Comparison of polyphenol, anthocyanin and antioxidant capacity in four varieties of Lonicera caerulea berry extracts

Food Chemistry 197 (2016) 522–529

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Comparison of polyphenol, anthocyanin and antioxidant capacity in four varieties of Lonicera caerulea berry extracts Yuehua Wang a, Jinyan Zhu a,b, Xianjun Meng a,⇑, Suwen Liu a,c, Jingjing Mu a, Chong Ning a a

College of Food Science, Shenyang Agricultural University, Shenyang, Liaoning 110866, China Food Inspection Monitoring Center of Zhuanghe, Dalian, Liaoning 116400, China c Department of Food Science and Technology, Hebei Normal University of Science and Technology, Qinhuangdao, Hebei 066004, China b

a r t i c l e

i n f o

Article history: Received 31 August 2015 Received in revised form 29 October 2015 Accepted 2 November 2015 Available online 3 November 2015 Chemical compounds studied in this article: Cyanidin-3-glucoside (PubChem CID: 441667) Peonidin-3-glucoside (PubChem CID: 443654) Delphinidin-3-glucoside (PubChem CID: 443650) Pelargonidin-3-glucoside (PubChem CID: 443648) Pelargonidin-3-rutinoside (PubChem CID: 44256626) Peonidin-3-rutinoside (PubChem CID: 44256842) Trolox (PubChem CID: 40634) Cyanidin-3-galactoside (PubChem CID: 44256700) Cyanidin-3-sambubioside (PubChem CID: 6602304) Gallic acid (PubChem CID: 370)

a b s t r a c t Four varieties of Lonicera caerulea berries—‘Wild’, ‘Beilei’, ‘No. 1’, and ‘No. 2’—were compared with respect to extraction yield, fruit weight, total soluble solids, polyphenol and anthocyanin contents, oxygen radical absorbance capacity (ORAC), and anthocyanin composition. Sixteen individual anthocyanins were identified in the selected varieties. Acylated anthocyanins, cyanidin 3-acetylhexoside and peonidin 3-acetylhexoside, were identified in L. caerulea berries for the first time. Cyanidin-3-glucoside was the most prominent anthocyanin in all four tested varieties. Wild type of L. caerulea fruit (‘Wild’), with the highest polyphenol content, contained 14 anthocyanins and the highest ORAC value. Eleven anthocyanins were found in ‘Beilei’ berries, which had a higher ORAC value than ‘No. 1’ and ‘No. 2’. The highest total soluble solid content and extraction yield were found in ‘No. 2’ and ‘Wild’ berries, respectively. Ó 2015 Elsevier Ltd. All rights reserved.

Keywords: Lonicera caerulea berries Polyphenols Anthocyanins Identification Antioxidant activity

1. Introduction Lonicera caerulea (blue honeysuckle, Caprifoliaceae) berries, which are native to Europe, are oval or elongated in shape and dark navy blue to purple in color, and are commonly used as folk medicine (Wu et al., 2015), but are less known as edible fruits because ⇑ Corresponding author. E-mail address: [email protected] (X. Meng). http://dx.doi.org/10.1016/j.foodchem.2015.11.006 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

of their bitterness and astringency. Currently, an increasing number of reports have suggested that polyphenolic compounds, especially anthocyanins, are the prominent functional components in L. caerulea berries. These compounds have a wide range of bioactive properties including antioxidant, anti-inflammatory, antimicrobial, anti-radiation, cardioprotective, gastroprotective, hepatoprotective, and other properties (Martin et al., 2014; Vostálová et al., 2013; Xie et al., 2011; Zhu, Zhang, & Lo, 2004). As a consequence, the berries are attracting more and more

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attention. In China, L. caerulea berries are mostly distributed in the northeast region. The ‘Wild’ (hereafter Wild) berries and a cultivar named ‘Beilei’ (hereafter Beilei) are well known. Other cultivars with larger fruit size and better flavor have been cultivated in recent years, but have not yet been registered. Anthocyanins, which possess potential health benefits that may be ascribed to their high antioxidant activities (Reig, Iglesias, Gatius, & Alegre, 2013), are a group of widely distributed plant pigments; they are derived from anthocyanidins and sugar through a glycoside bond (Szajdek & Borowska, 2008; Veberic, Slatnar, Bizjak, Stampar, & Mikulic-Petkovsek, 2015). The most commonly occurring anthocyanidins are petunidin, cyanidin, pelargonidin, delphinidin, peonidin, and malvidin, and the sugars are arabinose, galactose, glucose, rhamnose, and xylose (Latti, Kainulainen, Hayirlioglu-Ayaz, Ayaz, & Riihinen, 2009). The composition of anthocyanins varies among plant species and even different cultivars within a species and tissues within a plant (Kim, Kim, Kim, & Park, 2013). Previous studies have shown that the anthocyanins in L. caerulea berries include glucoside and rutinoside of cyanidin, peonidin, and delphinidin, along with 3,5-dihexoside of cyanidin and peonidin that have not been found in some other berries such as blueberry. This may be one reason why L. caerulea berries have a higher antioxidant capacity than blueberries (Myjavcová et al., 2010; Palíková et al., 2008). Moreover, research has shown that the anthocyanin composition found in L. caerulea berry extracts varies depending on the extraction medium (Myjavcová et al., 2010). There is little knowledge about anthocyanin composition in different genotypes of L. caerulea berries, especially the better-tasting new varieties cultivated in northeast China. Therefore, the objectives of this study were: (i) to measure the total polyphenol and total anthocyanin contents and the antioxidant capacity of extracts of berries from four L. caerulea varieties; (ii) to identify individual anthocyanins in these L. caerulea berry extracts; and (iii) to compare the antioxidant (total polyphenol and anthocyanin) content, antioxidant activity, and characteristic anthocyanins in the selected berry extracts. The results will be used to select one cultivar for future functional study, which will provide a theoretical reference for identifying functional fruits. 2. Materials and methods 2.1. Chemicals All of the chemical reagents used in the present study were analytical or high-performance liquid chromatography (HPLC) grade. The Folin–Ciocalteau reagent, gallic acid, and other compounds used to determine total polyphenols and anthocyanins; acetonitrile and formic acid used for the analysis of anthocyanins; and 2,20 -Azobis (2-amidinopropane) dihydrochloride (AAPH) (95% purity) and the Trolox standard used for ORAC value analysis were purchased from Dingguo Biological Technology Co., LTD (Liaoning, Shenyang, China). 2.2. Plants and fruit sampling Wild L. caerulea berries were harvested in Baishan City (41°560 17.7900 N, 126°250 6.8500 E), Jilin Province, China on June 20, 2014. Three cultivars, one identified as Beilei and two unidentified cultivars here called No. 1 and No. 2, were grown in an orchard in Hailin City (44°350 29.8100 N, 129°220 23.3700 E), Heilongjiang Province, China. The Beilei cultivar was cultivated along with an introduced variety from Russia. It was identified by experts in the field in July 2010 and recorded by the Non-Main Crop Varieties Register Office

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of Heilongjiang Province in April 2011. The Beilei fruits taste both sweet and sour with a little bitterness, and the individual fruits are much bigger than those of wild berries, which taste only bitter. No. 1 fruits, which taste similar to Beilei fruits, are elongated in shape with a maximum longitudinal diameter of about 2.2 cm and transverse diameter of 1.2 cm. No. 2 fruits, which taste sweet with a little sour, are bigger than Beilei fruits. Berries were harvested from plants grown in the orchard (three plots  ten plants for each cultivar) on June 28, 2015.

2.3. Extraction of anthocyanins Anthocyanin extraction was performed five times for each variety. For each extraction, approximately 300 g of L. caerulea berries was weighed accurately and homogenated, after which approximately 1000 mL acidized methanol (0.1% HCl) was added to a beaker containing the homogenate at a material-to-solvent ratio of 1:10. The material was incubated for 90 min in an ultrasonic bath and subjected to vacuum filtration. The residue was re-extracted by repeating the extraction procedure until the solvent remained clear. Extracting solutions were then combined for rotary evaporation (RE-5203A, Shanghai Bilon Instruments Co., Ltd., China) at 40 °C until no alcohol remained. The concentrated solution was filtered and then separated through a 400 mL glass column loaded with nonionic polystyrene–divinylbenzene resin (D101, Shanghai, China) at 4 °C. Deionized water was passed through the column to remove water-soluble substances, and methanol was used as the eluting solvent. After concentration in the rotary evaporator at 40 °C, the collected liquid was freeze-dried into powder using a vacuum freeze dryer (LGO.2, Shenyang Aerospace Xinyang Quick Freezing Equip. Manuf. Co., Ltd., China). The obtained powder was placed in sealed 2-mL centrifuge tubes and stored at 20 °C until analysis. Extraction yields (5 replicates) were calculated using the following formula:

extraction yield ð%Þ ¼ W t =W f  100% where Wt is the weight of extracts and Wf is the weight of fruit used. For the fruit weight assessment, 10 replicates were conducted, with 80 fully ripe berries from each plant per replicate. The fruit weight was determined immediately after harvesting. Total soluble solids (TSS) of each sample was determined with 10 replicates of each variety; for each determination the juice was extracted from ten berries of each plant and measured using a hand refractometer (PAL-1, Hangzhou Top Instrument co., Ltd., China). Each replicate used berries from a different plant.

2.4. Determination of total polyphenol content Total polyphenol content was assessed according to the protocol described by Singleton, Orthofer, and Lamuela-Raventos (1999), slightly modified. Sample tubes were loaded with 200 lL sample (diluted 1:10) and 1 mL Folin–Ciocalteu’s reagent, and covered with aluminum foil. After a 4-min incubation at room temperature, 800 lL of 75 g/L sodium carbonate solution was injected, and the mixture was incubated in the dark for 120 min at room temperature. The absorbance values of the reaction mixture were measured at 765 nm against a deionized water blank using a UV–Vis spectrophotometer (TU-1810, PuXi, China). A standard curve was created using gallic acid as the standard substance with a concentration range of 0–100 mg/L. Results were expressed in mg of gallic acid equivalent per g of extract (mg GAE/g DW).

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2.5. Determination of total anthocyanin

2.8. Statistical analysis

The pH-differential method, a classic method for total anthocyanin content determination, was conducted according to Denev et al. (2014). Briefly, extracts were weighed accurately and 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), then incubated in the dark for 20 min at room temperature. Then, the absorbances of the reacted mixtures were read at kmax and 700 nm. The total anthocyanin content, expressed as mg cyanidin-3-glucoside equivalents per liter, was calculated as follows:

To assess significant differences in fruit weight, TSS content, total polyphenols, total anthocyanins, and total antioxidant activity values (ORAC) among the four varieties, one-way analysis of variance (ANOVA) was performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Differences were considered significant at p < 0.05 and highly significant at p < 0.01. The correlations between antioxidant activity and the content of bioactive compounds (total polyphenols, total anthocyanins) were also analyzed. Anthocyanins were identified by molecular ions, fragment ions, and the UV/Vis spectra recorded by a DAD detector using HPLC–DAD–EIS–MS2 technology.

Anthocyanin content ðmg=LÞ ¼ ðDA  Mw  DF  1000Þ=ðe  lÞ where DA = (Akmax  A700) pH1.0  (Akmax  A700) pH4.5, Mw is the molecular weight of cyanidin-3-glucoside (449.2), DF is the dilution factor, e is the molar absorptivity (26900), and l is cell pathlength (usually 1 cm).

2.6. Oxygen radical absorbance capacity (ORAC) assessment A method developed by You et al. (2011) was slightly modified and used for ORAC determination. Briefly, a 50-lL sample, appropriately diluted with 75 mM phosphate buffer (pH = 7.4), was mixed with 50 lL of fluorescein (7.7 mM) in a 96-well microplate, then 150 mL of 221.3 mM AAPH was added to start the reaction. Control (Trolox + fluorescein + AAPH) and blank (fluorescein + AAPH) measurements were performed by replacing the samples with Trolox standards and buffer, respectively. Fluorescence, recorded at 490 nm (the excitation wavelength) and 530 nm (the emission wavelength) with a multi-functional fluorescence detector (ZDY-07, Shanghai Zhicheng Instrument co., Ltd., China), was measured every minute for 80 min, with temperature controlled at 37 °C. A standard curve was set up as the difference between the net area under the fluorescence decay curve (AUC) and that of the Trolox concentration (10–100 lM). ORAC values were calculated as lmol Trolox equivalent (TE) per gram of fresh sample (lmol TE/g FW). 2.7. Identification of anthocyanins 2.7.1. Sample preparation Ten milligrams of the extract was weighed accurately and diluted with 2 mL methanol, then filtered through a 0.45-lm filter, and analyzed using HPLC–DAD–ESI–MS2. 2.7.2. HPLC–DAD–EIS–MS2 analysis The analysis of anthocyanins was performed on a HPLC system (Agilent1100, USA) equipped with a DAD detector (G4212B). A Dikma Platisil C18 column (4.6 mm  250 mm, 5 lm) was used and operated at 25 °C. The optimized mobile phases used were acetonitrile (A), and 0.1% formic acid in water (B). The elution program was performed as follows: 0–45 min, 0–45% A; 45–50 min, 0% A. The injection column was 20 lL and the flow rate was maintained at 0.7 mL/min. Data were recorded at 520 nm. In-line MS2 data was collected by a mass spectrometer (SL, Agilent1100 series LC/MSD Trap, USA) equipped with electrospray ionization (ESI) set in positive ionization mode. Analysis was conducted with automatic secondary MS scanning and datadependent MS2 scanning from 50 to 1000 m/z. The capillary voltage was controlled at +3.5 kV. Nitrogen was used as the nebulizer gas at a pressure of 40 psi. Desolvation gas was heated to 350 °C and delivered at a flow of 12 L/min.

3. Results and discussion 3.1. Extraction yields, fruit weight, and TSS The results showed that extraction yields vary among varieties (Table 1). The Wild berries had the highest extraction yield (2.5%), which was 0.3% and 0.5% higher than that of the No. 2 and Beilei cultivars, respectively, although these differences were not significant (p > 0.05). No. 1 had the lowest extraction yield at 1.8%, which was significantly different from the other varieties (0.01 < p < 0.05). The extraction yields of aronia (chokeberry) and blueberry (14.2% and 8.7%, respectively) were higher than those of the L. caerulea varieties tested in this study (Hwang, Yoon, Lee, Cha, & Kim, 2014). Fruit weight and TSS content are basic values for fruit assessment. Average weights per 80 fruits are given in Table 1. The fruit weight of No. 2 berries (85 g) was significantly higher than that of Wild, Beilei, and No. 1 (p < 0.01). The Beilei 80-fruit weight of 68.3 g was 29.5 g and 13.3 g higher than that of Wild and No. 1 varieties, respectively. The Wild variety was characterized by the lowest fruit weight in the studied varieties. Fruit weight of L. caerulea berries is significantly different from cultivar to cultivar, as was also found in blueberry varieties (Scalzo, Stevenson, & Hedderley, 2013). Interestingly, the TSS content of the four varieties’ berries had a similar pattern as the fruit weight (Table 1). The No. 2 cultivar, which had the highest TSS content (16 Brix%) in the studied varieties, may be suitable for eating without processing. In addition, the TSS content (14.4 Brix%) of the identified cultivar Beilei was dramatically higher than that of Wild berries (p < 0.01), which had the lowest TSS value (6 Brix%). No. 1 berries were intermediate with a TSS value of 11 Brix%. 3.2. Total polyphenols, total anthocyanins, and antioxidant activity The tested varieties’ berries were extracted with acidic methanol, which may be a good extraction solvent for anthocyanins (Myjavcová et al., 2010). Polyphenols, which are secondary

Table 1 The fruit weight, total soluble solids and extraction yield of the four varieties of L. caerulea berries. Varieties

Fruit weight (g/80 fruits)

Total soluble solids (Brix%)

Extraction yield (% FW)

Wild Beilei No. 1 No. 2

38.8 ± 2.41Dd 68.3 ± 2.01Bb 55 ± 1.11Cc 85 ± 0.47Aa

6 ± 0.05Cd 14.4 ± 0.21Ab 11 ± 1.31Bc 16 ± 0.02Aa

2.5 ± 0.17Aa 2 ± 0.2Aa 1.8 ± 0.04Ab 2.2 ± 0.25Aa

Values are expressed as mean ± standard deviation. Mean ± standard deviation in the same column with different superscript lowercase letters denote statistically significant difference at p < 0.05, and different capital letters denote statistically significant difference at p < 0.01.

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metabolites, are found in a wide range of plants (Denev et al., 2014), and about 8000 polyphenols have been found in nature (Karaman, Tütem, Basßkan, & Apak, 2010). As shown in Fig. 1A, varieties varied in total polyphenol content. The highest content of total polyphenols (798 mg GAE/g DW) was found in Wild berry extract, followed by Beilei berry extract. The difference between the two was not significant (p = 0.192). Both were significantly higher than those of No. 1 and No. 2 cultivars’ berry extracts (p < 0.01), which were not different from one another (p = 0.174). The lowest total polyphenol content, at 470 mg GAE/g DW occurred in No. 2 berry extracts. Notably, the total polyphenol content in all of the L. caerulea variety extracts in the present study were higher than those in black aronia (chokeberry) (100 mg GAE/g DW) and blueberry (27.4 mg GAE/g DW) extracts (Hwang et al., 2014). Raudsepp et al. (2013) has reported that total polyphenol content of L. caerulea berries was 2-, 9-, and 0.8-fold higher than that of black currant, sea-buckthorn, and bilberry, respectively, which is consistent with our results. L. caerulea berries are characterized by relatively higher content of total polyphenols compared with other fruits. L. caerulea berries are known for their high anthocyanin content (Palíková, Valentova, Oborna, & Ulrichova, 2009; Svarcova et al., 2007). According to Chen et al. (2014) and Raudsepp et al. (2013), the total anthocyanin content of L. caerulea berries was 681 mg eqv. CYD-3-G/100 g FW and 360.0 mg eqv. CYD-3-G/L, respectively. In the present study, the total anthocyanin content of the tested cultivar extracts ranged from 400.1 to 457.5 mg eqv. CYD-3-G/g DW (in Fig. 1B are these results). The No. 1 berry extract had the highest total anthocyanin content, followed by Wild berries (415.8 mg eqv. CYD-3-G/g DW), and then No. 2 berries (404.2 mg eqv. CYD-3-G/g DW). Beilei berry extracts had the lowest total anthocyanin content. However, none of these values were significantly different from one another (p > 0.05). That Wild berries have higher anthocyanin content than cultivated (Beilei and No. 2) varieties may be because of the higher surface-to-volume ratio or different growth conditions, such as sunlight and rainfall. The results obtained in this study are consistent with those previously reported (Bakowska-Barczak, Marianchuk, & Kolodziejczyk, 2007; Deineka, Sorokopudov, Deineka, Shaposhnik, & Kol’tsov, 2005; Fan, Wang, & Liu, 2011); the total anthocyanin content of L. caerulea berries ranged from 116 to 1400 mg eqv. CYD-3-G/ 100 g FW in various cultivars. As a comparison, in maqui berry, cowberry, and bilberry, the total anthocyanin content was 5095 ± 348 mg eqv. CYD-3-G/100 g DW, 57 ± 0.1 mg eqv. CYD-3-

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G/100 g FW, and 330 mg eqv. CYD-3-G/100 g FW, respectively (Kähkönen, Heinämäki, Ollilainen, & Heinonen, 2003; Rodríguez et al., 2016). The ORAC assay, which closely reflects the antioxidant capacity in biological systems (Prior, Wu, & Schaich, 2005), is a method for assessing total antioxidant activity. The ORAC values of the selected varieties’ berry extracts are shown in Fig. 2. The ORAC value of Wild berry extracts was the highest at 68 lmol TE/g FW, followed by that of Beilei berry extracts (64.7 lmol TE/g FW), both were significantly higher than those of No. 1 (55.7 lmol TE/g FW) and No. 2 (52 lmol TE/g FW) berry extracts (p < 0.01). The ORAC value of No. 2 berry extracts was found to be lowest. There were no significant differences in ORAC values between Wild and Beilei berry extracts (p = 0.092) or between No. 1 and No. 2 berry extracts (p = 0.057). This indicates that the antioxidant activities of L. caerulea berry extracts vary among varieties. The ORAC value of Wild berry extracts was 0.8- and 0.4-fold higher than that of bayberry and blueberry, respectively (Huang, Sun, Lou, Li, & Ye, 2014; You et al., 2011). Additionally, there were significantly positive correlations between antioxidant activity (ORAC) and total polyphenol (R2 = 0.949) or total anthocyanin (R2 = 0.957) content, which indicates that the high oxidation resistance activity of L. caerulea berry extracts could be attributed to the high content of total polyphenols and anthocyanins; which is consistent with the observations by Hwang et al. (2014) and Zheng and Wang (2001). The positive correlations between ORAC and total polyphenol or total anthocyanin content have been found previously in other berry extracts (Wang & Stoner, 2008). 3.3. Identification of anthocyanins The anthocyanin composition in different varieties of other fruits has been studied extensively. Among seven cultivars of blue berries—‘Bluegold’, ‘Nui’, ‘Darrow’, ‘Legacy’, ‘Nelson’, ‘Hannah’s Choice’, and ‘Toro’—cyanidin-3-O-galactoside and paeonidin-3-Ogalactoside were the most common anthocyanins in ‘Legacy’ and ‘Toro’, respectively, but were not found at all in ‘Darrow’ (Bunea et al., 2013). Moreover, a large variation in anthocyanin content and composition have been found among different aronia (chokeberry) varieties (Wangensteen et al. (2014)), blueberries (Scalzo et al., 2013; Wang, Chen, Camp, & Ehlenfeldt, 2012), strawberries (Fernández-Lara et al., 2015), and currant berries (Šavikin et al., 2013). In the present study, 16 individual anthocyanins that belong to four groups of anthocyanidins (cyanidin, peonidin, pelargonidin,

Fig. 1. Total polyphenol (mg GAE/g DW) and total anthocyanin (mg CYD-3-G/L) content of the four varieties of L. caerulea berry extracts. The different superscript lowercase letters in the same column denote statistically significant difference at p < 0.05, and different capital letters denote statistically significant difference at p < 0.01.

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Fig. 2. The ORAC values of the four varieties of L. caerulea berry extracts. The different superscript lowercase letters in the same column denote statistically significant difference at p < 0.05, and different capital letters denote statistically significant difference at p < 0.01.

and delphinidin), were identified using HPLC–ESI–MS2 (Table 2). The prominent anthocyanin in all selected varieties of berry extracts was cyanidin-3-glucoside, which is consistent with the results described by Jurikova et al. (2012) and Chen et al. (2014). The ORAC value of cyanidin-3-glucoside was 3.5-fold higher than that of Trolox (Wang, Cao, & Ronald, 1997). Cyanidin-3galactoside and cyanidin-3-sambubioside were the main anthocyanins in aronia berries and elderberries, respectively (Veberic, Jakopic, Stampar, & Schmitzer, 2009; Wangensteen et al., 2014). The peak area percentage of cyanidin-3-glucoside ranged from 89.6% to 91.3%, which was slightly higher than the 79%–88% reported by Chaovanalikit, Thompson, and Wrolstad (2004); the highest peak area percentage of cyanidin-3-glucoside was found in No. 2 cultivar berry extracts. Although cyanidin-3-glucoside is found in many fruits, cyanidin-3-glucoside content is higher in L. caerulea berries than in other berries such as blueberry, blackberry, and raspberry (Chen et al., 2014). Moreover, 14 individual antho-

cyanins were identified in Wild berry extracts, while 11, 11, and 12 individual anthocyanins were found in Beilei, No. 1, and No. 2 berry extracts, respectively. Cyanidin-3,5-dihexoside (m/z 611), cyanidin-3-hexoside-catechin (m/z 737), dimer of cyanidinhexoside (m/z 897), peonidin-3,5-dihexoside (m/z 625), cyanidin3-glucoside (m/z 449), cyanidin-3-rutinoside (m/z 595), peonidin3-glucoside (m/z 463), peonidin-3-rutinoside (m/z 609), and dimer of cyanidin-3-hexoside (m/z 897) were detected in all four selected varieties. However, 5-Methylpyranocyanidin-3-hexoside (m/z 487), cyanidin-3-hexoside-ethyl-catechin (m/z 765) and delphinidin-3-glucoside (m/z 465), all of which have two or three hydroxyl groups exhibiting more activity as antioxidants than those with only one hydroxyl (Lohachoompol, Mulholland, Srzednicki, & Craske, 2008), were only found in Wild berries, which had the most categories of individual anthocyanins. This may be the reason for the highest antioxidant activity in Wild L. caerulea berries among the studied varieties. In addition, pelargonidin-3rutinoside with fragment ions at m/z 271, which was co-eluted with pelargonidin-3-glucoside in Wild berry extracts, was not found in Beilei, No. 1, or No. 2 berry extracts, and cyanidin-3acetylhexoside was found in all but Wild berry extracts. Peonidin-3-acetylhexoside (m/z 505) was found in No. 2 berry extracts only. The results suggest that the variation in composition and individual percentage of anthocyanins among L. caerulea varieties, as in blueberry cultivars (Bunea et al., 2013; You et al., 2011), may be due to the difference in the genotype, which is one of the major determinants of the anthocyanin composition in plants (Lv et al., 2015; Zheng et al., 2011). Only 13 individual anthocyanins were found in the chromatograms of the four varieties (Fig. 3), due to the two co-elu tions—cyanidin-3-glucoside with cyanidin-3-rutinoside and pelargonidin-3-glucoside with pelargoniadin-3-rutinoside—and the amounts of the anthocyanins, which was traceable by MS. This phenomenon was also observed in commercial rabbiteye blueberries and bilberries (Lohachoompol et al., 2008; Nakajima, Tanaka, Seo, Yamazaki, & Saito, 2004). Notably, disaccharide anthocyanins including cyanidin-3,5-dihexoside and peonidin-3,5-dihexoside, which were not identified in blueberry, were found in all four L. caerulea berry varieties. The present results supplement those reported by Myjavcová et al. (2010), Palíková et al. (2008), and Chaovanalikit et al. (2004). The acylated anthocyanins, cyanidin-3-acetylhexoside (found in the Beilei, No. 1, and No. 2 cultivars) and peonidin-3-

Table 2 The identification of anthocyanin in the four varieties of L. caerulea berry extracts and the corresponding peak area percentage. RT (min)

Molecular (m/z)

Fragment (m/z)

Tentative identification

17.699 18.477 18.585 19.346 20.644B

611 737 897 625 449 595 433 579 463 609 487 765 491 897 505 465

449, 575, 735, 463, 287 449, 271 433, 301 463, 325 603, 287 735, 301 303

Cyanidin-3,5-dihexoside Cyanidin-3-hexoside-catechin Dimer of cyanidin-hexoside Peonidin-3,5-dihexoside Cyanidin-3-glucoside Cyanidin-3-rutinoside Pelargonidin-3-glucoside Pelargonidin-3-rutinoside Peonidin-3-glucoside Peonidin-3-rutinoside 5-Methylpyranocyanidin-3-hexoside Cyanidin-3-hexoside-ethyl-catechin Cyanidin-3-acetylhexoside Dimer of cyanidin-3-hexoside Peonidin-3-acetylhexoside Delphinidin-3-glucoside

22.088C 22.748 23.364 24.206 25.808 26.087 26.374 27.988 28.005 A B C D

287 287 573, 287 301 287 271 301 475, 313, 287 573

Traceable amount was detected by MS. Cyanidin-3-glucoside coeluted with cyanidin-3-rutinoside. Pelargonidin 3-glucoside coeluted with pelargoniadin 3-rutinoside. ‘nd’ means not detected.

Peak area (%) Wild

Beilei

No. 1

No. 2

1.437 0.337 trA 0.093 89.796

2.346 0.143 tr 1.015 90.679

1.955 0.067 0.173 2.087 89.626

1.933 0.055 0.174 1.856 91.340

0.909

0.509 ndD 3.912 0.639 nd nd tr 0.757 nd nd

1.057 nd 3.987 tr nd nd 0.578 0.470 nd nd

0.612 nd 3.559 tr nd nd 0.143 0.300 0.028 nd

3.088 0.139 0.886 0.106 nd 2.046 nd 1.163

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Fig. 3. The HPLC chromatograms (detected at 520 nm) of the four varieties of L. caerulea berry extracts and ESI-MS spectra of cyanidin-3-acetylhexoside (MS = 505; MS2 = 301) and peonidin-3-acetylhexoside (MS = 491; MS2 = 287). (A) Wild, (B) Beilei, (C) No. 1, (D) No. 2. The mobile phases were A: acetonitrile, and B: 0.1% formic acid in water; the elution program was: 0–45 min, 0–45%A, 45–50 min, 0%A; the injection column was 20 lL and the flow rate was kept at 0.7 mL/min.

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