Investigation of Anthocyanin Profile of Four Elderberry Species and

Article pubs.acs.org/JAFC

Investigation of Anthocyanin Profile of Four Elderberry Species and Interspecific Hybrids Maja Mikulic-Petkovsek,*,† Valentina Schmitzer,† Ana Slatnar,† Biljana Todorovic,† Robert Veberic,† Franci Stampar,† and Anton Ivancic‡ †

Biotechnical Faculty, Department of Agronomy, Chair for Fruit, Wine and Vegetable Growing, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia ‡ Faculty of Agriculture and Life Sciences, Institute for Organic Farming, University of Maribor, SI-2000 Hoce, Slovenia ABSTRACT: A total of 19 different anthocyanins have been detected in four elderberry species and eight hybrids and quantified with the use of HPLC−MSn. The profile and content levels of anthocyanins varied considerably among the analyzed elderberry species and hybrids. Cyanidin-3-O-sambubioside and cyanidin-3-O-glucoside were the most abundant anthocyanins in Sambucus nigra fruits. On the other hand, the prevalent anthocyanin in S. javanica hybrids was identified as cyanidin-3-(E)-p-coumaroylsambubioside-5-glucoside. The highest content of total analyzed anthocyanins (TAA) was determined in berries of the interspecific hybrid S. javanica × S. racemosa, followed by S. nigra, (S. javanica × S. nigra) × cv. Black Beauty, and (S. javanica × S. nigra) × S. cerulea. Berries of S. nigra var. viridis contained significantly lower levels of TAA. Our results provide novel information for nutritional research in addition to breeding programs, which strive to create new hybrids or cultivars with enhanced levels of bioactive components. KEYWORDS: Sambucus cerulea, S. ebulus, S. javanica, S. nigra, S. racemosa, elderberry fruits, anthocyanins

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heart diseases,11,12 asthma, arthritis,13,14 cardiovascular diseases,15 and different viral infections.16,17 High anthocyanin content makes elderberry fruits useful for processing to concentrates, jams, jellies, juices, and dried powders.18,19 Because of their intense color, the concentrates are suitable for providing natural red/purple hues to different food products. Fruits of the cultivated elderberry are very dark purple, nearly black, but various species range from bright red to blue and dark purple shades.20 In Europe, cultivars derived from S. nigra are mainly cultivated, whereas in the U.S. most of the cultivars belong to S. Canadensis.21 The content and composition of anthocyanins vary between different species and cultivars22−27 and are also environmentally triggered. Factors such as light, temperature, and stress during cultivation significantly modify their synthesis and accumulation.28 Recently, a great deal of renewed interest in anthocyanins has emerged due to their potential health benefits as antioxidants and antiinflammatory agents. As the market for healthy products requires new genotypes with different processing and quality characteristics, there is a strong need for the selection of genotypes optimal for increasing the quality of food products. Data on the chemical composition of elderberry fruits are scarce; only a few studies revealed anthocyanin levels of various Sambucus species or cultivars.22−24 Although the content of fruit phenolics is affected by environmental conditions and the level of maturity at harvest, it is very important to know the chemical composition and the antioxidant capacity of different elderberry species to selectively

INTRODUCTION The Sambucus L. genus was formerly grouped in the Caprifoliaceae family, but novel genetic research places it into the Adoxaceae family.1,2 It includes between 5 and 30 species, depending on taxonomy, distributed in the temperate and subtropical regions of the Northern and Southern Hemisphere. The genus Sambucus mostly consists of perennial subshrubby herbs with creeping rhizomes, many-stemmed shrubs (sometimes rhizomatous), or small deciduous or semievergreen trees.3 The most important species are S. cerulea Raf. (blue elder), S. javanica Blume (Javanese elder, sin. Chinese elder), and S. nigra L. (common elderberry, syn. elderberry). The use of interspecific hybridization in the genetic breeding of the Sambucus species is a relatively novel approach. First systematic attempts to produce hybrid plants between S. nigra and S. racemosa were reported by Böcher4 in Denmark, Winge5 in North Zealand, and Nilsson6 in southern Sweden. The resulting F1 hybrids were usually sterile. Most of the phenotypical traits were intermediate between the parental species. According to Chia,7 a higher fertility level was achieved in crosses between S. canadensis and S. nigra, and S. cerulea and S. nigra. On the other hand,8 genetic sterility was reported in hybrids between S. nigra and S. ebulus. Presently, one of the most important trends in food and pharmaceutical industries is the growing demand for valuable natural sources of antioxidant compounds. Among common fruits and vegetables, elderberry is one of the richest in anthocyanins, flavonols glycosides, and other polyphenolics, which contribute to the high antioxidant capacity of its berries.9,10 Additionally, antioxidant properties of anthocyanins present in elderberries are associated with beneficial effects on human health due to their ability to protect against cancer or © XXXX American Chemical Society

Received: March 12, 2014 Revised: May 15, 2014 Accepted: May 16, 2014

A

dx.doi.org/10.1021/jf5011947 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

nigra) were highly self-incompatible, enabling pollination without emasculation. The hybrid origin of the majority of the interspecific hybrids was determined by molecular and morphological analyses.29 The hybrid origin of S. javanica × S. racemosa hybrids, which were created later, was determined only by morphological markers: by comparing leaf, inflorescence, floret, infrutescence, and berry characteristics. Hybrid plants are being maintained in the gene bank of the University of Maribor, Faculty of Agriculture and Life Sciences, near Hoce (Slovenia). For multiplication, a simple vegetative propagation by cuttings is used. Chemicals. The following standards were used for the quantification of anthocyanins: cyanidin-3-O-glucoside, cyanidin-3,5di-O-glucoside, and pelargonidin-3-O-glucoside from Sigma-Aldrich Chemie GmbH; and cyanidin-3-O-galactoside from Fluka Chemie GmBH. Methanol for the extraction of phenolics was acquired from Sigma-Aldrich Chemie. The chemicals for the mobile phases were HPLC−MS grade acetonitrile and formic acid from Fluka Chemie GmBH. Water for the mobile phase was double distilled and purified with the Milli-Q system (Millipore, Bedford, MA). Extraction of Phenolic Compounds. For each genotype, six independent repetitions were done (n = 6). Until extraction, they were kept in deep freeze (−80 °C). The extraction of berries was performed as described by Mikulic-Petkovsek et al.,30 with some modification. Elderberry fruits were ground to a fine paste with liquid nitrogen, and 3 g was extracted with 15 mL of methanol containing 3% (v/v) formic acid and 1% (w/v) 2,6-di-tert-butyl-4-methylphenol (BHT) in a cooled ultrasonic bath for 1 h. BHT was added to the samples to prevent oxidation. After extraction, the fruit extracts were centrifuged for 10 min at 10.000 rpm. Each supernatant was filtered through a Chromafil AO20/25 polyamide filter manufactured by Macherey-Nagel (Düren, Germany) and transferred to a vial prior to injection into the HPLC (high performance liquid chromatography) system. Determination of Individual Phenolic Compounds Using HPLC-DAD-ESI-MSn Analysis. Anthocyanins were analyzed on a Thermo Finnigan Surveyor HPLC system (Thermo Scientific, San Jose, CA) with a diode array detector at 530 nm. Spectra of the compounds were recorded between 200 and 600 nm. The column was a Gemini C18 (150 × 4.6 mm 3 μm; Phenomenex, Torrance, CA) operated at 25 °C. The elution solvents were aqueous 0.1% formic acid in double distilled water (A) and 0.1% formic acid in acetonitrile (B). Samples were eluted according to the linear gradient from 5% to 20% B in the first 15 min, followed by a linear gradient from 20% to 30% B for 5 min, then an isocratic mixture for 5 min, followed by a linear gradient from 30% to 90% B for 5 min, and then an isocratic mixture for 15 min before returning to the initial conditions.31 The injection amount was 20 μL, and the flow rate was 0.6 mL min−1. All phenolic compounds presented in our results were identified by an HPLC-Finnigan MS detector and an LCQ Deca XP MAX (Thermo Finigan, San Jose, CA) instrument with electrospray interface (ESI) operating in positive ion mode. The analyses were carried out using full scan data-dependent MSn scanning from m/z 110 to 1500. Column and chromatographic conditions were identical to those used for the HPLC-DAD analyses. The injection volume was 10 μL, and the flow rate was maintained at 0.6 mL min−1. The capillary temperature was 250 °C, the sheath gas and auxiliary gas were 60 and 15 units, respectively, the source voltage was 4 kV, and normalized collision energy was between 20−35%. Spectral data were elaborated using the Excalibur software (Thermo Scientific). The identification of compounds was confirmed by comparing retention times and their spectra as well as by adding the standard solution to the sample and by fragmentation. Concentrations of anthocyanins were calculated from peak areas of the sample and the corresponding standards and expressed in mg 100 g−1 fresh weight (FW) of elderberry fruits. For compounds lacking standards, quantification was carried out using compounds similar to the standards. Thus, pelargonidin-3-sambubioside was quantified in equivalents of pelargonidin-3-O-glucoside, and all cyanidin glycosides

use them in pharmaceutical and alimentary industry. To our knowledge, there have been no comparative studies on the chemical composition of a large number of elderberry genotypes grown under the same ecological conditions. Therefore, the purpose of the study was to determine a broad range of anthocyanins and their content in five genotypes belonging to four different elderberry species (Sambucus cerulea, S. ebulus, S. nigra − (var. nigra and var. viridis), and S. racemosa (var. miquelli)), and compare them to the anthocyanin profile of eight elderberry interspecific hybrids. The final aim is to select genotypes with favorable composition and content of anthocyanins. The biochemical evaluation of elderberry genetic resources and the identification of individual bioactive compounds and their properties as natural agents is of utmost significance to breeders. This is the first Article on anthocyanin composition of a large number of elderberry species and interspecific hybrids striving to select cultivars, which may prove interesting for different processing industries.

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MATERIALS AND METHODS

Plant Material. The biochemical investigation included four species of the Sambucus genus (1, Sambucus cerulea; 2, S. ebulus; 3, S. nigra; 4, S. racemosa var. miquelli) and three groups of their interspecific hybrids (1, three hybrids of S. cerulea; 2, four hybrids of S. nigra; 3, one hybrid of S. racemosa) (Table 1). The self-incompatible genotype of S. javanica originating from tropical regions did not produce a sufficient quantity of berries and could not be included in chemical analyses; however, this species was crucial in the hybridization process. All interspecific hybrids were developed at the University of Maribor, Faculty of Agriculture and Life Sciences, by Anton Ivancic using S. javanica as a genetic bridge. The female components (S. javanica and some of the hybrids S. javanica × S.

Table 1. Sambucus Species and Interspecific Hybrids Analyzed in the Study species

abbreviation

S. cerulea S. ebulus S. nigra var. nigra (common type) S. nigra var. viridis S. racemosa var. miquelli interspecific hybrids S. cerulea Group S. javanica × S. cerulea (S. javanica × S. nigra) × S. cerulea S. cerulea × S. nigra S. nigra Group S. javanica × S. nigra S. javanica × (S. javanica × S. nigra) (S. javanica × S. nigra) × S. nigra (S. javanica × S. nigra) × cv. Black Beauty S. racemosa Group S. javanica × S. racemosa

origin Slovenia (local cultivated genotype) Slovenia (wild-growing genotype) Slovenia (local population)

Slovenia (botanical garden Maribor) Slovenia (botanical garden Maribor) abbreviation origin JA × CER (JA × NI) × CER CER × NI JA × NI JA × (JA × NI) (JA × NI) × NI

JA × RAC

University of Maribor Gene Bank University of Maribor Gene Bank University of Maribor Gene Bank University of Gene Bank University of Gene Bank University of Gene Bank University of Gene Bank

Maribor Maribor Maribor Maribor

University of Maribor Gene Bank B



Article

Figure 1. Anthocyanin profile of elderberry fruits of Sambucus nigra, Sambucus cerulea, Sambucus racemosa var. miquelli, Sambucus ebulus, and Sambucus javanica × S. racemosa. Peak numbers correspond to the compounds listed in Table 2. (except for cyanidin-3-galactoside) were quantified in equivalents of cyanidin-3-O-glucoside. Statistical Analysis. The data were analyzed with the Statgraphics Plus 4.0 program (Manugistics, Inc.) using one-way analysis of variance (ANOVA). The differences among the species and interspecific hybrids were tested using the Duncan test at the 0.05 significance level. The means and the standard errors of the means are reported (mean ± SE).

standard). This cyanidin is one of the most common anthocyanins in plant tissue previously also confirmed in elderberry fruit.24,33 Cyanidin-3,5-diglucoside was detected in Sambucus nigra fruit and its interspecific hybrids. On the other hand, S. cerulea (CER), S. ebulus (EB), and S. racemosa var. miquelli (MIQ) did not contain this anthocyanin. Cyanidin-3,5diglucoside was also detected in two interspecific hybrids with S. javanica, JA × RAC and JA × CER. Although chemical analysis of S. javanica was not performed, it can be concluded that its fruits contain this anthocyanin as it is not present in RAC in CER fruit. Statistically the highest level of cyanidin-3,5diglucoside was detected in fruit of JA × (JA × NI) hybrid, which contained 37.2 mg 100 g−1 FW. It seems that S. javanica, similar to S. nigra, accumulates high levels of this cyanidin. The content level of cyanidin-3,5-diglucoside in analyzed elderberry fruits is in accordance with the results of Kaack and Austed.33 The second compound in peak 1 had an ion at [M+] m/z 743, which yielded MS2 fragment at m/z 581 ([M+] − 162, loss of a glucosyl unit) and MS3 fragment at m/z 449/287 ([M+] − 132, loss of a xylosyl and [M+] − 162, a glucosyl unit). This corresponds to the fragmentation pattern of cyanidin-3-Osambubioside-5-O-glucoside, a prior-identified elderberry anthocyanin.34 The highest content of this cyanidin was quantified in JA × CER (146.9 mg 100 g−1) hybrid, followed by (JA × NI) × cv. Black Beauty hybrid (136.8 mg 100 g−1). In the former, S. javanica is probably responsible for the accumulation cyanidin-3-O-sambubioside-5-O-glucoside as the

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RESULTS AND DISCUSSION All 13 elderberry genotypes were characterized by unique anthocyanin profiles. Because of limited space, only five representative chromatograms (species S. nigra, S. cerulea, S. racemosa var. miquelli, S. ebulus, and hybrid JA × RAC) are presented in Figure 1. A total of 19 different anthocyanins were detected in four elderberry species and eight hybrids. By comparing their MS data with recently published records,20,24 13 of them were identified. In all analyzed elderberries, cyanidin-based anthocyanins were the major anthocyanins, followed by glycosides of pelargonidin. Similar findings have previously been reported by Wu et al.24 and Veberic et al.32 Peak 1 contained two compounds. The first had a [M+] at m/z 611, which on MS2 produced a minor fragment at m/z 449 ([M+] − 162, loss of glucosyl unit) and a major fragment at m/ z 287 (cyanidin, [M+] − 162 corresponding to a glucosyl moiety). This compound was, therefore, identified as cyanidin3,5-diglucoside (also confirmed by the addition of an external C



Article

Table 2. List of Anthocyanins Identified in Different Accessions of Elderberry Fruits and MSn Specifications peak no.a

tRb

1

7.99

1 2

7.99 8.83

3

9.34

4

9.92

5

10.66

5 6 6 7 7

10.66 11.40 11.40 12.05 12.05

8 9

12.54 12.66

10 11

12.95 13.36

12 13

17.73 18.33

14

19.65

15

21.24

tentative identification cyanidin-3-O-sambubiosyl-5-Oglucoside cyanidin-3,5-O-diglucoside cyanidin-glycoside 1 (cyanidinpentoside-hexoside 1) cyanidin-glycoside 2 (cyanidinpentoside-hexoside 2) cyanidin-glycoside 3 (cyanidinpentoside-hexoside 3) cyanidin-glycoside 4 (cyanidinpentoside-hexoside 4) cyanidin-3-O-galactoside cyanidin-3-O-sambubioside cyanidin-3-O-glucoside cyanidin-3-O-rutinoside cyanidin-sambubiosidemalonylglucoside cyanidin-dipentoside pelargonidin-3-O-glucoside pelargonidin-3-O-sambubioside cyanidin-sambubiosideacetylglucoside cyanidin-xylosyl-dihexoside cyanidin-3-(Z)-p-coumaroylsambubioside-5-glucoside cyanidin-3-(E)-p-coumaroylsambubioside-5-glucoside cyanidin-3-p-coumaroylsambubioside

[M]+c (m/z)

MS2

identified in following species and interspecific hybridsd,e

MS3

743

581

449/287

in all, except CER, EB

611 581

449/287 449

287

in all, except CER, EB CER

581

449

287

CER

581

449

287

CER

581

449

287

in all, except JA × CER, JA × NI, VIR

449 581 449 595 829

287 449/287 287 449 667

287 449/287

in all, except JA × NI, CER, EB, MIQ, VIR in all in all JA × CER, (JA × NI) × NI, (JA × NI) × cv. Black Beauty, NI JA × CER, (JA × NI) × cv. Black Beauty, MIQ

551 433

419 271

565 785

433/271 623

449/287

CER, EB NI, (JA × NI) × NI, (JA × NI) × CER, JA × (JA × NI), (JA × NI) × cv. Black Beauty NI, CER, (JA × NI) × NI, JA × (JA × NI), (JA × NI) × cv. Black Beauty JA × RAC, JA × NI

905 889

743 727

449/287 449/287

JA × RAC, (JA × NI) × CER, JA × (JA × NI), JA × NI JA × RAC, (JA × NI) × CER, JA × (JA × NI), JA × NI, MIQ, CER × NI

889

727

449/287

727

433

287

JA × RAC, JA × CER, (JA × NI) × CER, JA × (JA × NI), JA × NI, (JA × NI) × cv. Black Beauty, MIQ, CER × NI JA × RAC, (JA × NI) × CER, JA × (JA × NI), JA × NI, MIQ, CER × NI

287

a

Peak number. bRetention time. cAnthocyanins were obtained in the positive ion mode. dAbbreviations: CER, Sambucus cerulean; EB, Sambucus ebulus; JA, Sambucus javanica; NI, Sambucus nigra var. nigra; VIR, Sambucus nigra var. viridis; MIQ, Sambucus racemosa var. miquelli. eAbbreviations for interspecific hybrids are in Table 1.

fruit of S. cerulea does not contain it. However, research on S. javanica biochemistry is very limited, and no data comparison with literature could be made. Fruits of S. ebulus also lacked cyanidin-3-O-sambubioside-5-O-glucoside, but a high content of this anthocyanin was measured. Cyanidin-3-O-sambubioside5-O-glucoside constituted from 3% to 44% total anthocyanins in elderberry fruit analyzed. Contrary to our results, Bridle and Garcia Viguera35 reported cyanidin 3-O-sambubioside-5-Oglucoside as the major anthocyanin in the S. nigra sample, but Bronnumhansen and Hansen36 reported cyanidin 3glucoside as the prevalent pigment of S. nigra. The variations in results may be explained by differences of individual genotypes as well as specific growing conditions. It is often difficult to compare anthocyanin levels of elderberries in recent studies mainly because of the different methods and variable expression of the results (e.g., fresh weight vs dry weight). MS data of peaks 2, 3, 4, and 5 shared the same mass spectral pattern: a molecular ion at 581 ([M]+ m/z 581),a MS2 fragment ion at m/z 449, and MS3 fragment ion at m/z 287. The fragment loss of 132 (a pentose unit) and 162 (a hexose unit) suggests the anthocyanins to be cyanidin-pentosidehexosides, and they were thus tentatively identified as cyanidin glycosides 1, 2, 3, and 4. Cyanidin glycosides 1−3 were only present in berries of Sambucus cerulea, but cyanidin glycoside 4 was identified in some others species and hybrids analyzed. An exceptionally high content of cyanidin glycoside 4 was detected in S. ebulus fruit (345.8 mg 100 g−1, representing 97% TAA), followed by S. cerulea fruit (78.9 mg 100 g−1, representing 53%

TAA). Other analyzed species and hybrids contained less than 1 mg 100 g−1 of this anthocyanin. No cyanidin glycoside 4 could be detected in hybrids between S. nigra and S. javanica (JA × NI) (Table 3). In addition to a [M+] ion at m/z 581, peak 5 yielded a [M+] ion at m/z 449. MS2 fragmentation produced a cyanidin ion at m/z 287 ([M+] − 162, loss of a hexose moiety). This peak was thus identified as cyanidin-3-galactoside and further confirmed by cochromatography with a cyanidin-3-galactoside standard. A relatively high content of this anthocyanin was measured in fruit of JA × CER (32.2 mg 100 g−1) hybrid, whereas other analyzed species and hybrids only contained traces of cyanidin3-galactoside (less than 0.7 mg 100 g−1). Peak 6 contained an ion at m/z 581, which fragmented on MS2 to produce a minor ion at m/z 449 ([M]+ − 132, loss of a xylosyl unit) and a major fragment at m/z 287 ([M]+ − 132 − 162, loss of xylosyl and glucosyl units). This compound was present in all studied elderberry species and hybrids and has been identified as cyanidin-3-O-sambubioside, previously confirmed in elderberry by Chandra et al.37 and Lee and Finn.20 Statistically the highest level of cyanidin-3-sambubioside was detected in S. nigra fruit (560 mg 100 g−1), representing 61% TAA. Consequently, several interspecific S. nigra hybrids ((JA × NI) × CER and (JA × NI) × NI) also contained high levels of this anthocyanin. Similar content levels of cyanidin-3sambubioside were reported by Veberic et al.23 and Wu et al.24 in various Sambucus species. S. cerulea fruit was also rich in this anthocyanin (representing 42% TAA); however, S. racemosa D


0.18 6.48 0.06

4.08 42.19 0.09

SE 1.22 2.70 0.83 1.60 4.62 0.40 1.02 3.41

x̅ 95.54 146.91 13.97 13.88 57.71 12.91 97.39 136.87

a c a

eb g b b d b e f

5.91 0.02

x̅ 32.49 33.64 17.86 8.74 37.26 4.52 3.26 25.34

0.91 0.01

0.42 0.62 1.06 1.01 2.34 0.14 0.03 0.52

SE

C-3,5-diglu

bc a

f f d c g b b e 0.12 0.06 0.02 0.06 0.09 4.70 5.44 0.16 0.32

0.47 0.30 0.08 0.72 0.31 78.99 345.82 0.17 1.08

SE 0.02

x̅ 0.51

C-pent-hex 4

a a b c a a

a a a

a

0.32

0.35 0.39

x̅ 0.64 32.27 0.13 0.00 0.29

0.09

0.03 0.12

0.03 0.62 0.03 0.00 0.08

SE

C-3-gal

a

a a

a b a a a

x̅ 32.37 31.91 59.05 1.14 131.15 6.62 181.76 91.40 2.85 0.49 0.13 190.63 0.55 1.06 0.61 5.89 0.17 11.05 0.13 2.81 0.64 0.16 0.01 0.01 15.98 0.34

SE

C-3-glu b b c a e a f d a a a f a

x̅ 10.68 13.78 111.64 2.45 18.22 5.63 104.69 24.63 63.43 6.56 1.36 344.48 1.03 0.35 0.26 11.14 0.36 1.78 0.11 1.62 2.11 3.64 0.18 0.08 20.65 0.64

SE

C-3-sam ab ab d a ab a d b c a a e a 9.36

3.00 0.14

2.16

x̅

1.11

0.34 0.01

0.09

SE

C-3-rut

c

b a

b

E

0.06

0.01

0.02

0.14

0.23

SE

1.48

x̅

a

a

bb

3.53

x̅ 10.45

0.19

0.35

SE

C-sam-acetylglu

a

b

SE

0.10 0.48 0.39

12.05 15.94

0.09

5.30

x̅ 2.66

C-xyl-dihex

c d

b

a

2.59

1.43 4.93 1.92 4.65

x̅ 11.92

0.09

0.03 0.43 0.13 0.11

0.41

SE

b

a c ab c

d

C-3-(Z)-cou-sam-5-glu

11.31

0.37

x̅ 583.04 0.83 162.89 49.22 246.08 191.23

0.28

0.13

5.68 0.32 5.41 5.09 9.81 3.78

SE

a

a

f a c b e d

C-3-(E)-cou-sam-5-glu

0.61

68.96 2.38 8.18 8.64

x̅ 8.03

0.05

4.69 0.26 0.36 0.26

0.14

SE

C-3-cou-sam

a

c a b b

b

x̅ 834.67 331.88 444.04 80.15 505.25 253.66 391.17 479.23 148.63 353.37 20.47 560.48 1.69

7.66 4.16 22.79 2.17 17.62 5.28 4.16 26.22 8.75 5.38 0.57 59.38 1.05

SE

total anthocyanins j e g b h d f gh c ef a i a

a Anthocyanin: C-sam-malonylglu, cyanidin-sambubioside-malonylglucoside; C-sam-acetylglu, cyanidin-sambubioside-acetylglucoside; C-xyl-dihex, cyanidin-xylosyl-dihexoside; C-3-(Z)-cou-sam-5-glu, cyanidin-3-(Z)-p-coumaroyl-sambubioside-5-glucoside; C-3-(E)-cou-sam-5-glu, cyanidin-3-(E)-p-coumaroyl-sambubioside-5-glucoside; C-3-cou-sam, cyanidin-3-p-coumaroyl-sambubioside. bDifferent letters (a−j) denote statistically significant differences in individual anthocyanins among elderberry species or interspecific hybrid by Duncan multiple range test at P < 0.05.

JA × RAC JA × CER (JA × NI) × CER CER × NI JA × (JA × NI) JA × NI (JA × NI) × NI (JA × NI) × cv. Black Beauty Sambucus cerulea Sambucus ebulus Sambucus racemosa var. miquelli Sambucus nigra Sambucus nigra var. viridis

C-sam-malonylglua

Table 4. Individual Quantities (mean ± SE, mg 100 g−1 FW) of Each Anthocyanin Determined in Different Elderberry Species or Interspecific Hybrid

a Anthocyanin: C-3-sam-5-glu, cyanidin-3-O-sambubiosyl-5-O-glucoside; C-3,5-diglu, cyanidin-3,5-O-diglucoside; C-pent-hex 4, cyanidin-pentoside-hexoside 4; C-3-gal, cyanidin-3-O-galactoside; C-3-glu, cyanidin-3-O-glucoside; C-3-sam, cyanidin-3-O-sambubioside; C-3-rut, cyanidin-3-O-rutinoside. bDifferent letters (a−g) denote statistically significant differences in individual anthocyanins among elderberry species or interspecific hybrid by Duncan multiple range test at P < 0.05.

JA × RAC JA × CER (JA × NI) × CER CER × NI JA × (JA × NI) JA × NI (JA × NI) × NI (JA × NI) × cv. Black Beauty Sambucus cerulea Sambucus ebulus Sambucus racemosa var. miquelli Sambucus nigra Sambucus nigra var. viridis

C-3-sam-5-glua

Table 3. Individual Quantities (mean ± SE, mg 100 g−1 FW) of Each Anthocyanin Determined in Different Elderberry Species or Interspecific Hybrid

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presented), which is in accordance with Wu et al.24 and their study on S. nigra fruit. The compound in peak 11 showed a molecular ion at m/z 785 and MS2 fragment at m/z 623 ([M]+ − 162, loss of a hexose residue) and MS3 fragment at m/z 449/287 ([M]+ − 42 loss of acetyl; − 162 hexose and − 132 pentose moieties). This fragmentation pattern is similar to that observed for the compound in peak 6. Thus, peak 11 was identified as cyanidin 3-xylosylhexoside-acetyl-hexoside. Because sambubioside (i.e., 2-O-β-D-xylosyl-D-glucose) is the xylosylglucoside most frequently found as an anthocyanin substituent,39 peak 11 could be tentatively identified as cyanidin-sambubioside-acetylglucoside. A similar fragmentation pattern of a cyanidin-based anthocyanin has previously been reported in Sambucus samples by Lee and Finn.20 In our samples, it was merely present in two hybrids, JA × RAC (10.4 mg 100 g−1) and JA × NI (3.5 mg 100 g−1). Interestingly, hybrids of a similar origin (S. javanica as a mother plant) did not contain cyanidin-sambubioside-acetylglucoside. Peak 12 presented a molecular ion [M − H]− at m/z 905, which yielded MS2 fragment ion at m/z 743 ([M]+ − 162]−, loss of a hexoside moiety), and MS3 fragment at m/z 449 ([M]+ − 132, loss of a pentoside and − 162 a hexoside or caffeoyl moiety) and 287 (cyanidin, − 162 loss of a hexoside moiety). The fragmentation pattern is similar to that obtained for peak 1; the difference is one hexose or caffeoyl unit. The peak was tentatively identified as cyanidin-xylosyl-dihexoside or cyanidin-caffeoyl-sambubiosyl-glucoside. This anthocyanin was quantified in range of 2.6−15.9 mg 100 g−1 berries in the following hybrids: JA × RAC, (JA × NI) × CER, JA × (JA × NI), and JA × NI. As all hybrids share the same species, S. javanica, the latter potentially represents the genetic source for the accumulation of this specific anthocyanin. Peaks 13 and 14 were characterized by the same mass spectral pattern. Both compounds had a molecular ion at m/z 889 that fragmented on MS2 to produce ion at m/z 727 ([M]+ − 162, loss of a glucosyl moiety) and on MS3 produce the ions at m/z 449 ([M]+ − 132 a pentosyl and − 146 a coumaroyl moiety) and 287 (cyanidin, − 162 a glucosyl moiety). According to the study of Lee and Finn20 and Inami et al.,40 this fragmentation pattern matches with cyanidin-3-(Z)-pcoumaroyl-sambubioside-5-glucoside and cyanidin-3-(E)-p-coumaroyl-sambubioside-5-glucoside. The results indicate that both anthocyanins are characteristic for S. javanica and S. racemosa fruit as they are only present in hybrids, where these species were used as parent plants. Statistically highest level of cyanidin-3-(Z)-p-coumaroyl-sambubioside-5-glucoside (11.9 mg 100 g−1) and cyanidin-3-(E)-p-coumaroyl-sambubioside-5glucoside (583 mg 100 g−1) was determined in JA × RAC hybrid in which they combined to represent up to 71% total analyzed anthocyanins. On the other hand, S. nigra, S. cerulea, and S. ebulus fruit did not contain these two anthocyanins as reported also Jordheim et al.41 Peak 15 contained [M]+ ion at m/z 727, which yielded MS2 fragment at m/z 433 ([M]+ − 132 and − 162, loss of xylosyl and glucosyl units) and MS3 fragment at m/z 287 ([M]+ − 146, loss of a coumaroyl unit). This corresponds to the fragmentation pattern of cyanidin-3-p-coumaroyl-sambubioside, which has previously been detected in elderberry fruit.20,40 It was only detected in hybrids involving S. javanica, S. cerulea, or S. racemosa var. miquelli parent. The highest level of cyanidin-3p-coumaroyl-sambubioside has been determined in (JA × NI) × CER (68.9 mg 100 g−1) fruit. In accordance with our results,

var. miquelli (6% TAA) and S. ebulus only contained low amounts of cyanidin-3-sambubioside (less than 2% TAA). The second anthocyanin in peak 6 was identified as cyanidin-3-Oglucoside by its [M]+ at m/z 449, which on MS2 yielded a cyanidin fragment ion at m/z 287 formed by the cleavage of a 162 amu glucosyl unit. It was further confirmed by the addition of an external standard. S. nigra was characterized by highest levels of this anthocyanin (representing one-third of all anthocyanin analyzed in the fruit), followed by its hybrids: (JA × NI) × NI, JA × (JA × NI), and (JA × NI) × cv. Black Beauty. S. ebulus and S. racemosa var. miquelli contained only minor amounts of this anthocyanin. In accordance with our results, S. nigra samples analyzed by Watanabe et al.38 contained more cyanidin-3-sambubioside than cyanidin-3glucoside. On the other hand, Kaack and Austed33 report higher levels of cyanidin-3-glucoside. In S. nigra berries analyzed in our study, cyanidin-3-sambubioside and cyanidin3-glucoside combined represented approximately 95% TAA. These results are similar to those already reported in the literature by Seabra et al.10 and Nakajima et al.34 Peak 7 showed a molecular ion at m/z 595, which fragmented to produce MS2 ions at m/z 449 ([M]+ − 146, loss of a rhamnosyl moiety) and m/z 287 ([M]+ − 308, cleavage of rhamnosyl and glucosyl units), suggesting the presence of cyanidin-3-O-rutinoside. It was only present in specific hybrids; (JA × NI) × cv. Black Beauty was particularly rich in this anthocyanin (64.7 mg 100 g−1) (Table 3). As one of the parents of the hybrid is Sambucus nigra, the results confirm the findings of Veberic et al.,23 who reported 9.6 mg of cyanidin-3-rutinoside 100 g−1 in fruits of S. nigra ‘Haschberg’. The second anthocyanin in peak 7 had an ion at m/z 829, which yielded a MS2 fragment at m/z 667 ([M]+ − 162, loss of a hexose unit) and MS3 at m/z 449/287 ([M]+ − 132 − 162 −86, loss of xylosyl, glucosyl, and malonyl units). This compound is thus a cyanidin-pentosylhexoside-malonylhexoside, tentatively identified as cyanidin-sambubioside-malonylglucoside and present only in specific hybrids (JA × RAC, JA × CER, (JA × NI) × cv. Black Beauty, and MIQ). This is the first report of this compound in elderberry berries. Peak 8 showed a molecular ion of m/z 551 and a neutral loss of 264 u, corresponding to two pentoses, so it was tentatively identified as a cyanidin dipentoside. It was detected in S. cerulea and S. ebulus fruit in levels below 0.5 mg 100 g−1. This is the first report of cyanidin dipentoside ([M]+ at m/z 551) in elderberry berries. Peak 9 had [M]+ at m/z 433 and on MS2 yielded a fragment ion at m/z 271 (pelargonidin, [M]+ − 162, loss of a hexosyl unit). On the basis of the mass spectra data, HPLC elution properties, and the addition of an external standard, peak 9 was identified as pelargonidin-3-O-glucoside. It was only detected in traces in berries of S. nigra and its hybrids (JA × NI) × NI, (JA × NI) × CER, JA × (JA × NI), and (JA × NI) × cv. Black Beauty (data not presented). Similarly, Lee and Finn20 and Wu et al.24 report low levels of pelargonidin-3-glucoside in S. nigra fruit. The anthocyanin with a molecular ion of m/z 565 was identified as pelargonidin-3-O-sambubioside (peak 10). On MS2 it produced a minor fragment at m/z 433 ([M]+ − 132, loss of a xylosyl unit) and a major fragment at m/z 271 ([M]+ − 162, loss of a glucosyl unit). It has been confirmed in the following species and hybrids: S. nigra and S. cerulea, (JA × NI) × NI, JA × (JA × NI), and (JA × NI) × cv. Black Beauty. Only low levels of this anthocyanin were quantified (data not F



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Lee and Finn20 reported that S. nigra fruit lacks cyanidin-3-pcoumaroyl-sambubioside. Interestingly, berries of the CER × NI hybrid contained the last three mentioned anthocyanins (Table 4). The absence of the anthocyanins cyanidin-3-(Z)-p-coumaroyl-sambubioside-5glucoside, cyanidin-3-(E)-p-coumaroyl-sambubioside-5-glucoside, and cyanidin-3-p-coumaroyl-sambubioside in S. cerulea and S. nigra, and their presence in their hybrid CER × NI, could be explained by homo- and heterozygosity in association with pollination system of elderberries, and genetic inheritance of these two anthocyanins (the interactions of two or more loci with dominant and recessive alleles and/or the involvement of inhibiting genes). Elderberries can be pollinated by wind and insects, and cross-pollination is probably more important than self-pollination.3,42 Elderberries are predominantly used as processed food or dietary supplements; therefore, it is useful to know their content of phenolic compounds, especially anthocyanins, which represent the highest share in elderberry fruit. The major anthocyanins detected in fruit of different species and interspecific hybrids were cyanidin-3-O-sambubioyl-5-O-glucoside, cyanidin-3,5-diglucoside, cyanidin-3-sambubioside, cyanidin-3-glucoside, and cyanidin-3-(E)-p-coumaroyl-sambubioside-5-glucoside. Conversely, in S. javanica and S. racemosa, hybrids cyanidin-3-(Z)-p-coumaroyl-sambubioside-5-glucoside and cyanidin-3-(E)-p-coumaroyl-sambubioside-5-glucoside prevailed. Total analyzed anthocyanins (TAA) varied greatly from hybrid to hybrid. Promising species or interspecific hybrids with TAA higher than 400 mg 100 g−1 were (JA × NI) × CER, (JA × NI) × cv. Black Beauty, Sambucus nigra, and, in particular, JA × RAC hybrid, which contained 834 mg 100 g−1 of anthocyanins. Berries of S. nigra var. viridis contained significantly lower levels of TAA, which is closely correlated with the pale color of their fruits; they are predominantly green with a slight hint of red color. Several minor anthocyanins have also been confirmed in different elderberries. Pelargonidin-3glucoside, pelargonidin-3-sambubioside, cyanidin-3-galactoside, cyanidin-dipentoside ([M]+ at m/z 551), and different cyanidin glycosides 1−3 ([M]+ at m/z 581) were quantified in traces. Our results indicate that elderberry fruits are a superior source of anthocyanins to other fruit species, for example, apples, peaches, and strawberries.43−45 However, a great variation in anthocyanin profile has been observed among elderberry species and hybrids. These differences may contain important implications and information, which may become evident in health effects studies. Some elderberry genotypes could be used to design new raw materials with high contents of anthocyanins, which can serve to produce elderberry products with health-promoting properties based on their positive health effects. Data from our research present new information for nutritional research in addition to breeding programs, which have turned their focus to fruit quality and strive to improve nutritional or health value to create new hybrids or cultivars with enhanced levels of bioactive components.

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Investigation of Anthocyanin Profile of Four Elderberry Species and

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