Transfer and Mass Balance of Ellagitannins, Anthocyanins, Flavan-3

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Transfer and Mass Balance of Ellagitannins, Anthocyanins, Flavan-3-ols and Flavonols During the Processing of Red Raspberries (Rubus ideaus L.) to Juice. Micha# Sójka, Jakub Macierzy#ski, Wojciech Zaweracz, and Maria Buczek J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01590 • Publication Date (Web): 11 Jun 2016 Downloaded from http://pubs.acs.org on June 12, 2016

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Journal of Agricultural and Food Chemistry

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Transfer and Mass Balance of Ellagitannins, Anthocyanins, Flavan-3-ols and Flavonols During

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the Processing of Red Raspberries (Rubus ideaus L.) to Juice.

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Michał Sójka*†, Jakub Macierzyński†, Wojciech Zaweracz‡, Maria Buczek‡

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Corresponding author

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* Phone: +48 42 631 2788. E-mail: [email protected]

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†

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90-924 Łódź, Poland

Lodz University of Technology, Institute of Food Technology and Analysis, ul. Stefanowskiego 4/10,

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‡

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Poland

Research Institute of Horticulture, Experimental Station in Brzezna, Brzezna 1, 33-386 Podegrodzie,

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ACS Paragon Plus Environment


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Abstract

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The putative health benefits of raspberries and raspberry-based products are potentially attributable to

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the presence of polyphenolic compounds, such as ellagitannins, anthocyanins, flavanols, and

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flavonols. Their content in the products of raspberry processing into juice may be affected by the fruit

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cultivar, technological process parameters, and the properties of the polyphenolics themselves. The

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objective of the study was to investigate the composition and quantity of the above polyphenolics in

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raspberries and the products of their processing, that is, juice and press cake (including its seed and

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seedless fractions). The study also examined the relationship between the molecular mass of

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ellagitannins and their transfer to juice. The average percentage contributions of ellagitannins,

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anthocyanins, flavanols, and flavonols to total polyphenolics in the fruits were 64.2%, 17.1%, 16.9%,

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and 1.8% respectively. Analysis of raspberry products showed that the dominant compounds in juice

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were anthocyanins, with 65.1% contribution to total polyphenolics while in raspberry press cake they

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were tannins 98.0% (mainly ellagitannin including lambertianin C and sanguiin H-6). As shown by

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our mass balance calculations, on average 68.1% of ellagitannins and 87.7% of flavanols were

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retained in the press cake, and especially in its seedless fraction. In addition, a significant negative

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correlation was found between the molecular mass of ellagitannins and their transfer to juice. An

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increase in molecular mass from 1568 Da to 2805 Da resulted in more than a 10-fold decrease in

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ellagitannin transfer.

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Keywords: raspberry, press-cake, phenolics, ellagitannins, lambertianin C, sanguiin H-6

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Introduction

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Due to their high content of bioactive compounds and desirable sensory qualities, there has been

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growing interest in raspberry fruits and raspberry-based products, both on the part of consumers and

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scientists. This has also been reflected in the data on crops, which show an upward trend in the

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countries which are the main producers of raspberries, such as Russia, Serbia, Poland, and the USA.1

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Among the bioactive substances in raspberries and raspberry-based products, polyphenolics are a

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major group of compounds offering potential health benefits.2,3 Of particular importance are

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ellagitannins, which are the predominant raspberry polyphenolics, as well as anthocyanins and

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flavanols.4,5 Raspberries vary greatly in terms of anthocyanin composition. According to the studies by

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Ancos et al.6 and Krüger et al.7, the major anthocyanins in the studied cultivars of red raspberries are

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cyanidin 3-O-sophoroside, cyanidin 3-O-glucoside, and cyanidin 3-O-glucosyl-rutinoside. Depending

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on the cultivar and growing conditions, anthocyanin content ranges from 29-116 mg/100 g fresh

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weight (FW).8,9 Gasperotti et al.5 reported that the predominant ellagitannins in raspberries are

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sanguiin H-6, 1, and lambertianin C, 2 (Figure 1), which on average account for 81% of the total.

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Similarly to anthocyanins, total ellagitannin content depends on many factors and ranges from 90 to

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164 mg/100 g. Flavanols, including proanthocyanidins, constitute another major group of raspberry

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polyphenolics. According to Gu et al.10, raspberry proanthocyanidins are B-type polymers with a

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relatively low degree of polymerization (DP 2.7). They are mostly built of catechin, epicatechin, and,

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to a smaller extent, epiafzelechin. The content of these compounds in raspberries amounts to 79

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mg/100 g FW.11 In contrast, raspberries are not a rich source of flavonols. As reported by Wang et

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al.12, their concentration in ripe red raspberries is 3.5 mg/100 g FW, with the main compounds being

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quercetin glycosides.

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The health benefits of the above-mentioned polyphenolics have not been sufficiently

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researched to date. Selected studies on laboratory animals show that raspberry polyphenolics,

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including ellagitannins, may prevent hepatic lesions as well as arthritis.2,13 A study by Seeram et al.14

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showed that extracts from fruits such as raspberries may inhibit the proliferation of human carcinoma



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cells and stimulate their apoptosis in vitro. The ellagitannins and other polyphenolics present in berries

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also exhibit some antimicrobial properties. According to Puupponen-Pimia et al.15 extracts from these

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fruits have a strong bactericidal effect on some Gram-negative bacteria. However, it should be noted

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that in vivo the health effects of polyphenolics result from the activity of their metabolites rather than

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the native molecules. In the case of ellagitannins, the main metabolites are the products of their

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decomposition, that is, ellagic acid and urolithin.16

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Due to their short shelf life, raspberries are usually preserved by deep freezing or processed

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into juice, jam, or syrup.17 The processing of raspberries into juice may significantly affect the

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qualitative and quantitative composition of polyphenolics in the products. This may be caused by

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process conditions, resulting in polyphenolic degradation or transformation as well as by the

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morphological features of the fruits and the properties of the various compounds. Additionally, those

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polyphenolics which occur predominantly in the seeds and skins are largely retained in the press

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cake.18 The influence of the production process on the transfer of raspberry ellagitannins, including

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lambertianin C, 2, and sanguiin H-6, 1 (Figure 1) has not been fully elucidated. In the literature, there

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is scarce information about ellagitannin transformation in raspberries, in contrast to blackberries.19 The

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study by Hager et al.20 showed that up to 43% of ellagitannins are transferred to juice, while the

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remaining part is retained in the press cake and undergoes some degree of degradation. Moreover,

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Gancel et al.21 reported variation in the transfer of different ellagitannins from blackberry fruit to juice:

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compounds with higher molecular mass were more likely to be retained in the press cake. This was

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also observed for highly polymerized proanthocyanidins. In their study of cranberries, White et al.18

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reported that proanthocyanidins with a degree of polymerization greater than 10 are mostly retained in

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the press cake. Due to high polyphenolic retention and the presence of other compounds, such as

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dietary fiber and unsaturated fatty acids, raspberry press cake is increasingly often treated as a

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valuable starting material for interesting products.22,23 Of particular importance are seeds, which

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account for almost 80% of the press cake by weight.24 Some studies have shown that phenolics

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compound found in raspberry seed may confer considerable health benefits.25,26 Also the seedless

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fraction of the press cake, consisting of flesh remains and skins, may be valuable, but that issue has

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not been addressed in the literature.


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The objective of this work was to determine the polyphenolic composition of raspberry fruits

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and to investigate the distribution (transfer and retention) of selected polyphenolics in the products of

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raspberry processing, such as juice and press cake, including the seed and seedless fractions. The study

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also examined the relationship between the molecular weight of ellagitannins and their transfer to

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juice.

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Materials and methods

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Plant Material. Raspberry fruits of the cultivars 'Laszka', 'Benefis', 'Polka', and 'Polana' from the

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harvest seasons 2012 and 2013 were supplied by the Experimental Station of the Research Institute of

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Horticulture in Brzeźna. In terms of cultivation and harvest conditions, the above cultivars belong to

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two groups: summer raspberries ('Laszka' and 'Benefis'), which bear fruit only once a year on

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floricanes, and everbearing fall raspberries ('Polka' and 'Polana'), which produce fruit twice a year, the

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first time on floricanes and the other time on primocanes, in the fall.27

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The fruits were harvested when they were fully ripe i.e. in the harvest maturity. Fruits during the

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harvest were well, evenly colored, and were easily separable from the core (receptacle). 'Laszka' and

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'Benefis' were harvested from floricanes at the beginning of July, while 'Polka' and 'Polana' from

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primocanes at the beginning of September. Following harvest, the fruits were immediately frozen,

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packed in polyethylene bags, and stored at -18 °C until processing. The fruits were processed into

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juice no longer than 2 weeks after harvest.

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Chemicals and standards. For polyphenolic extraction or purification, all solvents or reagents were

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puriss p.a. grade or HPLC grade. For HPLC and LC-MS analysis all solvents were HPLC gradient or

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LC-MS grade. Acetone and ascorbic acid were purchased form POCH (Gliwice, Poland). HPLC grade

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methanol and phosphoric acid (purity 85%) were purchased from J.T. Baker (Deventer, Netherlands).

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Acetonitrile, LC-MS grade methanol, formic acid, phloroglucinol, and sodium acetate were purchased

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from Sigma-Aldrich Chemie (Steinheim, Germany). Glacial acetic acid was purchased from Chempur



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(Piekary Śląskie, Poland). Ethanol (purity 96.3% (v/v)) was purchased form Nord-Clas (Łódź,

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Poland). Ultrapure water for extraction and purification was obtained from an Elix3 System

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(Millipore, Vienna, Austria) was used. Ultrapure water for HPLC analysis was obtained from a

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Hydrolab HLP5 System (Straszyn, Poland).

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Standards for the quantitation of ellagitannins were produced in our laboratory according to the

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procedure described below. Ellagic acid, anthocyanin, and flavonol standards, i.e. cyanidin 3-O-

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glucoside, cyanidin 3-O-rutinoside, quercetin 3-O-glucoside, quercetin 3-O-rutinoside, quercetin 3-O-

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galactoside,

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rutinoside, and kaempferol were purchased from Extrasynthese (Genay, France). (-)-Epicatechin and

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(+)-catechin were purchased from Sigma-Aldrich Chemie (Steinheim, Germany).

quercetin 3-O-glucuronide,

quercetin,

kaempferol 3-O-glucoside,

kaempferol 3-O-

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Food processing. Prior to processing, 1 kg of fruit was defrosted and ground using a model 886.9

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food grinder (Zelmer, Rzeszów, Poland). The pulp was heated to 50 °C and treated with the enzyme

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preparation Rohapect 10L (AB Enzymes, Darmstadt, Germany) at a dose of 0.2 mL per 1 kg of pulp.

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Enzymatic treatment was conducted at 50 °C for 1 h with stirring every 10 min. Subsequently, juice

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was pressed using a laboratory hand screw press (home-built, Lodz University of Technology,

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Poland). To simulate industrial conditions, the resulting press cake was additionally extracted with

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water at a ratio of 2 parts of press cake to 1 part of water by weight. Extraction was conducted at 50 °C

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for 15 min, after which a second pressing was performed to obtain secondary juice and press cake. The

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juices from the first and second pressings were combined, and the press cake was dried at 70 °C for 4–

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5 h in a KC-100/200 1.6 kW drying oven (WAMiE, Warsaw, Poland). The juice (unclarified,

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unfiltered) was immediately analyzed in terms of polyphenolic content and soluble solids (°Brix)

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using a PR-32α refractometer (Atago, Tokyo, Japan). The dry weight of the fruits and dried press cake

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was determined in accordance with AOAC 920.151, by the gravimetric method.

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The dried press cake was fractionated using a Analysette 3 multi-deck sieve shaker (Fritsch, Idar-

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Oberstein, Germany) with mesh sizes 0.2 mm, 0.4 mm, 0.6 mm, 0.8 mm, 1 mm, 1.25 mm, 1.6 mm,

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and 2 mm. Sieving was conducted at a vibration amplitude of 1.5 for 5 min. The resulting fractions

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were weighed. The seed fraction was obtained by combining fractions with particles larger than 1 mm,


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while the seedless fraction was obtained by combining fractions with particles smaller than 1 mm. The

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yield of juice and press cake, including the seed and seedless fractions, is presented in Table 1.

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Polyphenolic extraction and preparation of HPLC samples. Prior to extraction, samples of fruits

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(c.a. 50 g) and press cake (c.a. 30 g) were ground using liquid nitrogen in an analytical mill (IKA,

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Staufen, Germany). Ellagitannins were extracted from the fruits and press cake, according procedure

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described by Klimczak and Król,28 using 70% acetone containing 1% acetic acid. First, 0.5 g of

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ground press cake or 2 g of ground fruit was placed in a 7 mL test tube and 4 mL of a solvent was

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added. Then, the tube content was mixed using a vortex and sonicated for 5 min in an ultrasonic bath

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IS-4 (Intersonic, Olsztyn, Poland). Following, sonication the solution was left in the dark for 15 min

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for extraction. The solution was centrifuged at 10,000 × g in an MPW-260R centrifuge (Med.

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Instruments, Warsaw, Poland), and poured into a flask. The centrifugation residue was subjected to

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additional double extraction using 3 mL of the solvent, in accordance with the above procedure. The

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obtained extracts were combined in a 10 mL volumetric flask. Each sample was extracted in triplicate.

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The extracts thus obtained were diluted 1:1 (v/v) with mobile phase A, centrifuged at 12,000 × g, and

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transferred into vials.

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Anthocyanins and flavonols were extracted from the fruits and press cake, in the same way as

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ellagitannins, except that the solution used was methanol:water:formic acid (50:48:2, v/v/v).

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Extraction procedure of anthocyanins and flavonols was based on the method described by

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Kapsakalidis et al..29

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Prior to ellagitannin, anthocyanin, and flavonol analysis, the samples were diluted 1:1 (v/v) with

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mobile phase A and centrifuged at 12,000 × g.

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Extraction and identification of sanguiin H-6 and lambertianin C standards. Sanguiin H-6, 1, and

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lambertianin C, 2 (Figure 1) were isolated from the press cake left over from juice pressing. Fresh

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press cake (200 g) was placed in a 2 L polyethylene bottle and 1 L of 60% acetone was added. In the

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first step, extraction was conducted at ambient temperature for 8 h. The extraction process was

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augmented using orbital shaker set to 150 rpm. Subsequently, raw extract was filtered through cotton

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filter cloth in order to separate solids, while in the second step, the post-extraction residue was



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extracted according to the same procedure. The extracts obtained from both steps were combined and

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filtered using a 3.6 mm thick Hobrafilt S40N cellulose filter with 5 µm nominal retention (Hobra-

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Školnik S.R.O., Broumov, Czech Republic). Acetone was removed from the raw extract using a rotary

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vacuum evaporator at 60 °C and under decreasing pressure of 450–72 mbar. The extract without

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acetone was again filtered through a Hobrafilt S40N filter, and then purified on a 90 × 1.6 cm column

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with Amberlite XAD 1600N resin (DOW, Midland, MI). The extract was loaded onto the column at a

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rate of approx. 15 mL/min. Subsequently, the column was eluted at a rate of approx. 10 mL/min using

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a series of aqueous ethanol solutions at an ethanol concentration of 10%, 20%, 30%, 40%, 50%, and

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60%. The volume of solutions used in elution, at each ethanol concentration, was equal to the column

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volume. The eluate with the highest content of lambertianin C and sanguiin H-6 was collected using a

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40% ethanol solution. Subsequently, ethanol was removed and the eluate was concentrated up to

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approx. 5°Brix using a rotary vacuum evaporator at 60 °C and under decreasing pressure of 135–72

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mbar. The concentrated extract was then freeze-dried at -32 °C for 48 h. The obtained dry preparation

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of raspberry ellagitannins, red in color, was dissolved in water and individual ellagitannins were

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isolated using preparative HPLC. The isolation of lambertianin C and sanguiin H-6 was carried out

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using a chromatograph from Knauer (Berlin, Germany) composed of two gradient pumps (Knauer K-

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501), a 250 mm x 21.2 mm i.d., 10 µm, AXIA-packed Luna C18(2) 100Å column, with a 15 mm x

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21.2 mm i.d. guard column of the same material (Phenomenex, Torrance, CA), a UV–Vis detector, a

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fraction collector (Teledyne ISCO, Lincoln), and Eurochrom 2000 chromatographic software. Two

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eluents were used for separation: eluent A: 0.1% formic acid in water, eluent B: 75% methanol. The

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flow rate was 15 mL/min. The following gradient was used: 0–5 min, 10% B; 5–30 min, 10–25% B;

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30–50 min, 25–35% B; 50–65 min, 35–40% B; 65–70 min, 40–10% B; and 70–75 min, 10% B. The

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injection volume was 500 µL. Detection wavelength was set to 260 nm. Lambertianin C and sanguiin

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H-6 peaks were collected from 20 separations and combined; methanol was removed from the

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obtained solutions using a rotary vacuum evaporator at 60 °C under a pressure of 100 mbar, and the

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preparations were freeze-dried. The molecular masses of lambertianin C and sanguiin H-6 were

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verified by a Q Exactive Orbitrap mass detector (Thermo Fisher Scientific, Waltham, MA). Aqueous

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solutions of these substances were directly infused into a heated electrospray ionization (H-ESI)


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source via a syringe pump with a flow of 20 µL/min. Analyses were carried out in the negative ion

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mode. The source parameters were as follows: vaporizer temperature 50 °C, ion spray voltage 3 kV,

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capillary temperature 300 °C; sheath gas and auxiliary gas flow rates 5 and 0 units, respectively. To

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generate MS/MS data, the precursor ions were fragmented in a high energy collision induced

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dissociation (HDC) cell with energy collision optimized to obtain an intensity of the precursor ion

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close to 10% of the relative scale of the spectrum. The results of identification of sanguiin H-6 and

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lambertianin C were identical to those presented in Table 2. The obtained standards of lambertianin C

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and sanguiin H-6 were characterized by HPLC purity of more than 90% (210 nm, using HPLC

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conditions as described in the section Quantitation of ellagitannins).

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Identification of ellagitannins. A Dionex Ultimate 3000 high performance liquid chromatograph

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(HPLC) coupled with a DAD and Q Exactive Orbitrap mass spectrometer (MS) (Thermo Fisher

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Scientific, Waltham, MA) was used for the identification of ellagitannins. The solvents used for

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separations were as follows: solvent A: 1% (v/v) formic acid in water and solvent B: 80:20 (v/v)

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acetonitrile:water solution. The following gradient was used: 0–6.5 min, 5% (v/v) B; 6.5–12.5 min, 5–

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15% (v/v) B; 12.5–44 min, 15–45% (v/v) B; 44–45 min, 45–75% (v/v) B; 45–50 min, 75% (v/v) B;

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50–52 min, 75–5% (v/v) B; 52–65 min, 5% (v/v) B. The column used was a 250 mm x 4.6 mm i.d., 5

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µm, Luna C18(2) 100Å, with a 4 mm x 3 mm i.d. guard column of the same material (Phenomenex,

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Torrance, CA). The column temperature was set to 35 °C, the flow rate was 1 mL/min, and the

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injection volume was 20 µL. Chromatographic data were collected using Xcalibur software (Thermo).

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The MS system coupled to the HPLC was an Orbitrap mass spectrometer equipped with an H-ESI

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probe used in the negative mode. The source parameters were as follows: vaporizer temperature 500

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°C, ion spray voltage 4 kV, capillary temperature 400 °C; sheath gas and auxiliary gas flow rate 75

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and 20 units, respectively. The detector was operated in either the full MS or full MS/dd-MS2 scan

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modes. In the full MS mode, the scan rage of m/z 200–2000 was used. To generate MS2 data, the full

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MS/dd-MS2 scan mode was used. In this mode, the selected precursor ions entered into an HDC

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collision cell, where they were fragmented with normalized collision energy (NCE) to obtain product

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ion spectra (MS2). In our experiments, the NCE used to generate MS2 spectra was set to 20. Tuning



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and optimization were performed using direct injection of a raspberry ellagitannin preparation diluted

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in an 80:20 (v/v) mixture of mobile phases A and B at a flow of 0.25 mL/min. The results of

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ellagitannin identification are given in Table 2.

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Quantitation of ellagitannins. The content of ellagitannins was determined using a Smartline

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chromatograph (Knauer, Berlin, Germany), composed of a degasser (Manager 5000), two pumps

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(P1000), autosampler (3950), thermostat, and PDA detector (2800). Ellagitannins were separated on a

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250 mm x 4.6 mm i.d., 5 µm, Gemini C18 110Å column (Phenomenex, Torrance, CA) by gradient

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elution with solvent A: 0.05% (v/v) phosphoric acid:water and solvent B: 83:17 (v/v)

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acetonitrile:water with 0.05% phosphoric acid. The column temperature was set to 35 °C, the flow rate

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was 1.25 mL/min, and the gradient program was as follows: 0–5 min, 5% (v/v) B; 5–10 min, 5–15%

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(v/v) B; 10–35 min, 15–40% (v/v) B; 35–40 min, 40–73% (v/v) B; 40–44 min, 73% (v/v) B; 44–46

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min, 73–5% (v/v) B; 46–54 min, 5% (v/v) B. The injection volume was 20 µL. Data were collected

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using ClarityChrom v. 3.0.5.505 software (Knauer, Berlin, Germany). Ellagitannins were detected at

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250 nm, and standard curves for lambertianin C, sanguiin H-6, and ellagic acid, in the ranges of 0.5 –

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300 mg/L, 0.5 – 225 mg/L, 0.5 – 20 mg/L respectively, were used for quantitation. The sanguiin H-6

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curve was used for the quantitation of sanguiin H-10 isomers, 4 (Figure 2). The lambertianin C curve

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was used for the quantitation of lambertianin C derivatives, 5 (Figure 3) and lambertianin D, 3 (Figure

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1). The ellagic acid curve was used for ellagic acid, 6, and its conjugates 7, 8, 9 (Figure 4).

271 272

Identification of anthocyanins. The equipment used for the identification of anthocyanins was the

273

same as that described for identification of ellagitannins. The solvents used for separations were as

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follows: solvent A: 1% (v/v) formic acid in water and solvent B: 1% (v/v) formic acid in methanol.

275

The following gradient was used: 0–30 min, 20–65% (v/v) B; 30–31 min, 65–100% (v/v) B; 31–33

276

min, 100% (v/v) B; 33–34 min, 100–20% (v/v) B; 34–45 min, 20% (v/v) B. A column 150 mm x 4.6

277

mm i.d., 3 µm, Gemini–NX C18 110Å was used with a Gemini-NX C18, 4 mm x 3 mm i.d. pre-

278

column (Phenomenex, Torrance, CA). The column temperature was set to 35 °C, the flow rate was 0.5

279

mL/min, and the injection volume was 10 µL. The source parameters were as follows: vaporizer


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temperature 400 °C, ion spray voltage 3.8 kV, capillary temperature 380 °C; sheath gas and auxiliary

281

gas flow rate 60 and 20 units, respectively. The detector was operated in either the full MS or full

282

MS/dd-MS2 scan modes. In the full MS mode, the scan rage of m/z 250–1000 was used. To generate

283

MS2 data, the NCE parameter was set to 30. Tuning and optimization were performed using direct

284

injection of cyanidin 3-O-glucoside standard diluted in a 75:25 (v/v) mixture of mobile phases A and

285

B at a flow of 0.25 mL/min. The results of anthocyanin identification are given in Table 2.

286 287

Quantitation of anthocyanins and total flavonols. The quantitation of anthocyanins and flavonols

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was performed according to the methodology described in another work by Sójka et al..37 The same

289

separation conditions and apparatus were used for determination. The standard curves were plotted on

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the basis of external standards of cyanidin 3-O-glucoside (in the range of 1.5 – 156 mg/L) and

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quercetin 3-O-rutinoside (in the range of 1.5 – 50 mg/L). The cyanidin 3-O-glucoside curve was used

292

for the quantitation of each anthocyanin. The quercetin 3-O-rutinoside curve was used to calculate the

293

total flavonols.

294 295

HPLC analysis of flavanols (total proanthocyanidins and catechins). The quantitation of flavanols

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(a sum of monomeric and polymeric flavan-3-ols) was carried out by acid-catalyzed degradation of

297

polymeric proanthocyanidins in excess of phloroglucinol, as described in a previous work by Sójka et

298

al..37 The same separation conditions and apparatus were used for determination. Prior to analysis,

299

fruit, juice, and press cake samples were subjected to freeze-drying at -36 °C for 48 h.

300 301

Statistics. All the results were statistically examined by one-way analysis of variance and the post-hoc

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Duncan test with a statistical significance of p ≤ 0.05. To illustrate differences in the content of the

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studied components between the fruit cultivars, k-means clustering was performed. Statistical analysis

304

was carried out using Statistica Version 10 software (StatSoft, Tulsa, OK).

305 306

Results and Discussion

307



308

Fruit polyphenolics. Polyphenolic content in the studied fruits largely depended on the cultivar and

309

ranged from 1511 to 2363 mg/100 DW (Table 3). Ellagitannins were the dominant polyphenolic

310

group, with their percentage contribution to total polyphenolics (where 100% is the sum of all

311

measured polyphenols) ranging from 53.5% to 75.9% (on average 64.2%); the average contributions

312

of the other compounds, that is, anthocyanins, flavanols, and flavonols, were 17.1%, 16.9%, and 1.8%,

313

respectively. The cultivars with low ellagitannin content ('Polana' and 'Polka') were characterized by a

314

higher concentration of anthocyanins and flavonols.

315

In order to facilitate polyphenolic content comparison with the results reported by other authors, who

316

presented their data relative to fresh weight (FW), without specifying the dry weight of the fruit, we

317

adopted an average value of 14.2% based on the water content of raspberries given by USDA in the

318

National Nutrient Database for Standard Reference.38 The data converted using the above formula are

319

marked with an asterisk (DW* - dry weight).

320

A comparison of the ellagitannin content of raspberries presented herein with previous studies is not

321

straightforward as most data are based on determination of ellagic acid formed by hydrolysis of native

322

ellagitannins. There are few publications in which ellagitannins were determined directly. In the

323

present study, the directly quantitated ellagitannin content of fruit ranged from 853 to 1793 mg/100 g

324

DW. These results are similar to those reported by Gasperotti et al.5 and Vrhovsek et al.39, that is, 581–

325

956 mg/100 g DW* for the cultivars 'Polka' and 'Polana'. A similar ellagitannin content of raspberries

326

(1831–2295 mg/100 g DW*) was reported by Koponen et al..40

327

The ellagitannin profile of raspberry fruit mostly consisted of two compounds, lambertianin C and

328

sanguiin H-6, which accounted for almost 90% of total ellagitannins, with lambertianin C content

329

being on average 50 ± 3% for all cultivars. The remaining 10% comprised derivatives of the above-

330

mentioned ellagitannins, lambertianin D, sanguiin H-10 isomers, as well as ellagic acid and its

331

conjugates. Among these ellagitannins, of particular note is lambertianin D (casuarictin tetramer), 3

332

(Figure 1), which was present only in the summer cultivars ('Laszka' and 'Benefis') with an average

333

concentration of 64 mg/100 g DW. Similarly, Gasperotti et al.5 reported that lambertianin C and

334

sanguiin H-6, present in the 'Polka' and 'Polana' cultivars, accounted for 64% of total ellagitannins.

335

The slightly lower content of these compounds may be attributable to the higher concentration of


Page 12 of 43

Page 13 of 43


336

ellagic acid and its conjugates in that study. According to Gasperotti et al.5, along with the dominant

337

ellagitannins, raspberries also contained sanguiin H-10 isomers, sanguiin H-6 minus one gallic moiety,

338

and sanguiin H-6 plus one gallic acid moiety. Our study partially corroborated those results, and in

339

particular the presence of sanguiin H-10 isomers, 4 (Figure 2), and lambertianin C minus one ellagic

340

moiety, 5 (Figure 3). A chromatogram presenting the ellagitannin profile of the studied cultivars is

341

given in Figure 5. The content of ellagic acid and its conjugates was in the range of 13–26 mg/100 g

342

DW, with the predominant compound being ellagic acid pentose conjugate, 7 (Figure 4). In the study

343

by Gasperotti et al.5, the content of those compounds in various raspberry cultivars was greater by an

344

order of magnitude and amounted to 114–254 mg/100 g DW*.

345

Statistical analysis revealed significant differences in ellagitannin content between the summer

346

cultivars ('Laszka' and 'Benefis') and the everbearing cultivars ('Polka' and 'Polana'). The summer

347

cultivars contained almost twice as much ellagitannins than the everbearing ones.

348

Anthocyanin content depended on the cultivar and ranged from 201 to 405 mg/100 g DW (Table 3).

349

'Polana' was the richest in anthocyanins, with their concentration being almost double that of 'Laszka'.

350

Among the studied anthocyanins, the predominant compounds were cyanidin 3-O-sophoroside and

351

cyanidin 3-O-glucoside (59.2% and 27.4%, respectively). 'Benefis' differed slightly from the other

352

cultivars in terms of anthocyanin profile: the relative contribution of cyanidin 3-O-sophoroside was

353

only 41.1%, while that of cyanidin 3-O-rutinoside and cyanidin 3-O-glucosyl-rutinoside was high

354

(14.3% and 15.9%, respectively). Anthocyanins have been well characterized in the literature.

355

Numerous studies indicate that the content of those compounds and their relative proportions are

356

mostly cultivar-dependent. The study by Ancos et al.8, involving the cultivars grown in Spain, showed

357

variation in anthocyanins both in terms of their content and profile. The early cultivars were

358

characterized by a lower content of those compounds (252 mg/100 g DW), in contrast to the fall-

359

bearing ones (712 mg/100 g DW). The anthocyanin profile was cultivar-dependent, with the dominant

360

compounds being cyanidin 3-O-sophoroside and cyanidin 3-O-glucoside, similarly to our findings.

361

Chen et al.35, who studied 15 cultivars of various species of raspberries suggested that these fruits

362

could be classified into three groups in terms of their anthocyanin profiles. The dark-red cultivars

363

(group one) and black raspberries (group two) are characterized by high anthocyanin content (4633



364

mg/100 g DW*), with the predominant compounds being cyanidin 3-O-glucoside and cyanidin 3-O-

365

rutinoside. In contrast, anthocyanin content in red and pink cultivars (group three; Rubus ideaus L.)

366

was only 704 mg/100 g DW*, with high proportions of cyanidin 3-O-sophoroside and cyanidin 3-O-

367

glucosyl-rutinoside. The cultivars investigated in the present paper were similar to the third group.

368

Statistical analysis of anthocyanin content showed that among the studied cultivars, 'Polana'

369

raspberries from seasons 2012 and 2013 and 'Polka' raspberries from season 2012 exhibited the

370

highest values and were significantly different from the others. There is also some variation in total

371

anthocyanins between the seasons, probably mostly due to climatic conditions.9 The greatest

372

difference in this respect was observed for 'Polka' (more than 100 mg/100 g DW).

373

In the studied cultivars, the content of flavanols (proanthocyanidins and monomeric catechins

374

combined) was similar to that of anthocyanins and ranged from 271 to 363 mg/100 g DW as

375

determined by HPLC based on phloroglucinolysis products. 'Benefis' from season 2012 and 'Polka'

376

from season 2013 were the richest in these compounds, as confirmed by statistical analysis. According

377

to Gu et al.10, raspberry flavanols are mostly B-type proanthocyanidins, predominantly (85%)

378

consisting of catechin and epicatechin, as well as a small amount of epiafzelechin. The average degree

379

of proanthocyanidin polymerization ranges from 1 to 10. In the study by Rzeppa et al.41, the average

380

flavanol content in raspberries was 140 mg/100 g DW, with the prevalent compounds being

381

proanthocyanidin B4 and (-)-epicatechin. Hellström et al.11 reported a slightly higher proanthocyanidin

382

content in raspberries (562 mg/100 g DW). Hosseinian et al.42 found that raspberries were much richer

383

in proanthocyanidins, in some cases containing more than 4400 mg/100 g DW. On the other hand, the

384

raspberry fruits investigated by Kähkönen et al.43 contained as little as 4 mg of flavanols/100 g DW.

385

The above discrepancies may be attributable to differences in analytical techniques and the standards

386

used for calculation, which makes comparison very difficult.

387

In this study, total flavonols were the last of the analyzed polyphenolic groups. In this paper, total

388

flavonols comprised quercetin and kaempferol glycosides. Their content ranged from 16.9 to 43.8

389

mg/100 g DW, which is much less than in other Rosaceae fruits, such as blackberries (approx. 220

390

mg/100 g DW* assuming 11.85% dry matter) and chokeberries (approx. 110 mg/100 g DW* assuming

391

25% dry matter).37 Our study is consistent with the study of Kähkönen et al.43, which reported flavonol


Page 14 of 43

Page 15 of 43


392

content in raspberries at 15–30 mg/100 g DW. An in-depth investigation by Mikulic-Petkovsek et al.44

393

showed that in edible raspberries, flavonols consist mostly of quercetin 3-O-glucuronide and quercetin

394

3-O-arabinoside, smaller amounts of other quercetin glycosides, as well as kaempferol and

395

isorhamnetin glycosides. Among the studied cultivars, 'Polana' and 'Benefis' were the richest in

396

flavonols, with an average content of 40 mg/100 g DW, which is almost twice as much as in the

397

cultivar 'Laszka' (22 mg/100 g DW). This difference is statistically significant.

398

Differences in the polyphenolic composition of raspberry fruit between the summer cultivars ('Laszka'

399

and 'Benefis') and the everbearing ones ('Polka' and 'Polana') were confirmed by statistical methods

400

using k-means cluster analysis on standardized data. Figure 6 shows the results of this analysis with

401

two clusters, which are differentiated based on distances between points representing the studied

402

substances (along the Y axis). The long distances between the points confirm considerable variation in

403

respect of the content of ellagitannins and selected anthocyanins. The compounds exhibiting the least

404

differences between the clusters were cyanidin 3-O-glucosyl-rutinoside, cyanidin 3-O-rutinoside, as

405

well as total flavonols and flavanols.

406 407

Juice polyphenolics. The polyphenolic content of juice is presented per dry weight (DW) in Table 4

408

and ranged from 562 to 927 mg/100 g DW. The average percentage contribution of ellagitannins,

409

anthocyanins, flavonols, and flavonols to total polyphenolics amounted to 39%, 50%, 9%, and 2%

410

respectively. In contrast to fruit, the predominant polyphenolics in the juice obtained from the cultivars

411

'Polka' and 'Polana' were anthocyanins, accounting for 53.2–65.1% of total polyphenolics. Also in the

412

case of 'Laszka' and 'Benefis', the share of anthocyanins was high (38.9–41.5%). The relative

413

proportions of the various polyphenolic groups in juice were primarily influenced by their content in

414

the starting material, and so they depended on the processed fruit cultivar. For instance, the juice

415

obtained from the cultivars 'Laszka' and 'Benefis' contained the highest proportion of ellagitannins,

416

while that obtained from 'Polka' and 'Polana' had the highest share of anthocyanins. The differences

417

between the studied products were statistically significant. Due to the fact that in the juice one of large

418

group of polyphenolics were anthocyanins, they had a significant effect on its total polyphenolic

419

content. As a result, among the studied cultivars, juices from 'Polka' and 'Polana' were found to have



420

the highest polyphenolic content despite the fact that the fruits from which they were made contained

421

much less polyphenols than 'Benefis' and 'Laszka'. These data clearly show that in the production

422

process, the transfer of the various compounds to the juice is of critical importance to its polyphenolic

423

profile.

424

The ellagitannin profile of juices was mostly influenced by the presence of sanguiin H-6, whose

425

percentage contribution to total ellagitannins was on average 66.1%. In contrast, the average

426

contribution of lambertianin C was only 14.2%. This indicates the poor transfer of the latter compound

427

from the fruit to the juice during the production process. Other compounds with a significant presence

428

in the juice included sanguiin H-10 isomers, which on average accounted for 11.3% of total

429

ellagitannins. The content of ellagic acid and its conjugates ranged from 10.9 to 20.8 mg/100 g DW,

430

with the predominant forms being free ellagic acid and ellagic acid pentose conjugate. In the study by

431

Rommel and Wrostland 45, the combined content of ellagitannins and ellagic acid in raspberry juice

432

was from 20 to 80 mg/100 g DW, depending on the cultivar, assuming 10% soluble solids. As reported

433

by Bermúdez-Soto and Tomás-Barberán,46 the content of all ellagitannin derivatives in concentrated

434

juice was approx. 600 mg/L (no data on dry weight). In terms of the content of individual

435

ellagitannins, there is more available information in the literature on blackberry fruit. According to

436

Gancel et al.21, juice obtained by dilution of blackberry purée contained approx. 1945 mg/100 g DW of

437

lambertianin C and sanguiin H-6 combined, as well as 106 mg/100 g DW of ellagic acid. However, the

438

production process of that juice differed significantly from that of a typical fruit juice. In the study of

439

Hager et al.47, unclarified and unpasteurized blackberry juice (assuming 10% soluble solids) contained

440

109.1 mg/100 g DW of ellagitannins, with the predominant compounds being lambertianin C and

441

sanguiin H-6.

442

In the present study, the content of anthocyanins in raspberry juice was 227 to 603 mg/100 g DW,

443

depending on the cultivar and their relative proportion in total polyphenolics was higher than in the

444

fruits. In this respect, 'Polana' was remarkable for the high share of anthocyanins in total polyphenolics

445

(65.1%). The differences in anthocyanin content between cultivars were statistically significant.

446

Similarly, as in the case of fruits, the predominant anthocyanins were cyanidin 3-O-sophoroside and

447

cyanidin 3-O-glucoside, which accounted for 65.1% and 24.5% of total anthocyanin content. In


Page 16 of 43

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448

quantitative terms, a similar anthocyanin content (369–470 mg/100 g DW assuming 10% soluble

449

solids) was reported by Versari et al.48, with the same predominant compounds.

450

The content of the other two polyphenolic groups, that is, flavanols and flavonols, was much lower

451

and amounted to 40–80 mg/100 g DW and 11–30 mg/100 g DW, respectively. Among the studied

452

cultivars, 'Polka' from season 2012 and 'Polana' from 2013 exhibited the highest content of these

453

polyphenolics. Differences between the cultivars were found to be statistically significant. There is

454

scarce information in the literature concerning flavanol content in raspberry juices and the data seem

455

to be contradictory. According to Viljanen et al.49, raspberry juice does not contain these compounds,

456

while Hosseinian et al.42 reports as much as 7700 mg/100 g DW (assuming 10% soluble solids).

457 458

By-product polyphenolics. In the process of fruit juice production, press cake is an essential by-

459

product. In our case, fresh press cake amounted to 9.9–12.0% of the weight of the processed

460

raspberries. Subsequently, using a multi-deck sieve shaker, dried press cake was divided into two

461

fractions (seed and seedless), accounting for 92.4–95.0% and 5.0–7.6% of total press cake weight,

462

respectively (Table 1).

463

The polyphenolic composition of the above two fractions is given in Tables 5 and 6. The average total

464

polyphenolic content in the seed and seedless fractions of the press cake was 3317 and 6895 mg/100 g

465

DW, almost 1.8 and 3.7 times higher than in the fruits. The press cake was characterized by a

466

particularly high percentage contribution of tannins (ellagitannins and proanthocyanidins) to total

467

polyphenolics (98% for both fractions). The average share of ellagitannins, flavanols, anthocyanins,

468

and flavonols in total polyphenolics was 72.9%, 25.1%, 1.4%, and 0.6% for the seed fraction, and

469

93.1%, 4.4%, 2.0%, and 0.5% for the seedless fraction, respectively. These data show that

470

ellagitannins were the predominant polyphenolics in the seedless fraction of raspberry press cake,

471

while flavanols were mostly accumulated in the seeds. The content of the various compounds in the

472

press cake depended on the fruit cultivar, with statistically significant differences.

473

The most abundant press cake ellagitannins were lambertianin C and sanguiin H-6, whose average

474

percentage contribution to total ellagitannins was 51.0% and 33.8% in the seeds and 58.8% and 29.0%

475

in the seedless fraction, respectively. In addition, in the cultivars 'Laszka' and 'Benefis', the



476

ellagitannin profile of the press cake was affected by the presence of lambertianin D, which accounted

477

for 13.5% of total ellagitannins in the seeds, but only 1.5% in the seedless fraction. Apart from the

478

above three compounds, lambertianin C isomer contributed significantly to the ellagitannin profile of

479

the seedless fraction accounting on average for 7.3% of total ellagitannins. The average content of

480

ellagic acid in the press cake was 28 mg/100 g DW and 45 mg/100 g DW in the seed and seedless

481

fractions, respectively.

482

In the literature, there are no data on the ellagitannin content of raspberry press cake. However, there

483

are some reports concerning blackberries, whose ellagitannin profile is similar to that of raspberries.20

484

According to these reports, ellagitannin content in blackberry press cake is 23.5 mg/100 g FW.

485

Assuming that the process of blackberry juice production would lead to 10% press cake and that press

486

cake would contain 40% dry matter, then total ellagitannin content in blackberry press cake would

487

amount to 587 mg/100 g DW, which is much less than the results obtained herein for raspberry press

488

cake. However, it should be noted that ellagitannin content in the processed blackberry fruit is several-

489

fold lower.

490

In terms of flavanols, the seeds were found to contain approx. 3 times as much of these compounds as

491

the seedless fraction. The average flavanol content in the seed and seedless fractions was 818 mg/100

492

g DW and 291 mg/100 g DW, respectively. This means that flavanols are mostly accumulated in the

493

seeds, which is also the case e.g., in grapes.50 The opposite was found for chokeberry press cake in our

494

previous work i.e. the seedless fraction contained more flavanols than the seeds.36

495

Due to the high water solubility of anthocyanins, a considerable part of these compounds is transferred

496

to the juice. As a result, their content in the press cake is low, amounting on average to 129 mg/100 g

497

DW in the seedless fraction and 43 mg/100 g DW in the seeds. The presence of anthocyanins in the

498

seeds is attributable to the fact that after sieving the seed fraction still contains some residue from the

499

seedless fraction in the form of clumped skins and particles which are similar in size to the seeds;

500

additionally, some minute particles of flesh may adhere to the seeds. In the press cake, the prevalent

501

anthocyanins were cyanidin 3-O-sophoroside and cyanidin 3-O-glucoside, accounting for 47.7% and

502

44.0% of the total, respectively, both in the seed and seedless fractions.


Page 18 of 43

Page 19 of 43


503

As compared to the other polyphenolic groups, the average flavonol content was low, amounting to

504

19.6 mg/100 g DW for the seeds and 35.0 mg/100 g DW for the seedless fraction.

505 506

Percentage contribution and retention of polyphenolics in raspberry products. Based on the

507

weight of processed fruits and products as well as polyphenolic content, a mass balance was drawn up

508

to illustrate the transfer of the various compounds. The balance was calculated using the formula given

509

below and expressed in terms of percentage retention (PR) of individual compounds or groups of

510

compounds in the studied product:

PR =

511

C prod ⋅ m prod C fruit ⋅ m fruit

⋅100 %

512

where:

513

C prod – content of a given compound or group of compounds in the studied product

514

m prod – weight of the studied product

515

C fruit – content of a given compound or group of compounds in the processed fruits

516

m fruit – weight of the processed fruits

517 518

Figure 7 shows the average retention of the various groups of polyphenolics in the juice and press

519

cake, including its seed and seedless fractions. As can be seen, in the course of fruit processing

520

ellagitannins were mostly retained in the seeds (68.0%). Approx. 11.8% of ellagitannins were

521

transferred to the juice, 10.5% were retained in the seedless fraction of the press cake, while the

522

remainder (approx. 10%) were degraded (marked as unaccounted losses). Similar ellagitannin

523

retention in the press cake (67%) was found by Hager et al.20 in the process of blackberry juice

524

production. The press cake also exhibited high retention of flavanols in the seeds (87%). Many studies

525

18,19,37

526

the seeds and insoluble parts of skins are a rich source of these compounds. In the present study, the

527

behavior of anthocyanins and flavonols differed from that of tannins. Their transfer to juice was

528

significantly higher, at 67.8% and 31.1%, respectively. The retention of anthocyanins in the press cake

529

was very low (5.4% for the seeds and 0.9% for the seedless fraction). In the case of flavonols, approx.

have reported that high retention of flavanols, especially proanthocyanidins, in the press cake as



530

25% were retained in the press cake. Unaccounted losses occurred both in the case anthocyanins and

531

flavonols, amounting to 25.7% and 41.9%, respectively. According to Hager et al.47, this effect may be

532

linked to the degradation of these compounds in the course of fruit processing or press cake drying.

533

This has also been corroborated by Gancel et al.21, who reported that the concentration of both

534

anthocyanins and ellagitannins may decrease by over 50% as a result of high process temperatures.

535

Figure 8 presents the distribution of individual ellagitannins between the juice and the seed and

536

seedless fractions of the press cake in the course of raspberry juice production. The obtained results

537

indicate a significant correlation between the molecular mass of ellagitannins and their transfer to

538

juice. Low molecular mass compounds (1568 Da), such as sanguiin H-10 isomers, were transferred to

539

juice at a rate of over 40%. Sanguiin H-6 (1870 Da), whose mass is greater than that of sanguiin H-10

540

isomers due to an additional ellagic acid moiety (302 Da), exhibited 20.6% transfer to juice. The

541

compound with a molecular mass of 2503 Da, identified as lambertianin C minus one ellagic acid

542

moiety, revealed 6.6% transfer, while the transfer of lambertianin C, and its isomers with a mass of

543

2805 Da amounted to 3.2%. The above-mentioned ellagitannins were largely retained in the press

544

cake, and especially in the seedless fraction. Similarly, Hager et al.20 reported that 22% and 35% of

545

sanguiin H-6 and lambertianin C were transferred to juice, while the remaining ellagitannins were

546

mostly retained in the seeds. Unusual results were observed for lambertianin D, with a mass of 3740

547

Da, which exhibited extremely low transfer to juice (1.6%) and retention of over 200% in the seeds.

548

The incorrect mass balance shows that this compound was probably not sufficiently extracted from the

549

fruits.

550

The transfer of conjugates to juice was varied (32.0 – 45.5%). It was the highest for methyl ellagic

551

acid pentose conjugate, for other conjugates was above 32.0%. However, it should be noted that

552

ellagic acid acetylpentose conjugate, was not retained in the press cake, which may be due to the

553

hydrolysis of this compound under the processing conditions. Different behavior was observed for

554

ellagic acid, which exhibited 68.1% transfer to juice and more than 200% retention in the press cake.

555

The inconsistent mass balance for ellagic acid suggests that a large proportion of this compound may

556

be generated due to enzymatic and thermal processes in the course of juice production resulting in

557

hydrolysis of ellagitannins and subsequent release of free ellagic acid. This has been corroborated by


Page 20 of 43

Page 21 of 43


558

the study of Gancel et al.21, which indicates that in the course of juice production some ellagitannins

559

(approx. 20%) may be hydrolyzed to ellagic acid due to thermal treatment as early as at the stage of

560

fruit blanching and crushing.

561

The studied raspberry fruits of the cultivars 'Laszka', 'Polka', 'Polana', and 'Benefis' varied in

562

terms of polyphenolic content (from 1511 to 2362 mg/100 g DW). The average percentage

563

contributions of the studied groups of compounds to total phenolics were, in descending order,

564

ellagitannins: 64.2%, anthocyanins and flavanols: 17%, and flavonols: 1.8%. The summer cultivars

565

('Laszka' and 'Benefis') exhibited the highest polyphenolic content and the highest share of

566

ellagitannins (75%). In turn, the everbearing cultivars ('Polka' and 'Polana') revealed the highest

567

anthocyanin content. The analysis and mass balance of the products of raspberry processing (juice and

568

press cake) showed that many polyphenols, mostly tannins, are 90% retained in the press cake, and

569

especially in the seeds. As a result, the content of ellagitannins and flavanols in juice is low and

570

amounts to 217–324 mg/100 g DW and 40–80 mg/100 g DW, respectively. The seedless fraction of

571

the press cake is characterized by very high content of ellagitannins, reaching 8000 mg/100 g DW for

572

the cultivars rich in these compounds. This means that the press cake left over from juice processing

573

may be a valuable raw material for the production of preparations rich in ellagitannins such as

574

sanguiin H-6 and lambertianin C. The mass balance indicated that the transfer of ellagitannins to juice

575

was affected by their molecular mass: an increase from 1568 Da to 2805 Da led to a more than 10-fold

576

decrease in ellagitannin transfer.

577 578

Acknowledgments

579 580

This study was financially supported by the Polish Ministry of Science and Higher Education as a part

581

of the resources allocated for science in 2010–2013 under research project No. NN312360139, and

582

Statute Funds of Institute of Food Technology and Analysis.

583

Author Contributions: Michał Sójka designed the research, supervised, performed the experiments

584

and wrote the manuscript; Jakub Macierzyński performed the experiments and analyzed the data,

585

Wojciech Zaweracz and Maria Buczek were responsible for breeding plant material and its delivery.



586

References

587

1. FAO. FAO Crop Database. Food and Agriculture Organisation. URL: faostat.fao.org (accessed

588

November 5, 2013).

589

2. Liu, Y.; Liu, M.; Li, B.; Zhao, J-L.; Zhang, C-P.; Lin, L-Q.; Chen, H-S.; Zhang, S-J.; Jin, J-C.;

590

Wang, L.; Li, L-J.; Liu, J-R. Fresh raspberry phytochemical extract inhibits hepatic lesion in Wistar rat

591

model. Nutr. Metab. 2010, 7, 84.

592

3. Ross, H.A.; McDougall, G.J.; Stewart, D. Antiproliferative activity is predominantly associated

593

with ellagitannins in raspberry extracts. Phytochemistry 2007, 68, 218-228.

594

4. Bobinaitė, R.; Viškelis, P.; Venskutonis, P.R. Variation of total phenolics, anthocyanins, ellagic

595

acid and radical scavenging capacity in various raspberry (Rubus spp.) cultivars. Food Chem. 2012,

596

132, 1495-1501.

597

5. Gasperotti, M.; Masuero, D.; Vrhovsek, U.; Guella, G.; Mattivi, F. Profiling and accurate

598

quantification of Rubus ellagitannins and ellagic acid conjugates using direct UPLC-Q-TOF HDMS

599

and HPLC-DAD analysis. J. Agric. Food Chem. 2010, 58, 4602-4616.

600

6. Ancos, B.; Gonzalez, E.; Cano M.P. Differentiation of raspberry varieties according to anthocyanin

601

composition. Z. Lebensm.-Unters. -Forsch. A. 1999, 208, 33-38.

602

7. Krüger, E.; Dietrich, H.; Schöpplein, E.; Rasim, S.; Kürbel P. Cultivar, storage conditions and

603

ripening effects on physical and chemical qualities of red raspberry fruit. Postharvest Biol. Technol.

604

2011, 60, 31-37.

605

8. Ancos, B.; Ibañez, E.; Reglero, G.; Cano, M.P. Frozen storage effects on anthocyanins and volatile

606

compounds of raspberry fruit. J. Agric. Food Chem. 2000, 48, 873-879.

607

9. Remberg, S.F.; Sønsteby, A.; Aaby, K.; Heide, O.M. Influence of postflowering temperature on

608

fruit size and chemical composition of Glen Ample raspberry (Rubus ideaus L.) J. Agric. Food Chem.

609

2010, 58, 9120-9128.


Page 22 of 43

Page 23 of 43


610

10. Gu, L.; Kelm, M.A.; Hammerstone, J.F.; Beecher, G.; Holden, J.; Haytowitz, D.; Prior, R.L.

611

Screening of foods containing proanthocyanidins and their structural characterization using LC-

612

MS/MS and thiolytic degradation. J. Agric. Food Chem. 2003, 51, 7513-7521.

613

11. Hellström, J.K.; Törrönen, A.R.; Mattila, P.H. Proanthocyanidins in common food products of

614

plant origin. J. Agric. Food Chem. 2009, 57, 7899-7906.

615

12. Wang, S.Y.; Chen, C-T.; Wang, C.Y. The influence of light and maturity on fruit quality and

616

flavonoid content of red raspberries. Food Chem. 2009, 112, 676-684.

617

13. Jean-Gilles, D.; Li, L.; Ma, H.; Yuan, T.; Chichester, C.O.; Seeram N.P. Anti-inflamatory effects

618

of polyphenolics-enriched red raspberry extract in an antigen arthritis rat model. J. Agric. Food Chem.

619

2012, 60, 5755-5762.

620

14. Seeram, N.P.; Adams, L.S.; Zhang, Y.; Lee, R.; Sand, D.; Scheuller, H.S.; Heber, D. Blackberry,

621

black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and

622

stimulate apoptosis of human cancer cells in vitro. J. Agric. Food Chem. 2006, 54, 9329-9339.

623

15. Puupponen-Pimiä, R.; Nohynek, L.; Hartmann-Schmidlin, S.; Kähkönen, M.; Heinonen, M.;

624

Määttä-Riihinen, K.; Oksman-Celdentey, K.M. Berry phenolics selectively inhibit the growth of

625

intestinal pathogens. J. Appl. Microbiol. 2005, 98, 991-1000.

626

16. Tomás-Barberán, F.A.; Garcia-Conesa, M.T.; Larossa, M.; Cedrá, B.; González-Barrio, R.;

627

Bermúdez-Soto, M.J.; González-Sarrias, A.; Espin J.C. Bioavailability, metabolism, and bioactivity of

628

food ellagic acid and related polyphenols. In Recent advances in polyphenol research. Daayf, F.;

629

Lattanzio, V.; Eds. Wiley-Blackwell UK, 2008, 263-277.

630

17. Belitz, H-D.; Grosch, W.; Schieberle, P. Fruits and fruit products. In Food chemistry 4th revised

631

and extended edition; Springer-Verlag Berlin Heidelberg, Germany 2009, Ch. 18, 807-861.

632

18. White, B.L.; Howard, L.R.; Prior, R.L. Impact of different stages of juice processing on the

633

anthocyanin, flavonol, and procyanidin contents of cranberries. J. Agric. Food Chem. 2011, 59, 4692-

634

4698.



635

19. Howard, L.R.; Prior, R.L.; Liyanage, R.; Lay, J.O. Processing and storage effect on berry

636

polyphenols: challenges and implications for bioactive properties. J. Agric. Food Chem. 2012, 60,

637

6678-6693.

638

20. Hager, T.J.; Howard, L.R.; Prior, R.L. Processing and storage effects on the ellagitannin

639

composition of processed blackberry products. J. Agric. Food Chem. 2010, 58, 11749-11754.

640

21. Gancel, A-L.; Feneuil, A.; Acosta, O.; Pérez, A.M.; Vaillant, F. Impact of industrial processing

641

and storage on major polyphenols and the antioxidant capacity of tropical highland blackberry (Rubus

642

adenotrichus). Food Res. Int. 2011, 44, 2243-2251.

643

22. Górecka, D.; Pachołek, B.; Dziedzic, K.; Górecka, M. Raspberry pomace as a potential source for

644

cookies enrichment. Acta Sci. Pol., Technol. Aliment. 2010, 9, 451-462.

645

23. Pieszka, M.; Tombarkiewicz, B.; Roman, A.; Migdał, W.; Niedziółka, J. Effect of bioactive

646

substances found in rapseed, raspberry and strawberry seed oils on blood lipid profile and selected

647

parameters of oxidative status in rats. Environ. Toxicol. Pharmacol. 2013, 36, 1055-1062.

648

24. McDougal, N.R. In The evaluation of raspberry pomace as a feedstuff for growing pigs. M.Sc.

649

thesis. The University of British Columbia, Vancouver, Canada 1990.

650

25. Kang, I.; Espín, J.C.; Carr, T.P.; Tomás-Barberán, F.A.; Chung, S. Raspberry seed flour attenuates

651

high-sucrose diet-mediated hepatic stress and adipose tissue inflammation. J. Nutr. Biochem. 2016, 32,

652

64-72.

653

26. Kosmala, M.; Zduńczyk, Z.; Juśkiewicz, J.; Jurgoński, A.; Karlińska, E.; Macierzyński, J.;

654

Jańczak, R.; Rój, E. Chemical composition of defatted strawberry and raspberry seeds and the effect of

655

these dietary ingredients on polyphenol metabolites, intestinal function, and selected serum parameters

656

in rats. J. Agric. Food Chem. 2015, 63, 2989-2996.

657

27. Gwozdecki, J.; Lisek, J.; Łabanowska, B.H.; Meszka, B.; Mochecki, J.; Treder, W. In Metodyka

658

integrowanej produkcji malin, edition no. 3; Mochecki, J. Ed; Main Inspectorate of Plant Health and

659

Seed Inspection, Warszawa, 2014, 3-5.


Page 24 of 43

Page 25 of 43


660

28. Klimczak, E.; Król, B. Determination of different forms of ellagic acid in by-products form

661

strawberry processing. Zywn., Technol., Jakosc. 2010, 4, 81-94

662

29. Kapasakalidis, P.G.; Rastall, R.A.; Gordon, M.H. Extraction of polyphenols from processed black

663

currant (Ribes nigrum L.) residues. J. Agric. Food Chem. 2006, 54, 4016-4021.

664

30. McDougall, G.J.; Conner, S.; Pereira-Caro, G.; Gonzalez-Barrio, R.; Brown, E.M.; Verrall, S.;

665

Stewart, D.; Moffet, T.; Ibars, M.; Lawther, R.; O’Connor, G.; Rowland, I.; Crozier, A.; Gill C.I.R.

666

Tracking (poly)phenol components from raspberries in ileal fluid. J. Agric. Food Chem. 2014, 62,

667

7631-7641.

668

31. Kähkönen, M.; Kylli, P., Ollilainen, V.; Salminen, J-P.; Heinonen, M. Antioxidant activity of

669

isolated ellagitannins from red raspberries and cloudberries. J. Agric. Food Chem. 2012, 60, 1167-

670

1174.

671

32. Tanaka, T.; Tachibana, H.; Nonaka G-I.; Nishioka, I.; Hsu, F-L.; Kohda, H.; Tanaka, O. Tannins

672

and related compounds. CXXII. New dimeric, trimeric and tetrameric ellagitannins, lambertianins A-

673

D, from Rubus lambertianus. Chem. Pharm. Bull. 1993, 41, 1214-1220.

674

33. Hager, T.J.; Howard, L.R.; Liyanage, R.; Lay, J.O.; Prior, R.L. Ellagitannin composition of

675

blackberry as determined by HPLC-ESI-MS and MALDI-TOF-MS. J. Agric. Food Chem. 2008, 56,

676

661-669.

677

34. Mullen, W.; Yokota, T.; Lean M.E.J.; Crozier, A. Analysis of ellagitannins and conjugates of

678

ellagic acid and quercetin in raspberry fruits by LC-MSn. Phytochemistry. 2003, 64, 617-624.

679

35. Chen, L.; Xin, X.; Zhang, H.; Yuan, Q. Phytochemical properties and antioxidant capacities of

680

commercial raspberry varieties. J. Funct. Foods 2013, 5, 508-515.

681

36. Sójka, M.; Kołodziejczyk, K.; Milala, J. Polyphenolic and basic composition of black chokeberry

682

industrial by-products. Ind. Crops Prod. 2013, 51, 77-86.

683

37. Sójka, M.; Klimaczak, E.; Macierzyński, J.; Kołodziejczyk, K. Nutrient and polyphenolic

684

composition of industrial strawberry press cake. Eur. Food Res. Technol. 2013, 237, 995-1007.



685

38. USDA. National Nutrient Database of Standard Reference. URL: ndb.nal.usda.gov (accessed

686

December, 2015).

687

39. Vrhovsek, U.; Giongo, L.; Mattivi, F.; Viola, R. A survey of ellagitannins content in raspberry and

688

blackberry cultivars grown in Trentino (Italy). Eur. Food Res. Technol. 2008, 226, 817-824.

689

40. Koponen, J.M.; Happonen, A.M.; Matilla, P.H.; Törrönen, A.R. Contents of anthocyanins and

690

ellagitannins in selected foods consumed in Finland. J. Agric. Food Chem. 2007, 55, 1612-1619.

691

41. Rzeppa, S.; Bargen, C.V.; Bittner, K.; Humpf, H-U. Analysis of flavan-3-ols and procyanidins in

692

food samples by reversed phase high-performance liquid chromatography coupled to electrospray

693

ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS). J. Agric. Food Chem. 2011, 59,

694

10594-10603.

695

42. Hosseinian, F.S.; Li, W; Hydamaka, A.W.; Tsompo, A.; Lowry, L.; Friel, J.; Beta, T.

696

Proanthocyanidin profile and ORAC values of Manitoba berries, chokecherries, and seabuckthron. J.

697

Agric. Food Chem. 2007, 55, 6970-6976.

698

43. Kähkönen, M.P.; Hopia, A.I.; Heinonen, M. Berry phenolics and their antioxidant activity. J.

699

Agric. Food Chem. 2001, 49, 4076-4082.

700

44. Mikulic-Petkovsek, M.; Slatnar, A.; Stampar, F.; Veberic, R. HPLC-MSn identification and

701

quantification of flavonol glycosides in 28 wild and cultivated berry species. Food Chem. 2012, 135,

702

2138-2146.

703

45. Rommel, A.; Wrostland, R.E. Ellagic acid content of red raspberry juice as influenced by cultivar,

704

processing, and environmental factors. J. Agric. Food Chem. 1993, 41, 1951-1960.

705

46. Bermúdez-Soto, M.J.; Tomás-Barberán, F.A. 2004. Evaluation of commercial red fruit juice

706

concentrates as ingredients for antioxidant functional juices. Eur. Food Res. Technol. 2004, 219, 133-

707

141.


Page 26 of 43

Page 27 of 43


708

47. Hager, T.J.; Howard, L.R.; Prior, R.L. Processing and storage effects on monomeric anthocyanins,

709

percent polymeric color, and antioxidant capacity of processed blackberry products. J. Agric. Food

710

Chem. 2008, 56, 689-695.

711

48. Versari, A.; Biesenbruch, S.; Barbanti, D.; Farnell, P.J.; Galassi, S. Effect of pectolytic enzymes

712

on selected phenolic compounds in strawberry and raspberry juices. Food Res. Int. 1997, 30, 10:811-

713

817.

714

49. Viljanen, K.; Halmos, A.L.; Sinclair, A.; Heinonen, M. Effect of blackberry and raspberry juice on

715

whey protein emulsion stability. Eur. Food Res. Technol. 2005, 221, 602-609.

716

50. Kammerer, D.; Claus, A.; Carle, R.; Schieber, A. Polyphenol screening of pomace from red and

717

white grape varieties (Vitis vinifera L.) by HPLC-DAD-MS/MS. J. Agric. Food Chem. 2004, 52,

718

4360-4367.

719



720

FIGURE CAPTIONS:

721

Figure 1. Structures of sanguiin H-6, 1, lambertianin C, 2, and lambertianin D, 3. These three major

722

Rubus ideaus L. ellagitannins correspond to peaks 7, 6, 4 respectively in Table 2.

723

Figure 2. Structures of all possible sanguiin H-10 isomers, 4. Peaks 1 and 3 from Table 2 correspond

724

to these structures.

725

Figures 3. Structures of lambertianin C without ellagic acid moiety isomers, 5, (corresponding to peak

726

2 in Table 2).

727

Figure 4. Structures of ellagic acid, 6, and ellagic acid conjugates: ellagic acid pentose, 7, ellagic acid

728

acetylpentose, 8, and methyl ellagic acid pentose, 9. These structures correspond to peaks 9, 8, 11 and

729

10 respectively in Table 2.

730

Figure 5. Chromatogram of raspberry ellagitannins for the summer cultivars ('Laszka' and 'Benefis')

731

and the everbearing cultivars ('Polka' and 'Polana').

732

Figure 6. Cluster analysis of raspberry fruit cultivars with the use of k-means clustering; cluster 1 –

733

'Polka' and 'Polana' cultivars, cluster 2 – 'Laszka' and 'Benefis' cultivars.

734

Peak numbers correspond to those in Table 2..total ET, total ellagitannin; total EAC, total ellagic acid conjugates; total

735

ET+EAC, total ellagitannin and ellagic acid conjugates; total ACY, total anthocyanins; total FLAVO, total flavonols; total

736

FLAVA, total flavanols; total PH, total polyphenols.

737

Figure 7. Percentage retention of polyphenolics (ellagitannins, anthocyanins, flavanols, and flavonols)

738

in red raspberry juice and press cake fractions.

739

total ET+EAC, total ellagitannin and ellagic acid conjugates; total ACY, total anthocyanins; total FLAVO, total flavonols;

740

total FLAVA, total flavanols; total PH, total polyphenols.

741

Figure 8. Percentage retention of ellagitannins in red raspberry juice and press cake fractions.

742

Peak numbers correspond to those in Table 2.


Page 28 of 43

Page 29 of 43


Table 1. Average Juice and Press Cake Yield in Fruit Processing and Contribution of the Seed and Seedless Fractions to the Press Cake Cultivar

'Laszka' [%]

'Polka' [%]

'Polana' [%]

'Benefis' [%]

93.4 9.9 5.3

91.9 11.2 5.5

93.6 11.5 6.1

93.7 12.0 6.5

95.0 5.0

94.5 5.5

92.4 7.6

93.7 6.3

Fruit processing Juice yielda Fresh press-cake yielda Dry press-cake yielda,b Press-cake fractionation Seed fraction Seedless fration

a – process yield, calculated on the basis of the weight of the products obtained relative to the weight of the raw starting material. b – means the product obtained after drying of fresh press-cake.



Page 30 of 43

Table 2. LC-MS Identification of Ellagitannins and Anthocyanins in Raspberry Polyphenolic Extracts Peak no.

tR

MS data

[min]

MS/MS value

1

14.57

[1567.15]-1 [783.07]-2

783

2

15.13

[1250.60]-2 [833.40]-3

1250

3

17.72

[1567.15]-1 [783.07]-2

783

4

18.88

[1246.43]-3 [934.07]-2

1246

5

19.25

[1401.01]-2 [934.07]-3

1401

6

19.75

[1401.01]-2 [934.07]-3

1401

7

20.57

[934.07]-2 [1869.14]-1

934

8 9 10 11

22.52 24.28 26,70 27.38

[433.04]-1 [301.00]-1 [447.06]-1 [475.05]-1

433 447 475

12 13 14 15 16 17

3.97 4.70 5.32 5.72 6.63 7.20

[611.16]+ [611.16]+ [757.22]+ [449.11]+ [595.16]+ [433.11]+

611 611 757 449 595 433

MS/MS data Ellagitannins and ellagic acid conjugates [1235.07]-1 [935.08]-1 [633.07]-1 [469.01]-1 [301.00]-1 [2200.19]-1 [1867.14]-1 [1567.14]-1 [1235.07]-1 [933.06]-2 [633.07]-1 [301.00]-1 [1265.14]-1 [1103.09]-1 [935.08]-1 [933.07]-1 [633.07]-1 [469.01]-1 [301.00]-1 [1869.14]-1 [1567.14]-1 [1235.07]-1 [935.08]-2 [633.07]-1 [301.00]-1 [1869.14]-1 [1567.14]-1 [1235.07]-1 [935.08]-1 [633.07]-1 [301.00]-1 [1869.14]-1 [1567.14]-1 [1235.07]-1 [935.08]-1 [633.07]-1 [301.00]-1 [1567.14]-1 [1235.07]-1 [935.08]-1 [633.07]-1 [301.00]-1 [301.00]-1 [315.02]-1 [301.00]-1 Anthocyanins [287.05]+ [287.05]+ [287.05]+ [287.05]+ [287.05]+ [271.06]+

tR – retention time; (standard) – identification based on the standard compound


Tentative structural assignment

Reference

Sanguiin H-10 isomer

5, 30, 31

Lambertianin C without ellagic moiety

5

Sanguiin H-10 isomer

5, 30, 31

Lambetrianin D (standard)

32,33

Lambertianin C isomer

33

Lambertianin C (standard)

5, 30, 31, 32, 33

Sanguiin H-6 (standard)

5, 30, 31, 32, 33

Ellagic acid pentose conjugate Ellagic acid (standard) Methyl ellagic acid pentose conjugate Ellagic acid acetylpentose conjugate

5, 30, 34

Cyanidin 3,5-O-diglucoside Cyanidin 3-O-sophoroside Cyanidin 3-O-glucosyl-rutinoside Cyanidin 3-O-glucoside (standard) Cyanidin 3-O-rutinoside (standard) Pelargonidin 3-O-glucoside

30

5, 30, 31, 33, 34 5, 34 5, 34

30, 35 30, 35 30, 35 30, 35 30, 35

Page 31 of 43


Table 3. Ellagitannin, Anthocyanin, Flavanol, and Flavonol Content (mg/100g DW) in Different Cultivars of Raspberry Fruits

Peak no.

'Laszka' 2012 Mean

SD

'Polka' 2012 Mean

1 2 3 4 5 6 7 8 9 10 11 total ETa total EACb total ET+EACc

'Polana' 'Benefis' 'Laszka' 2012 2012 2013 Mean SD Mean SD Mean Ellagitannins and ellagic acid conjugates

SD

12.3abc 25.6c 19.8b 41.9b 98.7d 991.5c 580.9b 10.9c 5.1c 3.3b 2.9b 1770.8b 22.2b 1793.0b

1.1 2.7 0.7 9.0 4.3 104.2 53.6 0.7 0.3 0.2 0.2 157.4 1.4 158.8

9.8a 12.2a 16.9b 0.0a 14.1a 402.4a 383.8a 6.7b 2.9a 2.2a 1.7a 839.1a 13.5a 852.7a

0.5 0.5 0.3 0.0 2.0 14.9 3.8 0.3 0.1 0.0 0.0 21.0 0.2 20.8

13.2bc 13.6a 10.7a 0.0a 14.8a 422.4a 371.5a 3.9a 2.8a 2.2a 3.7cd 846.3a 12.6a 858.9a

12 13 14 15 16 17 total anthocyanins

5.1c 166.0c 21.8c 64.3b 13.2c 0.9a 271.1b

0.0 1.4 0.1 0.5 0.1 0.2 1.8

1.3ab 237.1d 1.5a 111.3e 0.8a 4.6c 356.6c

0.1 7.3 0.1 3.1 0.0 0.1 10.7

0.5a 261.7e 38.2e 83.7c 18.6d 2.4b 405.1d

0.0 0.2 0.6 0.0 0.0 0.1 0.4

total flavonols total flavan-3-ols

16.9a 281.7a

0.2 5.1

29.3b 272.7a

1.3 1.2

43.8d 288.5a

0.9 14.3

Sum

2362.7b

162.0

1511.4a

31.6

1596.3a

1.0 1.9 1.0 0.0 4.4 44.2 33.8 0.3 0.1 0.2 0.4 86.2 1.1 87.3

15.0c 22.8bc 31.4c 104.3c 60.0c 785.6b 585.9b 10.5c 7.1d 4.2c 3.8cd 1605.1b 25.7bc 1630.8b

SD

'Polka' 2013 Mean

SD

'Polana' 2013 Mean

SD

1.3 1.3 3.8 22.0 9.0 69.4 31.9 0.8 0.7 0.1 0.2 138.7 1.8 140.5

13.9bc 22.4bc 19.2b 44.4b 90.4d 851.1bc 526.5b 13.7d 4.8bc 4.0c 3.8cd 1567.9b 26.3c 1594.1b

0.9 1.5 1.2 1.7 6.4 48.3 29.8 0.5 0.0 0.2 0.3 89.8 1.0 90.8

14.9c 17.6ab 18.3b 0.0a 38.9b 500.9a 383.6a 10.5c 4.9bc 4.3c 3.0bc 974.2a 22.7bc 996.9a

1.2 0.4 0.8 0.0 0.8 4.2 6.9 1.0 0.1 0.2 0.0 12.6 1.3 13.9

10.8ab 15.8a 10.6a 0.0a 34.2b 424.9a 356.2a 4.9a 4.1b 3.3b 3.9d 852.5a 16.2a 868.7a

2.3 5.2 1.7 0.0 13.9 101.0 64.9 1.0 0.6 0.4 0.6 189.0 2.6 191.5

0.0 0.8 0.3 0.4 0.0 0.0 1.5

4.8c 135.8b 8.9b 60.6ab 7.0b 0.5a 217.6a

0.5 12.7 0.8 6.7 0.7 0.0 21.5

2.4b 146.0b 0.9a 100.7d 0.6a 2.4b 252.9b

1.3 4.2 0.7 2.6 0.1 0.2 9.1

0.2a 238.5d 47.1f 82.7c 22.9e 2.3b 393.8d

0.3 5.4 2.3 2.3 1.2 0.1 11.5

0.9 1.4

27.6b 271.0a

2.3 12.1

26.4b 342.5c

0.5 3.0

38.5c 323.2b

1.9 5.5

144.4

2110.2b

54.9

1618.6a

20.5

1624.1a

210.5

Anthocyanins 2.1b 82.5a 32.0d 54.9a 28.6f 0.6a 200.7a

Other 41.9d 363.7d

Sum of polyphenols 74.2

2237.2b

Values are means ± standard deviation (SD); n = 3; the results in the individual lines marked by the same letter do not differ statistically at p < 0.05. Peak numbers correspond to those in Table 2. a Total ET, total ellagitannin content. bTotal EAC, total ellagic acid conjugates content. cTotal ET+EAC, total ellagitannin and ellagic acid conjugates content.



Page 32 of 43

Table 4. Ellagitannin, Anthocyanin, Flavanol, and Flavonol Content (mg/100g DW) in Raspberry Juices from Different Cultivars

Peak no.

'Laszka' 2012 Mean

SD

'Polka' 2012 Mean

'Benefis' 'Laszka' 2012 2013 SD Mean SD Mean Ellagitannins and ellagic acid conjugates


7.9a 5.2c 17.5b 1.4c 3.0c 55.4d 216.7c 5.3ab 2.5a 1.9a 1.3a 307.2c 10.9a 318.1c

0.0 0.7 0.4 0.0 0.2 1.0 3.0 0.1 0.0 0.0 0.0 5.3 0.1 5.4

10.4b 1.7a 17.4b 0.0a 1.2a 32.9b 164.0b 6.6c 6.0d 2.8c 1.6c 227.6b 16.9c 244.5b

0.3 0.1 0.1 0.0 0.0 0.8 1.5 0.0 0.1 0.0 0.1 2.8 0.1 2.9

7.9a 2.6b 20.6c 0.7b 2.1b 45.4c 160.1b 5.0a 4.4b 2.3b 1.5b 239.4b 13.1b 252.5b


5.5e 154.6b 12.7b 53.1a 8.4b 0.8a 235.1a

0.1 1.8 0.1 0.3 0.1 0.0 2.5

1.8c 269.3d 2.1a 105.2c 1.2a 6.2c 385.8c

0.0 7.7 0.0 3.2 0.1 0.4 11.4

1.4b 121.9a 33.1c 49.4a 20.9c 0.6a 227.4a


11.0a 40.8a

0.0 4.6

15.4b 80.0c

0.1 0.0

17.9c 64.4b

Sum

604.9a

1.6

725.8c

14.4

SD

'Polka' 2013 Mean

12.4c 2.8b 25.6e 2.3d 2.4b 44.8c 216.8c 7.1d 5.8d 2.5bc 1.6c 307.0c 17.0c 324.0c

0.4 0.0 0.7 0.0 0.0 0.9 5.1 0.2 0.0 0.0 0.0 7.0 0.1 7.1

0.0 0.5 1.0 0.5 0.3 0.0 1.3

7.5f 187.2c 11.0b 72.9b 7.6b 0.8a 287.0b

0.3 2.1

0.4 0.0 0.2 0.1 0.1 0.7 1.1 0.0 0.1 0.1 0.0 2.7 0.2 2.9

SD

'Polana' 2013 Mean

SD

10.6b 1.1a 22.0d 0.0a 1.5a 25.4a 136.0a 7.4d 8.2e 3.7d 1.5b 196.5a 20.8e 217.3a

0.7 0.2 0.2 0.0 0.0 4.2 7.3 0.2 0.2 0.2 0.0 12.7 0.2 12.9

9.8b 1.6a 13.5a 0.0a 3.5d 24.1a 154.5b 5.5b 5.5c 3.7d 3.4d 207.0a 18.2d 225.2a

0.5 0.0 0.2 0.0 0.2 0.8 4.3 0.1 0.0 0.2 0.0 6.0 0.4 6.3

0.4 5.2 0.5 2.1 0.3 0.0 8.5

2.8d 249.4d 1.1a 125.0e 0.7a 4.0b 383.0c

0.1 6.4 0.0 3.3 0.0 0.1 10.1

0.7a 369.0e 79.3d 115.6d 35.2d 3.9b 603.6d

0.1 20.2 4.7 7.1 2.3 0.2 34.6

13.7b 67.7b

0.4 0.4

14.7b 42.4a

0.6 0.7

29.9d 68.4b

1.9 2.6

692.3bc

2.3

657.4b

3.0

927.1d

40.2

Anthocyanins

Other

Sum of polyphenols 562.2a

2.4



Page 33 of 43


Table 5. Ellagitannin, Anthocyanin, Flavanol, and Flavonol Content (mg/100g DW) in the Seed Fraction of Raspberry Press Cake

Peak no.

'Laszka' 2012 Mean

SD

'Polka' 2012 Mean

'Benefis' 'Laszka' 2012 2013 SD Mean SD Mean Ellagitannins and ellagic acid conjugates


11.6a 24.4a 17.3ab 430.6c 190.3e 1810.2d 685.0a 7.6bc 27.8b 5.6b 0.0 3169.4c 41.0bc 3210.4c

0.0 0.7 0.3 11.5 4.9 33.8 12.3 0.0 0.8 0.1 0.0 62.9 0.9 63.8

22.2c 29.3b 24.3c 0.0a 12.9a 852.4a 796.0bc 5.7a 25.0a 4.8a 0.0 1737.1a 35.6a 1772.7a

1.5 1.6 0.5 0.0 0.8 0.5 1.1 0.1 0.3 0.0 0.0 5.1 0.3 4.8

15.3b 25.1a 15.3a 386.3b 108.6c 1657.8c 826.0c 6.9b 29.9b 4.7a 0.0 3034.5c 41.5c 3076.0c


0.5d 14.7ab 0.9b 13.8ab 1.2b 0.1ab 31.2a

0.1 1.2 0.0 1.1 0.1 0.0 2.3

0.2b 25.3cd 0.2ab 20.2bc 0.2a 0.7d 46.9abc

0.0 2.3 0.0 1.8 0.0 0.1 4.3

0.1b 12.3a 2.7c 11.7a 3.3c 0.2b 30.4a


18.4abc 945.1c

2.3 102.4

15.2a 777.4b

1.3 10.2

19.0abc 1175.9d

Sum

4205.0c

161.6

2612.2a

11.0

SD

'Polka' 2013 Mean

12.8ab 28.8b 18.9b 419.4c 123.1d 1483.0b 768.0b 13.9d 35.6c 4.7a 0.0 2854.0b 54.1d 2908.1b

1.1 0.3 1.8 22.6 11.6 70.1 21.4 0.7 1.8 0.4 0.0 128.9 2.8 131.7

0.0 0.2 0.0 0.1 0.1 0.0 0.5

0.3c 17.8abc 0.5ab 19.2b 1.0b 0.0a 38.7ab

0.1 54.9

0.3 0.3 0.1 3.2 2.1 17.0 12.8 0.1 0.3 0.2 0.0 35.9 0.6 36.5

SD

'Polana' 2013 Mean

SD

21.3c 33.2c 30.6d 0.0a 60.4b 877.3a 710.1a 8.0c 25.6a 4.4a 0.0 1732.9a 38.0ab 1770.9a

1.9 1.4 0.7 0.0 1.2 11.7 23.3 0.2 0.5 0.2 0.0 40.3 0.9 41.2

26.6d 29.3b 23.1c 0.0a 66.9b 884.5a 814.3c 7.4bc 24.3a 4.8a 0.0 1844.7a 36.5a 1881.1a

0.2 0.5 0.3 0.0 1.6 10.7 11.1 0.0 0.6 0.1 0.0 1.9 0.6 2.5

0.0 3.6 0.2 3.6 0.2 0.0 7.6

0.0a 21.8bc 0.0a 27.2c 0.0a 0.7d 49.6bc

0.0 2.9 0.0 4.2 0.0 0.1 7.2

0.0a 30.8d 4.0d 20.8bc 3.4c 0.5c 59.5c

0.0 6.0 0.7 3.9 0.6 0.1 11.2

25.7c 622.3a

5.4 1.6

15.9ab 704.7ab

1.4 25.7

23.1bc 684.5ab

4.0 35.9

3594.8b

117.1

2541.2a

58.3

2648.3a

18.2

Anthocyanins

Other

Sum of polyphenols 4301.3c

92.0




Page 34 of 43

Table 6. Ellagitannin, Anthocyanin, Flavanol, and Flavonol Content (mg/100g DW) in the Seedless Fraction of Raspberry Press Cake

Peak no.

'Laszka' 2012 Mean

SD

'Polka' 2012 Mean

'Benefis' 'Laszka' 2012 2013 SD Mean SD Mean SD Ellagitannins and ellagic acid conjugates


28.6a 117.6ab 22.8ab 107.9c 853.4b 5191.5c 1715.5ab 15.4ab 42.2a 5.2a 1.4a 8037.2c 64.1a 8101.3d

2.4 11.1 1.9 17.2 136.0 551.7 158.6 1.2 5.1 0.4 0.1 878.9 6.9 885.8

68.2c 139.2b 38.0c 0.0a 378.3a 3745.9b 2192.8b 25.3c 64.0b 8.7b 1.9ab 6562.4bc 99.9b 6662.3bc

14.5 26.2 6.7 0.0 91.7 627.9 370.8 4.2 11.0 1.5 0.5 1137.9 17.2 1155.1

32.1ab 102.9ab 29.5bc 71.4b 481.9a 3887.7b 1736.2ab 11.2a 29.9a 4.3a 1.9ab 6341.7abc 47.3a 6389.0abc


1.6d 47.2b 2.6c 45.1b 3.7b 0.7b 100.8b

0.2 7.3 0.5 7.3 0.5 0.2 15.8

0.4b 87.4d 0.6a 69.6d 0.5a 2.6d 161.1cd

0.0 1.2 0.0 1.0 0.0 0.0 2.2

0.3b 32.1a 6.8d 30.3a 8.0c 0.4a 77.8a


34.7ab 227.3a

5.1 10.6

35.7ab 300.3b

0.7 16.3

34.7ab 235.5a

Sum

8464.1d

875.6

7159.3bc

1141.7

6737.0abc

1.8 2.8 0.6 1.1 25.3 43.1 17.4 0.1 0.6 0.2 0.2 92.0 0.8 92.9

'Polka' 2013 Mean

SD

'Polana' 2013 Mean

SD

35.7ab 114.1ab 28.9bc 147.9d 468.2a 4162.7b 1976.0ab 20.2bc 43.2a 4.8a 2.4b 6933.5bc 70.6a 7004.1bc

1.0 1.2 0.8 11.3 8.6 52.8 27.9 0.4 0.9 0.1 0.1 100.0 1.4 101.4

47.3b 132.6ab 37.1c 0.0a 390.7a 3235.9ab 1769.6ab 23.3c 60.9b 7.7b 2.5b 5613.3ab 94.4b 5707.7ab

5.4 22.0 5.9 0.0 61.9 519.4 284.9 3.7 9.3 1.3a 0.1 899.5 14.4 913.9

39.5ab 98.7a 17.4a 0.0a 313.2a 2659.1a 1595.9a 10.5a 32.1a 5.1a 4.6c 4723.8a 52.3a 4776.1a

0.3 1.4 0.2 0.0 1.8 17.6 2.3 0.2 0.3 0.1 0.0 16.1 0.5 16.6

1.2c 51.6b 1.8b 52.3bc 3.0b 0.6ab 110.4b

0.1 3.2 0.1 3.7 0.2 0.0 7.3

0.0a 66.9c 0.0a 81.0e 0.0a 2.0c 149.9c

0.0 2.7 0.0 3.8 0.0 0.1 6.6

0.0a 90.9d 11.4e 61.0cd 11.0d 2.0c 176.3d

0.0 3.8 0.4 2.2 0.5 0.1 7.1

34.2a 228.2a

2.8 0.4

29.9a 399.4d

1.3 0.0

40.9b 353.9c

1.9 0.7

7377.0bc

111.8

6286.9ab

921.9

5347.1a

7.0

Anthocyanins 0.0 0.7 0.2 0.8 0.2 0.0 2.0

Other 1.0 10.2

Sum of polyphenols 79.6



Page 35 of 43


Figure 1.



Figure 2.

Compound Sanguiin H-10 Sanguiin H-3 Sanguiin H-10 isomer

R1 R1’ HHDP H H HHDP

Moiety R2 R2’ H H HHDP HHDP

R3 R3’ HHDP HHDP H H


Page 36 of 43

Page 37 of 43


Figure 3.

Compound Lambertianin C without ellagic moiety 1 Lambertianin C without ellagic moiety 2 Lambertianin C without ellagic moiety 3 Lambertianin C without ellagic moiety 4

R1 H

R1’ H

HHDP

Moiety R2 R2’ R3 R3’ HHDP HHDP H

H

HHDP

HHDP

HHDP

HHDP

HHDP H

H

HHDP

R4 R4’ HHDP HHDP HHDP H

HHDP – hexahydroxydiphenic acid moiety


H


Page 38 of 43

Figure 4.

.

Compound Methyl ellagic acid pentose conjugate 1 Methyl ellagic acid pentose conjugate 2 Methyl ellagic acid pentose conjugate 3


R1 CH3

Moiety R2 H

R3 H

H

CH3

H

H

H

CH3

Page 39 of 43


Figure 5.



Page 40 of 43

Figure 6. Plot of Means for Each Cluster Cluster 1: 'Polka' and 'Polana' cultivars Cluster 2: 'Laszka' and 'Benefis' cultivars 1,5

1,0

0,5

0,0

-0,5

-1,0


total PH

total FLAVA

total FLAVO

total ACY

17

16

15

14

13

12

total ET+EAC

total EAC

total ET

11

10

9

8

7

6

5

4

3

2

1

-1,5

Page 41 of 43


Figure 7.

100

[%]

80 60 40 20 0

Total ET+EAC Juice

Total ACY

Total FLAVO

Pomace seed fraction

Total FLAVA

Pomace seedless fraction


Total PH


Page 42 of 43

Figure 8. 100 90 80 70

[%]

60 50 40 30 20 10 0

9

8

10

11

1

3

7

2

5

Increase of molecular mass Juice

Pomace seed fraction

Pomace seedless fraction


6

4

Page 43 of 43


84x47mm (96 x 96 DPI)


Transfer and Mass Balance of Ellagitannins, Anthocyanins, Flavan-3

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