Highly Selective Separation and Purification of Anthocyanins from

Mar 18, 2015 - Powdered bilberry extract (United States Pharmacopoeia, USP35-NF30), which is prepared from ripe bilberry fruits (Vaccinium myrtillus L...
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Highly Selective Separation and Purification of Anthocyanins from Bilberry Based on a Macroporous Polymeric Adsorbent Lijuan Yao, Na Zhang, Chenbiao Wang, and Chunhong Wang* Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, People’s Republic of China ABSTRACT: Powdered bilberry extract (United States Pharmacopoeia, USP35-NF30), which is prepared from ripe bilberry fruits (Vaccinium myrtillus L.), is the main ingredient of drugs alleviating visual fatigue and diabetic retinopathy because of the rich anthocyanins (purity of 36%). In this study, a method based on a macroporous polymeric adsorbent was established to obtain anthocyanin compounds from bilberry, in which the purity of the anthocyanins was improved to 96%, conducive to further pharmacological research and improvement of the efficiency of the drug. On the basis of the structure of anthocyanins, we designed a series of macroporous polymeric adsorbents based on the copolymerization of divinylbenzene (DVB) and ethylene glycol dimethyl acrylate (EGDMA). In this situation, EGDMA not only regulated the polarity of the adsorbent but also acted as the cross-linking agent to ensure the matrix structure of the adsorbent, which had a high specific surface area and could provide more interaction sites during adsorption with anthocyanins. Among the synthesized polymeric adsorbents with different contents of EGDMA, the one with 20% EGDMA content (DE-20) was demonstrated to exhibit optimal adsorption capacity and selectivity to anthocyanins compared to various commercial adsorbents through static adsorption and desorption experiments. In addition, the optimum condition of the dynamic adsorption−desorption experiment was further explored. The results indicated that the purity of anthocyanins after rinsing with 20% ethanol was determined to be approximately 96% at a desorption ratio of 83%, which was clearly higher than that in powdered bilberry extract. The established separation and purification method of anthocyanins with high purity is expected to be applied in industrial production. KEYWORDS: bilberry, anthocyanin, polymeric adsorbent, purification, adsorption

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It is worth mentioning the contribution by He and Giusti,18 which is about a method for separating anthocyanins depleted in a phenolic mixture content from fruit or vegetable feedstock containing anthocyanins and phenolic mixtures. They have applied a novel cation-exchange/reversed-phase combination solid-phase extraction (SPE) technique to purify crude extracts of various representative anthocyanin sources, such as bilberry, black currant, black raspberry, blueberry, etc., and substantially elevate anthocyanin purity. Simmons19 strengthened the potential of this technique for application on a commercial scale. Its main mechanism is the use of a combination of cation exchange and hydrophobic interaction. In our paper, we provided a method based on a macroporous adsorbent to achieve similar results, which uses less toxic organic solvents and is suitable for industrial production. We aimed at the purification of anthocyanins from bilberry fruit and not crude extracts. Macroporous resins have been employed in the purification of anthocyanins, including cyanidin 3-glucoside,20 anthocyanins from blueberries,21 anthocyanins from black bean,22 anthocyanins from jamun (Syzygium cumini L.),23 and anthocyanins from the calyx extract of roselle (Hibiscus sabdariffa L.),24 because of their relatively low cost and easy regeneration. Different commercial macroporous resins were evaluated for

INTRODUCTION

Recently, there has been growing global interest in the fruits of bilberry (Vaccinium myrtillus L.) because of its high anthocyanin content.1−3 Anthocyanins are glycosides of polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium or flavylium salts. Anthocyanins are valued as pigments but are also widely used as nutraceuticals in food and pharmaceutical preparations because of their antioxidant property and health benefit,4,5 including anticancer, antiinflammatory, and vasoprotective effects,6,7 and especially the suggested positive effect on night vision.8 Myrtocyan (Indena, Milan, Italy) is a typical anthocyaninbased product obtained from bilberry fruits.9 Drugs that used powdered bilberry extract (United States Pharmacopoeia, USP35-NF30) as main ingredients, such as Difrarel (Laboratoires Leurquin Mediolanum, France), etc., have significant effects on alleviating visual fatigue and diabetic retinopathy.10−12 However, the content of anthocyanins in commercially available powdered bilberry extract accounts for only approximately 36%, which is usually prepared from the ripe fruits of bilberry using suitable solvents, such as alcohol, methanol, or water, or mixtures of these solvents and then refined using polymeric adsorption technology.1,12−15 Because there are many unknown components in the extract, it is not conducive to the biological effects of anthocyanin.16,17 In addition, there is no doubt that these additional compounds increase the drug dose. Therefore, to prepare anthocyanins of high purity from bilberry is of great significance. © XXXX American Chemical Society

Received: December 18, 2014 Revised: March 18, 2015 Accepted: March 18, 2015

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the adsorption properties of anthocyanins; however, the purity of anthocyanins has not yet been significantly improved. This lack of improvement is most likely because the matrix of the most commercial adsorbent is hydrophobic (e.g., polystyrene), and the adsorption mechanism is mainly based on the hydrophobic interaction between adsorbents and anthocyanins. The hydrophobic affinity is non-specific, especially in aqueous solution. There are multiple phenolic hydroxyl or hydrophilic sugar moieties in the structure of anthocyanins, which leads to the increasing hydrophilicity of anthocyanins. Therefore, hydrophilic adsorbents with amide, hydroxy, or other polar functional groups, such as polyamide or dextran, were selected for the preparation of anthocyanins with high purity, which were expected to have better adsorption selectivity toward anthocyanin.25,26 However, in practical applications, these adsorbents did not exhibit good adsorption performance, because the extraction and separation of natural products based on the adsorption method usually occurs in aqueous solution, in which water molecules significantly destroy the polar affinity between adsorbents and adsorbates.27−29 Hence, in aqueous solution, a rigid hydrophobic structure remains necessary, which is consistent with the results concerning the adsorption performance of peptide molecules in aqueous solution. In this consideration, the copolymerization of a monomer containing polar functional groups with styrene or divinylbenzene (DVB) becomes the most advantageous method. Commercial macroporous resins AB-8 and ADS-17 are examples of copolymerization of methacrylate and DVB. Nevertheless, to maintain skeleton stability, a sufficient cross-linking degree is indispensable for resin synthesis, which indicates that the content ratio of the polar monomer and hydrophobic monomer can only be modulated in a much smaller range, which definitely limits the application of these resins. In addition, the large difference in polarity between the two monomers means that the monomers do not readily copolymerize with each other to produce a uniform cross-linked network in free-radical polymerization. Therefore, some of the functional groups derived from the polar monomers were not uniformly distributed on the pore surface of the adsorbent, which indicates why the adsorption selectivity of the weak polar commercial adsorbent was improved at the expense of a decrease in adsorption capacity.28 To solve this problem, in the present study, we selected ethylene glycol dimethyl acrylate (EGDMA) as the monomer providing functional groups. EGDMA not only regulates the polarity of the adsorbent but also acts as cross-linking agent to copolymerize with DVB because it contains two polymerizable double bonds. Therefore, the necessary network structure of the adsorbent was maintained, whereas the concentration of the weak polar monomer can be adjusted across a larger scale. In this paper, a series of poly(EGDMA-co-DVB) with different EGDMA contents was synthesized. The resins with the best adsorption performance were screened out through static adsorption and desorption experiments. On this basis, the parameters of the dynamic adsorption and desorption experiments were optimized to achieve anthocyanins with high purity of 96%. The high-performance liquid chromatography (HPLC) fingerprint of the products is consistent with USP35-NF30. The established method of separation and purification of anthocyanins from bilberry will hopefully be applied in industrial production.

Article

MATERIALS AND METHODS

Materials. EGDMA [analytical-reagent (AR) grade] and DVB (purity of 80%) were obtained from the Chemical Plant of Nankai University (Tianjin, China). 2,2′-Azobis(isobutyronitrile) (AIBN), toluene, 200 gasoline, poly(vinyl alcohol) (PVA), ethanol, hydrochloric acid, and sodium chloride were purchased from Tianjin Chemical Co. (Tianjin, China) and were all AR grade. Bilberry fruits (frozen) was purchased from Sweden. Bilberry dry extract CRS was purchased from European Directorate for the Quality of Medicines & HealthCare (EDQM). The percentage content of cyanidin 3-O-glucoside chloride in bilberry dry extract CRS is 3.44 wt %. HPLC-grade anhydrous formic acid, acetonitrile, and methanol were purchased from Concord Technology (Tianjin, China). All of the solutions prepared for HPLC were filtered through 0.45 μm nylon membranes before use. Pretreatment of Commercial Adsorbents. The macroporous adsorbent Amberlite XAD-7 was purchased from Rohm and Hass (Philadelphia, PA). The macroporous adsorbents XAD-4, AB-8, and ADS-17 were purchased from Nankai Hecheng S&T (Tianjin, China). Before the adsorption experiments, the adsorbents were soaked in ethanol and then thoroughly washed with deionized water. Synthesis of Adsorbents with Functional Groups. The beads were prepared using a suspension polymerization method. An organic solution composed of DVB, EGDMA, porogenic agent (toluene/200 gasoline = 70:30, w/w), and the initiator AIBN (0.5%, w/w) were mixed with the aqueous solution composed of poly(vinyl alcohol) (PVA, 1%, w/w) and sodium chloride (5%, w/w) in a 1000 mL threenecked round-bottomed flask equipped with a mechanical stirrer, reflux condenser, and thermometer. The round-bottomed flask was heated using a programmed heater. The mixture was stirred to yield a suspension of oil beads with a suitable size in the aqueous solution (100−120 rpm), heated at 65 °C for 4 h, and maintained at 85 °C for 6 h. The copolymer beads were filtered out, washed with a large amount of hot water followed by ethanol, then packed in a Soxhlet extractor, and eluted with petroleum ether. The proportion between EGDMA and DVB was changed in the synthesis process to obtain polymeric adsorbents with different hydrophobic affinities. The adsorbents were named DE-5, DE-10, DE-20, DE-30, DE-40, corresponding to 5, 10, 20, 30, and 40% EGDMA contents in weight, respectively. The synthesis process is shown in Figure 1. Determination of Pore Structure Parameters of Adsorbents. The pore structure parameters of the adsorbents were measured using an automatic surface area and pore size analyzer (Autosorb-1 MP,

Figure 1. Synthesis process of poly(DVB-co-EGDMA) adsorbent, namely, DE resin. B

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Journal of Agricultural and Food Chemistry Quantachrome Instruments, Boynton Beach, FL) based on the Brunauer−Emmett−Teller (BET) nitrogen adsorption method. Determination of the Moisture Content Imbedded in the Adsorbents. The hydrated adsorbents disposed of deionized water were weighed accurately and then dried in an oven at 105 °C until a constant weight was attained. The following equation was used to calculate the moisture content (α, %) imbedded in the adsorbent:

α=

Wwet − Wdry Wwet

× 100%

Table 2. Anthocyanins/Anthocyanidins and Corresponding Retention Time in the HPLC Chromatogram

(1)

where Wwet and Wdry are the weights (g) of the hydrated and dry adsorbents, respectively. Characterization of Adsorbents. Infrared spectra of DE adsorbent were obtained from a Nicolet Magna 560 Fourier transform infrared (FTIR) spectrometer with a pellet of potassium bromide and adsorbent in the range of 500−4000 cm−1. The inner surface morphology of the copolymer particles was investigated using a scanning electron microscope (SEM, Hitachi 3500N). The appearance of adsorbent beads was observed by an optical microscope (Olympus BX5). HPLC Analysis of Anthocyanins. The HPLC analysis method of anthocyanins was based on USP35-NF30 with some adjustments. First, 0.1250 g of bilberry dry extract CRS was dissolved in the mixed solvent (hydrochloric acid/methanol = 2:98, v/v) and diluted to 25.0 mL. The reference solution was obtained by diluting 5.0 mL of the above solution to 20.0 mL with 10% phosphoric acid. The concentration of prepared reference solution is 1.25 mg/mL. The analysis was performed on an Agilent 1200 series HPLC system with a 1200 diode array detector and 1200 quaternary pump with a degasser. A reversed-phase column (250 × 4.6 mm inner diameter) packed with Venusil ASB C18 (5 μm) was used for chromatographic analysis. The mobile phase was composed of mobile phase A (anhydrous formic acid/H2O = 8.5:91.5, v/v) and mobile phase B (anhydrous formic acid/acetonitrile/methanol/H2O = 8.5:22.5:22.5:41.5, v/v/v/v) and was used by the gradient program shown in Table 1. The flow rate was 1.0 mL/min. The detection wavelength was 535 nm. The entire column system was at a constant 30 °C using an Agilent 1200 thermostated column compartment.

mobile phase A (%)

mobile phase B (%)

elution

0−20 20−35 35−50 50−51 51−55 55−56 56−70

82 → 78 78 → 72 72 → 35 35 → 0 0 0 → 82 82

18 → 22 22 → 28 28 → 65 65 → 100 100 100 → 18 18

linear linear linear linear isocratic linear isocratic

The peaks corresponding to the anthocyanins and anthocyanidins were identified by the chromatogram supplied with bilberry extract CRS and USP35-NF30. The retention times and elution orders of the peaks are shown in Table 2. The chromatogram of bilberry extract CRS was used as a standard to calculate the concentration of anthocyanins in the tested solution, expressed as cyanidin 3-Oglucoside chloride, using the following expression:

c=

AcrwVr A rV

anthocyanins/anthocyanidins

retention time (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

delphinidin 3-O-galactoside chloride delphinidin 3-O-glucoside chloride cyanidin 3-O-galactoside chloride delphinidin 3-O-arabinoside chloride cyanidin 3-O-glucoside chloride petunidin 3-O-galactoside chloride cyanidin 3-O-arabinoside chloride petunidin 3-O-glucoside chloride delphinidin chloride peonidin 3-O-galactoside chloride petunidin 3-O-arabinoside chloride peonidin 3-O-glucoside chloride malvidin 3-O-galactoside chloride peonidin 3-O-arabinoside chloride malvidin 3-O-glucoside chloride cyanidin chloride malvidin 3-O-arabinoside chloride petunidin chloride peonidin chloride malvidin chloride

10.7 12.6 14.8 15.9 18.0 20.2 21.6 24.2 27.0 27.7 29.1 32.1 33.7 36.4 38.3 41.2 42.3 45.8 50.1 51.4

solution, V and Vr are injection volumes (μL) of test solution and reference solution, respectively, and w is the percentage content of cyanidin 3-O-glucoside chloride in bilberry extract CRS. Preparation of Bilberry Crude Extraction Solution. According to the literature,20,30,31 as the molecular forms of anthocyanins change with the pH value in aqueous solution, their stability also changes. The experimental results suggest that the stability of anthocyanins is optimal when the pH is 2.0; thus, the aqueous solution environment was maintained at pH 2.0 during the entire experimental process. The preparation of bilberry crude extraction was as follows: first, 500 g of bilberry frozen fruit was crushed into pulp in a cooking machine. Then, the pulp was transferred into a round-bottom flask with acidified 60% ethanol aqueous solution (adjust pH 2.0 with hydrochloric acid). The solid−liquid (fruit/extraction media, m/v) ratio was 1:3, and extraction was carried at 50 °C for 1 h twice. Collected supernatants were concentrated by rotary evaporation at 50 °C under vacuum for 1 h. The crude extraction solution was filtered using talcum powder to remove slime before use. The concentration of the solution obtained was 3.5 mg/mL, determined by the HPLC method. Static Adsorption and Desorption Tests. The static adsorption tests were performed as follows: 1.0 g of hydrated test adsorbents was placed into a 100 mL flask with a lid, and 20 mL of the crude extract solution was added. The flask was then shaken (100 rpm) at a constant temperature of 25 °C until the adsorption equilibrium was reached. The concentrations of the anthocyanins in the solution before and after the adsorption were analyzed by HPLC. The following equation was used to quantify the equilibrium adsorption capacity (Qe, mg/g of dry resin) of the adsorbent:

Table 1. Gradient Program of HPLC Analysis of Anthocyanins time (min)

peak number

Qe =

(C0 − Ce)V Wwet(1 − α)

(3)

where C0 and Ce are the initial and equilibrium concentrations of anthocyanins in the solutions, respectively (mg/mL), V is the volume of the solution (mL), Wwet is the weight of the hydrated adsorbent (g), and α is the moisture content of the adsorbent (%). After adsorption, the beads were washed with deionized water and then desorbed with 60% ethanol aqueous solution. The desorption ratio (Dd, %) was calculated using the following equation:

(2)

where c is the concentration (mg/mL) of anthocyanins (peaks 1−8, 10−15, and 17) in the chromatogram obtained with the test solution, cr is the concentration (mg/mL) of reference solution, A is the sum of the areas of the peaks corresponding to the anthocyanins (peaks 1−8, 10−15, and 17) in the chromatogram obtained with the test solution, Ar is the area of the peak corresponding to cyanidin 3-O-glucoside chloride (peak 5) in the chromatogram obtained with reference

Dd = C

CdVd × 100% (C0 − Ce)V

(4) DOI: 10.1021/jf506107m J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 3. Structure Parameters of Adsorbents adsorbent

DVB content (wt %)

structure

functional group

specific surface area(m2/g)

moisture content (%)

XAD-4 AB-8 ADS-17 XAD-7 DE-5 DE-10 DE-20 DE-30 DE-40

100 95 60 0 95 90 80 70 60

poly(DVB) poly(MA-co-DVB) poly(MA-co-DVB) poly(EGDMA) poly(EGDMA-co-DVB)

none ester group ester group ester group ester group ester group ester group ester group ester group

780 750 625 450 854 850 842 837 828

61.8 67.5 69.6 51.2 67.0 68.7 70.1 71.9 73.4

where Cd is the concentration of anthocyanins in the desorption solution determined by HPLC (mg/L), Vd is the volume of the desorption solution (mL), and C0, Ce, and V are the same as the parameters in eq 3. To calculate the purity of the extract, the solid content was measured using the following method: the desorption solution measured accurately by HPLC was placed into a weighing bottle of constant weight, which was then placed in an oven to dry to constant weight at 105 °C to completely remove the solvent. The purity of the extract was calculated using the following equation:

purity =

CdVd × 100% W − W0

suggested that EGDMA plays a supporting role for the adsorbent matrix instead of lowering the specific surface area because of the introduction of a weak polar monomer. In addition, we also noticed that the parameter of the adsorbent XAD-7 was unique. Although XAD-7 was composed entirely by the polymer of EGDMA and should have exhibited a high cross-linking degree, its specific surface area was reduced greatly because the chain of EGDMA is more flexible than that of DVB because of the existence of benzene. The weaker skeleton rigidity could cause a collapse of the pore structure of the adsorbents, thus reducing the specific surface area of the resin. It is inferred that the content of EGDMA should be controlled in a certain range. In addition, the moisture content of the DE adsorbent increased with increasing the EGDMA content, indicating the increasing hydrophilicity of this series of adsorbents. The structure of adsorbents with different EGDMA contents was characterized by FTIR spectroscopy. The strong stretching vibration band at 1732 cm−1observed in Figure 2 indicates that

(5)

where W is the total weight of the weighing bottle and residue solid after drying (g), W0 is the weight of the weighing bottle (g), and Cd and Vd are the same as the parameters in eq 4. Dynamic Adsorption and Elution Tests. Dynamic adsorption tests were performed as follows: the hydrated test adsorbent was packed into a glass column (Ø = 15 mm), and the bed volume (BV) was 30 mL. The solution prepared as described flowed through the adsorbent column. The elution tests were performed as follows: after the adsorption equilibrium was reached, the adsorbate-laden column was first washed with deionized water and then eluted by desorption agents (pH 2.0). The concentration of anthocyanins in the effluent solution was monitored by HPLC analysis, and the desorption ratio and purity of desorption solution were calculated using eqs 4 and 5. Acquirement of Products and Repeated Tests. After the optimized process of dynamic adsorption and desorption tests was confirmed, experiments based on the selected adsorbent were performed 4 times under the optimized conditions.

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RESULTS AND DISCUSSION Characterization of Selected Commercial Adsorbents and Synthesized DE Adsorbents. The structural parameters of the commercial adsorbents and synthesized adsorbents were characterized, and the results are presented in Table 3. We know that a higher specific surface area of the adsorbent provides more sites for the adsorbate, thus improving the adsorption capacity. As demonstrated in Table 3, for the selected commercial adsorbents, with the increase of the polar moiety content, the DVB content decreased. The moisture content data reveals that the hydrophilicity of these adsorbents increased (except XAD-7), whereas the corresponding specific surface area declined remarkably, indicating the damage of the high cross-linking structure of the adsorbent. In contrast, the synthesized adsorbents with similar DVB content exhibited completely different trends; their specific surface area gradually decreased, but all remained at a relatively high level. From Table 3, it can be observed that, for the adsorbents AB-8 and DE-5 or ADS-17 and DE-40, whose DVB contents were almost equivalent, the specific surface area of the latter was greatly increased in comparison to that of the former. This finding

Figure 2. Infrared (IR) spectra of DE adsorbents with various EGDMA contents.

ester groups were successfully introduced into the DE adsorbents. The intensity of ester groups increased with increasing the EGDMA content, which indicates that adsorbents with different EGDMA contents were synthesized. The optical microscope and SEM results of DE-20 are shown in Figure 3. The exterior of the beads had an intact spherical appearance, whereas the interior consisted of a distinct porous structure, which also indirectly demonstrated that the necessary high cross-linking structure of DE adsorbent was successfully sustained. D

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Figure 3. Images of adsorbent DE-20: (A) appearance of adsorbent beads observed under an optical microscope and (B) inner surface of the adsorbent under a SEM.

Results of Static Equilibrium Adsorption Experiments. Several representative commercial adsorbents were selected, and the static equilibrium adsorption and desorption experiment was conducted. The results are presented in Figure 4. The

exhibited better adsorption selectivity compared to commercial adsorbents, which was demonstrated by the purity of the anthocyanins in the eluent. The purity increased gradually and then decreased with increasing the EGDMA content because the interaction between the adsorbents and adsorbate also plays an important role in the adsorption selectivity in addition to the specific surface area. In addition, the synergistic dipolar and hydrophobic interactions can be used to explain this phenomenon very well. When the content of EGDMA exceeded 20%, the hydrophobic interaction decreased significantly caused by the decline of the percentage of DVB. There was not a sufficient driving force for the adsorbent to overcome the obstacles of the water molecules to bring the adsorbate molecule closer to the adsorbent matrix; the synergistic effect weakened, and thus, the adsorption ability decreased. Therefore, it is suggested that the synergistic dipolar and hydrophobic interaction between DE-20 and anthocyanins comes to an optimum point. All of these results confirmed that our strategy to regulate the adsorption selectivity of adsorbents by introducing EGDMA was successful. Consideration must be given to both the adsorption capacity and purity. The DE-20 adsorbent possessed the best selectivity, and its adsorption capacity and desorption ratio were acceptable. Hence, further experiments were performed using the DE-20 adsorbent. Effect of the Adsorption Rate on the Adsorption Property of DE-20 Adsorbent. To further investigate the adsorption property of the DE-20 adsorbent toward anthocyanins, adsorption curves were measured under different adsorption rates, and the results are presented in Figure 5. If the solution flows through the column too fast, parts of the anthocyanins rapidly rush out of the column before interacting with the adsorbent, resulting in a decrease of the adsorption capacity. Reducing the rate of adsorption is beneficial for the adsorbate to thoroughly contact the adsorbents; however, when the flow rate is too low, the production period will be extended and the efficiency is reduced. In Figure 5, anthocyanins leaked early at 80 mL at a flow rate of 3 BV/h. When the flow rate was reduced to 2 BV/h, the leak point moved to 120 mL. In comparison to the leakage point corresponding to the adsorption rate of 1 BV/h, there was no obvious difference between the two adsorption curves. Considering both the experiment period and adsorption capacity, we select the flow rate of 2 BV/h, and the corresponding volume of the adsorption solution is 120 mL (4 BV).

Figure 4. Adsorption capacity of four commercial resins (XAD-4, AB8, ADS-17, and XAD-7) and purity of anthocyanins obtained through static adsorption experiments on these resins.

purity of the anthocyanins in the desorption solution increased significantly with increasing hydrophilicity of the selected adsorbents, indicating better adsorption selectivity. However, the adsorption capacity declined notably because of the decreasing specific surface area. Therefore, regulating the hydrophilicity of the resin on the premise of maintaining a high specific surface area is of great importance. As demonstrated in Table 4, for DE resins, the adsorption capacity results agreed with those of the specific surface area, both showing slight declines with increasing polarity. Because of the introduction of EGDMA, the synthesized adsorbents Table 4. Results of Static Adsorption and Desorption of DE Resins eluent adsorbent

Qe (mg/g)

purity (%)

desorption ratio (%)

DE-5 DE-10 DE-20 DE-30 DE-40

213.2 210.9 208.5 194.6 193.2

32.2 34.5 37.6 35.3 34.1

96.7 95.4 97.2 97.4 96.5 E

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Results of Isocratic Elution on DE-20 Adsorbent. Increasing the ethanol concentration in the eluent is one solution to effectively solve the trailing phenomenon observed in Figure 6. Therefore, we selected 20, 30, and 40% ethanol aqueous solution as the desorption solvent, and its suitable consumption is 3 BV. Figure 7 shows the results of isocratic elution with different desorption solvents on DE-20 adsorbent.

Figure 5. Dynamic leakage curves of anthocyanins on adsorbent DE20 at different adsorption rates of 1, 2, and 3 BV/h.

Results of Gradient Dynamic Elution on DE-20 Adsorbent. The dynamic desorption experiments were conducted as described. After reaching the adsorption equilibrium, the adsorbent column was washed with deionized water and then desorbed by gradient elution with 10, 20, 30, and 60 vol % ethanol aqueous solutions in sequence; the results are presented in Figure 6. Figure 7. Purity and desorption ratio of anthocyanins eluted by ethanol aqueous solution of different concentrations (20, 30, and 40 vol %) in isocratic elution mode on DE-20 resin.

The desorption ratio and purity of the anthocyanins obtained are the most important factors in dynamic desorption experiments. According to Figure 7, we found that the desorption ratio was obviously improved to above 80% after increasing the concentration of ethanol in desorption solvent. A total of 83.2, 84.7, and 85.2% of the total anthocyanins can be obtained by eluting with 20, 30, and 40% ethanol solution, corresponding to purities of 96, 74, and 57%, respectively. It can be observed that the desorption ratio improved slightly under different elution conditions; however, the purity decreased remarkably with increasing the ethanol concentration in the desorption agents. These results can be explained by the different affinities of different components in bilberry toward adsorbents. The affinity between anthocyanin and the adsorbent was relatively weaker because anthocyanin is more hydrophilic, whereas the hydrophobic interaction between other impurities and adsorbents was much more intensive. In addition, the latter interaction was easier to break down using a higher ethanol concentration; thus, more impurities flowed out along with anthocyanins simultaneously, leading to the lower purity of anthocyanins. In summary, the 20% ethanol aqueous solution (3 BV, pH 2.0) was selected as the most suitable desorption solvent, and the anthocyanin product was recycled at a high desorption ratio of 83% and high purity of 96%. Results of Enrichment and Purification of Anthocyanins from Bilberry Based on DE-20 Resin. Briefly, the optimization of the purification process of anthocyanin on DE20 adsorbent can be summarized as follows: First, the hydrated adsorbent DE-20 is packed into a glass column (Ø = 15 mm), and the BV is 30 mL. Then, the crude extraction solution of anthocyanins (3.5 mg/mL) flows through the adsorbent DE-20 column at a flow rate of 2 BV/h. The adsorption capacity is 120 mL (4 BV). The adsorbate-laden column is then washed with 3 BV deionized water (pH 2.0),

Figure 6. Purity and desorption ratio of anthocyanins obtained from gradient elution by ethanol aqueous solution at different concentrations of 10, 20, 30, and 60 vol % in sequence, as a function of the BV. The experiment was conducted on DE-20 resin, and consumption of ethanol aqueous solution of each concentration is 3 BV.

During the gradient elution process, the anthocyanins and other components laden on the adsorbent gradually migrated down according to their affinity to the adsorbents and elution. Figure 6 shows that approximately 40% of the anthocyanins can be desorbed when eluted by 10% ethanol solution in the second BV, which indicated that the affinity between anthocyanins and adsorbents was relatively weak, such that a low concentration of ethanol solution could break the bonding interaction. The purity of the anthocyanins in the desorption solution declined greatly with increasing the desorption volume, from 96% for the highest drops to less than 10%. Therefore, we concluded that a low concentration of ethanol solution was more suitable for obtaining anthocyanins at high purity. Considering the relatively low desorption ratio of 10% ethanol, we decided to perform the dynamic experiments in isocratic mode using ethanol aqueous solution of higher concentration as the desorption solvent. F

DOI: 10.1021/jf506107m J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

are achieved via separation technology based on macroporous polymeric adsorbents, which represents both a novel breakthrough and a method involving simple operation, low cost, and high efficiency, which is suitable for industrial-scale production. The product of anthocyanins with high purity is beneficial for further research concerning their medicinal and pharmacological applications.

followed by the elution of 20% ethanol aqueous solution (pH 2.0), at a flow rate of 2 BV/h. Finally, anthocyanins are successfully obtained at a high purity of 96% and high desorption rate of 83%. Figure 8 shows a HPLC chromatogram of the highly pure anthocyanins and crude extraction solution of bilberry. The

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AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: +86-2223503935. E-mail: wch2004@nankai. edu.cn. Funding

The authors thank the financial support by the Tianjin Municipal Science and Technology Commission (Grant 13JCZDJC32900) and the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) (IRT1257). Notes

The authors declare no competing financial interest.

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Figure 8. HPLC chromatograms for anthocyanins/anthocyanidins obtained from the final product (red line) and crude extraction solution (blue line), which are consistent with the HPLC chromatogram of USP35-NF30 (powdered bilberry extract). Peaks are numbered according to Table 2. Inset figures are magnified chromatograms for anthocyanidins numbered 19 and 20.

peaks of both of the chromatograms are consistent with USP35-NF30 (powdered bilberry extract), indicating that, in the entire process of extraction and purification, the composition of anthocyanins remains the same as in the fruit, except that the purity is improved remarkably and the amount of contained anthocyanidins is lower, which is also consistent with USP35-NF30. Results of Repeatability of Anthocyanins on the Adsorbent DE-20. The results of repeatability of anthocyanins on the adsorbent DE-20 are listed in Table 5. Different Table 5. Results of Purification and Repeatability of DE-20 test number

desorption ratio (%)

purity (%)

1 2 3 4 average standard deviation (SD)

83.2 84.0 82.4 82.7 83.1 0.61

96.2 95.9 96.8 98.1 96.7 0.84

REFERENCES

batches all exhibited good purification effects on the anthocyanins, which indicates that the method based on the synthesized macroporous adsorbent DE-20 was entirely practical for the purification of anthocyanins in bilberry. Through a simple extraction and separation process, the content of anthocyanins increased from the original 0.5% in bilberry frozen fruits to high purity of 96% in the final product, which illustrates the effectiveness and practicality of the separation and purification method based on macroporous resin DE-20. For the first time, anthocyanins with high purity G

DOI: 10.1021/jf506107m J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/jf506107m J. Agric. Food Chem. XXXX, XXX, XXX−XXX