Article pubs.acs.org/JAFC
Characterization of Phenolic Compounds of Thorny and Thornless Blackberries Joanna Kolniak-Ostek,*,† Alicja Z. Kucharska,† Anna Sokół-Łętowska,† and Izabela Fecka§ †
Department of Fruit, Vegetables and Cereals Technology, Wrocław University of Environmental and Life Sciences, Chełmońskiego 37/41, 51-630 Wrocław, Poland § Department of Pharmacognosy, Wrocław Medical University, Borowska 211A, 50-140 Wrocław, Poland S Supporting Information *
ABSTRACT: The aim of this study was to identify and compare the contents of phenolic acids, tannins, anthocyanins, and flavonoid glycosides in thorny and thornless blackberries. Five thorny and nine thornless cultivars were used for this study. Thirty-five phenolic compounds were determined in the examined fruits, and one phenolic acid, three ellagic acid derivatives, one anthocyanin, and six flavonols were characterized for the first time in blackberries. The thornless fruits were characterized by a higher content of anthocyanins (mean = 171.23 mg/100 g FW), ellagitannins (mean = 3.65 mg/100 g FW), and ellagic acid derivatives (mean = 2.49 mg/100 g FW), in comparison to thorny ones. At the same time, in thorny fruits, the contents of hydroxycinnamic acids (mean = 1.42 mg/100 g FW) and flavonols (mean = 5.70 mg/100 g FW) were higher. KEYWORDS: phenolics, UPLC-MS/MS, blackberries, thorny, thornless
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naturally have varying numbers of “thorns” on their canes. In general, the new cultivars of erect and semierect blackberries are thornless. The thornlessness of blackberries is a genetic feature and was designed and developed to reduce thorn contamination in machine-harvested fruits. The thornless chimera form is unstable and commonly reverts to thorny canes with environmental or mechanical injury.8 The major phenolic compounds in berries are anthocyanins, flavonols, and flavan-3-ols, including proanthocyanidins, ellagitannins, and hydroxycinnamic acids.9,10 Anthocyanins are the predominant group present in berries, and these natural pigments give blackberries their characteristic red, violet, and blue colors. Blackberries are a good source of anthocyanins in which the anthocyanin contents were reported to be 67.4−230 mg/100 g fresh weight.11,12 Major anthocyanins in blackberries have been identified as cyanidin-based glycosides.13 Anthocyanins from berries have been shown to have anti-inflammatory effects in both in vitro and in vivo models.14 They possess also antioxidant, anticancer, and neuroprotective properties.15 The purpose of this research was to identify phenolic compounds and quantify main anthocyanins, flavonoids, ellagitannins, and phenolic acids that were found to be characteristic of thorny and thornless blackberries.
INTRODUCTION
Ellagitannins are generally esters of hexahydroxydiphenic (HHDP) and gallic acids, with monosaccharides (especially with glucose), which are easily hydrolyzed. During the hydrolysis, they are liberated to HHDP residue, which spontaneously lactonizes to ellagic acid. Some ellagitannins are known, which also possess a modified HHDP group with an additional galloyl moiety, for example, valoneoyl, sanguisorboyl, and tergalloyl. Most of the ellagitannin structures that have been observed in Rubus species fruits and leaves are oligomeric forms of the galloyl-bis-HHDPglucose with the sanguisorboyl group formed between monomers (the m-GOD-type oligomers with a β- or α-glucose core).1−3 Four dimers of sanguiins H-6 and H-10, lambertianins A and B, a trimer lambertianin C, and a tetramer lambertianin D and various monomeric ellagitanins (e.g., sanguiin H-2, casaurictin, pedunculagin) and ellagic acid derivatives (metylated or glycosylated ellagic acid) have been identified in the Rubus genus.4 However, in most of the studies there are still unidentified ellagitannins due to the diverse and complex nature of the structures. In addition, there is still very limited information on the ellagitannin composition of blackberries.5 Blackberries are currently promoted as being a rich source of polyphenols. Genetic and environmental factors, such as cultivar, maturity, UV light exposure, and harvesting method, play an important role in berry composition.6 It is well-known that levels of phenolics and the antioxidant capacity of blackberries are influenced by maturity and that there is pronounced variation among cultivars.7 Blackberries belong to the genus Rubus and bear aggregate fruit consisting of a number of fleshy drupelets, each containing a single seed (pyrene) around the central torus or receptacle. The Rubus spp. (Rosaceae family) is cultivated worldwide, but primarily in the northern hemisphere. Three main types of blackberries have been developed into commercial crops: trailing, erect, and semierect blackberries. Blackberry species © XXXX American Chemical Society
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MATERIALS AND METHODS
Chemicals. Pure standards of ellagic, p-coumaric, chlorogenic, cryptochlorogenic, neochlorogenic, and quinic acid, cyanidin 3-Ogalactoside, cyanidin 3-O-arabinoside, cyanidin 3-O-rutinoside, kaempferol 3-O-glucoside, quercetin 3-O-glucoside, quercetin 3-O-galactoside, quercetin 3-O-glucuronide, quercetin 3-O-rutinoside, and quercetin Received: August 18, 2014 Revised: February 23, 2015 Accepted: February 27, 2015
A
DOI: 10.1021/jf5039794 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
tR
2.40 3.73
4.12
4.79 5.12
5.14
5.15
5.17 5.61
5.82
6.13 6.35 6.45
6.57
6.60
6.63
6.96
7.07
7.11
7.26
7.38
7.82
7.94
7.99
8.34
8.51
peak
1 2
3
4 5
6
7
8 9
10
11 12 13
14
15
B
16
17
18
19
20
21
22
23
24
25
26
265/347
253/360
256/354
256/348
256/354
256/354
257/354
256/354
518
256/354
253/362
254/364
518
516 254/357 253/361
245
517 246
512
516
515 246
515
325 314
UV λmax (nm)
447.0959
447.0570
505.0979
433.0735
505.0979
463.0879
477.0665
463.0879
593.1520
609.1483
300.9999
489.1042
535.1084
419.0980 433.0415 491.0434
1103.0897
595.1713 1869.1028
494.8155
419.0987
449.1063 [1401.0981]‑2
449.1083
353.0880 325.0949
[M − H]−/[M + H]+ (m/z)b
C20H18O10 C23H25O12
348.8967 [M − rha + H] , 287.0502 [M − Meth − Meth + H]
448.3338
C20H16O12 C21H20O11
285.0394 [M − hex − H]−
C20H18O11
C23H22O13
448.3769
434.3503
506.1060
464.3763
478.3598
C21H18O13 C21H20O12
464.3763
C21H20O12
506.1060
592.4161
610.5175
302.1926
490.3705
C23H22O13
C25H20O17
C27H30O16
C14H6O8
C22H18O14
534.4231
418.3509 434.3073 492.3433
C20H18O10 C19H14O12 C21H16O14 C24H22O14
1104.7491
C48H32O31
594.5181 1871.2750
493.4374
418.3509
448.3769 2805.9045
448.3769
354.3087 326.2986
exact mass
301.0359 [M − hex − H]−, 178.9965 [M − hex − C7H8O2 − H]−, 151.0017 [M − hex − C7H8O2 − CO − H]− 301.0354 [M − glcA − H]−, 178.9965 [M − glcA − C7H8O2 − H]−, 151.0042 [M − glcA − C7H8O2−CO − H]− 301.0277 [M − hex − H]−, 178.9920 [M − hex −C7H8O2 − H]−, 151.0042 [M − hex − C7H8O2 − CO − H]− 301.0354 [M − Ac − hex − H]−, 178.9938 [M − Ac − hex − C7H8O2 − H]−, 151.0042 [M − Ac − hex − C7H8O2 − CO − H]− 301.0354 [M − pent − H]−, 178.9938 [M − pent − C7H8O2 − H]−, 151.0042 [M − pent − C7H8O2 − CO − H]− 301.0354 [M − Ac − hex − H]−, 178.9938 [M − Ac − hex − C7H8O2 − H]−, 151.0042 [M − Ac − hex − C7H8O2 − CO − H]− 315.0113 [M − pent − H]−, 300.9999 [M − pent − Meth − H]−
300.9999 [M − Ac − Me − pent − H]−, 257.0208 [M − Ac − Me − pent − O − CO − H]−, 229.0137 [M − Ac − Me − pent − O − CO − CO − H]− 285.0425 [M − O − H]−, 257.0208 [M − O − CO − H]−, 229.0137 [M − O − CO − CO − H]− 301.0277 [M − rha − hex − H]−, 178.9938 [M − rha − hex − C7H8O2−H]−, 151.0042 [M − rha − hex − C7H8O2 − CO − H]− 287.0571 [M − diox − hex + H]+
287.0571 [M − rha − hex + H]+ 935.0878 [gall/bis-HHDP/glc − H]−, 633.0853 [gall/HHPD/glc − H]−, 469.0005 [sanguisorbic acid dilactone − H]−, 300.9999 [EA − H]− 935.0878 [gall/bis-HHDP/glc − H], 633.1234 [gall/HHPD/glc − H]−,469.0005 [sanguisorbic acid dilactone -H]−, 300.9999 [EA − H]− 287.0536 [M − xyl + H]+ 300.9999 [M − pent − H]−, 229.0137 [M − pent − O − CO − CO − H]− 315.0113 [M − glcA − H]−, 300.9999 [M − glcA − O − H]−, 257.0208 [M − glcA − O − CO − H]−, 229.0137 [M − glcA − O − CO − CO − H]− 287.0536 [M − mal − hex + H]+ C27H30O15 C82H53O52
C21H20O11 C123H80O78
287.0536 [M − hex + H]+ 935.0669 [gall/bisHHDP/glc − H]−, 633.0727 [gall/HHPD/glc − H]−, 469.0005 [sanguisorbic acid dilactone − H]−, 300.9999 [EA − H]− 287.0571 [M − pent + H]+ +
C21H20O11
287.0557 [M − hex + H]+
+
C16H18O9 C15H18O8
proposed formula
191.0552 [M − caff − H]− 163.0778 [M − hex − H]−
MS/MS (m/z)b
Table 1. Identification of Phenolic Compounds in Thorny and Thornless Fruitsa
448.1038
448.0649
506.1058
434.0814
506.1058
464.0958
478.0744
464.0958
592.1441
610.1562
302.0078
490.1121
534.1005
418.0901 434.0494 492.0513
1104.0976
594.1634 1870.1107
493.8076
418.0908
448.0984 1402.106
448.1004
354.0959 326.1028
measured mass
quercetin 3-Oacetylhexoside 2c methylellagic acid pentoside kaempferol glucosided
quercetin 3-Oglucosided quercetin 3-Oacetylhexoside 1c quercetin pentoside
quercetin 3-Orutinosided cyanidin 3-Odioxalylglucoside quercetin 3-Ogalactosided quercetin glucuronided
cyanidin 3-O-xyloside ellagic acid pentoside methylellagic acid glucuronide 1 cyanidin 3-O-(6-Omalonyl-β-D)glucoside ellagic acid acetyl methylpentosidc ellagic acidd
sanguiin H-2
cyanidin 3-Oarabinosided cyanidin dimethoxyrhamnosidec cyanidin 3-O-rutinosided sanguiin H-6
neochlorogenic acidc,d p-coumaric acid hexosided cyanidin 3-Ogalactosided cyanidin 3-O-glucoside lambertanin C
tentative identification
0.27
0.27
0.00
0.27
0.00
0.28
0.29
0.28
0.27
0.36
0.18
0.26
0.32
0.26 0.26 0.29
0.65
0.35 1.16
0.37
0.26
0.28 1.01
0.28
0.21 0.20
Δm (ppm)
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/jf5039794 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article 0.19 302.0433 302.2357 C15H10O7 301.0354 255/373 11.10 35
Abbreviations: tR, retention time; Ac, acetyl; caff, caffeoyl; diox, dioxallyl; EA, ellagic acid; gall, galloyl; glc, glucose; glcA, glucuronic acid; he, hexoside; HHDP, hexahydroxydiphenic acid; Me, methyl; meth, methoxy; mal, malonyl; pent, pentoside; rha, rhamnoside; xyl, xyloside. bExperimental data. cChemicals identified for the first time in Rubus f ruticosus L. dIdentification confirmed by commercial standards.
490.1121 490.3705 C20H16O12
447.0959 [M − Ac − H]−, 315.0149 [M − Ac − pent − H]−, 300.9999 [M − Ac − pent − Me − H]− 178.9938 [M − glcA − C7H8O2 − H]−, 151.0042 [M − glcA − C7H8O2 − CO − H]− 489.1042 10.30 34
252/369
490.1138 490.4136 C23H22O12 285.0394 [M − Ac − hex − H] 489.1059 10.26 33
265/347
508.2332 508.4289 C23H24O13 361.1638 [M − rha − H]−,301.0277 [M − Meth − Meth − H]− 507.2253 9.91 32
256/354
−
490.1138 9.37 31
265/347
489.1059
C23H22O12
490.4136
490.1121 490.3705 8.94 30
252/369
489.1042
C20H16O12
506.1058 506.1060 8.81 29
256/354
505.0979
C23H22O13
492.2055 492.3433 C21H16O14 491.1976 253/360 8.70 28
were purchased from Extrasynthese (Lyon, France). Formic acid and methanol were purchased from Sigma-Aldrich (Steinheim, Germany). Acetonitrile was from Merck (Darmstadt, Germany). Plant Material. Blackberry fruits (Rubus fruticosus L.) of five thorny cultivars (‘Darrow’, ‘Gazda’, ‘Leśniczanka’, ‘Early Wilson’, and ‘Zagroda’) and nine thornless cultivars (‘Black Beauty’, ‘Black Satin’, ‘Chester Thornless’, ‘Hull Thornless’, ‘Loch Ness’, ‘Orkan’, ‘Smoothstern’, ‘Tayberry’, and ‘Thornfree’) were used for this study. Fruits were collected from the Research Station for Cultivar Testing in Masłowice and hand-harvested at the stage of consumption (full color) during the growing season of July−August 2012. Before analysis, fruits were cleaned, frozen with liquid nitrogen, and stored overnight at −20 °C. Extraction of Polyphenols for Quantitative and Qualitative Analysis. Frozen fruits of blackberries were homogenized. A 5 g portion of homogenate was extracted twice with 80% aqueous methanol (v/v) acidified with 1% HCl (ultrasound bath, 10 min). Combined solution was then filtered through a Schott funnel with Whatman no. 1 filter paper, and filtrates were collected into 50 mL flasks. Each sample was prepared three times in order to have three technical replicates. For HPLC/UPLC-MS analysis, portions of extracts (2 mL) were centrifuged at 19000g for 15 min at 4 °C, and the supernatant was filtered through a Hydrophilic PTFE 0.22 μm membrane (Millex Samplicity Filter, Merck) and used for analysis. UPLC-MS/MS Analysis of Polyphenols. Identification of blackberry polyphenols was carried out by using an ACQUITY Ultra Performance LC system (UPLC) with binary solvent manager (Waters Corp., Milford, MA, USA) and a Micromass Q-Tof Micro mass spectrometer (Waters, Manchester, UK) equipped with an electrospray ionization (ESI) source operating in negative and positive modes. Separations of individual polyphenols were carried out using a UPLC BEH C18 column (1.7 mm, 2.1 mm × 50 mm, Waters Corp., Milford, MA, USA) at 30 °C. The elution solvents were aqueous 0.1% formic acid (A) and 100% acetonitrile (B). Samples (10 μL) were eluted according to the linear gradient described previously by Kolniak-Ostek et al.16 Analysis was carried out using full scan, data-dependent MS scanning from m/z 100 to 2500. 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 a flow rate of 300 L/h. HPLC-PDA Analysis of Polyphenol. The contents of anthocyanins, flavonols, ellagitannins, and phenolic acids were calculated on the basis of assays by HPLC-PDA described previously by Sokół-Łet̨ owska et al.17 The compounds were monitored at 254 nm (ellagitannins and ellagic acid derivatives), 320 nm (hydroxycinnamic acids), 360 nm (flavonols), and 520 nm (anthocyanins). The amounts of anthocyanins, flavonols, ellagitannins, including ellagic acid derivatives, and hydroxycinnamic acids were calculated as an external standard of regression equations, determined experimentally and converted into cyanidin 3-O-glucoside (anthocyanins), quercetin (flavonols), chlorogenic acid (hydroxycinnamic acids), and ellagic acid (ellagitannins and ellagic acid derivatives). Statistical Analysis. Results were presented as the mean ± standard deviation of three technical replications. All statistical analyses were performed with Statistica version 10.0 (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 p < 0.05. Principal component analysis (PCA) was performed using XLSTAT on mean values of 14 samples (thorny and thornless blackberries) and 5 variables (anthocyanins, ellagic acid derivatives, ellagitannins, flavonols and hydroxycinnamic acids).
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RESULTS AND DISCUSSION Qualitative Analysis of Polyphenols. General. Table 1 lists the 35 compounds identified through UPLC-MS/MS (with PDA and Q/TOF detectors) experiments along with their retention times (tR), UV−vis spectral profiles at 200−600 nm, and comparison with standard reference compounds, when available. Molecules that were certainly or putatively identified in negative ion mode belong to the compound groups of flavonols,
a
0.26
0.30
0.20
0.30
0.26
0.00
0.14
0.27
ellagic acid methylpentoside methylellagic acid glucuronide 2 quercetin 3-Oacetylhexoside 3c methylellagic acid acetylpentoside 1c kaempferol acetylgalactosidec quercetin dimethoxyrhamnosidec kaempferol acetylglucosidec methylellagic acid acetylpentoside 2c quercetind 448.0649 448.3338 C20H16O12
300.9999 [M − Me − pent − H]−, 257.0208 [M − Me − pent − O − CO − H]−, 229.0137 [M − Me − pent − O − CO − CO − H]− 315.0113 [M − glcA − H]−, 300.9999 [M − glcA − O − H]−, 257.0208 [M − glcA − O − CO − H]−, 229.0137 [M − glcA − O − CO − CO − H]− 301.0354 [M − Ac − hex − H]−, 178.9938 [M − Ac − hex − C7H8O2−H]−, 151.0042 [M − Ac − hex − C7H8O2 − CO − H]− 447.0959 [M − Ac − H]−, 315.0149 [M − Ac − pent − H]−, 300.9999 [M − Ac − pent − Me − H]− 285.0394 [M − Ac − hex − H]− 447.0570 253/360 8.59 27
UV λmax (nm) tR peak
Table 1. continued
[M − H]−/[M + H]+ (m/z)b
MS/MS (m/z)b
proposed formula
exact mass
measured mass
tentative identification
Δm (ppm)
Journal of Agricultural and Food Chemistry
C
DOI: 10.1021/jf5039794 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Structures of identified ellagitannins.
identified as cyanidin 3-O-rutinoside and peak 7, with m/z at 494.8155, as cyanidin dimethoxyrhamnoside. Peaks 6 and 11, with identical psudomolecular cation (m/z 419.0987) and mass spectrometric fragmentation patterns, were identified as cyanidin 3-O-arabinoside and cyanidin 3-O-xyloside on the basis of their retention times and UV−vis absorption spectra, compared with those of corresponding authentic standards. Peak 14, with [M + H]+ at m/z at 535.1084, was identified as cyanidin 3-O-(6-Omalonyl-β-D)-glucoside, whereas peak 18, with [M + H]+ at m/z at 593.1520, was identified as cyanidin 3-O-dioxalylglucoside. Flavonols. The examination of the chromatograms in TOF-MS mode revealed the presence of quercetin (peak 35, m/z 301.0354). The MS/MS fragmentation showed further dissociation of the main fragment and produced the typical ion cluster of quercetin with large abundance of m/z 178.9938 and 151.0042.22 Examination of the chromatograms in TOF-MS mode of thorny and thornless blackberries also revealed the presence of 10 quercetin glycosides (peaks 17, 19−24, 29, and 32). Peak 17 had a pseudomolecular ion at m/z 609.1483 that fragmented in m/z 301.0354, owing to the loss of rhamnose (146 Da) and glucose (162 Da) residues. This compound was therefore tentatively identified as quercetin 3-O-rutinoside (rutin) on the basis of its retention time and UV−vis spectra, compared with those of corresponding authentic standards. Peaks 19 and 21, with identical pseudomolecular anions at m/z 463.0879 that fragmented in m/z 301.0359 owing to the loss of a hexose residue (162 Da), were identified as quercetin 3-Ogalactoside (hyperoside) and quercetin 3-O-glucoside (isoquercitrin) on the basis of their retention times, UV−vis spectral profiles, and accurate standards. Peak 20 showed an [M − H]− at m/z 477.0665, which produced fragments at m/z 301.0354,
ellagitannins, ellagic acid derivatives, and hydroxycinnamic acids. Positive ionization was used for the identifiction of anthocyanins. Seven anthocyanins (peaks 3, 4, 6, 8, 11, 14, and 18), seven flavonols (17, 19, 20, 21, 23, 26, and 35), three ellagitannins (5, 9, and 10), six ellagic acid derivatives (12, 13, 16, 25, 27, and 28), and one hydroxycinnamic acid (peak 2) were reported previously in blackberry fruits.13,18−20 The following compounds, one phenolic acid (peak 1), one anthocyanin (peak 7), six flavonols (22, 24, 29, 31, 32, and 33), and three ellagic acid derivatives (15, 30, and 34) were identified for the first time in blackberries. Anthocyanins. The analysis in the TOF-MS revealed the presence of eight anthocyanins in the investigated cultivars of blackberries, using positive ionization mode (Table 1). As previously stated, the identification of anthocyanins was performed by using the complementary information on chromatographic behavior and mass fragmentation, together with UV−vis profile and retention time. The MS/MS fragmentation demonstrated that the pseudomolecular cations of identified anthocyanins (peaks 3, 4, 6, 7, 8, 11, 14, and 18) were the precursors of cyanidin ([M + H]+ at m/z 287.0557), indicative of cyanidin -glycosides. Peaks 3 and 4, with identical pseudomolecular cation (m/z 449.1183) and mass spectrometric fragmentation patterns, were identified as cyanidin 3-O-galactoside and cyanidin 3-O-glucoside on the basis of their retention times and UV−vis absorption spectra, compared with those of corresponding authentic standards. According to Long-Ze and Harnly,21 the elution order of phenolic compounds for C18 columns is invariant. Glycosylated flavonoids with a monosaccharide at the same position elute in the order galactoside, glucoside, xyloside, arabinopyranoside, arabinofuranoside, rhamnoside, and then glucuronide. Peak 8, with m/z at 595.1713, was D
DOI: 10.1021/jf5039794 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 2. Segment from 2.25 to 11.25 min of LC-DAD chromatogram at 280 nm of ‘Tayberry’ thornless blackberry. Peak number identities are displayed in Table 1.
acid dilactone, which is formed from the liberated sanguisorboyl group (Figure 1), and a fragment with m/z at 300.9999, corresponding to ellagic acid. Peak 5 had an [M − 2H]−2 at m/z 1401.0981 and was identified as trimer lambertianin C. The MS/MS fragmentation gave an ion with [M − H]− at m/z at 935.0669, owing the loss of galloyl-HHDP-glucose (466 Da), and fragments with m/z at 633.0727, 469.0005, and 300.9999, corresponding to galloyl-HHDP-glucose, sanguisorbic acid dilactone, and ellagic acid, respectively. Peak 10 had a [M − H]− ion at m/z 1103.0897 and a principal MS/MS fragment at m/z 934.5746 derived from the loss of a gallic acid unit (169 Da) and was identified as monomeric sanguiin H-2. The MS/MS fragmentation also gave fragments with m/z at 633.1234, 469.0005, and 300.9999, as described above. Ellagic Acid and Its Derivatives. The examination of the UPLC chromatograms in TOF-MS mode of thorny and thornless blackberries revealed the presence of free ellagic acid (peak 16, m/z 300.9999), MS/MS fragments at m/z 285.0425, 257.0208, and 229.0137, which are characteristic for ellagic acid,25 and eight ellagic acid derivatives (peaks 12, 13, 15, 25, 27, 28, 30, and 34). Peak 12 has a pseudomolecular ion at m/z 433.0415 and MS/MS fragments at m/z 300.9999 and 229.0137; this dissociation pattern was previously observed by Simirgiotis and Schmeda-Hirschann26 in Fragaria chiloensis berries and attributed to an ellagic acid pentoside. The TOFMS analysis revealed the presence of ellagic acid acetylmethylpentoside (peak 15, m/z 489.1042). The MS/MS mass spectrum showed peaks at m/z 300.9999, corresponding to the loss of acetyl (42 Da) and methylpentoside (146 Da); 257.0208, corresponding to the loss of carbon oxide (28 Da); and 229.0137, corresponding to the loss of the second carbon
corresponding to the loss of a glucuronic acid residue (176 Da), and at 178.9938 and 151.0042, which are characteristic for quercetin. This pattern of fragmentation is in agreement with quercetin glucuronide, which was previously reported in Rubus species.22 Peaks 23 (m/z 433.0735) and 32 (m/z 507.2253) were characterized as quercetin pentoside and quercetin dimethoxyrhamnoside, on the basis of their fragmentation pattern, with a loss of pentose (132 Da) and rhamnose (146 Da), respectively. Peaks 22, 24, and 29, with an identical pseudomolecular anion (m/z 505.0979) and mass spectrometric fragmentation patterns, were identified as quercetin acetylhexosides. Peak 26 had a pseudomolecular ion at m/z 447.0959, which fragmented to m/z 285.0394, owing to the sugar moiety (162 Da) and corresponding formation of the aglycone.23 This fragmentation was in agreement with those elsewhere and was attributed to kaempferol glucoside.24 Peaks 31 and 33 have pseudomolecular ions at m/z 489.1059 and are fragmented at 285.0394, owing to the loss of the acetylhexose unit (204 Da). These compounds may be identified as kaempferol acetylgalactoside and acetylglucoside. Ellagitannins. High MW ellagitannins based on galloyl-bisHHDP-glucose units, such as sanguiins and lambetianins, have been commonly found in different kinds of berries, including blackberries.18,19 The structures of identified ellagitannin are shown in Figure 1. The analysis in the TOF-MS revealed the presence of sanguiin H-6, a dimeric ellagitannin (peak 9, m/z 1869.1028). The MS/MS fragmentation gave an ion with [M − H]− at m/z at 935.0878, owing to the loss of galloyl-bisHHDP-glucose (935 Da), a fragment with m/z at 633.0853, corresponding to the galloyl-HHDP-glucose structure, a fragment with m/z at 469.0005, corresponding to sanguisorbic E
DOI: 10.1021/jf5039794 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 3. Segment from 2.25 to 11.25 min of LC-DAD chromatogram at 280 nm of ‘Zagroda’ thorny blackberry. Peak number identities are displayed in Table 1.
monoxide (28 Da). Peak 25, which showed [M − H]− at m/z 447.0570 and fragmentation with MS/MS ions at m/z 315.0113, owing to the loss of a pentose residue (132 Da) and m/z 300.9999 (further loss of methyl), can be attributed to methylellagic acid pentoside. Peak 27 had an [M − H]− at m/z 447.0570 that fragmented in the MS/MS at m/z 300.9999, corresponding to the loss of rhamnose residue (146 Da); 257.0208, owing to the loss of carbon oxide (28 Da); and 229.0137, corresponding to the loss of the second carbon monoxide (28 Da). This compound was identified as ellagic acid methylpentoside.19 The analysis in the TOF-MS revealed the presence of two methylellagic acid glucuronide isomers (peak 13 with m/z at 491.0434 and peak 28 with m/z at 491.1976). The MS/MS mass spectrum showed peaks at m/z 315.0113 and 300.9999, corresponding to the loss of glucuronide (176 Da) and methyl (15 Da). Peaks 30 and 34 were identified as methylellagic acid acetylpentosides.19 They had a pseudomolecular ion at m/z 489.1042 and produced MS/MS fragments at m/z 447.0959, corresponding to the loss of acetyl (42 Da); 315.0149, owing to the loss of pentose (132 Da); and 300.9999, corresponding to the loss of methyl (15 Da). Hydroxycinnamic Acids and Others. Two hydroxycinnamic acids were identified by monitoring precursor and product ion scans and their retention times and UV−vis absorption spectra, by comparing them with those of corresponding authentic standards. Hydroxycinnamic acid (peak 1) yielded [M − H]− at m/z 353.0880, which was fragmented to generate m/z at 191.0552 (quinate ion). This component was identified as neochlorogenic acid by comparing it with the retention times and UV−vis profile of authentic standards of chlorogenic,
cryptochlorogenic, and neochlorogenic acids. Hydroxycinnamic acid (peak 2) was identified as the p-coumaric acid hexoside by comparing it with the retention times and UV−vis profile of authentic standards of p-coumaric acid. Its MS/MS produced an [M − H]− of m/z 325.0949, which fragmented to 163.2778, corresponding to the loss of hexose residue (162 Da). Quantitative Analysis of Polyphenols. General. Figures 2 and 3 show the LC-DAD chromatograms of thornless blackberries ‘Tayberry’ and thorny blackberries ‘Zagroda’. Tables 2 and 3 show the contents of individual phenolic compounds of thorny and thornless blackberries. The amounts of anthocyanins, flavonols, ellagitannins, ellagic acid derivatives, and hydroxycinnamic acids were converted into cyanidin 3-Oglucoside (anthocyanins), quercetin (flavonols), chlorogenic acid (hydroxycinnamic acids), and ellagic acid (ellagitannins and ellagic acid derivatives). The qualitative compositions were similar for all cultivars but were quantitatively very different (Tables 2 and 3). Anthocyanins. In thorny blackberries, the dominant compound among anthocyanins was cyanidin 3-O-glucoside (mean = 73.85 mg/100 g fresh weight). The highest amount of this compound (122.54 mg/100 g FW) has been found in ‘Zagroda’, whereas the lowest (25.15 mg/100 g FW) was reported in the ‘Gazda’ variety. The ‘Zagroda’ variety was also characterized by the highest contents of cyanidin methylpentoside, 3-O-arabinoside, 3-O-xyloside, and 3-O-(6-O-malonylβ-D)-glucoside (11.00, 0.41, 6.37, and 3.34 mg/100 g FW, respectively). Cyanidin 3-O-rutinoside has been found only in ‘Early Wilson’ blackberries (4.66 mg/100 g FW) and cyanidin 3-O-dioxalylglucoside only in the ‘Leśniczanka’ variety. F
DOI: 10.1021/jf5039794 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 2. Phenolic Compound Contents of Thorny Blackberries (mg/100 g FW)a,b peak
compoundc
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Cy 3-glc Cy diMeth-rham Cy 3-rut Cy 3-ara Cy 3-xyl Cy 3-mal-glc Cy 3-diox-glc lambertianin C sanguiin H-6 sanguiin H-2 EA pent MeEA glcA EA Ac-Me-pent EA MeEA pent EA Me-pent MeEA Ac-pent NChA p-cum hex Q 3-rut Q 3-gal Q glcA + Q 3-glc Q 3-Ac-hex Q pent
‘Darrow’ 88.78 ± 0.41h 8.51 ± 0.04f
‘Early Wilson’ 55.17 3.89 4.66 0.08
± ± ± ±
0.98j 0.49h 0.32b 0.00efg
2.04 ± 0.01h
0.96 ± 0.08k
2.70 ± 0.11j 1.17 ± 0.14j 0.05 ± 0.01f
3.27 1.00 0.03 0.19
± ± ± ±
0.19i 0.14j 0.00f 0.01c
0.10 2.21 0.16 0.22 0.39
± ± ± ± ±
0.00c 0.37c 0.03f 0.00a 0.00b
0.24 ± 0.00c
0.46 1.56 0.90 0.95 0.36
± ± ± ± ±
0.02b 0.04b 0.06d 0.03fg 0.00e
0.32 0.72 1.22 0.69 0.26
± ± ± ± ±
‘Leśniczanka’ 81.59 ± 1.82i 7.42 ± 0.05g
2.37 ± 0.01j 2.56 ± 0.14h 0.17 ± 0.02de 0.15 ± 0.00fg
0.22 ± 0.01ef
2.30 0.67 0.10 0.52
0.00c 0.09e 0.05c 0.00i 0.03f
± ± ± ± ± ± ±
0.05 3.06 1.48 3.03 3.11 2.21 0.13
1.15 ± 0.09j
0.32 ± 0.01bcd 1.36 ± 0.03fg
‘Gazda’ 25.15 ± 1.30k 1.67 ± 0.31i
2.56 1.67 1.73 0.56
± ± ± ±
± ± ± ±
0.01bc 0.02a 0.01c 0.03a
0.01a 0.06b 0.09b 0.02e
2.71 0.18 0.08 0.37
0.91 1.58 1.86 1.01
± ± ± ±
± ± ± ±
0.00g 0.09i 0.04i 0.04g 0.28i 0.14i 0.04e
0.28b 0.01ef 0.00d 0.01b
0.10d 0.03b 0.00a 0.04a
‘Zagroda’ 122.54 ± 1.32f 11.00 ± 0.23d 0.41 ± 0.04a 6.37 ± 0.08e 3.34 ± 0.04e 4.56 4.82 0.29 0.76 0.19
± ± ± ± ±
0.09g 0.14e 0.01c 0.02a 0.05efg
0.66 ± 0.03h 0.51 ± 0.04b
0.95 0.47 0.59 1.49 1.79 1.11
± ± ± ± ± ±
0.05c 0.02a 0.04a 0.03b 0.02a 0.08e
mean 74.65 6.50 0.93 0.11 1.89 1.79 0.61 3.20 2.35 0.13 0.19 0.18 0.02 1.85 0.30 0.08 0.30 0.19 0.09 0.27 1.45 1.43 1.27 0.44
a
Abbreviations: Cy 3-glc, cyanidin 3-O-glucoside; Cy diMeth-rham, cyanidin dimethoxyrhamnoside; Cy 3-rut, cyanidin 3-O-rutinoside; Cy 3-ara, cyanidin 3-O-arabinoside; Cy 3-xyl, cyanidin 3-O-xyloside; Cy 3-mal-glc, cyanidin 3-O-(6-O-malonyl-β-D)-glucoside; Cy 3-diox-glc, cyanidin 3-Odioxalylglucoside; EAd, ellagic acid derivative; EA pent, ellagic acid pentoside; MeEA glcA, methylellagic acid glucuronide; EA Ac-Me-pent, ellagic acid acetylmethylpentoside; MeEApen, methylellagic acid pentoside; EA Me-pent, ellagic acid methylpentoside; MeEA Ac-pent, methylellagic acid acetylpentoside; EA, ellagic acid; NChA, neochlorogenic acid; p-cum hex, p-coumaric acid hexoside; Q 3-rut, quercetin 3-O-rutinoside; Q 3-gal, quercetin 3-O-galactoside; Q glcA, quercetin glucuronide; Q 3-glc, quercetin 3-O-glucoside; Q 3-Ac-hex, quercetin 3-O-acetyl-hexoside; Q pent, quercetin pentoside. bValues are means ± standard deviation, n = 3. Mean values within a row with different letters are significantly different at p < 0.05. cAmounts of anthocyanins, flavonols, ellagitannins, including ellagic acid derivatives, and hydroxycinnamic acids were converted into cyanidin 3-O-glucoside (anthocyanins), quercetin (flavonols), chlorogenic acid (hydroxycinnamic acids), and ellagic acid (ellagitannins and ellagic acid derivatives).
‘Smoothstern’ and ‘Orkan’ fruits (0.14 and 0.18 mg/100 g FW, respectively). The analysis shows that the concentrations of flavonols in thorny and thornless blackberries were significantly different. In thornless blackberries the sum of flavonols of individual varieties was lower by 86% in comparison to thorny ones. Ellagitannins. In thorny blackberries, the dominant compound was lambertianin C (mean = 3.47 mg/100 g FW). The highest amount of this compound (4.56 mg/100 g FW) has been found in ‘Zagroda’, whereas the lowest (2.37 mg/100 g FW) is in ‘Gazda’ blackberries. The ‘Zagroda’ variety was characterized by the highest content of all ellagitannins. The amounts of sangiuiins H-6 and H-2 in those fruits were 4.82 and 0.29 mg/100 g FW, respectively. The lowest concentration of ellagitannins has been found in ‘Early Wilson’ blackberries. The amounts of sanguiins H-6 and H-2 in those varieties were 1.00 and 0.03 mg/100 g FW, respectively. In thornless blackberries, similar to in thorny ones, lambertianin C was dominant (mean = 6.30 mg/100 g FW). The highest amount of this compound (8.65 mg/100 g FW) was found in ‘Thornfree’, whereas the lowest (3.95 mg/100 g FW) was in ‘Hull Thornless’ blackberries. The ‘Thornfree’ variety was also characterized by the highest concentration of other ellagitannins. The amounts of sanguiins H-6 and H-2 in those fruits were 7.58 and 0.47 mg/100 g FW, respectively. The lowest amounts of sanguiin H-6 (2.48 mg/100 FW) and
The dominant compound among anthocyanins in thornless blackberries was cyanidin 3-O-glucoside (mean = 134.77 mg/ 100 g FW). The highest amount of this compound (188.77 mg/100 g FW) was found in ‘Loch Ness’ fruits, whereas the lowest (80.76 mg/100 g FW) was in ‘Hull Thornless’. Simultaneously, cyanidin 3-O-rutinoside (11.14 mg/100 g FW) has been found only in ‘Hull Thornless’ blackberries. The analysis revealed statistically significant differences in the anthocyanin concentrations of thorny and thornless blackberries. The sum of those compounds of individual thornless varieties was higher by 47% in comparison to thorny ones. Flavonols. In thorny blackberries, the dominant compound among flavonols was quercetin 3-O-galactoside (mean = 1.64 mg/100 g FW). The highest amount of this compound was found in ‘Gazda’ fruits (2.56 mg/100 g FW), whereas the lowest (0.72 mg/100 g FW) was reported in the ‘Early Wilson’ variety. The ‘Zagroda’ variety was characterized by the highest contents of quercetin 3-O-rutinoside and glucuronide (0.59 and 1.79 mg/100 g FW) simultaneously; quercetin 3-O-pentoside was not found in those fruits. In thornless fruits, the dominant compound among flavonols was quercetin 3-O-acetyl-hexoside (1.13 mg/100 g FW). The highest concentration of this compound has been determined in the ‘Chester Thornless’ variety (1.60 mg/100 g FW), whereas the lowest (0.66 mg/100 g FW) was in ‘Hull Thornless’ blackberries. Quercetin 3-O-rutinoside has been found only in G
DOI: 10.1021/jf5039794 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
H
Cy 3-glc Cy diMethrham Cy 3-rut Cy 3-ara Cy 3-xyl Cy 3-mal-glc Cy 3-diox-glc lambertianin C sanguiin H-6 sanguiin H-2 EA pent MeEA glcA EA Ac-Mepent EA MeEA pent EA Me-pent MeEA Ac-pent NChA p-cum hex Q 3-rut Q 3-gal Q glcA + Q 3-glc Q 3-Ac-hex Q pent
compoundc
1.01 ± 0.03bc 0.33 ± 0.03b 0.25 ± 0.00h 0.87 ± 0.00g 0.46 ± 0.00d
1.55 ± 0.02a 0.19 ± 0.01c
0.52 ± 0.02fg 0.75 ± 0.02e
1.34 ± 0.04d 0.45 ± 0.01d
0.01c 0.03b 0.01g 0.01b 0.07d 0.16b 0.02ab
1.21 ± 0.01g 0.19 ± 0.00ef
± ± ± ± ± ± ±
2.14 ± 0.11 cd 0.18 ± 0.02ef
0.33 8.47 2.58 12.99 6.99 6.52 0.43 0.16 ± 0.00fg 0.16 ± 0.00b
0.00fg 0.00g 0.01c 0.04c 0.16c 0.10g 0.00d
0.44 ± 0.13a
± ± ± ± ± ± ±
138.94 ± 0.46e 12.21 ± 0.02c
147.65 ± 0.37d 12.40 ± 0.05c
0.07 5.00 4.37 9.97 7.40 3.29 0.19
‘Black Satin’
‘Black Beauty’
± ± ± ± ± ± ± 0.00ef 0.12f 0.08d 0.06e 0.13f 0.04h 0.03e
1.60 ± 0.06c 0.71 ± 0.02b
0.62 ± 0.11ef 0.75 ± 0.13e
0.57 ± 0.02e
1.49 ± 0.44efg 0.18 ± 0.01ef
0.27 ± 0.01cde
0.09 5.88 4.19 8.46 5.93 2.48 0.12
166.89 ± 2.45b 14.83 ± 0.22a
‘Chester Thornless’
± ± ± ± ± ± ± 0.02d 0.16c 0.05e 0.09f 0.03h 0.01h 0.01e
± ± ± ± 0.23a 0.01ef 0.00b 0.03c
0.66 ± 0.03i 0.16 ± 0.02g
4.02 0.18 0.17 0.23
0.11 ± 0.00g
0.24 7.24 3.35 6.76 3.95 2.56 0.13
80.76 ± 1.52i 7.04 ± 0.11g
‘Hull Thornless’ ‘Loch Ness’
± ± ± ± 0.36g 0.14h 0.06de 0.04b
0.67 ± 0.01i
1.12 ± 0.01c 0.60 ± 0.05f
0.20 ± 0.01f
1.81 ± 0.36def 0.16 ± 0.00f 0.16 ± 0.00b
4.43 2.63 0.17 0.45
11.14 ± 0.26a 0.25 ± 0.01d 6.41 ± 0.18de
188.77 ± 6.83a 13.54 ± 0.83b
‘Orkan’
± ± ± ± ± ± ± 0.00ef 0.10h 0.18b 0.05d 0.07b 0.04d 0.01c
0.78 ± 0.00h 0.23 ± 0.01f
0.18 ± 0.00d 0.61 ± 0.00fg 0.46 ± 0.01g
1.69 ± 0.02defg 0.24 ± 0.00 cd
0.35 ± 0.00abc
0.11 4.69 4.90 9.59 7.88 5.24 0.31
113.26 ± 2.29g 9.87 ± 0.27e
‘Smoothstern’
± ± ± ± ± ± ± 0.01e 0.09d 0.10a 0.04d 0.10e 0.01f 0.00c
1.01 ± 0.00f 0.29 ± 0.02ef
0.14 ± 0.01d 0.49 ± 0.00g 0.39 ± 0.01gh
1.35 ± 0.01fg 0.24 ± 0.00 cd
0.37 ± 0.01ab
0.11 6.63 5.80 9.74 6.40 4.51 0.27
151.07 ± 2.23c 13.20 ± 0.14b
‘Tayberry’
± ± ± ± ± ± ± 0.02b 0.19a 0.03h 0.14c 0.14gh 0.06c 0.00b
0.91 ± 0.00g 0.29 ± 0.08ef
0.22 ± 0.00h 0.28 ± 0.01h
0.74 ± 0.04d 0.25 ± 0.03c
1.18 ± 0.02g 0.28 ± 0.04c
0.22 ± 0.02def 0.22 ± 0.02a
0.37 8.98 2.18 10.07 4.21 5.59 0.38
143.97 ± 1.54d 12.27 ± 0.07c
‘Thornfree’
± ± ± ± ± ± ±
0.02c 0.03c 0.00f 0.02a 0.03a 0.08a 0.03a
0.73 ± 0.01hi 0.23 ± 0.01f
0.11 ± 0.01i
1.08 ± 0.07b 0.38 ± 0.00b
1.90 ± 0.24cde 0.21 ± 0.00df
0.23 ± 0.00def 0.23 ± 0.00a
0.31 6.99 2.75 13.52 8.65 7.58 0.47
113.91 ± 0.48g 9.98 ± 0.00e
mean
0.95 0.31
1.87 0.21 0.04 0.03 0.57 0.13 0.04 0.44 0.36
1.24 0.21 6.70 3.35 9.01 6.20 4.49 0.27 0.05 0.24 0.07
138.36 11.70
a Abbreviations: Cy 3-glc, cyanidin 3-O-glucoside; Cy diMeth-rham, cyanidin dimethoxyrhamnoside; Cy 3-rut, cyanidin 3-O-rutinoside; Cy 3-ara, cyanidin 3-O-arabinoside; Cy 3-xyl, cyanidin 3-O-xyloside; Cy 3-mal-glc, cyanidin 3-O-(6-O-malonyl-β-D)-glucoside; Cy 3-diox-glc, cyanidin 3-O-dioxalylglucoside; EAd, ellagic acid derivative; EA pent, ellagic acid pentoside; MeEA glcA, methylellagic acid glucuronide; EA Ac-Me-pent, ellagic acid acetylmethylpentoside; MeEApen, methylellagic acid pentoside; EA Me-pent, ellagic acid methylpentoside; MeEA Ac-pent, methylellagic acid acetylpentoside; EA, ellagic acid; NChA, neochlorogenic acid; p-cum hex, p-coumaric acid hexoside; Q 3-rut, quercetin 3-O-rutinoside; Q 3-gal, quercetin 3-O-galactoside; Q glcA, quercetin glucuronide; Q 3-glc, quercetin 3-Oglucoside; Q 3-Ac-hex, quercetin 3-O-acetyl-hexoside; Q pent, quercetin pentoside. bValues are means ± standard deviation, n = 3. Mean values within a row with different letters are significantly different at p < 0.05. cAmounts of anthocyanins, flavonols, ellagitannins, including ellagic acid derivatives, and hydroxycinnamic acids were converted into cyanidin 3-O-glucoside (anthocyanins), quercetin (flavonols), chlorogenic acid (hydroxycinnamic acids), and ellagic acid (ellagitannins and ellagic acid derivatives).
23 24
14 15 16 17 18 19 20 21 22
3 4 5 6 7 8 9 10 11 12 13
1 2
peak
Table 3. Phenolic Compound Contents of Thornless Blackberries (mg/100 g FW)a,b
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/jf5039794 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry sanguiin H-2 (0.12 mg/100 g FW) were found in ‘Chester Thornless’ variety. The analysis revealed statistically significant differences in the total ellagitannins concentration of thorny and thornless blackberries. The sum of those compounds of individual thornless varieties was higher by 44% in comparison to thorny ones. Ellagic Acid and Its Derivatives. In thorny blackberries, the dominant compound among ellagic acid derivatives was ellagic acid (mean = 1.69 mg/100 g FW). The highest amount of ellagic acid (2.71 mg/100 g FW) was determined in ‘Leśniczanka’, whereas the lowest (0.66 mg/100 g FW) was in ‘Zagroda’ variety. ‘Early Wilson’ variety was characterized by the greatest diversity of ellagic acid derivatives. Ellagic acid acetyl methylpentoside (0.10 mg/100 g FW) was found in only those fruits. Ellagic acid pentoside was found only in ‘Zagroda’ and ‘Early Wilson’ varieties (0.76 and 0.19 mg/100 g FW, respectively). In thornless blackberries, ellagic acid was also the dominant compound (mean = 2.60 mg/100 g FW). The highest content of this compound (4.02 mg/100 g FW) was found in ‘Hull Thornless’ blackberries, whereas the lowest (1.18 mg/100 g FW) was in the ‘Tayberry’ variety. Ellagic acid pentoside was found only in ‘Loch Ness’ variety (0.45 mg/100 g FW), whereas methylellagic acid acetylpentoside 2 was found only in ‘Hull Thornless’ (0.23 mg/100 g FW). The analysis revealed statistically significant differences in the sum of ellagic acid derivatives of thorny and thornless blackberries. The sum of those compounds of individual thornless varieties was higher by 27% in comparison to thorny ones. Hydroxycinnamic Acids. In thorny blackberries, hydroxycinnamic acids were found only in the ‘Zagroda’ variety. The amount of neochlorogenic acid was 0.95 mg/100 g FW, and that of p-coumaric acid hexoside acid was 0.47 mg/100 g FW. In thornless blackberries, the highest amount of neochlorogenic acid has been determined in ‘Black Beauty’ fruits (1.55 mg/100 g FW). The highest amount of p-coumaric acid hexoside (0.38 mg/100 g FW) was found in the ‘Thornfree’ variety. In ‘Hull Thornless’, ‘Smoothstern’, and ‘Orcan’ blackberries, no hydroxycinnamic acids were found. The analysis shows that the concentrations of the sum of hydroxycinnamic acids in thorny and thornless blackberries were significantly different. In thornless blackberries, the sum of hydroxycinnamic acids of individual varieties was lower by 21% in comparison to thorny ones. The concentrations of ellagic acid derivatives (mean = 2.92 mg/100 g FW in thorny and 2.49 mg/100 g FW in thornless blackberries) were quite similar to those reported by other authors. Siriwoharn and Wrolstad9 reported that blackberries had ellagic acid derivative contents varying from 1.64 to 3.62 mg/100 g FW, whereas Gancel et al.27 reported 7.57 mg of ellagic acid in 100 g FW of highland blackberry. Hager et al.5 reported that ellagitannins content in blackberries varied from 2.06 to 9.78 mg/100 g FW, whereas Siriwohran et al.7 reported anthocyanins content varying from 131 to 256 mg/100 g FW, which was higher than our research (mean = 86.47 mg/100 g FW in thorny and 171.23 mg/100 g FW in thornless blackberries) and similar to values of Gancel et al.,27 who reported 126.3 mg/100 g FW for cyanidin 3-Oglucoside and 11.73 mg/100 g FW for cyanidin 3-Omalonylglucoside in highland blackberry. Bilyk and Sapers28 reported a mean of 1.55 mg/100 g FW of flavonols, which was lower than in our research (5.70 mg/100 g FW in thorny and 2.09 mg/100 g FW in thornless blackberries), whereas
Heinonen et al.29 and Siriwoharn and Wrolstad9 described higher concentrations of these compounds: 8.3 mg/100 g FW and 11.6−17.8 mg/100 g, respectively. Sellappan et al.12 reported that blackberries and blueberries had hydroxycinnamic acid contents varying from 0.40 to 2.08 mg/100 g FW, which was quite similar to our results (1.42 mg/100 g FW in thorny and 0.7 mg/100 g FW in thornless blackberries). According to Wang and Lin30 and Jiao and Wang,31 the levels of phenolics are influenced by maturity and there is pronounced variation among cultivars. The results of the present study demonstrate that the proportion and content of anthocyanins, flavonols, ellagitannins, and phenolic acids varied greatly among cultivars and types of blackberries. The large standard deviation in some of the results underscores the difficulty in obtaining evenly matured fruit samples for analysis. Thornlessness has long been a priority in almost all blackberry breeding programs and remains a major goal today; the second one is pest and disease resistance.32 In plants, flavonoids play an important role in biological processes. Polyphenols, which can be acquired in tissues under stress, are key components of active and potent defense mechanisms against pests and pathogens.33 In many works34−36 there is a strong correlation between a high phenolic concentration and resistance against herbivores including insect pests. The higher concentration of polyphenols in thornless blackberries may be the result of development of cultivars resistant to pests and diseases. At the same time, thornless blackberries could develop new strategies of physiological defense by the production of larger amounts of flavonoids, including tannins. Tannins are derivatives with a bitter taste and toxic properties that can discourage or deter animals from feeding. The incorporation of novel resistance to pests and diseases is regarded as essential for the development of cultivars suitable for processing. In conclusion, total phenolic and total anthocyanin contents varied greatly among cultivars. The ‘Loch Ness’ and ‘Chester Thornless’ cultivars contained the highest total anthocyanin and total phenolic contents among the 14 cultivars studied. ‘Gazda’ berries had the highest flavonol concentrations, whereas ‘Thornfree’ and ‘Hull Thornless’ selections were highest in ellagitannins and ellagic acid derivatives, respectively. Thorny and thornless blackberries are a good source of antioxidants that can be used in foods and nutritional supplement formulations.
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ASSOCIATED CONTENT
S Supporting Information *
Representative MS spectra for all group of compounds, comparison table, and PCA of the polyphenolic content of thorny and thornless blackberries. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(J.K.-O.) Phone/fax: +48 71 3207 706. E-mail: joanna.
[email protected]. Funding
This work was financially supported by Wroclaw Centre of Biotechnology, program The Leading National Research Centre (KNOW) for years 2014−2018. Notes
The authors declare no competing financial interest. I
DOI: 10.1021/jf5039794 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
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DOI: 10.1021/jf5039794 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
