Food Science and Technology Research, 25 (1), 29_38, 2019Copyright © 2019, Japanese Society for Food Science and Technologydoi: 10.3136/fstr.25.29

http://www.jsfst.or.jp

Original paper

Isolation and Purification of Anthocyanin from Blueberry Using Macroporous Resin Combined Sephadex LH-20 Techniques

Hongkun Xue, Liuyang Shen, Xiaorui Wang, Chenghai Liu, Chai Liu, Han Liu and Xianzhe Zheng*

College of Engineering, Northeast Agricultural University, Harbin, 150030, China

Received June 14, 2018 ; Accepted September 12, 2018

Abstract: The objectives of this study were to isolate and purify blueberry anthocyanin by combination of macroporous resin and Sephadex LH-20 and identify anthocyanin composition after isolation and purification. The static adsorption/desorption experiments of anthocyanin on ten different types of resins were compared. The results showed that the highest AB-8 macroporous resin adsorption rate of 97.73 % and desorption rate of 81.52 % from blueberry were achieved under optimal conditions (Adsorption conditions: a flow rate of feed 1.0 mL/min, anthocyanin concentration of 1.0 mg/mL, pH of 3.0; Desorption conditions: an eluent flow rate of 1.5 mL/min, ethanol concentration of 60 %, pH of 3.0). The anthocyanin purity increased 19.86-fold from 4.58 % to 90.96 % after one run treatment with macroporous resin combined Sephadex LH-20 method. Delphinidin-3-glucoside and cyanidin-3-glucoside were identified by HPLC-ESI-MS/MS. The results of this study may effectively promote the purification of anthocyanin from most blueberry varieties as well as from other plant materials.

Keywords: Blueberry anthocyanin, macroporous resin, seperation, identification

*To whom correspondence should be addressed. E-mail: zhengxz@neau.edu.cn

IntroductionRecently, blueberry (Vaccinium spp.) has attracted the

interest of consumers due to abundant anthocyanin contents. Anthocyanin is naturally occurring phenolic secondary metabolites that belong to the flavonoid family (Ongkowijoyo et al., 2018). As water-soluble natural pigments, anthocyanin is promising alternatives to synthetic food colorings (Diaconeasa et al., 2015). Anthocyanin exhibits potential health benefits owing to their particular chemical structure and colorant properties. Unfortunately, the low yields achieved during extraction, the instability of anthocyanin and difficulties in obtaining high-purity anthocyanin products hamper further bioactivity research on anthocyanin (Zheng et al., 2015). Most of the researches on anthocyanin are focused on the use of crude anthocyanin extracts from berry. However, the presence of non-anthocyanin phenolic compounds and other impurities inevitably interferes with the evaluation of the biological activities of crude anthocyanin extracts (Wang et al., 2014). Considering these issues, isolation and purification of

anthocyanin from blueberry to obtain high-purity anthocyanin products are a promising endeavor.

Anthocyanin possesses two benzene rings joined by a linear three carbon chain (C2-C4), and its basic structure is the C6-C3-C6 system, which make them highly radical-scavenging activity. Extensive researches suggested that wild blueberries have a higher antioxidant capacity than strawberries, raspberries and mulberries (Flores et al., 2015). Anthocyanin has high benefits for human health with respect to its free radical scavenging (Jiao et al., 2017), antibacterial (Pertuzatti et al., 2016), inhibition cancer cell increment (Yun et al., 2010) and protection eyesight (Zielinska et al., 2016). Therefore, the blueberry fruit is considered as potential functional food to protect against many types of diseases. Nevertheless, previous researches on the biological activities of blueberry anthocyanin are mostly based on the crude anthocyanin extracts from blueberry, which is unable to further study the internal relationship between biological activities of blueberry anthocyanin and their structures. Thus, the method established

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to obtain a large number of high-purity anthocyanin monomer has great significance for deep study on structure-activity relationship of anthocyanin.

The preliminary enrichment and purification of blueberry anthocyanin are the key steps in obtaining a single high-purity anthocyanin monomer. Currently, the separat ion of anthocyanin from plants has been studied using ion exchange, solvent and high-speed counter-current chromatography and so on (Chang et al., 2012; Sheng et al., 2014; Smidova et al., 2017). However, these methods exhibit many disadvantage including time-consuming, laborious and only suitable for small-scale production. Accordingly, macroporous resin emerges as an alternative candidate for the separation of anthocyanin from blueberry. To begin with, macroporous resin is poor cost and easily obtained. In addition, macroporous resin has good selectivity, high adsorption and desorption capacity. Therefore, it has been widely used in the separation and purification of polysaccharide (Xu et al., 2016), polyphenols (Xi et al., 2015) and other active components. To sum up, some researchers have applied macroporous resin to the separation and purification of anthocyanin from blueberry (Zheng et al., 2015; Buran et al., 2014). But the complex blueberry anthocyanin was isolated and purified by using only macroporous resin, which could not obtain high-purity anthocyanin components. This has also become an urgent problem that limits the widespread use of blueberry anthocyanin. For this reason, the anthocyanin of blueberry was isolated and purified using a combination of macroporous resin and Sephadex LH-20 methods to solve the problem on low purity of anthocyanin purified by macroporous resin.

Materials and MethodsChemical reagents All other solvents were of HPLC grade

and all chemicals were of analytical grade (>99 %). The standard of cyanidin-3-glucoside and delphinidin-3-glucoside were purchased from Sigma Chemical Co. (St. Louis, Mo, U.S.A).

Preparation of crude anthocyanin extract from blueberry The cooling stored blueberries were smashed by a fruit pulping machine (Philips-30, Zhuhai, China) to acquire the blueberries purees. Then, the blueberries purees were frozen in a TD-50 freezing-vacuum dryer (Shanghai Pudong freeze drying equipment Co., Ltd., Shanghai, China) at temperature of ‒18 ℃ for 48 h. The blueberry powders were prepared using a plant grinder and screening over 40 meshes. The blueberry powders were packed in a seal dark plastic bag and stored in the refrigerator at ‒20 ℃ for the latter experiments.

Microwave assisted extraction was applied to extract blueberry anthocyanin according to the method published by our previous study (Zheng et al., 2013). The lyophilized blueberry powders (2.0000±0.0005 g) were weighed to put into extraction vial and were added to 60 mL of 60 % ethanol solution to dissolved blueberry powders. Then, the mixtures

were placed in the center of the microwave workstation (MWS, FISO Technologies Inc., Quebec, Canada). Microwave extraction conditions are set as follows: Microwave power was 600 W and extraction time was 50 s. After the extraction, the mixtures were centrifuged at 10,000 g for 15 min at 4 ℃ and filtrated Whatman No.1 filter paper. The filter residue was extracted 2 times under the same condition and collected the filtrate to obtain the crude blueberry extracts (CBE), and it was evaporated through a rotary evaporator to remove the ethanol at temperature not exceeding 40 ℃ for 2 h, and then freeze-dried to obtain anthocyanin crude extracts powders (ACEP)

Determination of anthocyanin content The total anthocyanin content in extracts was directly determined using the pH differential method (Wang et al., 2014). The absorbance of anthocyanin was measured at 513 nm and 700 nm, respectively. The anthocyanin content was calculated by the Eq. 1.

c = A×Mω×DFε×L ······Eq. 1

Where, A=[(A513‒A700)pH1.0‒(A513‒A700)pH4.5], is the molecular weight of anthocyanin (449.2 g/mol), DF is the dilution factor, is the molar extinction coefficient (26,900 L/cm mol), L is the path length (1 cm).

The anthocyanin purity can be calculated by taking the calculated anthocyanin concentration into the Eq. 2 (Zhao et al., 2017).

P = c×V×DFm × % ······Eq. 2

Where, P is anthocyanin purity (%), c is anthocyanin content (g/mL), V is the volume of anthocyanin extracts (mL), m is anthocyanin mass (g).

Static adsorption and desorption properties of the resins The ACEP (1.0000±0.0005 g) was accurately weighed to put into a 50 mL beaker and dissolved with 60 % ethanol solution. Then the mixture was set up in a 1000 mL brown volumetric flask with pH 3.0 citric acid buffer to obtain ACEP of the sample solution. The resins were pretreated according to the method of Mariotti-Celis et al. (2017) with modification. Activated macroporous resins (1.0000±0.0005 g dry weight) were added to 50 mL the ACEP of the sample solution in 100 mL flask while agitating on a vibratory shaker at 25 ℃ in the dark for 12 h to reach the adsorption equilibrium. After adsorption, the mixtures (the ACEP of sample solution and macroporous resins) were filtrated to obtain the filtrate, and the ACEP of the sample solution and the absorbance of the filtrate were measured by using a UV/Vis spectrometer (Lambda 35, Perkinelmer, Singapore) with a variable wavelength detector at 500 nm wavelength. The absorbance was recorded as A0 and A1, respectively. After reaching the adsorption equilibrium, the macroporous resins adsorbed anthocyanin were washed by deionized water for 3 times and then desorbed by 20 mL of 60 % ethanol solution in 100 mL flask while agitating on a vibratory shaker at 25 ℃ in the dark for 12 h to reach the

Isolation and Purification of Anthocyanin 31

desorption equilibrium. The absorbance of filtrate after ethanol elution was determined at 500 nm wavelength, and it was recorded as A2. The adsorption rate and desorption rate were calculated by Eqs. 3 and 4, respectively.

Y1 / % = A0‒A1A0

×100 ······Eq. 3

Y2 / % = A2A0‒A1

×100 ······Eq. 4

Where, A0 is the absorbance of ACEP of the sample solution, A1 is the absorbance value of the filtrate after resin adsorption, A2 is the absorbance of filtrate after ethanol elution.

Experimental design Single factor experiment on adsorption of AB-8

macroporous resin According to the adsorption and desorption properties of macroporous resin, AB-8 macroporous resin was selected as the most suitable resin for purification of blueberry anthocyanin. Activated macroporous resins (1.0000±0.0005 g dry weight) were employed under different adsorption conditions as shown in Table 1. The effect of three factors in terms of the flow rate of feed, anthocyanin concentration and pH on adsorption rate of AB-8 macroporous resin was determined by single factor experiment. The absorbance values of filtrate before and after adsorption were measured at 500 nm per 10 mL. The adsorption rate of AB-8 macroporous resin was calculated according to the Eq. 3.

Single factor experiment on desorpt ion of AB-8 macroporous resin Adsorption saturated AB-8 macropores resin was used under different desorption conditions as shown in Table 2. The effect of three factors in terms of the elution flow rate, ethanol concentration and pH on desorption rate of AB-8 macroporous resin was determined by single factor experiment. The absorbance values of filtrate before and after desorption were measured at 500 nm per 10 mL. The desorption rate of AB-8 macroporous resin was calculated according to the Eq. 4.

Separation and purification of anthocyanin from blueberry by Sephadex LH-20 The extracts obtained by purification of AB-8 macroporous resin (macroporous resin extracts) were concentrated to achieve the high concentration extracts, and it was extracted by the Sep-Pak C18 solid phase extraction column, which was eluted with acidified deionized water and 60 % ethanol solution (0.01 % HCl) at elution flow rate of 0.5 mL/min, respectively. The concentrated extracts obtained by collecting and concentrating the eluent in this process, and it was further separated and purified using the Sephadex LH-20 (Φ16×60 cm). After the anthocyanin were fully adsorbed, and then it was eluted with 60 % methanol solution. The filtrate was collected 1 tube for pre 3 mL. The 2 components were obtained after concentration and freeze drying of the filtrate. The obtained components were recorded as component 1 and component 2.

Identification of anthocyanin by HPLC-DAD-ESI-MS/MS Component 1 and component 2 obtained by Sephadex LH-20 separation and purification were analyzed using HPLC-DAD-ESI-MS/MS. Component 1 and component 2 were dissolved in HPLC-grade methanol and filtered through a 0.45 μm filter membrane (Fisher Scientific, pittsburgh, PA) prior to the analysis. An HPLC using a 1100 Series liquid chromatography system (Agilent Technologies Inc., U.S.A) equipped with adiode array detector (DAD) and Zorbax Eclipse XDB-C18 column (4.6 mm×150 mm, 5 μm, Agilent) was used to measure the contents of anthocyanin component 1 and component 2. The mobile phase was composed of two solvents, 1 % methane acid (mobile phase A) and 1 % formic acid acetonitrile (mobile phase B). The gradient was: 0 min 15 % B; 0‒20 min, 15‒30 % B; 20‒25 min, 30‒35 % B; 25‒35 min, 35‒40 % B; 35‒42 min, 40 % B; 42‒43 min, 40‒100 % B; 43‒48 min, 100 % B; and 48‒49 min, 100‒15 % B, followed by equilibration for 5 min at 15 % B. The mobile phase was pumped through the system at a rate of 0.8 mL/min and injected 20 µL of sample, and the oven

Table 1 . Factors and levels of single factor experiment of AB-8 macroporous resin adsorption

Factors Levels

Adsorption flow rate (mL/min) 0 .5 1 .0 1 .5 2 .0 2 .5Anthocyanin concentration (mg/mL) 0 .5 1 .0 1 .5 2 .0 2 .5

pH 1 .0 2 .0 3 .0 4 .0 5 .0

Table 2 . Factors and levels of single factor experiment of AB-8 macroporous resin desorption

Factors Levels

Elution flow rate (mL/min) 0 .5 1 .0 1 .5 2 .0 2 .5Ethanol concentration (%) 40 50 60 70 80

pH 1 .0 2 .0 3 .0 4 .0 5 .0

Note: The eluent of different pH values was prepared with a citric acid/ sodium citrate buffer solution

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temperature was set to 25 ℃. Detection of anthocyanin was conducted at wavelength of 530 nm by the ultraviolet with diode array detector. Standard samples were injected for identification and quantitative analysis. Conditions of mass spectrometry detection was determined according to the method of Wang et al. (2014) with modification. The mass detector used was an ABI Q-Trap mass spectrometer equipped with an electrospray ionization (ESI) source and then interfaced to a computer running Applied Biosystems Analyst version 1.4 software. HPLC-DAD-MS/MS system were determined from a test using cyanidin-3-glucoside as a standard prior to sample detection.

The MS/MS parameters were as follows: ESI source, positive mode; drying and nebulizing gas, nitrogen; nebulizer pressure, 30 psi; dry gas flow, 12 L/min; temperature, 400 ℃; capillary voltage, 4.5 kV; scan range, from 100 m/z to 2000 m/z.

Determination of color of anthocyanin in blueberry Anthocyanin samples (0.0500±0.0002 g) were dissolved with pH 3.0 citric acid buffer solution and set up in a 100 mL brown volumetric flask, and it was took out 2 mL and diluted with pH 3.0 citric acid buffer solution, and then removed 2 mL from the diluent and added 10 mL color reagent. The mixture was place into a 30 ℃ water bath for 30 min in the dark. The absorbance of mixture was measured at 500 nm. The color value of blueberry anthocyanin (Y3) was calculated using Eq. 5 (Chen et al., 2016).

Y3 = A×rm ······Eq. 5

Where, r is the dilution factor, A is the absorbance of mixture, m is sample mass (g).

Statistical analysis All experiments were performed in triplicate, and analysis of variance (ANOVA) for each set of data was performed. The least significant difference (LSD) at P < 0.05 was calculated by using Duncan's Multiple Range Test to analyze the significant differences in results using SAS software (8.0, SAS Institute Inc., NC, USA).

Results and DiscussionsScreening of macroporous resin It may be noted that

adsorption and desorption efficiencies correlate with the properties of the resin (polarity, surface area, average pore diameter) and chemical features to the solute (Sandhu et al., 2013). The adsorption of anthocyanin on macroporous resin is a physical action through van der Waals force or hydrogen bonding. Besides, the π-π conjugation between anthocyanin and benzene rings of resins is also important (Lin et al., 2012). Anthocyanin contains benzene rings and and hydrogen groups with non-polar or polar. Therefore, ten resins were screened to adsorb and desorb blueberry anthocyanin and results are presented in Table 3.

As seen in Table 3, the static absorption and desorption experiments of blueberry anthocyanin on 10 kinds of resins were compared. According to the adsorption and desorption properties of macroporous resin, AB-8 macroporous resin shows higher adsorption capacity (6.85 mg/g) /desorption rate (91.89 %) for blueberry anthocyanin and ratios than 9 kinds of resins, which could be attributed to its similar polarity with anthocyanin. According to the rule “likes dissolve likes”, the hydroxyl groups of anthocyanin are high prone to the formation of hydrogen bonds by macroporous resins. In addition, it may be due to the large surface area of AB-8 macroporous resin, which leads to great adsorption sites and high mass exchange rate on the resin, which is conducive to the adsorption of anthocyanin on AB-8 macroporous resin (Chandrasekhar et al., 2012). Similar results were reported in case of purple-fleshed potato and jamun anthocyanin (Heinonen et al., 2016; Jampani et al., 2014). In the desorption tests, the desorption rate (78.65 %) was the maximum in case of AB-8 macroporous resin compared to that of other nine resins. Polarity was one of the most obvious variables that affected the desorption capacity of resin. In other words, the higher the polarity of resin, the weaker the desorption capacity (Buran et al., 2014).

The above results showed that the material, polarity, surface area and average pore diameter were critical for the adsorption and desorption capacities of blueberry anthocyanin

Table 3 . Comparison of the adsorption and desorption efficiencies using ten macroporous resins

Model Polarity Surface area (m2/g)

Average pore diameter (nm)

Adsorption rate (%)

Desorption rate (%)

Adsorption capacity (mg/g)

HPD-700 Non-polar 650~700 8 .5~9 88 .56 75 .33 6 .47HPD-100 Non-polar 650~700 8 .5~9 88 .37 76 .97 6 .19

D101 Non-polar 500~550 9~10 85 .38 73 .66 6 .25D4020 Non-polar 450~500 12~16 83 .29 70 .13 5 .38D3520 Non-polar 480~520 8 .5~9 81 .32 69 .43 5 .11D1400 Non-polar 530~570 6 .5~7 .5 72 .56 64 .31 3 .64NKA Non-polar 570~590 20~22 65 .85 68 .46 4 .52S-8 Polar 100~120 28~30 88 .76 70 .55 4 .75

DA201 Polar 250~300 20~30 84 .33 68 .53 6 .16AB-8 Weak-polar 450~520 13~14 91 .86 78 .65 6 .85

Isolation and Purification of Anthocyanin 33

on ten resins. Weak-polar AB-8 macroporous resin showed better adsorption and desorption capacities than those resins. Hence, further experiments have been performed using AB-8 macroporous resin as an adsorbent.

Dynamic adsorption curve on AB-8 macroporous resin Process parameters such flow rate of feed, anthocyanin concentration and pH on absorption of blueberry anthocyanin on AB-8 macroporous resin were also investigated to obtain optimum dynamic absorption conditions. The results of dynamic absorption curves of anthocyanin on AB-8 macroporous resin are shown in Fig.1.

The dynamic breakthrough curve on AB-8 resin was obtained based on the volume of eluent liquid and the concentration of solute therein and is given in Fig. 1(a). From the Fig. 1(a) we can know that the adsorption rate remarkably decreased with the column volume under different flow rates of feed (P < 0.05). The optimal adsorption performance was observed at the flow rate 1.0 mL/min. The decrease of adsorption rate was above 1.0 mL/min flow rate, which

attributed to the reason anthocyanin have no sufficient time to undergo interactions with active sites at the surface of resins. In contrast, although the low flow rate allows more time for the anthocyanin to interact with the active sites of the adsorbent, the low flow rate may cause anthocyanin chromatography and deposition on macroporous resin, which seriously affected the stability of the column bed and the purification of anthocyanin. These results were in a good agreement with Wang et al.’s (2013) results. In general, the eluent concentration on reaching the 5 % of the inlet concentration is defined as the breakthrough point (Sandhu et al., 2013). Hence, it can be inferred that 22.0 BV of solution and flow rate of 1 mL/min are the standardized conditions for dynamic adsorption.

Effect of anthocyanin concentration on adsorption rate of AB-8 macroporous resin is shown in Fig.1 (b). As can be seen from Fig.1 (b), anthocyanin concentrations at 0.5 and 1.0 mg/mL had higher adsorption rates than those with higher concentrations. Anthocyanin concentration above 1.0 mg/mL, increasing anthocyanin concentration has a negative effect on

Fig. 1. Dynamic absorption curves of anthocyanin on AB-8 macroporous resin at different flow rates, anthocyanin concentrations of 0.5 mg/mL and pH 3.0 (a); anthocyanin concentrations, flow rate of 1.0 mL/min and pH 3.0 (b); pH, flow rate of 1.0 mL/min and anthocyanin concentrations of 1.0 mg/mL (c)Note: Error bars represent standard deviation (SD) in the manuscript.

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dynamic absorption capacity anthocyanin on AB-8 resin because the higher anthocyanin concentration contains more impurities than those with lower concentrations. The phenomena would prevent the adsorption of anthocyanin on AB-8 resin due to impurities. In addition, impurities seriously affected the flow and diffusion rate of anthocyanin on macroporous resins, which led to appearance an earlier leak point. Moreover, the high anthocyanin concentrations were prone to appear flocculation and precipitation, which induced pollution and blockage of macroporous resin (Zhang et al., 2011). But low anthocyanin concentration caused a reduction in resin utilization and improvement the adsorption of impurities (Zhang et al., 2011). Hence, it can be inferred that anthocyanin concentration of 1.0 mg/mL was the optimum conditions for dynamic adsorption.

Effect of pH on adsorption rate of AB-8 macroporous resin is shown in Fig.1 (c). It can be observed that the adsorption rate was highest when the pH was 3.0 and decreased by either raising or lowing pH under the experimental condition. This phenomenon may be explained on the basis of the structural transformations of anthocyanin as a function of pH (Yang et al., 2015). In acidic aqueous solutions anthocyanin exist as four main equilibrium species: the quinonoidal base A, the flavylium cation AH+, the carbinol or pseudobase B, and the chalcone C. A decrease of both the color intensity and the concentration of the flavylium cation transforms the colorless neutral carbinol form at pH 3.0. The highest adsorption capacity is presumably due to the highest of the hydrophobicity in equilibrium solution at pH 3.0. Similar results were obtained by other authors in case of anthocyanin from pulp wash of pigmented oranges and anthocyanin from blueberry fruits using macroporous resin separation (Di et al., 2002; Li et al., 2009). When the pH increased further above 3.0, more carbinol form is yielded through ring-opening, and more carbinol is irreversibly changed to the colorless chalcone, which does not absorb visible light and obviously decrease adsorption capacity.

Dynamic desorption curve on AB-8 macroporous resin In devising a potential industrial production process for anthocyanin product, it is necessary to take into consideration not only the adsorbent, but also the eluent solvent and its concentration, pH, and elution flow rate. In order to elucidate the effect of elution flow rate, ethanol concentration and pH on the elution of anthocyanin, studies were carried out with elution flow rate (varying from 0.5 to 2.5 mL/min), ethanol concentration (concentration varying from 40 % to 80 %, v/v) , pH (varying from 1.0 to 5.0) and the results are presented in Fig. 2.

As shown in Fig. 2(a), at the elution flow rate 1.5 mL/min, the best elution performance was obtained because the eluent solvent have sufficient time to undergo interactions with anthocyanin at the surface of AB-8 resin. In general, further increasing elution flow rate has a negative effect on dynamic desorption capacity of eluent solvent on resin. On the contrary,

an lower elution flow rate prolonged the working period (Yang et al., 2012). Therefore, 1.5 mL/min was selected as the best elution flow rate in consideration of the short working time and lower volume consumption for further experiments.

Ethanol is the preferable desorbent for macroporous resin because it can be easily removed from the solution and recycled and has low cost and no toxicity to the samples (Zhao et al., 2011). In addition, acidic solutions have been used to elute anthocyanin from resins with the objective of maintaining the flavylium cation form, which is red and stable (Castañeda-Ovando et al., 2009). As shown in Fig. 2(b), the desorption efficiency of anthocyanin significantly increased with the increase of column volume, reached its peak value, and then sharply decreased with the column volume under different ethanol concentrations (P < 0.05). The best and worst desorption occurred at 60 % and 80 % ethanol concentration, respectively. Desorption of blueberry anthocyanin from macroporous resin is the result of competing interactions between the intermolecular forces of adsorption on the macroporous resin and dissolution in the solvent. When intermolecular forces are recessive, blueberry anthocyanin desorb from the resin into the solvent. The 60 % ethanol solution might be the best matching of polarity between desorption solvent and anthocyanin (Chen et al., 2008), thus facilitating desorption. Therefore, 60 % ethanol solution was selected as the optimal desorption solution and employed in the dynamic desorption experiment. It was reported that the presence of 60 % ethanol solution required for eluent anthocyanin (Yang et al., 2015), which supported the above observation. Hence, it can be inferred that high ethanol concentration is not preferable as an eluent solvent.

The pH value of eluent solvent is very critical for the adsorption and desorption properties of resins, since the pH value determines the extent of ionization of anthocyanin molecules, thereby affecting their adsorption affinity. As shown in Fig. 2(c), the desorption efficiency initially increased remarkably, and then decreased obviously with column volume under different pH (P < 0.05). For the desorption process, increase of pH value accelerated the ionization processes of phenolic hydroxyl groups in anthocyanin. Hence, the adsorption affinity between anthocyanin and resins was weaken, and anthocyanin could be desorbed easier from AB-8 resin. As a result, the desorption ratio was increased. When the pH value exceeded 3.0, a sharp peak and peak ares obviously decreased. Because the impurities were also desorbed easier from the resin and the relative content of anthocyanin in desorption solution was decreased at higher pH value (Fu et al., 2006). Thus, the pH value of eluent solvent was adjusted to 3.0 in consideration of the adsorption and desorption process.

Purity and color value of blueberry anthocyanin before and after purification Blueberry powders (2.0000±0.0005 g) were extracted by microwave assisted extraction method to obtain the crude blueberry extracts (CBE), and CBE was

Isolation and Purification of Anthocyanin 35

evaporated through a rotary evaporator to remove the ethanol at temperature not exceeding 40 ℃ for 2 h, and then freeze-dried to obtain anthocyanin crude extracts powders (ACEP). The ACEP were extracted using acetic ether for 2-3 times to remove fat soluble impurities. After the extraction, the remainder was decompressed and concentrated to obtain the atropurpureus crude extracts, which was purified by the optimal adsorption and desorption conditions of macroporous resin to obtain macroporous resin extracts. It was further purified by Sep-Pak C18 solid phase extraction and Sephadex LH-20. The purity and color value of the extracts obtained by different purification methods were calculated by Eqs. 2 and 5, respectively. The results are shown in Table 4.

From Table 4, after being treated with AB-8 macroporous under the optimum separation conditions, the color value and purity of anthocyanin increased 8.84-fold from 13.56 to 119.81 and 9.96-fold from 4.58 % to 45.62 %, respectively. After purification with Sephadex LH-20, the purity of anthocyanin increased to 90.96 %, 19.86-fold of 4.58 % in crude blueberry extracts, and the color value of anthocyanin increased to

152.23, 11.23-fold of 13.56 in crude blueberry extracts. The results indicated that the combination macroporous resin and Sephadex LH-20 method can obviously improve the color value and purity of blueberry anthocyanin.

Quantitative and qualitative analysis of anthocyanin in blueberry After purification of AB-8 macroporous resin, the purity of blueberry anthocyanin increased from 4.58 % to 45.62 %. The macroporous resin extracts were further purified using Sep-Pak C18 solid phase extraction and Sephadex LH-20. Finally, the 2 components were obtained under different purification methods. Component 1, component 2, the standard of cyanidin-3-glucoside and delphinidin-3-glucoside were analyzed by HPLC. The results are shown in Fig. 3.

As shown in Fig. 3, the retention time of components 1 and 2 were 20.07 min and 34.91 min, respectively. The retention time of stand delphinidin-3-glucoside and cyanidin-3-glucoside were 20.06 min and 34.92 min, respectively. The retention time components 1 and 2 were similar to two standard products, and hence component 1 and 2 were preliminarily determined to be delphinidin-3-glucoside and cyanidin-3-glucoside, respectively.

Fig. 2. Dynamic desorption curves of anthocyanin on AB-8 macroporous resin at different elution flow rates, ethanol concentrations of 50 % and pH 2.0 (a); ethanol concentrations, elution flow rate of 1.5 mL/min and pH 2.0 (b); eluent solvent pH, elution flow rate of 1.5 mL/min and ethanol concentrations of 60 % (c)

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The standard curvilinear equations of delphinidin-3-glucosid and cyanidin-3-glucosid were obtained by the relationships of standard concentration with peak area of HPLC spectra.

Delphinidin-3-glucoside: Y=65,231X‒47,223 ······Eq. 6

Cyanidin-3-glucoside: Y=61,537X‒33,452 ······Eq. 7

According to the Eq. 2, the purity of component 1 and component 2 obtained by Sephadex LH-20 purification were 90.88 % and 91.37 %, respectively.

Identification of anthocyanin structure To understand the basic structure as well as the structural stability of anthocyanin, the chemical structures of the component 1, component 2 and two standard samples were determined by HPLC-ESI-MS/MS, and the results are shown in Fig. 4.

The mass spectrograms of the standard delphinidin-3-glucoside and anthocyanin component 1 are shown in Fig. 4(a) and Fig. 4(b), respectively. Both component 1 (retention time (tR) = 20.07 min) and the standard delphinidin-3-glucoside produced a molecular ion at m/z 465 and a fragment ion at 303, indicating that it is a delphinidin derivative (Nicoué et al., 2007). The neutral loss of 162 mass units corresponded to a hexose molecule (Liu et al., 2011). Hence, component 1 tentatively identified as delphinidin-3-glucoside. The mass spectrograms of the standard cyanidin-3-glucoside and anthocyanin component 2 were shown in Fig. 4(c) and Fig. 4(d), respectively. Both component 2 (retention time (tR) =

34.91 min) and the stand cyanidin-3-glucoside produced a molecular ion at m/z 449 and a fragment ion at 287, indicating that it is a cyanidin derivative (Bochi et al., 2015). The neutral loss of 162 mass units corresponded to a hexose molecule (Li et al., 2016). Therefore, component 2 tentatively identified as cyanidin-3-glucoside.

ConclusionsThis study has provided insights into the separation and

purification of anthocyanin from blueberry extract using the combination of (AB-8) macroporous resin and Sephadex LH-20. AB-8 macroporous resin showed higher adsorption/desorption capacities for blueberry anthocyanin and ratios than other resins. The adsorption capacity was 6.85 mg/g, while the ratios of adsorption and desorption were 91.86 % and 78.56 %, respectively. The optimum adsorption/desorption parameters to achieve the highest macroporous resin adsorption rate of 97.73 % and desorption rate of 81.52 % from blueberry powders were obtained under a flow rate of feed of 1.0 mL/min, anthocyanin concentration of 1.0 mg/mL, pH of 3.0 (adsorption conditions) and an eluent flow rate of 1.5 mL/min, ethanol concentration of 60 %, pH of 3.0 (desorption conditions). The anthocyanin purity increased 9.96-fold from 4.58 % to 45.62 % and 19.86-fold from 4.58 % to 90.96 % after one run treatment with AB-8 macroporous resin and Sephadex LH-20, respectively. Delphinidin-3-glucosid and cyanidin-3-glucosid were identified by HPLC-ESI-MS/MS. The results presented a valuable and practical strategy for separating high-purity anthocyanin and help to promote the purification of anthocyanin from blueberry fruits as well as from other plant.

Acknowledgments The authors gratefully thank the financial support provided by the National Natural Science Foundation of China (31571848) for this research project.

Conflict of interestThe authors declare that they have no conflict of interest.

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