Composition and antioxidant activity of anthocyanins from Aronia melanocarpa cultivated in Haicheng, Liaoning, China

Food Bioscience 30 (2019) 100413

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Composition and antioxidant activity of anthocyanins from Aronia melanocarpa cultivated in Haicheng, Liaoning, China

T

Lingshuai Menga, Jinyan Zhua,b, Yan Mac, Xiyun Suna, Dongnan Lia, Li Lia, Hongqi Baid, Guang Xina,∗∗, Xianjun Menga,∗ a

College of Food Science, Shenyang Agricultural University, Shenyang, 110866, Liaoning, PR China Food Inspection Monitoring Center of Zhuanghe, Dalian, 116400, Liaoning, PR China c Experimental Teaching Center, Shenyang Normal University, Shenyang, 110034, Liaoning, PR China d Liaoning Institute for Food Control, Shenyang, 110015, Liaoning, PR China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Aronia melanocarpa Black chokeberry Lonicera caerulea Phenols Anthocyanins Lard

Aronia melanocarpa fruits are high in phenolic substances-mainly anthocyanins. The anthocyanins, as a watersoluble plant pigments with strong antioxidant activity, have been used in food and medical industries. The fresh weight, total soluble solids, total anthocyanins and total phenols of Aronia melanocarpa were determined. Anthocyanins were purified using ion exchange resin coupled with chromatography, and identified using HPLC and mass spectrometry. Then, the antioxidant activities of anthocyanins were determined using different kit methods, and the potential application of purified anthocyanins was tested in lard. The results showed the anthocyanins content increased from 280 to 930 mg/g after being purified using semi-preparative reverse phase HPLC. After anthocyanins were purified using absorbent resin, a total of 7 different anthocyanins, including cyanidin-3,5-dihexoside, the dimer of cyanidin-hexoside, cyanidin-3-O-(galactoside, glucoside, arabinoside, xyloside) and delphinidin-3-O-rutinoside, were identified using HPLC-MS, cyanidin-3,5-dihexoside and a dimer of cyanidin-hexoside were first found in A. melanocarpa. In addition, cyanidin-3-O-(galactoside, glucoside, arabinoside, xyloside) were identified using HPLC from anthocyanins purified using absorbent resin coupled with semi-preparative reverse phase HPLC. Furthermore, the antioxidant tests showed that the antioxidant capacity of anthocyanins could be significantly improved by increasing the purity of anthocyanins, and purified anthocyanins were a good antioxidant for lard. A. melanocarpa cultivated in Haicheng is high in anthocyanins and cyanidin-3-O-(galactoside, glucoside, arabinoside, xyloside) were the main anthocyanins. Purified anthocyanins could be separated and may have further applications in food.

1. Introduction Aronia melanocarpa, whose common name is black chokeberry, is a member of the Rosaceae family and originates from the eastern parts of North America (Bannasch, 2012). A. melanocarpa is documented as one of the best plant sources of bioactive phenolic substances, like anthocyanins, phenolic acids, flavonols and procyanidins (Simić et al., 2016). With the high levels of phenolic compounds, black chokeberry berry may have many beneficial health properties, such as antioxidant, antidiabetic, and immunomodulatory effects. Related research (Gawryś, Zawada, & Wawer, 2012; Hwang, Yoon, Lee, Cha, & Kim, 2014; Xu & Mojsoska, 2013) showed that A. melanocarpa had been beneficial in improving disorders or diseases associated with oxidative stress based

∗

on evidence from in vitro studies and animal experiments (Chrubasik, Li, & Chrubasik, 2010). Therefore, in some European and North American countries, black chokeberry has been used as both a traditional medicine and health food for many years (Białek, Rutkowska, & Hallmann, 2012). The total amount of anthocyanins in fresh A. melanocarpa varies between 357 and 461 mg/100 g fresh weight (FW) (Benvenuti, Pellati, Melegari, & Bertelli, 2004; Jakobek et al., 2007a, 2007b). For other natural sources (Cesoniene, Jasutiene, & Sarkinas, 2009; Kalt, Mcdonald, Ricker, & Lu, 1999; Kivi, Khavarinejad, Dehgan, Najafi, & Hajilo, 2013; Toromanovic et al., 2008; Wang et al., 2016), the total amount of anthocyanins in fresh blueberries varies between 83 and 370 mg/100 g (FW), the total amount of anthocyanins in fresh raspberry varies between 35 and 45 mg/100 g (FW), the average

Corresponding author. Corresponding author. E-mail addresses: [email protected] (G. Xin), [email protected] (X. Meng).

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https://doi.org/10.1016/j.fbio.2019.100413 Received 14 April 2018; Received in revised form 1 May 2019; Accepted 2 May 2019 Available online 07 May 2019 2212-4292/ © 2019 Elsevier Ltd. All rights reserved.

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Hailin, Heilongjiang, China) in June 2016, transported fresh and then stored at −80 °C (for a maximum of three months) before use. The Lonicera caerulea fruit tastes both sweet and sour with a little bitterness, the pericarp is blue-black, the pulp is bright red, the shape is spherical with a maximum longitudinal diameter of about 2.2 cm and transverse diameter of 1.2 cm.

content of anthocyanins in 4 varieties of blue honeysuckle varies between 400 and 458 mg/100 g (FW), and the total anthocyanins content of cranberry varies between 41 and 207 mg/100 g (FW). The total amount of anthocyanins in fresh A. melanocarpa and blue honeysuckle (Lonicera caerulea) are very close. Compared to other berries, the A. melanocarpa anthocyanins profile is very simple, consisting almost exclusively of cyanidin glycosides, which makes it easier to get highpurity anthocyanins and different anthocyanins monomer. Related research had shown that anthocyanins have a strong antioxidant capacity and the antioxidant capacity has a high positive correlation with the purity of anthocyanins (Kim et al., 2007). Therefore, the isolation of high-purity anthocyanins from A. melanocarpa may be beneficial. The separation methods for anthocyanins range from simple solvent extraction to various forms of chromatography. The separation of anthocyanins from plant materials had been studied using techniques such as solid-phase extraction (Saito et al., 1995), high-speed countercurrent chromatography (Degenhardt, Knapp, & Winterhalter, 2000), column chromatography (CC) (Giuliana, Fossen, & Anderson, 1998), and preparative high-performance liquid chromatography (HPLC) (Takeoka et al., 1997). CC, as an important purification technique, has been used to isolate flavonoids, tannins, and monomeric anthocyanins, especially with the extensive application of a polar ion exchange resin (Beninger & Hosfield, 2003). However, CC cannot meet the requirements of purfication because of low purification efficiency. Therefore, simple and effective methods for large scale purification of pure anthocyanins from natural products are needed. Related research had suggested that high purity anthocyanins mixtures and monomers could be isolated from wild blueberries using a combination of CC and semipreparative reverse phase HPLC (RPLC) (He & Giusti, 2011; Wang, Yan, Xu, & Liu, 2014). Therefore, conventional methods such as CC coupled with chromatographic separations such as RPLC can be used to fractionate or isolate pure products from plants, such as anthocyanins, and the combination of various methods also provides new opportunities for the extraction, isolation, and purification of anthocyanins. This study aimed to analysis the composition of anthocyanins in A. melanocarpa cultivated in Haicheng, Liaoning, China, isolate their highpurity anthocyanins, evaluate the antioxidant activity of these anthocyanins and investigate their potential application in food.

2.3. Determination of fresh weight and total soluble solids The fresh weight of black chokeberry was measured immediately after the fruit was harvested at the farm. The fruits have no pits. The 80 fruits harvested from different plants were randomly selected, and the average was taken. The total soluble solids of black chokeberry was measured immediately after the fruit was harvested at the farm, the fruit juice of black chokeberry was obtained using hand squeezing (wearing gloves) and the juice was used to measure the total soluble solid (TSS) using a hand-held refractometer (LB90T, Guangzhou Speed Electronic Technology Co., Ltd., Guangzhou, Guangdong, China). The test was repeated 10 times and the average was taken. 2.4. Extraction and determination of anthocyanins and polyphenols Approximately 250 g of black chokeberry were homogenized using a juicer (Joyoung Co., Ltd., Hangzhou, Zhejiang, China), after which approximately 1.25 l was acidified: 80% (v/v) ethanol with 0.1% HCl (v/v) was added to the homogenate at a material-to-solvent ratio of 1:5. The material was sonicated for 35 min in an ultrasonic bath (80 W) (SB25-12DTN, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, Zhejiang, China) and filtered using a qualitative filter paper (Hangzhou Special-paper Co., Ltd., Hangzhou, Zhejiang, China) using a vacuum pump (Yuhua Instrument Co., Ltd., Gongyi, Henan, China). The residue was re-extracted by repeating the extraction procedure about 3 times until the solvent remained clear. The extraction solvent was removed using rotary evaporation (RE-5203A, Shanghai Bilon Instruments Co., Ltd., Shanghai, China) at 28 °C until no alcohol remained and the anthocyanins extract was stored at 4 °C (for a maximum of 72 h) before use. Then, the anthocyanins content was measured using a pH differential method (Giusti & Wrolstad, 2001). Briefly, extracts were dissolved using distilled water, 1 ml of the sample (diluted 1:10) was mixed with 9 ml of potassium chloride buffer (pH = 1.0) and 9 ml of sodium acetate buffer (pH = 4.5), respectively, then incubated in the dark for 20 min at room temperature (20 °C). Then, the absorbances of the reacted mixtures were read at 520 and 700 nm, respectively, using the ELISA microplate reader (iMarkTM, Bio-Rad, Hercules, CA, USA). The total anthocyanin content, expressed as mg cyanidin-3-glucoside equivalents/g of black chokeberry fresh fruit, was calculated as follows:

2. Materials and methods 2.1. Chemicals The Folin–Ciocalteu reagent and, gallic acid were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). The acetonitrile, formic acid, T-AOC assay kit, ABTS assay kit, FRAP assay kit, dibutylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) were purchased from Dingguo Biological Technology Co., Ltd. (Shenyang, Liaoning, China). Cyanidin-3-O-(galactoside, glucoside, arabinoside, xyloside) standards were purchased from Tokiwa Phytochemical Co., Ltd. (Chiba, Chiba Prefecture, Japan).

(mg/g) = {[A520-A700) Anthocyanin content V × n × 1000}/(ε × m)

pH=1.0-(A520-A700)

pH=4.5] ×

Where V is the volume, n is the dilution factor, ε is the molar absorptivity (26900) and m is the fruit weight. In addition, the anthocyanins in Lonicera caerulea were extracted according to Wang et al. (2016). Briefly, ∼300 g of L. caerulea berries were homogenized, after which approximately 1 l acidified methanol (0.1% HCl, v/v) was added to the homogenate at a material-to-solvent ratio of 1:10. The material was incubated for 90 min in the ultrasonic bath and filtered. The residue was re-extracted by repeating the extraction procedure about 3 times until the solvent remained clear. The concentration and storage of this material was the same as the chokeberry. The total phenolic components in A. melanocarpa were extracted, and the total phenolic content was determined according to D’Alessandro, Kriaa, Nikov, and Dimitrov (2012). Briefly, tubes with 200 μl sample (diluted 1:10) and 1 ml Folin–Ciocalteu's reagent, were covered with aluminum foil. After a 4-min incubation at room

2.2. Plants and fruit sampling Black chokeberry cultivar “Fu Kangyuan No.1” was harvested (total soluble solids > 16% Brix) and transported (4 °C) from Liaoning Fu Kangyuan Black Chokeberry Technology Co., Ltd. (40°47′41″N, 122°40′42″E, Haicheng, Liaoning, China) in September 2016, and then stored at the College of Food Science in Shenyang Agricultural University at −80 °C (for a maximum of three months) before use. The black chokeberry fruit tastes sweet and sour with a slight astringency, the pericarp is purple-black, the pulp is dark-red, the shape is spherical and the diameter is about 1.4 cm “Beilei” Lonicera caerulea berries, which have similar anthocyanins content as A. melanocarpa, were chosen as a control. They were harvested (total soluble solids > 14% Brix) from an orchard in Hailin City (44°35′29.81″N, 129°22′23.37″E 2

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temperature, 800 μl of 75 g/l sodium carbonate solution was added, and the mixture was incubated in the dark for 120 min at room temperature. The absorbances were measured at 765 nm against a deionized water (Nongfu Spring Co., Ltd., Hangzhou, Zhejiang, China) blank using the ELISA microplate reader. A standard curve was prepared using gallic acid, and the results were expressed as mg gallic acid equivalents/g black chokeberry fresh fruit.

2.6.2. HPLC–DAD–EIS–MS2 analysis The analysis of anthocyanins was done using an HPLC system (Agilent1100, Agilent Technologies Co., Ltd., Palo Alto, CA, USA) equipped with a DAD detector. A Dikma Platisil C18 column (4.6 mm × 250 mm, 5 μm) (Agilent Technologies Co., Ltd.) was used at 25 °C. The optimized mobile phases were acetonitrile (A) and 0.1% formic acid in water (B). The following gradient was used: 0–45 min, 0–45% A; 45–50 min, 0% A. The injection volume was 20 μl, and the flow rate was maintained at 0.7 ml/min. Data were obtained at 520 nm. The MS2 data were collected using a mass spectrometer (Agilent5975E, Agilent Technologies Co., Ltd.) equipped with electrospray ionization (ESI) set in positive ionization mode. MS parameters were as follows: Automatic secondary MS scanning and data-dependent MS2 scanning from 50 to 1000 m/z; capillary voltage was controlled at +3.5 kV; nitrogen was used as the nebulizer gas at a pressure of 40 psi; nitrogen was heated to 350 °C at a flow rate of 12 l/min.

2.5. Purification of anthocyanins 2.5.1. Purification of anthocyanins using absorbent resin Anthocyanins extracted from A. melanocarpa were first purified using a NKA-9 absorbent resin, which is a polar ion exchange resin (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). Briefly, the activated resin was introduced into a 1.8 × 30 cm column, the anthocyanins were loaded into the column using a digital constant flow pump (Shanghai Huxi Analysis Instrument Factory Co., Ltd., Shanghai, China), the flow rate was 6 ml/min, the dilution ratio of the sample solution was 4, the loading amount was 425 ml and the time for adsorption saturation was 1 h. After that, the distilled water (2 times the column volume) was used to remove water soluble impurities. Then, ethanol (50%, v/v) was used to elute anthocyanins, the elution flow rate was 6 ml/min. The anthocyanins extracted from Lonicera caerulea were purified according to Wang et al. (2016). The concentrated solution was filtered using a qualitative filter paper and then separated through a 400 ml glass column loaded with nonionic polystyrene–divinylbenzene resin (D101, Shanghai Mosu Science Equipment Co., Ltd., Shanghai, China) at 4 °C. Briefly the anthocyanins were loaded into the column using the digital constant flow pump, the flow rate was 5 ml/min, the dilution ratio of the sample solution was 2, the loading amount was 490 ml and the time for adsorption saturation was 2 h. After that, the distilled water (2 times the column volume) was used to remove water soluble impurities. Then, methanol (100%, v/v) was used to elute anthocyanins, the elution flow rate was 5 ml/min. Finally, the crude extracts of anthocyanins from A. melanocarpa and Lonicera caerulea were obtained, solvents were eliminated using rotary evaporation, and the compounds were transferred to water and freezedried using a vacuum freeze-drier (Labconco Co., Kansas City, MO, USA). The anthocyanins powders were stored at −20 °C (for a maximum of 4 weeks) before use and the anthocyanins content was measured using the pH differential method.

2.7. Quantification and identification of anthocyanins using HPLC The total anthocyanins extract (32 μg) purified using RPLC was diluted with 2 ml methanol, filtered using a 0.45-μm filter, then anthocyanins were quantified and identified using HPLC at 520 nm. It was assumed that the peak areas for each peak was identical on a molecular basis regardless of the chemistry of molecule, the anthocyanins were identified based on the cyanidin-3-O-(galactoside, glucoside, arabinoside, and xyloside) standards and quantified based on the calibration curves of cyanidin-3-xyloside. The standards concentrations were 2, 4, 8, 10 and 20 μg/ml, and the detailed conditions were described in the HPLC–DAD–EIS–MS2 analysis section above. 2.8. Total antioxidant capacity assessment The total antioxidant capacity of different sample, including A. melanocarpa juice (A. melanocarpa), A. melanocarpa anthocyanins extracts purified using absorbent resin (A-anthocyanins-A), A. melanocarpa anthocyanins extracts purified using absorbent resin and semipreparative RPLC (A-anthocyanins-R), L. caerulea juice (L. caerulea), and L. caerulea anthocyanins extracts purified using absorbent resin (Lanthocyanins-A) were evaluated using T-AOC, ABTS and FRAP kits, respectively. 2.8.1. T-AOC assay A. melanocarpa anthocyanins could reduce Fe3+ to Fe2+, which could form a stable complex with phenanthrolines. The total antioxidant capacity of anthocyanins was determined using a T-AOC kit based on colorimetry. Briefly, a 10.00 mg sample was diluted with 2 ml methanol. Based on the manufacturer's instruction, 1 ml reagent 1 was added into the test tube and control tube, 0.2 ml sample was added, 2 ml reagent 2 and 0.5 ml reagent 3 were added, and hand shaken before placing in a water bath at 37 °C for 30 min. Then 0.2 ml reagent 4 was added, 0.2 ml sample was added into the control tube, and then 0.2 ml reagent 5 was added and kept at room temperature (20 °C), the reaction systems were blended and placed at room temperature for 10 min. The absorbance was measured at 520 nm using the ELISA microplate reader, and one T-AOC unit (U) was defined as mg of sample/ min at 37 °C for each 0.01 increase in the absorbance of the reaction system. The antioxidant capacity was expressed as T-AOC U/mg.

2.5.2. Purification of anthocyanins using RPLC The RPLC using a LC3000 HPLC (Beijing Tong Heng Innovation Technology Co., Ltd., Beijing, China) was used to further isolate and purify the A. melanocarpa anthocyanins. The column was a Daisogei C18 (30 cm × 250 mm × 10 μm, 120 A) (Beijing Tong Heng Innovation Technology Co., Ltd.), coupled with a UV-vis detector (Beijing Tong Heng Innovation Technology Co., Ltd.). Mobile phases were methanol (A) and 2.5% formic acid (B) with the following gradient: 22% isocratic A for 10 min, from 22 to 27% A over 7 min, from 27 to 30% A over 50 min, and from 30 to 50% A over 53 min at a flow rate of 5 ml/min. The concentration and volume of the anthocyanins were 1.5 mg/ml and 4 ml, respectively. Detection was carried out at 520 nm, and all the anthocyanins absorption peaks were collected in a fraction collector. Solvents were removed using rotary evaporation, the compounds were transferred to water and freeze-dried, and the anthocyanins powders were stored at −20 °C (for a maximum of 4 wk) before use.

2.8.2. ABTS assay A 10.00 mg extract was diluted with 2 ml methanol, and trolox (Dingguo Biological Technology Co., Ltd., Shenyang, Liaoning, China) was selected as the standard solution. The ABTS radical was generated through a chemical oxidation reaction with potassium persulfate according to Stratil, Klejdus, and Kubán (2006). Briefly, 0.2 ml of 7.4 mM ABTS solution and 0.2 ml of 2.6 mM potassium persulfate solution were mixed and kept at room temperature for 12 h. The concentration of the

2.6. Identification of anthocyanins using HPLC-MS 2.6.1. Sample preparation A 10.00 mg purified anthocyanins sample was diluted with 2 ml methanol, filtered using a 0.45-μm filter (Dingguo Biological Technology Co., Ltd., Shenyang, Liaoning, China), and analyzed using HPLC–DAD–ESI–MS2 according to Wang et al. (2016). 3

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ABTS radical solution was adjusted with ethanol to an absorbance of 0.70 at 734 nm. Then, 0.2 ml sample, 0.2 ml 80% ethanol or 0.2 ml trolox were added to 0.8 ml of ABTS radical solution and incubated at room temperature for 6 min, and the absorbance at 734 nm was measured immediately using the ELISA microplate reader. The antioxidant capacity was calculated from the linear trolox calibration curve and expressed as trolox equivalents/mM). 2.8.3. FRAP assay A 10.00 mg extract was diluted with 4 ml methanol. The ability to reduce ferric ions was measured using a modified version of the method of Benzie and Strain (1996). An aliquot (5 μl) of each sample was added to 180 μl of FRAP reagent, and the reaction mixture was incubated at 38 °C for 5 min. The increase in absorbance at 593 nm was measured using the ELISA microplate reader. Fresh working solutions of FeSO4 were used for calibration. The antioxidant capacity was based on the ability to reduce the ferric ions in the samples calculated from the linear calibration curve and expressed as FeSO4·7H2O equivalents/mM. 2.9. Application of anthocyanins in food Lard was selected as a food model to evaluate the antioxidant capacity of selected and identified anthocyanins (A-anthocyanins-R), and the method was based on a modified version of the method described by Li, Ma, Meng, and Yu (2010). Lard was obtained from fresh pork (Nongda Non-staple Food Supermarket, Shenyang, Liaoning, China) by boiling for 10 min. Samples (20 mg) of BHA, BHT or anthocyanins were mixed with 200 g lard. In addition, 200 g lard was used as a control. Then all the samples were placed in a 50 °C incubator, stirred once every 10 min. The peroxide value (POV) as mmol/kg was used to evaluate the oxidation rate of lard and was determined every 2 h. Briefly, the determination of POV is based on iodometry, peroxides in lard could react with excess KI to form I2 in acid conditions, and the I2 could be titrated using Na2S2O3. The POV of lard could be calculated according to the amount of Na2S2O3, the equation was as follows.

Fig. 1. RPLC chromatogram of black chokeberry anthocyanins after purification using NKA-9 resin at 520 nm.

3.2. Purification and identification of anthocyanins using absorbent resin and HPLC-MS Anthocyanins, extracted from A. melanocarpa and L. caerulea, were purified using absorbent resin. The anthocyanins contents were very close and were about 280 ± 10 mg/g dry weight (DW). The HPLC-MS results are shown in Fig. 2. A total of 11 peaks were observed using HPLC at 520 nm (Fig. 2a), and 7 different anthocyanins were identified based on their HPLC retention times, elution order, spectroscopic characteristics, and fragmentation pattern (Table 1). These components were identified as 7 different anthocyanidins peaks 1, 2, 4–7, 10, including cyanidin-3,5-dihexoside (peak 1), the dimer of cyanidin-hexoside (peak 2), cyanidin-3-O-galactoside (peak 4), cyanidin-3-O-glucoside (peak 5), cyanidin-3-O-arabinoside (peak 6), cyanidin-3-O-xyloside (peak 7) and delphinidin-3-O-rutinoside (peak 10). These anthocyanins were mainly divided into two categories, including cyanidin (peak 1, 2, 4–7) and delphinidin (peak 10).

POV (mmol/kg) = 1000 (V-V0) c/m where V is the volume of Na2S2O3 solution determined (ml), V0 is the volume of Na2S2O3 solution with the control (ml), c is the concentration of Na2S2O3 solution (mol/l), m is the sample weight (g). 2.10. Statistical analysis Each experiment was done in triplicate, and mean values with standard deviations were obtained using Excel software (Version 2003; Microsoft Corp., Redmond, WA, USA). The IBM Statistical Program for the Social Sciences (SPSS) Version 17.0 (IBM Corp., Armonk, NY, USA) was used to do the statistical analysis. The significance difference between any two mean values was determined using two-way ANOVA at the 95% confidence level (p ≤ 0.05), and significant difference among groups were determined using LSD and Duncan's multiple range test (p ＜0.05). The graphing was done using Excel Software Version 2003 (Kingsoft Corp., Beijing, China).

3.3. Quantification and identification of anthocyanins Four typical anthocyanins were successfully separated using RPLC (Fig. 1) and identified using HPLC (Fig. 3). The anthocyanins were cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, cyanidin-3-O-arabinoside and cyanidin-3-O-xyloside, and the anthocyanins content was 930 mg/g (DW) based on the cyanidin-3-xyloside calibration curves (Table 3). 3.4. Determination of antioxidant activity using T-AOC assay

3. Results A-anthocyanins-R had the highest T-AOC value, which reached 56.6 U/mg, and the T-AOC value of A-anthocyanins-R was significantly higher than the vitamin C (Vc), A. melanocarpa, and A-anthocyanins-A (p＜0.05), for which the T-AOC values were 17.0, 6.86, and 38.6 U/mg, respectively (Fig. 4). The antioxidant capacity increased by 46.5% after purification using RPLC. In addition, the T-AOC value of L-anthocyanins-A (37.8 U/mg) was slightly lower than A-anthocyanins-A, and the T-AOC value of L. caerulea (4.86 U/mg) was slightly lower than A. melanocarpa.

3.1. Determination of fresh weight, total soluble solids, total anthocyanins and total phenols The weight of black chokeberry cultivated in Haicheng, China was 1.2 ± 0.1 g/fruit (FW), the total soluble solid content was 16.6 ± 0.1 Brix%, The total anthocyanins and total phenol contents were 380 ± 20 mg/100 g (FW) and 1900 ± 100 mg/100 g (FW), respectively. 4

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Fig. 2. HPLC chromatogram of black chokeberry anthocyanins after purification using NKA-9 resin at 520 nm (a) and MS spectra of peak 1 (b), peak 2 (c), peak 4 (d), peak 5 (e), peak 6 (f), peak 7 (g), and peak 10 (h).

Fig. 2. (continued) 5

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Table 1 Anthocyanins identified in black chokeberry purified using NKA-9 resin. Peak

Peak area (%)

Retention time (min)

[M]+ (m/z)

[M]2+(m/z)

Anthocyanin

1 2 4 5 6 7 10

0.4 0.6 66.9 3.8 23.6 4.7 0.1

14.32 15.54 17.10 17.90 18.51 20.30 27.49

610.9 869.8 448.6 448.7 418.8 418.8 610.3

448.7/286.9 735.0/573.0/447.2 286.6 286.8 286.9 286.9 464.6/302.9

Cyanidin-3,5-dihexoside Dimer of cyanidin-hexoside Cyanidin-3-O-galactoside Cyanidin-3-O-glucoside Cyanidin-3-O-arabinoside Cyanidin-3-O-xyloside Delphinidin-3-O-rutinoside

3.5. Determination of antioxidant activity using ABTS assay

3.6. Determination of antioxidant activity using FRAP assay

The highest ABTS·+ scavenging ability was found for Vc (0.59 mM), the ABTS·+ scavenging ability of A-anthocyanins-R (0.53 mM) was significantly higher than A. melanocarpa (0.08 mM), A-anthocyanins-A (0.42 mM), L. caerulea (0.09 mM) and L-anthocyanins-A (0.35 mM) (p＜ 0.05) (Fig. 5). This order was similar to that of the T-AOC assay. The antioxidant capacity of anthocyanins increased by 29.3% after purification using RPLC.

The antioxidant abilities of A-anthocyanins-R (6.52 mM) was significantly higher than Vc (3.10 mM), A. melanocarpa (0.68 mM), A-anthocyanins-A (4.82 mM), L-anthocyanins-A (4.36 mM) and L. caerulea (0.42 mM) (p＜0.05) (Fig. 6). The antioxidant capacity of anthocyanins increased by 35.3% after purification using RPLC. 3.7. Application of anthocyanins in food The high-purity anthocyanins (A-anthocyanins-R), prepared using

Fig. 3. HPLC chromatogram of black chokeberry anthocyanins after purified using RPLC (A), HPLC chromatogram of standards mixed with cyanidin-3-O-(galactoside, glucoside, arabinoside, xyloside) (B), HPLC chromatogram of cyanidin-3-O-galactoside standard (C), HPLC chromatogram of cyanidin-3-O-glucoside standard (D), HPLC chromatogram of cyanidin-3-O-arabinoside standard (E) and HPLC chromatogram of cyanidin-3-O-xyloside standard (F). 6

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melanocarpa contains the highest amounts of polyphenols and anthocyanins in comparison to blackberries, red raspberries and strawberries according to Jakobek et al. (2007a). Previous studies reported that the total amount of anthocyanins in fresh A. melanocarpa varied between 357 and 461 mg/100 g (FW), and the total phenol content in fresh A. melanocarpa varied between 719 and 2560 mg/100 g (FW) (Benvenuti et al., 2004; Rop et al., 2010). In this study, the contents of total anthocyanins and total phenols were 380 mg/100 g (FW) and 1900 mg/ 100 g (FW), respectively. They were consistent with the previous findings. It can be seen that there is a high anthocyanins content in A. melanocarpa from Haicheng, China. It is important to analyze the anthocyanins composition, obtain a high-purity anthocyanins mixture or monomers and study the oxidation resistance of anthocyanins. The anthocyanins composition in A. melanocarpa from different places with different methods had been studied, and the identification of anthocyanins in A. melanocarpa is summarized in Table 2. There were many references that identified the structure of anthocyanins (Dembczyński et al., 2015; Jakobek et al., 2007a; Jan & Sapis, 2010; Kapci, Neradová, Čížková, & Rajchl, 2013; Oszmiański & Wojdylo, 2005; Slimestad, Torskangerpoll, Nateland, Johannessen, & Giske, 2005; Zheng & Wang, 2003). Their findings were consistent across the trials and the structure of anthocyanins in A. melanocarpa consisted of cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, cyanidin-3-O-arabinoside and cyanidin-3-O-xyloside. The 4 kinds of anthocyanins should be the main anthocyanins in A. melanocarpa. Furthermore, anthocyanins were identified by Wu, Gu, Prior, and Mckay (2004), except for the 4 anthocyanins in A. melanocarpa which were identified above. Pelargonidin-3-O-arabinoside was first found in A. melanocarpa. In addition, Rop et al. (2010) only found two anthocyanins, cyanidin-3-Oarabinoside and cyanidin-3-O-galactoside, in A. melanocarpa from Denmark, Sweden and Finland. There was also a study to identify anthocyanins in A. melanocarpa, Tao et al. (2017) identified cyanidin-3-Oglucoside, cyanidin-3-O-rutinoside and delphinidin-3-O-rutinoside in A. melanocarpa. Cyanidin-3-O-rutinoside and delphinidin-3-O-rutinoside were first identified in A. melanocarpa (Dembczyński et al., 2015; Jakobek et al., 2007a; Jan & Sapis, 2010; Kapci et al., 2013; Oszmiański & Wojdylo, 2005; Rop et al., 2010; Slimestad et al., 2005; Wu et al., 2004; Tao et al., 2017; Zheng & Wang, 2003). Anthocyanins extracted from A. melanocarpa were identified using HPLC-MS. A total of 11 anthocyanins peaks were observed using HPLC. Peaks 1, 2, 4–7, and 10 may be anthocyanins according to the molecular ion peak and ion fragment peak. The retention time of peak 1 was 14.32 min, the molecular ion peak was 610.9 (m/z), and the ion fragment peaks were 448.7/286.9 (m/z), while the retention time of peak 2 was 15.60 min, the molecular ion peak was 869.8 (m/z), and the ion fragment peaks were 735.0/573.0/447.2 (m/z). The anthocyanins followed a general retention order based on the degree of polarity of the molecular structure, which was primarily affected by three components of an anthocyanins within an HPLC system (Barnes, Nguyen, Shen, &

Table 2 Identification of anthocyanins in A. melanocarpa. Reference

Anthocyanins

Zheng and Wang (2003) Wu et al. (2004) Slimestad et al., 2005 Oszmiański & Wojdylo, 2005 Jakobek et al., 2007a Rop et al. (2010) Jan & Sapis, 2010 Kapci et al. (2013) Dembczyński et al., 2015 Tao et al., 2017 This study

A A A A A A A A A – A

B B B B B B B B B – B

C C C C C – C C C C C

D D D D D – D D D – D

– E – – – – – – – – –

– – – – – – – – – F –

– – – – – – – – – G G

– – – – – – – – – – H

– – – – – – – – – – I

Note: A, cyanidin-3-O-arabinoside; B, cyanidin-3-O-galactoside; C, cyanidin-3O-glucoside; D, cyanidin-3-O-xyloside; E, pelargonidin-3-O-arabinoside; F, cyanidin-3-O-rutinoside; G, delphinidin-3-O-rutinoside; H, cyanidin-3,5-dihexoside; I, dimer of cyanidin-hexoside; -, unidentified. Table 3 Quantification of anthocyanins purified by RPLC using HPLC. Standard

Time (min)

Concentration (ug/mL)

Peak area

Standard curve line

cyanidin-3-Oxyloside

21.53

2.0 4.0 8.0 10.0 20.0

40 74 157 195 381

y = 19.10x-9.478e−1

Sample

cyanidin-3-Ogalactoside cyanidin-3-Oglucoside cyanidin-3-Oarabinoside cyanidin-3-Oxyloside

Anthocyanin content(DW) 17.14

16

154

18.08

10

19.24

99

21.84

24

930 mg/g

absorbent resin coupled with semi-preparative RPLC, was used to evaluate the antioxidant properties when used with lard (Fig. 7). The results showed that anthocyanins and synthetic antioxidants BHT, BHA could effectively slow the oxidation rate of lard, and the antioxidant properties of anthocyanins were better than BHT and BHA. 4. Discussion Anthocyanins in A. melanocarpa have been attracting more and more attention as a result of the high antioxidant activity. A.

Fig. 4. Antioxidant activities of A. melanocarpa, A. melanocarpa anthocyanin extracts purified using absorbent resin (Aanthocyanins-A), A. melanocarpa anthocyanins extracts purified using absorbent resin and semi-preparative RPLC (A-anthocyanins-R), ‘Beilei’ L. caerulea (L. caerulea), ‘Beilei’ L. caerulea berry anthocyanin extracts purified using absorbent resin (L-anthocyanins-A), and VC (Vc) using the T-AOC assay. The values represent the mean ± SD of three independent experiments, *p < 0.05 was considered significant.

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Fig. 5. Antioxidant activities of A. melanocarpa, A. melanocarpa anthocyanins extracts purified using absorbent resin (A-anthocyanins-A), A. melanocarpa anthocyanins extracts purified using absorbent resin and semi-preparative RPLC (A-anthocyanins-R), ‘Beilei’ L. caerulea (L. caerulea), ‘Beilei’ L. caerulea berry anthocyanins extracts purified using absorbent resin (L-anthocyanins-A), and VC (Vc) using an ABTS assay. The values represent the mean ± SD of three independent experiments, *p < 0.05 was considered significant.

Fig. 6. Antioxidant activities of A. melanocarpa, A. melanocarpa anthocyanins extracts purified using absorbent resin (A-anthocyanins-A), A. melanocarpa anthocyanins extracts purified using absorbent resin and semi-preparative RPLC (A-anthocyanins-R), ‘Beilei’ L. caerulea (L. caerulea), ‘Beilei’ L. caerulea berry anthocyanins extracts purified using absorbent resin (L-anthocyanins-A), and VC (Vc) using a FRAP assay. The values represent the mean ± SD of three independent experiments, *p < 0.05 was considered significant.

Fig. 7. Antioxidant activity of anthocyanins, BHT, BHA in lard.

while the retention time of peak 5 was 17.90 min, the molecular ion peak was 448.7 (m/z), and the ion fragment peak was 286.8 (m/z). The sugar substituents of peak 4 and peak 5 were isomers and were glucose and galactose, respectively. Therefore, peak 4 and peak 5 should be cyanidin-3-O-galactoside and cyanidin-3-O-glucoside depending on the order of the attached glycosides consistent with the references. Similarly, peak 6 and peak 7 should be cyanidin-3-O-arabinoside and cyanidin-3-O-xyloside, respectively, and peak 10 should be delphinidin-3O-rutinoside. Therefore, 7 anthocyanins, including the 4 main anthocyanins that had been reported several times, were identified in A. melanocarpa from Haicheng, China. In addition, delphinidin-3-O-rutinoside that was reported only once previously was also identified. Cyanidin-3,5-dihexoside and cyanidin-hexoside were first found in A. melanocarpa, and the pelargonidin-3-O-arabinoside and cyanidin-3-O-

Schug, 2009; Nicoué, Savard, & Belkacemi, 2007; Wu & Prior, 2005). The different anthocyanidins would follow this elution series (from shortest to longest retention time): Delphinidin, cyanidin, petunidin, pelargonidin, peonidin, and malvidin. Those that differ only by their number and type of glycosides would follow this elution series (from shortest to longest retention time): 3,5-Diglucoside, 3-diglucoside, galactoside, sambubioside, glucoside, arabinoside, rutinoside, and xyloside. Those that differ only by their acylation would elute after any nonacylated species and would follow this elution series (from shortest to longest retention time): Malonoyl, acetoyl, and coumoroyl. So, peak 1 should be cyanidin-3,5-dihexoside and peak 2 should be the dimer of cyanidin-hexoside consistent with the references (Wang et al., 2016). In addition, the retention time of peak 4 was 17.10 min, the molecular ion peak was 448.6 (m/z), and the ion fragment peak was 286.6 (m/z), 8

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liquid chromatography-electrospray ionization-ion trap-time of flight-mass spectrometry. Journal of Chromatography A, 1216, 4728. Beninger, C. W., & Hosfield, G. L. (2003). Antioxidant activity of extracts, condensed tannin fractions, and pure flavonoids from Phaseolus vulgaris L. seed coat color genotypes. Journal of Agricultural and Food Chemistry, 27, 7879–7883. Benvenuti, S., Pellati, F., Melegari, M., & Bertelli, D. (2004). Polyphenols, anthocyanins, ascorbic acid, and radical scavenging activity of Rubus, Ribes, and Aronia. Journal of Food Science, 69, FCT164–FCT169. Benzie, I. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": The FRAP assay. Analytical Biochemistry, 239, 70–76. Białek, M., Rutkowska, J., & Hallmann, E. (2012). Black chokeberry (Aronia melanocarpa) as potential component of functional food. Zywnosc Nauka Technologia Jakosc, 85, 21–30. Cesoniene, L., Jasutiene, I., & Sarkinas, A. (2009). 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(2012). Aronia in the diet of diabetics. Diabetologia Kliniczna, 5, 196–200. Giuliana, C., Fossen, T., & Anderson, O. M. (1998). Petunidin 3-O-α-rhamnopyranoside-5O-β-glucopyranoside and other anthocyanins from flowers of Vicia villosa. Journal of Agricultural and Food Chemistry, 46, 4568–4570. Giusti, M. M., & Wrolstad, R. E. (2001). Characterization and measurement of anthocyanins by UV-visible spectroscopy. Food Analytical Chemistry, 2, 63–69. He, J., & Giusti, M. M. (2011). High-purity isolation of anthocyanins mixtures from fruits and vegetables–a novel solid-phase extraction method using mixed mode cation-exchange chromatography. Journal of Chromatography A, 1218, 7914–7922. Hwang, S. J., Yoon, W. B., Lee, O. H., Cha, S. J., & Kim, J. D. (2014). Radical-scavenginglinked antioxidant activities of extracts from black chokeberry and blueberry cultivated in Korea. Food Chemistry, 146, 71–77. Jakobek, L., Šeruga, M., Medvidovic´Kosanovic´, M., Novak, I., & Voc´A, N. (2007a). 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D., Mazza, G., Holub, B. J., & Wang, J. (2004). Anthocyanin metabolites in human urine and serum. British Journal of Nutrition, 91, 933–942. Kim, M. J., Hyun, J. N., Kim, J. A., Park, J. C., Kim, M. Y., Kim, J. G., et al. (2007). Relationship between phenolic compounds, anthocyanins content and antioxidant activity in colored barley germplasm. Journal of Agricultural and Food Chemistry, 55, 4802. Kivi, A. R., Khavarinejad, R. A., Dehgan, G., Najafi, F., & Hajilo, J. (2013). Antioxidant capacity and phytochemical properties of raspberry species in Iran. International Journal of Biosciences, 3, 145–152. Li, Y., Ma, C., Meng, X., & Yu, N. (2010). Antioxidation of anthocyanins from blueberry fruits (in Chinese). Journal of the Chinese Cereals & Oils Association, 2, 93–95. Nicoué, E. E., Savard, S., & Belkacemi, K. (2007). Anthocyanins in wild blueberries of Quebec: Extraction and identification. Journal of Agricultural and Food Chemistry, 55, 5626. Oszmiański, J., & Wojdylo, A. (2005). 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rutinoside were not found. Anthocyanins were purified using absorbent resin coupled with semi-preparative RPLC, and the anthocyanins content increased from 280 to 930 mg/g (DW). Then the total antioxidant capacity of anthocyanins with different purity were studied using T-AOC, ABTS and FRAP. The results showed that the antioxidant activity of anthocyanins increased significantly with the increase of anthocyanins purity. The total amount of anthocyanins in fresh A. melanocarpa and L. caerulea were similar, therefore, the antioxidant activity of anthocyanins in L. caerulea may help to evaluate antioxidant activity of anthocyanins in A. melanocarpa. Related research had shown that anthocyanins could be studied with humans (Kay et al., 2004, 2005). Cyanidin-3-glycosides could be rapidly metabolized and absorbed extensively following a moderate-tohigh oral dose, and the metabolites were identified as glucuronide conjugates, as well as methylated, oxidized derivatives of cyanidin-3galactoside and cyanidin-glucuronide, which have no negative effect on the human body. In this study, the high-purity anthocyanins (930 mg/g (DW)), prepared using absorbent resin coupled with semi-preparative RPLC, was further used to evaluate the antioxidant properties with lard. It was observed that anthocyanins were good antioxidants. Phenolic hydroxyl groups of anthocyanins, as a hydrogen donor, have the ability to capture free radicals, which can inhibit the action of reactive oxygen radicals. On the other hand, anthocyanins having an ortho-diphenolic hydroxyl group can bind metal ions and reduce the catalytic effect of metal ions on oxidation reaction. 5. Conclusion Overall, A. melanocarpa is a source of cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, cyanidin-3-O-arabinoside and cyanidin-3-Oxyloside, and may contain cyanidin-3,5-dihexoside, cyanidin-hexoside, delphinidin-3-O-rutinoside, cyanidin-3-O-rutinoside and pelargonidin3-O-arabinoside, depending on the A. melanocarpa varieties from different places and extraction methods. This is possibly because the main structure of anthocyanins was determined by genetic information, while the geographical location and the environment may lead to different anthocyanins. This study confirmed the previous research findings. However, the structure of anthocyanins in A. melanocarpa was further supplemented. In addition, the high-purity anthocyanins in A. melanocarpa cultivated in Haicheng, Liaoning, China could be prepared using absorbent resin coupled with semi-preparative RPLC, and the antioxidant capacity of the purified anthocyanins was higher. They had a strong antioxidant against lard. Conflicts of interest All the authors declare that they have no conflict of interest. Acknowledgements We acknowledge the financial support from the following sources: The “Thirteenth Five Year” National Key Research and Development Program of China (Contract No. 2017YFD0400704-4). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fbio.2019.100413. References Bannasch, N. (2012). Aronia melanocarpa (black chokeberry) – the next European superfruit. Wellness Foods Europe, 1, 46. Barnes, J. S., Nguyen, H. P., Shen, S. J., & Schug, K. A. (2009). General method for extraction of blueberry anthocyanins and identification using high performance

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Wang, Y., Zhu, J., Meng, X., Liu, S., Mu, J., & Ning, C. (2016). Comparison of polyphenol, anthocyanin and antioxidant capacity in four varieties of Lonicera caerulea berry extracts. Food Chemistry, 197, 522–529. Wu, X., Gu, L., Prior, R. L., & Mckay, S. (2004). Characterization of anthocyanins and proanthocyanidins in some cultivars of Ribes, Aronia, and Sambucus and their antioxidant capacity. Journal of Agricultural and Food Chemistry, 7846–7856. Wu, X., & Prior, R. L. (2005). Systematic identification and characterization of anthocyanins by HPLC-ESI-MS/MS in common foods in the United States: Fruits and berries. Journal of Agricultural and Food Chemistry, 53, 2589–2599. Xu, J., & Mojsoska, B. (2013). The immunomodulation effect of Aronia extract lacks association with its antioxidant anthocyanins. Journal of Medicinal Food, 16, 334–342. Zheng, W., & Wang, S. Y. (2003). Oxygen radical absorbing capacity of phenolics in blueberries, cranberries, chokeberries, and lingonberries. Journal of Agricultural and Food Chemistry, 51, 502.

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