl-rutinoside-5-O-glucoside

delphinidin-3-O-rutinoside-5-O-glucoside

2

11

3-caffeoylquinic acid

compd

1

peak

156.87 ± 3.87 13.67 ± 1.17 3.82 ± 0.66 1.37 ± 0.11 239.35***

0.91 ± 0.17 2.66 ± 0.11 0.24 ± 0.04 160.95***

1.62 ± 0.11

4.96 ± 0.87

63.26 ± 5.75

0.38 ± 0.05

0.44 ± 0.04

0.84 ± 0.07

5.67 ± 1.01

6.10 ± 1.20

0.98 ± 0.06

ndb

2.56 ± 0.41

21.08 ± 0.68

14.24 ± 1.51

15.95 ± 0.85

7.71 ± 0.64

7.80 ± 1.07

2.11 ± 0.26

1.68 ± 0.13

0.20 ± 0.04

0.29 ± 0.10

1.69 ± 0.15

7.81 ± 1.50

47.64 ± 2.47

0.24 ± 0.07

1.37 ± 0.08

0.24 ± 0.01

2.37 ± 0.12

0.82 ± 0.04

S. scabrum

0.83 ± 0.03

S. burbankii

Table 1. Qualitative and Quantitative (mg/100 g dm) Characterization of S. scabrum and S. burbankii Phenolic Compound Profiles with UPLC-PDA−MS/MSa

Journal of Agricultural and Food Chemistry Article

dx.doi.org/10.1021/jf4045233 | J. Agric. Food Chem. 2014, 62, 1512−1519

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Article

Figure 1. UPLC profile at 320 nm (phenolic acid and quercetin derivative) and 520 nm (anthocyanins) of Solanum berry extracts. For abbreviations of the peak labels, see Table 1 1515

dx.doi.org/10.1021/jf4045233 | J. Agric. Food Chem. 2014, 62, 1512−1519

Journal of Agricultural and Food Chemistry

Article

negative and positive mode before and after fragmentation. The data obtained from UPLC/MS were subsequently entered into the MassLynx 4.0 ChromaLynx Application Manager software. On the basis of these data, the software is able to scan different samples for the characterized substances. The runs were monitored at the following wavelengths: hydroxycinnamates at 320 nm, flavonol glycosides at 360 nm, and anthocyanins at 520 nm. Photodiode detector (PDA) spectra were measured over the wavelength range 200−600 nm in steps of 2 nm. Retention times and spectra were compared with those of pure standards within 200−600 nm. Calibration curves were run for the external standards 3-, 4-, and 5-O-caffeoylquinic acids, p-coumaric acid, quercetin-3-O-rutinoside and -3-O-glucoside, peonidin-3-O-rutinoside, delphinidin-3-O-rutinoside, and petunidin-3-O-rutinoside at concentrations ranging from 0.05 to 5 mg/mL. Amounts of 3-, 4-, and 5-Ocaffeoylquinic acids were calculated for the quantification of the neochlorogenic, cryptochlorogenic, and chlorogenic acids and their isomers. The concentrations of p-coumaric and sinapic acid containing compounds were calculated by using p-coumaric and sinapic acid, respectively. The concentrations of delphinidin-, petunidin-, and malvidin-containing compounds were calculated by using delphinidin, petunidin, and malvidin aglycons, respectively. Obtained results was express as milligrams per 100 g of dry matter (dm). Analysis of Antioxidant Activity. The protocols for analysis were prepared as described previously by Wojdyło et al.19 The ABTS•+ activity of the sample was used for measurement of antioxidant activity. The total antioxidant potential of the sample was determined using a ferric reducing ability of plasma (FRAP) assay. 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 are expressed in millimole of Trolox per 100 g dm. Cyclic Voltammograms. The mixture of 50 mM ammonium formate (pH 7.4) and acetonitrile (50/50, v/v) was prepared as the supporting electrolyte. All experiments were performed on a ROXY EC system (Antec; Zoeterwoude, The Netherlands). Electrochemical conversions were accomplished in an electrochemical thin-layer cell (ReactorCell, Antec). The reactor cell consisted of a three-electrode arrangement including a working electrode, an auxiliary electrode, and a reference electrode. Glassy carbon was used as working electrode material. The accessible area of the working electrode was 15.1 mm2. The inlet block of the cell was employed as auxiliary electrode and the HyREF (Antec) electrode was used as reference electrode. The working electrode and the auxiliary electrode inlet block were separated by a 50-μm spacer, giving a cell volume of approximately 750 nL. Potentials (0−3000 mV) were applied using a purposive potentiostat (ROXY potentiostat, Antec). Shredded S. scabrum and S. burbankii berry extracts (∼2.5 g) were mixed with 3 mL of ammonium formate and then centrifuged for 10 min at 15 000 rpm (20.878g; MPW-360R; Warsaw, Poland). Prior to each run, the surface of the glassy carbon electrode was freshly abraded with 12 μm flattening plate and rinsed with redistilled water and methanol. The scan was taken in the potential range between 0 and 3000 mV with a scan rate 50 mVs−1. Samples were pumped through the cell at a flow rate of 5 μL/min by a syringe pump model 74900 (Cole Parmer; Vernon Hills, IL). Cyclic voltammograms were also recorded for 0.25 mM cyanidin, malvidin, and pelargonidin aglycon standards solutions.

for quercetin derivatives, and at 520 nm for anthocyanins and are shown in Figure 1, respectively. Eighteen phenolic acid derivatives and flavonoids were detected in the S. scabrum and S. burbankii berry extracts. Among them, there were three nonacylated anthocyanin hexosides and six acylated anthocyanin hexosides. Nine compounds other than antocyanin phenolic ones were tentatively identified in the S. scabrum and S. burbankii berry extracts for the first time. Among them, there were seven phenolic acid derivatives and two flavonol hexosides. Chlorogenic acid (5-caffeoylquinic acid, compound 1) and its isomers were found at different tR, showing the deprotonated molecule [M − H]− (m/z 353) and the ion corresponding to the deprotonated quinic acid (m/z 191) in the full scan mode. Reference substances neochlorogenic acid (3-caffeoylquinic acid, compound 3) and cryptochlorogenic (4-caffeoylquinic acid, compounds 4) were confirmed by injection of standards and the subfractions in neutral loss of m/z 162, which also corresponds to the loss of a caffeic acid unit. Several isomers of chlorogenic acid have been found in other plant sources.20,21 Compounds 6 and 8 possessing two malonyl-caffeoylquinic acid derivatives were detected in full-scan experiments showing m/z 439, 191, and 173 at the tR of 4.66 and 5.76 min in decreasing concentrations. The mass spectra of the peaks in full scan mode showed the typical fragmentation of the malonyl acid and quinic acid of chlorogenic acid derivatives at m/z 86 and 191, respectively. Acetylo-p-coumaroylquinic acid (9) and sinapoyl-malic acid (14) were found in the analyzed berries as well. The compound 9 (tR 6.24 min, λmax 310 nm) produced the parent ion at m/z 163 and compound 14 (tR 9.10 min, λmax 328 nm) produced an ion at m/z 233, corresponding to p-coumaric acid and sinapic acid, respectively. The chemical nature of the nonphenolic acid side chains was established using high resolution in source fragmentation experiments in a negative ion mode showing fragment ions with the neutral losses of 60 amu (m/z 379 − 60 = 319) and 116 amu (m/z 339 − 116 = 223) for the acetyl and maloyl residues, respectively (Table 1). Two glucosides of quercetin, namely, quercetin-3-O-rutinoside (10, rutin) and quercetin-3-O-glucoside (12, isoquercitrin), were identified. Quercetin-3-O-rutinoside was detected at the tR of 8.30 min, whereas quercetin-3-O-glucoside was found at the tR of 8.44 min, both of them exhibiting a mass spectrum in full-scan mode with m/z 609 and 463, respectively, and a precursor ion scan m/z 301 as quercetin aglycon. Comparison of the mass spectra and tR with those of the respective standards confirmed the occurrence of these glycosylated flavonols in the S. scabrum and S. burbankii berry extracts. The fragmentation pathway of O-glycosylated flavonoids starts with the cleavage of the glycosydic bonds and elimination of the sugar moieties with charge retention on the aglycon. In compounds containing two or more sugars at the same aglycon carbon, ions arising from the cleavage of the glycosidic bonds between sugar units are weak. Compounds such as quercetin-3O-glucosyl-rutinoside and quercetin-3-O-rutinoside were typical for the Solanaceae family and have been previously reported in potato.22 Quercetin, a major representative of the flavonol subclass found at high concentrations in fruit and vegetables, has recently received considerable attention. This flavonoid displays the ability to prevent the oxidation of LDL by scavenging free radicals and chelating transition metal ions. As a result, quercetin may aid in the prevention of certain diseases, such as cancer, atherosclerosis, and chronic inflammation by

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RESULTS AND DISCUSSION Characterization and Quantification of Phenolic Compounds. The data for retention times (tR), wavelengths of maximum absorbance (λmax), protonated/deprotonated molecules ([M + H]+/[M − H]−), diagnostic fragments, and identification for each peak of the phenolic compound detected in the S. scabrum and S. burbankii berry extracts are listed in Table 1. The compounds were characterized according to their retention time at 320 nm for phenolic acid derivatives, 360 nm 1516

dx.doi.org/10.1021/jf4045233 | J. Agric. Food Chem. 2014, 62, 1512−1519

Journal of Agricultural and Food Chemistry

Article

The concentrations of low molecular weight flavonols (quercetin) and hydroxycinnamic acid conjugates (caffeic, pcoumaric and sinapic acid) were higher in the S. burbankii berries (78.89 mg/100 g) than in the S. scabrum berries (33.13 mg/100 g) (Table 1). In both berries, 3-caffeoylquinic acid was dominant, and the concentration of 5-caffeoylquinic acid was higher than that of 4-caffeoylquinic acid. The content of acylated derivatives of anthocyanins showed the largest differences between the investigated berries. S. scabrum berries were much more richer sources of anthocyanins (206.22 mg/ 100 g) than S. burbankii berries (82.06 mg/100 g). Price et al.2 found more anthocyanin compounds in S. scabrum berries (350 mg/100 g). The concentration of acylated antocyanins was higher than that of the nonacylated antocyanins in Solanum berries. Petunidin-3-O-p-coumaroyl-rutinoside-5-O-glucoside was the dominant acylated antocyanin in the investigated berries. Figure 2 shows the spectral character of S. scabrum (A) and S. burbankii (B) berry extracts at pH 2.5 and 7.5, indicating that

retarding oxidative degradation and inducing enzymes that detoxify carcinogens and also by blocking cancer formation by deactivating at least 30 types of potentially carcinogenic agents.23,24 The UPLC method was applied for short run times combined with a photodiode array detector (PDA), and a quadrupole/time-of-flight mass spectrometer (Q/TOF-MS) enables better characterization of individual compounds, especially with high molecular mass, i.e., acetylated anthocyanins. Three nonacylated anthocyanins were presented as delphinidin-O-3-rutinoside-O-5-glucoside (2; m/z 773, [M + H]+), petunidin-3-O-rutinoside-5-O-glucoside (5; m/z 787, [M + H]+), and malvidin-3-O-rutinoside-5-O-glucoside (7; m/z 801, [M + H]+). Malvidin-3-O-rutinoside-5-O-glucoside was detected only in S. burbankii berry extract. The ESI mass spectrum of pigments showed fragment ions at [M − 162 (glucose)]+ corresponding m/z 611, 625, and 639, respectively, and the next fragment ions at [M − 146 (hexoside, probably rhamnoside)]+ m/z 465, 479, and 493, respectively. The last fragment ions at [M − 162 (hexoside, probably glucoside)]+ m/z 303 (dephinidin), 317 (petunidin) and 331 (malvidin) were aglycons, as listed in Table 1. Seven other compounds 11 and 13−18 (tR 8.37−10.78 min; molecular weights m/z 918− 976 amu) were also clearly detected in Solanum berry extracts (Table 1). Acylated anthocyanin glycosides were easily identified on the basis of the increase in the mass of the parent ions and the wavelength maxima (330−336 nm) of their UV−vis spectra. Thus, peaks with the tR at 8.37, 8.89, 9.30, 9.82, 10.28, and 10.75 min were identified as 3-O-p-coumaroylhexoside-5-O-hexoside (11) and 3-O-feruoyl-hexoside-5-Ohexoside (13) of delphinidin, 3-O-p-coumaroyl-hexoside-5-Ohexoside (15) and 3-O-feruoyl-hexoside-5-O-hexoside (16) of petunidin, and 3-O-p-coumaroyl-hexoside-5-O-hexoside (17) and 3-O-feruoyl-hexoside-5-O-hexoside (18) of malvidin, respectively. The ESI mass spectrum of the acylated pigment compound 11 showed a flavylium cation at m/z 919 [M + H]+ together with a fragment ion at m/z 757 [M − 162 (glucose)]+. On the basis of observations of two additional fragment ions at m/z 465 [M − 292 (the presence of hexoside, probably rhamnose, and p-coumaric acid)], and m/z 303 derived from successive losses of 162 amu, the presence of glucose moiety was confirmed, for structural elucidation. Similar fragmentation ions were observed for the remaining acetylated anthocyanins (Table 1). Although these compounds have already been presented in earlier works,1,2 to enable their complete identification, their structures should be fully elucidated by means of NMR or by comparison with authentic compounds, if existing. In addition, nonacylated anthocyanins were shown to be the most abundant compounds in wild species of berry and other fruits.25,26 The presence of acylated anthocyanins is typical of grapes, blueberry, blackberry, and some vegetables, i.e., red cabbage, radish, and red-fleshed potato.27 Recent research has shown that anthocyanins with acylating substituents are more stable during processing and storage than other natural pigments.6,9,27 Brouillard et al.28 explain that the effect of the improved stabilization of the acylated anthocyanins has been attributed to the stacking of the acyl groups with the pyrylium ring of the flavylium cation, thereby reducing the susceptibility of a nucleophile attack of water and subsequent formation of intramolecular copigmentation of a pseudobase or a chalcone.

Figure 2. Spectral character of S. scabrum (A) and S. burbankii (B) berry extracts at pH 2.5 and 7.5.

these berries contain acylated anthocyanin. Changing the pH from 2.5 to 7.5 did not result in any significant reduction in absorbance at a wavelength of 530 nm, as is observed in the case of nonacylated anthocyanins. The variation of pH values influences anthocyanin properties, such as color, stability, spectral shape, and absorption peak.29 In our research, it could be observed from Figure 2 that the anthocyanin extracts from S. scabrum (A) and S. burbankii (B) berries had the maximum absorption around 530 nm at pH 2.5 and 7.5. Anthocyanin solutions, at both pH values, exhibited bright red-violet color, and their absorbances at 530 nm varied slightly. Acylated 1517

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anthocyanins are also more resistant to color fading with increased pH than their unacylated analogs. Unacylated anthocyanins are only stable at pH values where the flavylium cation dominates. Cevallos-Casals et al.6 showed that vegetables such as sweet potato and purple carrot, which contain acylated anthocyanins, were more resistant to the solution pH than red grape rich, which is in unacylated anthocyanins. Other researchers confirmed the unusual stability of acylated anthocyanins at pH >5.0.7,8 Antioxidant Activity and Cyclic Voltammetry of Solanum Berries. The antioxidant activity of S. scabrum (a) and S. burbankii (b) berries was tested by measuring their activity to scavenge radical (Table 2). The ABTS radical Table 2. Antioxidant Activity (mmol Trolox/100 g dm) of Solanum Fruits ABTS FRAP

S. scabrum

S. burbankii

2.89 ± 0.37 1.65 ± 0.09

2.07 ± 0.42 0.71 ± 0.06

Mean values with standard deviation of n = 3.

Figure 3. Cyclic voltammograms of S. burbankii and S. scabrum.

other radicals generated in the human body; hence the duration of this radical is longer.12 In both cultivars, the major compound was found to be the anthocyanin petunidin-3-(p-coumaroyl-rutinoside)-5-glucoside. Eight additional anthocyanins in S. burbankii berries and 11 in S. scabrum berries were identified as petunidin, delphinidin, and malvidin with the same glycosidic substitution pattern as the major pigment and varying degrees of acylation with pcoumaric and ferulic acids. The presence of seven phenolic acid mainly caffeoylquinic derivatives and two quercetin derivatives was reported for the first time. S. scabrum extracts are richer in anthocyanins but have less phenolic acid than S. burbankii. This study demonstrated the usability of fast and effective electrochemical measurements and a spectrophotometric technique for determination of the phenolic content as well as antioxidant activity of Solanum berry extracts. S. scabrum exhibited the highest TEAC values according to both CV and ABTS and FRAP assay. From the consumers’ point of view, the analyzed fruits are good sources of bioactive compounds and colorants to be used in the food industry.

scavenging activity was higher in S. scabrum berry extract [2.89 mmol Trolox equiv (TE)/100 g dm] than in S. burbankii berry extract (2.07 mmol TE/100 g dm). After comparing the ABTS•+ activity of the Solanum berries with that of known food products we found that they exhibited a lower antioxidant activity than that of blueberries (4.59 mmol TE/100g dm), which constitute one of the richest reported sources of antioxidants.30 But, still, S. scabrum berry extract had a higher antioxidant activity than Rubus species (0−2.53 mmol TE/100 g dm), which have been recommended as improving the nutritive value due to its high antioxidant activities.31 In regard to the FRAP activity, S. scabrum had 1.65 mmol TE/100 g dm and S. burbankii had 0.71 mmol TE/100g dm, respectively. The differences in polyphenols between Solanum varieties could be preliminarily attributed to their different contents of antioxidant activity. S. scabrum berry extract contained much more phenolic compounds than S. burbankii berry extract. The cyclic voltammetric technique (CV) was employed to study the electrochemical behavior of S. scabrum and S. burbankii berry polyphenol extracts, and results are presented in Figure 3. The major current peak appears at approximately 2.4 V for S. scabrum and at 2.1 V for S. burbankii. According to literature data, this peak may be attributed to powerful, low formal potential phenolics characterized by an o-hydroxyphenol or di- or trihydroxyphenol group. Petuninidin and delphinidin anthocyanin derivatives were the main compounds of Solanum berry, but higher contents of these compounds were determined in S. scabrum. These anthocyanins possess three and two catechol groups on the B-ring. Cevallos-Casals at al.6 explained that oxidation of phenolic compounds involved the formation of a stable quinone, which can be reduced in the reverse scan, this process appearing as a cathodic peak of the current response. In the return scan, the electrochemical behavior of S. burbankii extract is irreversible. Yakovleva et al.32 present the irreversible oxidation of some phenolic acids and flavones in plants. However Tamura et al.12 found that anthocyanins acylated by p-coumaric acid are much better antioxidants than α-tocopherol or (+)-catechin. Besides the color and antioxidant activity, anthocyanins have free radical scavenging activity. The anthocyanin radical is more stable than

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AUTHOR INFORMATION

Corresponding Author

*Tel./fax: +48 71 320 7706. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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