Identification and quantification of anthocyanins of 62 blueberry cultivars via UPLC-MS

Abstract Blueberries are rich in anthocyanins, which are plant-specific secondary metabolites that are a good source of plant nutrients. Although anthocyanins have been investigated, there is a lack of comprehensive detailed research that includes large numbers of blueberry cultivars, and the anthocyanin compositions of some cultivars are still unknown. Consequently, we determined the anthocyanin compositions of 62 cultivars using ultra-performance liquid chromatography–mass spectrometry (UPLC–MS). In total, we identified 30 anthocyanins derived from five kinds of anthocyanidin. We also identified four glycosides having three kinds of modifications. Among the tested cultivars, ‘Rubel’ contained the highest anthocyanin content, at 534.158 mg/100 g fresh weight (FW), whereas ‘Puru’ contained the lowest anthocyanin content at 71.734 mg/100 g FW. ‘Malvidin-gal’ had the highest average content among all the anthocyanin monomers, at 48.95 mg/100 g FW. ‘HL9’ had the greatest methylation degree, at 79.39%, and ‘HL2’ had the greatest acetylation degree, at 25.14%. This study provides detailed anthocyanin compositions and modifications of different blueberry cultivars. These data will aid in breeding high-quality blueberry cultivars and in developing related healthy functional foods.


Introduction
Blueberry is an evergreen perennial or deciduous shrub plant that is famous for the high antioxidant content of its fruit. Blueberry belongs to Ericaceous, Vaccinium spp., and blueberry cultivars are divided into three groups, lowbush, highbush and rabbit-eye. Blueberries are cultivated worldwide, and they produce the second largest small-fruit yield.
The antioxidant activity of blueberry fruit depends on the contents of the main phytochemical secondary metabolic products, which are anthocyanins, procyanidins, chlorogenic acid and other flavonoid compound [1,2]. These physiologically active substances have extremely strong antioxidant capacities and promote retinoid re-synthesis and improve immunity, as well as having anti-inflammatory, anti-cardiovascular disease, anti-aging and anti-cancer effects, which greatly promote human health [3]. Some reports indicate that they potentially improve the cognitive performance in children and regulate the intestinal microbiota composition [4,5]. Color plays a very important role in the acceptability of foods, and fruit color in blueberry is represented by the direct organoleptic quality index. Cesa revealed that malvidin (Mv)-glycoside (gly) and cyanidin (Cy)-gly are expressed as red-purple, whereas delphinidin (Dp)-gly is expressed as red-blue [6].
Among phytochemicals, anthocyanins are the most important subclass of antioxidant substances [7], and they are polyphenolic pigments, classified in the flavonoid group. There are six primary anthocyanidins in natural plants, pelargonidin (Pg), Cy, peonidin (Pn), Dp, petunidin (Pt) and Mv. The differences among these individual anthocyanidins are the numbers of hydroxyl groups [8]. Anthocyanins are water-soluble pigments composed of an anthocyanidin aglycone and a sugar moiety mainly attached on the C-ring, or less frequently, at the 5 or 7 position on the A-ring ( Figure 1). Blueberry is a good source of anthocyanins. In total, 15 kinds of anthocyanins have been identified in blueberry fruit, including the five anthocyanidin glys: Dp-gly, Pt-gly, Pn-gly, Cy-gly and Mv-gly [9].
Anthocyanins have many benefits in regulating human health. In this study, ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) technique was used to analyze the anthocyanin compositions and modifications of 62 blueberry cultivars and to further study the molecular genetic mechanisms of anthocyanin synthesis and accumulation in blueberry. This information lays the foundation for cultivating high-anthocyanin-rich blueberry cultivars.

Samples and samples pre-treatment
Blueberry fruit samples were collected at the Engineering Center of Genetic Breeding and Innovative Utilization of Small Fruits of Jilin Province, China. For each cultivar, five plants of relatively uniform growth were selected, and 10 ripe-grade fruit were selected randomly that had no pest or mechanical damage. The repeated sampling method was used, and fruit from the same cultivar were mixed into one sample. These samples were rapidly frozen in liquid nitrogen and stored at −80 °C. Prior to analyses, all the samples were ground into powder in a mortar with a pestle and liquid nitrogen.

Extraction and purification of anthocyanins
Using previously published methods, with some modifications, samples were extracted [10]. Briefly, 10 g of freeze-milled fruit was weighed, and 25 mL of 80% methanol was added. The mixture was shaken and subjected to ultrasound for 20 min, Ultrasonic instrument purchased from Ningbo Scientz Biotechnology (China). It was then centrifuged at 10,000 g for 5 min, and the supernatant was then poured into a 50.0-mL brown volumetric flask. The extraction with 20 mL 80% methanol was repeated once, and the supernatants were extracted twice. Finally, the volume was adjusted to 50.0 mL with 80% methanol.
In total, 10 mL of the extraction solution was pipetted into a tube, and the methanol organic phase was evaporated at 40 °C in a rotary evaporator. The remaining aqueous phase was poured into an activated solid-phase extraction cartridge, which was rinsed with 5 mL of water twice. Then, 5 mL of methanol (containing 0.05% HCl) was eluted into the solid-phase extraction cartridge twice, resuspended in 5 mL, passed through a 0.22-µm organic phase filter and loaded on the UPLC-PDA-MS/MS-Electrospray ion source instrument for analysis. Each sample was replicated three times.

MS/MS conditions
The procedure was modified according to previous experimental methods [11,12]. The electrospray ion source was adopted, with positive ion detection mode and the multiple responses monitoring mode (MRM). The temperature of the ion source was 150 °C, the desolvent temperature was 500 °C, the flow was 800 L/h, and the tapered gas velocity was 50 L/h. High purity argon was used as the collision gas at a velocity of 0.13 mL/min.

Qualitative identification
In this test, the MS conditions of capillary voltages and cone energies for the 30 anthocyanins were optimized in the electrospray ion positive ion mode in accordance with the reference. The MRM mode was used to collect the precursor ions and strong product ions. The qualitative identification of anthocyanins in blueberry fruits was carried out using the standard retention time and MS/MS mass spectral information. To ensure detection sensitivity, data were collected in two sections and quantitatively analyzed.

Analysis of anthocyanins
At present, UPLC is more popular for anthocyanin research compared with high-performance liquid chromatography, because UPLC has a shorter analysis time, improved peak efficiency and uses less solvents [13]. Owing to improved signal-to-noise ratios, UPLC can detect analytes at low concentrations [14]. UPLC-MS/ MS combines the separation capability of UPLC and the excellent identification capability of MS, which results in greater test repeatability and reliability, as well as qualitative accuracy [15,16].
Although there has been previous research on anthocyanins in blueberries, there are limited studies that compare anthocyanin traits among multiple blueberry germplasms. In this study we used the UPLC-MS/ MS method to analyze the anthocyanin compositions and contents of 62 blueberry cultivars. The detailed anthocyanin data from different blueberry genotypes will establish a basis for subsequent breeding work aimed specifically at high exterior quality and nutritional value.

Statistical analysis
Here, the test data were mean values with standard deviation (±SD) from at least three replications. All the test results were analyzed using the Microsoft Excel program (2013), and the statistical significant of the differences were assessed by SPSS software.

Total anthocyanin contents of different cultivars
The total anthocyanin contents were determined by summing all of the anthocyanin levels. As shown in Figure 2, the total anthocyanin contents of different cultivars were varied. Among the 62 cultivars, the average total anthocyanin content was 275.86 mg/100 g fresh weight (FW), and 47 cultivars had total anthocyanin contents between 200-400 mg/100 g FW, accounting for 76% of all the cultivars. Of all cultivars, 'Rubel' had the highest anthocyanin content, at 534.158 mg/100 g FW, and 'HL12' and 'Nui' had relatively high anthocyanin contents, exceeding 400 mg/100 g. 'Puru' had the lowest anthocyanin content at only 71.734 mg/100 g FW. The average total anthocyanin content of highbush blueberries was relatively higher than that of lowbush blueberries, at 276.66 ± 83.24 mg/100 g and 214.81 ± 71.55 mg/100 g respectively.

Identification of anthocyanin types
The anthocyanin chromatographic fingerprints of the 62 cultivars were preliminarily analyzed using UPLC-MS/MS. In this study, we adopted the MRM mode, collected parent ions and strong signal daughter ions, used standard substance retention times and MS/MS information (Table 1) to qualitatively identify anthocyanin components in blueberry fruit.

Anthocyanin compositions of different cultivars
The average content of each anthocyanin monomer is presented in Table 2, and Mv-gal was present at the highest level among all the cultivars, with a value of 48.95 ± 22.05 mg/100 g FW. Cy-ac-gal had the lowest average of 0.22 ± 0.36 mg/100 g FW. Mv-xy, Cy-ac-glu and eight other components had relatively low contents of less than 1 mg/100 g FW in blueberry fruit, and 13 components had medium contents, ranging from 1 to 10 mg/100 g FW. Dp-gal, Dp-glu, Mv-ara and nine other compounds had contents greater than 10 mg/100 g FW in blueberry fruit.
As shown in Figure 4, Mv-gly, Dp-gly and Pt-gly were present at relatively high levels compared with Cy-gly and Pn-gly in most blueberry cultivars. Mv-gly was the component with the highest total content in most blueberry cultivars, except for nine cultivars, including 'Puru' , 'Rubel' and 'Bluegold' , in which Dp-gly was the main component. The same anthocyanidin derivatives had different contents in various blueberry cultivars. Mv-gly, the most important fruit anthocyanin component, which had the highest contribution to total anthocyanin content and fruit color, had content levels that ranged widely. 'Nui' was the cultivar with the highest Mv-gly content, at 171.543 mg/100 g FW, whereas 'HL9' had the highest mass fraction of Mv-gly, at 55.64%. 'Puru' ranked last among all the cultivars for Mv-gly content, at 23.551 mg/100 g FW, and 'Bluegold' had the minimum percentage composition value, at 29.74%. Dp-gly was the anthocyanin component with the second highest level in most cultivars' fruit, at 207.66 mg/100 g FW, whereas 'Rubel' had the highest mass fraction at 38.88%. The minimum content and mass fraction of 19.792 mg/100g FW and 13.21%, respectively, were detected in 'HL9' .
Pt-gly also represented a high percentage of anthocyanins in blueberries, with 'Rubel' having the maximum total content, at 117.105 mg/100 g FW. However, the level of Pt-gly in 'Puru' was only one-seventh of that in 'Rubel' , at 16.446 mg/100g FW. Additionally, the changes in mass fractions ranged from 16.07% to 28.82%, and the relative proportion in 'Smallfruit' was 28.82%, which represented the largest ratio among all the cultivars and was 1.8 times that of 'HL11' , which had the minimum ratio.
Among the five of anthocyanidin derivatives, the Cy-gly and Pn-gly contents were relatively low. In all the cultivars, 'HL11' fruit had the highest contents, at 61.223 mg/100 g FW and 27.262 mg/100 g FW, respectively, and 'Puru' had the lowest contents, at 4.515 mg/100 g FW and 2.987 mg/100 g FW, respectively.

Anthocyanidin modifications
The data confirmed that there were three modifications in blueberry fruit anthocyanin biosynthesis. Because of these modifications, blueberry anthocyanin components show high diversity levels. The first modification is glycosylation, in which a sugar substituent usually replaces C-5 H + . Here, we detected that four glys, Glu, Gal, Ara and Xy, participate in these modifications. Among the test cultivars, the Xy-modified anthocyanidins were the lowest among the four anthocyanidin modifications, and 'Duke' , 'Northsky' , 'Bluejay' and 'H75' did not contain detectable Xy levels ( Figure 5).
The total gly content was calculated by summing all the different kinds of anthocyanidin-gly values, which varied among cultivars. The anthocyanidin-glu content of 'HL4' was 185.214 mg/100 g FW, which was the highest of all the cultivars. The glu content of 'Duke' was only 4.637 mg/100 g FW, which was the minimum value among all the samples. 'Rubel' had the highest anthocyanidin-gal content of 282.613 mg/100 g FW, whereas Puru had the lowest content among all the cultivars. The content of ara in 'Rubel' was the highest, at 122.577 mg/100 g, which was six times that of 'Patriot' , at 20.536 mg/100 g FW. Xy was the lowest component in all four kinds of glys, and 'Smallfruit' had the highest content of Xy, at 32.302 mg/100 g FW (Table 3).
Here, we found that in blueberry fruit, the methylation degree was relatively high. The methylation degrees of all the cultivars are presented in Figure 6, with most cultivars having a methylated anthocyanin degree of 60% to 68%. A few cultivars had methylation levels that were relatively low, with the range being 55% to 59%. In total, 8 cultivars, including 'Bluecrop' , 'Earliblue' and 'Ivanhoe' , had relatively high degrees of methylated anthocyanins, greater than 70%. 'HL9' had the highest methylated anthocyanin percentage, reaching up to 79.39% of the total anthocyanin content, whereas 'Brigitta' had the lowest methylation degree of all the cultivars, at 55.11%. Acetylation occurs in sugar moieties, but in our test, we only detected acetylated modifications. Two ac-gly were detected, ac-glu and ac-gal, and the acetylation degrees are shown in Figure 6. As with methylation, acetylation also showed large differences among the various cultivars. Some cultivars, such as 'Northsky' , 'Coville' , 'Bluejay' and 'Weymouth' , did not show any acetylated anthocyanins, and eight cultivars, including 'Rubel' , 'Rubel' and 'Grower' , were classified as having trace levels of acetylated anthocyanins, with the degree ranging from 0.029% to 0.66%. Rubel had the lowest acetylation degree value. 'Herbert', 'Hardyblue' and 20 other cultivars were in the low-acetylate modification group, which had acetylated anthocyanin percentages ranging from 1.15% to

Discussion
Total anthocyanin content is an important and direct evaluation standard for most berry fruits. Here, we found a large variation in the total anthocyanin contents of blueberry cultivars, with the change between the maximum and minimum being approximately 7 times. In a previous study [17], determined the anthocyanin contents of blueberries at different maturity stages. As the fruit matured, the anthocyanin content gradually increased, indicating that the total content is related to seasonal conditions and fruit maturity levels. Here, the total anthocyanin content of 'Rubel' was much greater than those of the other cultivars.
This cultivar was selected from wild blueberry, which usually has a high anthocyanin content compared with other blueberries [18]. In addition to genetic background, the anthocyanin content is also affected by different cultivation conditions. Zhang indicated that the same cultivars have different anthocyanin contents when produced in different cultivation areas [19].
In this study, we divided all the anthocyanins into five groups on the basis of their anthocyanidins. Through comparisons, we found that the basic composition of blueberry is similar to that of grape, and some studies have shown that the basic anthocyanidins in grape are Dp, Cy, Pt, Pn and Mv [20,21]. In our experiment, the average percentages of Mv and Dp were 38.34% and 28.71%, respectively, which are higher than those of the other anthocyanidins, and the percentage of each anthocyanidin present influenced the final fruit color. Compared with strawberry, cranberry and lingonberry [22][23][24], we found that in most blue-, violet-or black-colored fruit, the Mv, Dp and Pt levels were always relatively high. Routray  reported that Dp, Mv and Pt are linked to the blue-red color of blueberry fruit, whereas Cy and Pt are linked to an orange-red color [25]. Cy and Pn levels result in the presentation of different colors, and these two pigments are the main anthocyanidins of lingonberry and cranberry, resulting in their bright red colors. In blueberry, the Cy and Pn contents are relatively low compared with those of other components. In particular, Pn-based anthocyanins have been invariably mentioned as trace, or even undetectable, compounds [26,27]. In our study, the average proportion of Pn was only 3.50%, which may be why blueberry skins are blue or dark violet rather than red [28,29].
Anthocyanidins are flavonoid pigments that are stabilized by many modifications, and they are highly diverse. Here, we found three modifications, glycosylation, methylation and acetylation [30]. A previous study showed that the main gly modifications in blueberry are Glu, Gal, Ara and Xy [31], which is consistent with our results. In this study, we found many acetylated anthocyanins and acetylate is a common modification in most plant anthocyanin metabolic pathways.  Without acetylated anthocyanins, decolorization in neutral or acidic aqueous solutions easily occurs [32]. Our test results were consistent with those of Gao and Mazza [33], with three acetylated anthocyanins, 3-glucosides, 3-galadosides and 3-arabinosides, resulting from the five anthocyanidins in Lowbush Blueberry. They found that in 'Fundy' fruit, acetylated anthocyanins made up over 32% of the total anthocyanins, and the two major acetylated anthocyanins were Mv ac-glu and Mv ac-gal [34]. However, some cultivars, such as 'Bluecrop' , 'Brigitta' , 'Coville' , 'Elliot' and 'Rubel' , do not have detectable levels of acetylated anthocyanins [35,36]. In our study, seven cultivars, including 'Coville' , 'Chippwa' and 'Northsky' , did not present detectable levels of any acetylated anthocyanin. The Simeone et al. study revealed that acetylation is connected to the planting area [37]. They found that in Rome and Warsaw, the same cultivars showed different degrees of acetylate modifications, and they found that in cold areas, blueberry may have a high acetylated anthocyanin content. The modification degrees in 'Bluecrop' and 'Patriot' are 11.1% and 23.8%, respectively, in Warsaw. Their results were similar to ours. Compared with a previous study of Li et al. [21], we also found that plantings of some cultivars, such as 'Northland' , 'Northblue' and 'Bluecrop' , have higher acetylated degrees in Changchun than plantings in Zhuanghe, Dandong and Qingdao. Consequently, we speculated that the production of acetylated anthocyanins is a protective mechanism of plants used to maintain a stable fruit color [38]. A study revealed that acetylation causes hydrophilic anthocyanins to become lipophilic, and acetylated anthocyanins have higher inhibitory effects on lipid peroxidation than non-acetylated anthocyanins. Therefore, acetylated anthocyanins may be more likely to cross cell plasma membranes and be absorbed by human intestinal cells [39].
In total, seven cultivars, 'Blueray' , 'AF3' , 'AF4' , 'Smallfruit' , 'HL5' , 'HL13' and 'DF1' , attracted our attention, having relatively high degrees of both methylation and acetylation. Compared with most samples, they not only had high total anthocyanin contents but also had higher anthocyanin richness levels. Thus, these cultivars may have relatively high antioxidant activity levels, and, moreover, their extracts may be better absorbed by human intestinal cells. This indicates that these cultivars are good sources of anthocyanins, which can protect cells from oxidative deterioration. They can be used in potential functional foods to prevent diseases related to oxidative stress, and the higher modification degree means that the color may remain stable during processing.

Conclusions
In summary, we identified 30 anthocyanins derived from 5 anthocyanidins, Dp, Mv, Cy, Pt and Pn, as well as four glys, Glu, Gal, Ara and Xy. Among all the tested cultivars, 'Rubel' had the highest total anthocyanin content of 534.158 mg/100 g FW. Mv and Gal were the main anthocyanidin and gly of most cultivars, respectively, and in all the tested blueberries, Mv-gal had the highest average content of 48.95 mg/100 g FW. 'HL9' had the greatest methylation degree, at 79.39%, and 'HL2' had the greatest acetylation degree, at 25.14%. This study provides details of blueberry anthocyanin contents and compositions. The compositional characteristics of different cultivars will assist researchers in breeding blueberries with greater nutritional values and high anthocyanin levels.