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Feb 28, 2017 - Zi-Xuan Huang,. ‡. Jian Zhou,. ‡. Xue-Hui Li,. ‡. Min-Hua Zong,. †,‡ and Wen-Yong Lou*,†,§. †. Lab of Applied Biocatalysis, School of Food ...

Article pubs.acs.org/JAFC

Highly Efficient Enzymatic Acylation of Dihydromyricetin by the Immobilized Lipase with Deep Eutectic Solvents as Cosolvent Shi-Lin Cao,†,∥,# Xiao Deng,†,# Pei Xu,† Zi-Xuan Huang,‡ Jian Zhou,‡ Xue-Hui Li,‡ Min-Hua Zong,†,‡ and Wen-Yong Lou*,†,§ †

Lab of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, No. 381 Wushan Road, Guangzhou 510640, China ‡ School of Chemistry and Chemical Engineering, South China University of Technology, No. 381 Wushan Road, Guangzhou 510640, China § State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, No. 381 Wushan Road, Guangzhou 510640, China ∥ Department of Food Science, Foshan University, No. 18 Jiangwan Yi Road, Foshan 528000, China S Supporting Information *

ABSTRACT: A novel deep eutectic solvent (DES)−DMSO cosolvent system has been, for the first time, successfully used as the reaction medium for the enzymatic acylation of dihydromyricetin (DMY) catalyzed by the immobilized lipase from Aspergillus niger (ANL). The cosolvent mixture, ChCl:Glycerol−DMSO (1:3, v/v) proved to be the optimal medium. With the newly developed cosolvent, the initial reaction rate of enzymatic acylation of DMY achieved 11.1 mM/h and the conversion of DMY was 91.6%. [email protected] is stable and recyclable in this cosolvent, offering 90% conversion rate after repeated use of 5 times. The lipid-solubility of DMY-16-acetate was 10 times higher than that of its raw materials DMY. The results showed that the DMY-16-acetate product exhibits good antioxidative activity. The present research illustrated that the use of DES−DMSO cosolvent may become a feasible alternative for the synthesis of DMY ester. KEYWORDS: dihydromyricetin, deep eutectic solvent, Aspergillus niger lipase, acylation, antioxidant ability

INTRODUCTION Dihydromyricetin (DMY), also known as ampelopsin, is a natural flavanonol and shows numerous bioactivities, including antioxidant, anti-inflammatory, analgesic, antitussive, antibacterial, antithrombotic, and antitumor activities.1−3 However, the highly hydrophilic nature of DMY significantly limits its potential application. Compared with DMY, DMY-acetate was reported to have much higher liposolubility as well as comparable and even improved antioxidative ability.4 According to the previous literature, the useful DMY fatty acid esters were industrially synthesized by chemical routes using acid or alkaline catalysts.5 But there are a lot of drawbacks with these chemical methods, such as unsatisfactory yields, low regioselectivities, harsh reaction conditions, as well as timeconsuming and arduous purification processes.4 From this aspect, the competitive enzyme-catalyzed organic synthesis, for example, using lipase to replace chemical catalysts, has attracted growing interest in the hydrolysis,6 epoxidation,7 aldol addition,8 acylation,4 and alcoholysis,9 because of the excellent regioselectivities, wide substrate specificity, environmentally friendly nature, and mild reaction conditions.10 Moreover, the stability, reusability, and catalytic performances of enzyme can be generally enhanced through immobilization of enzyme.11 In the enzymatic acylation of DMY, a major problem in choosing solvents is the incompatibility between keeping a high enzyme activity and dissolving substrates well.4 This polar substrate is scarcely soluble in enzyme-friendly weak-polar and nonpolar solvents such as hexane, while the enzymes are prone © 2017 American Chemical Society

to deactivation in high-polar solvents in which the substrates can be scarcely soluble.12 Hence, the development of a suitable reaction medium is critical for the enzymatic acylation of DMY.13 Lipase-catalyzed synthesis of DMY fatty acid esters has been carried out in traditional organic solvents including acetonitrile, acetone, tetrahydrofuran (THF), tert-butanol, and dimethyl sulfoxide (DMSO).14 DMSO is a polar solvent that has the ability to dissolve both polar and nonpolar compounds. Therefore, it is usually used as the solvent or cosolvent for enzymatic synthesis of biodiesel and ester derivatives of polar compounds.15 Deep eutectic solvents (DESs) are considered as green solvents9 and catalysts.16,17 DESs can be prepared in high purity from low-cost starting materials, typically by mixing choline chloride (ChCl) with an hydrogen donor, e.g. an amine, amide, alcohol, or carboxylic acid.18 To date, some investigations on enzyme-catalyzed biotransformations in DEScontaining systems have been reported, including lipasecatalyzed transesterification19 and alcoholysis,9 protease-catalyzed peptide synthesis,20,21 and epoxide hydrolase-catalyzed asymmetric hydrolysis of 1,2-epoxyoctane.22 Due to the nontoxicity, biodegradability, and low-cost of DES, it is of Received: Revised: Accepted: Published: 2084

January 2, 2017 February 23, 2017 February 28, 2017 February 28, 2017 DOI: 10.1021/acs.jafc.7b00011 J. Agric. Food Chem. 2017, 65, 2084−2088


Journal of Agricultural and Food Chemistry

water bath (200 rpm). Samples (20 μL) were withdrawn at specified time intervals from the reaction mixture and diluted 20-fold with the methanol solution mobile phase of HPLC prior to HPLC analysis. In order to investigate the effect of the molar ratio of vinyl acetate to DMY, the enzymatic reaction was conducted as follows: 60 U of the [email protected] was mixed with 2 mL of the Ch-Gly/DMSO (1/3, v/v) cosolvent system. Subsequently, DMY (20 mM) and vinyl acetate (50−500 mM) were added, and the reaction was performed at 40 °C in a shaking water bath (200 rpm). For the effect of the enzyme amount, the enzymatic reaction was conducted as follows: 20−70 U of the [email protected] was mixed with 2 mL of the Ch-Gly/DMSO (1/ 3, v/v) cosolvent system. Then, DMY (20 mM) and vinyl acetate (200 mM) were added, and the reaction was carried out at 40 °C in a shaking water bath (200 rpm). Samples (20 μL) were withdrawn and determined by HPLC according to the above description.

great interest to investigate the lipase-catalyzed acylation of DMY by using DESs as alternative solvents or cosolvents.19,23 In this work, we for the first time reported a new and green cosolvent system consisting of DMSO and DES (choline chloride:glycerol) as the reaction medium for the efficient enzymatic acylation of DMY with Aspergillus niger lipase (ANL). In this novel cosolvent system, not only DMY had moderate solubility, but also the immobilized ANL showed much higher catalytic activity than that in either DMSO or DES solvent alone. Besides, the DMY ester product has better lipidsolubility and excellent antioxidant activity. Therefore, the newly developed biocatalytic system with the immobilized ANL and the DES-based cosolvent medium is very promising for efficient enzymatic acylation of DMY.

RESULTS AND DISSCUSSION Enzymatic Acylation of DMY in the ChCl:Glycerol (1:2) (Ch-Gly) and DMSO Cosolvent System. In the present study, the recovery of lipase activity after immobilization was around 83.6% and the enzyme protein loading of the [email protected] PD-MNPs was about 138 mg protein/g PD-MNPs carrier. The [email protected] exhibit spherical morphology with the diameter 99%) was purchased from Aladdin (Shanghai, China). Other chemicals, purchased from Guangzhou Chemical Reagent Co. Ltd., were of analytical grade unless stated otherwise. Immobilization of Aspergillus niger Lipase. The ANL (Aspergillus niger lipase) was immobilized according to our previous study.24 First, the magnetic nanoparticles (MNPs) were prepared according to the conventional coprecipitation method with some modifications.24 Fifteen milliliters of dopamine hydrochloride (2.5 mg/mL) was added to the MNP suspension. The pH of the mixture was adjusted to 8.5 by the addition of NaOH solution (0.1 mol/L). After vigorous stirring for 1 h, the polydopamine-coated magnetic nanoparticles (PD-MNPs) were separated with an external magnet and washed three times with deionized water. In order to immobilize ANL, an aqueous solution of ANL (1.5 mg/mL) was prepared first by dissolving the ANL powder in sodium phosphate solution (50 mmol/ L, pH = 8.0). Then the freshly prepared PD-MNPs solution was added to the ANL solution at 4 °C in an ice bath. After stirring at 100 rpm for 12 h, the immobilized ANL ([email protected]) was washed with deionized water and then collected. Synthesis of DESs. In this work, three kinds of DESs based on choline chloride were used as the cosolvents in enzymatic acylation of DMY. And three different hydrogen-bond donors (HBDs), such as glycerol, urea, and xylitol, were selected for the synthesis of these DESs. A 150 mL jacketed glass vessel and a magnetic stirrer were employed to mix choline chloride with different HBDs in different molar ratios at 80 °C and 300 rpm until a homogeneous and colorless liquid formed.23 The above-mentioned procedure was performed under nitrogen atmosphere. Enzyme Activity Assay. Lipase activity was assayed as described as follows:6 Briefly, the enzyme was added into a solution containing 0.6 mL of phosphate buffer (50 mM, pH 8.0) and 0.1 mL of pnitrophenyl acetate (80 mM) in 2-propanol. The reaction was carried out at 40 °C and 200 rpm for 5 min, and then stopped by adding 5.3 mL of ethanol. The mixture was centrifuged at 12,000g for 5 min (4 °C), and the absorbance of the supernatant at 405 nm was measured. The control experiments were carried out to determine the spontaneous hydrolysis of p-nitrophenyl acetate under the abovementioned conditions without enzyme.6 Enzymatic Acylation of DMY. In a typical experiment, 60 U of the [email protected] was mixed with 2 mL of pure DES or the DMSO−DES cosolvent system. Then, DMY (20 mM) and vinyl acetate (200 mM) were added to the above reaction system. The enzymatic acylation of DMY was performed at 40 °C in a shaking 2085

DOI: 10.1021/acs.jafc.7b00011 J. Agric. Food Chem. 2017, 65, 2084−2088


Journal of Agricultural and Food Chemistry

Figure 1. Effect of substrate molar ratio on acetylation of DMY by [email protected]

Figure 3. Recycling ability of [email protected] in the Ch-Gly/DMSO (1/3, v/v) cosolvent system.

The initial reaction rate and conversion rate increased with the increasing substrate molar ratio. When it reached 10:1 (vinyl acetate:DMY), the conversion rate and initial reaction rate were optimal, 91.3% and 11.2 mM/h, respectively. The effect of the amount of [email protected] on the [email protected] PD-MNPs catalytic DMY acetylation reaction was also investigated. The results were shown in Figure 2, when the

Moreover, [email protected] still kept above 56.7% of the relative activity even after being repeatedly used for 10 cycles. Certainly, the partial leakage of lipase from the support materials was observed with increasing batches of [email protected] reuse from 5 to 10 cycles, resulting in the gradual decrease of the relative activity. Also, the thermal stability of [email protected] was investigated by incubating [email protected] in the Ch-Gly/DMSO (1/3, v/v) cosolvent system at the operational temperature (40 °C) for 24 h, and it was found that [email protected] maintained more than 98% of their initial activity, indicating the relatively good thermal stability under the operational conditions. Accordingly, the [email protected] acetylation of DMY has a good prospect of industrial application. Lipid-Solubility Determination of DMY and DMY-16acetate. The solubility of DMY and acetylated products in the oil phase was shown in Table S4. The lipid-solubility of DMY16-acetate was 0.635 g/100 g oil, 10 times higher than the solubility of DMY (0.067 g/100 g oil). Antioxidant Ability of DMY and DMY-16-acetate. As shown in Figure 4, the effect of DMY and DMY-16-acetate

Figure 2. Effect of enzyme dosage on [email protected] acetylation of DMY.

enzyme amount was less than 50 U, the initial reaction rate and conversion rate increased rapidly with the increasing amount of enzyme. For example, when the amount of enzyme was 20 U, the initial reaction rate was 4.8 mM/h and the conversion rate was 70.6%. As the amount of enzyme increased to 50 U, the initial reaction rate was 11.1 mM/h and the conversion rate was 91.6%, respectively. When the amount of enzyme was more than 50 U, the conversion rate remained the same, and the initial reaction rate increased slightly. According to our previous study,24 the conversion of the lipase-catalyzed regioselective acylation of DMY was about 79.3% in DMSO. In contrast, a remarkable enhancement in the conversion was observed in the DES/DMSO cosolvent system (91.6%). This demonstrated that the addition of DES into DMSO could significantly improve the enzymatic regioselective acylation of DMY, which was attributable to the following reasons: (1) both the substrate DMY and the examined acylating reagents could be well dissolved in the DES-based cosolvent system; (2) the DES showed good biocompatibility with the enzyme. The operational stability (recycle-ability) of [email protected] was studied in the Ch-Gly/DMSO (1/3, v/v) cosolvent system. As shown in Figure 3, the immobilized lipase [email protected] PD-MNPs retained more than 90% of the relative activity after successive reuse of 5 cycles, exhibiting excellent recyclability.

Figure 4. Effect of antioxidant concentration on DPPH radical scavenging rate.

DPPH free radicals clearance was presented. It showed that the DPPH radical scavenging rate of both the DMY and DMY-16acetate increased constantly during the concentration range 0.25−9 mg/mL. When the concentration of the standard control Vc was 4.00 mg/mL, DPPH free radical clearance reached 100%. In terms of IC50 (the concentration of antioxidants when the DPPH clearance rate was 50%), DMY and DMY-16-acetate were 3.71 and 4.16 mg/mL, respectively. Both of them were higher than the IC50 of the standard control Vc, 0.51 mg/mL. It is interesting to note that, after DMY 2086

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acetate increased, their Fe2+-chelating ability increased accordingly. The IC50 of EDTA, DMY, and DMY-16-acetate Fe2+ chelating were 0.18 mg/mL, 0.10 mg/mL, and 0.13 mg/mL, respectively. This showed that DMY and DMY-16-acetate exhibited better Fe2+-chelating ability than EDTA. The lower chelating ability of DMY-16-acetate may be attributed to the reduction of its hydroxyl groups after acetylation.29 In this paper, we have demonstrated that the cosolvent mixture of Ch-Gly DES and DMSO is much more suitable for the enzymatic acylation of DMY than traditional organic solvents. Moreover, this novel cosolvent system can also be used for improving enzymatic synthesis of ester derivatives of polar polyhydroxylated compounds except DMY esters. In the cosolvent, DMSO played an important role in the enhancement of enzymatic acylation by increasing the affinity between enzyme and substrates. Moreover, the Ch-Gly DES mainly promoted the dissolution of the polar substrate and prevented the inactivation of the enzyme in a high concentration of strong-polar organic solution. This cosolvent mixture is apparently superior to the recently reported pure organic reaction media for the enzymatic synthesis of DMY esters, since the use of DMSO usually leads to significant inactivation of the enzyme as well as tedious workup procedures in the downstream purification. The DMY-16-acetate product exhibits good oxidation resistance: the IC50 values of DPPH scavenging, hydroxyl radical scavenging, and chelating Fe2+ were 4.16 mg/ mL, 1.79 mg/mL, and 0.13 mg/mL, respectively. Given the above advantages, the novel Ch-Gly DES and DMSO cosolvent developed in this work may find promising application in enzymatic catalysis.

acetylation, its ability to remove DPPH free radical was improved. Hydroxyl radical has high electronegativity and strong oxidizing ability.27 With a phenolic hydroxyl group and a strong electron donating group, DMY and their acetylated products can reduce hydroxyl radicals. As shown in Figure 5,

Figure 5. Effect of antioxidant concentration on hydroxyl radical scavenging rate.

with the increase of DMY and DMY-6-acetate concentration, the clearance rate of hydroxyl radical increased accordingly. When the DMY and DMY-16-acetate concentration reached 2.7 mg/mL, the hydroxyl radical clearance rate was 94.6% and 98.1%, respectively. Compared with DMY and DMY-16acetate, the effect of the Vc on the hydroxyl radicals clearance has a different pattern. When the concentration of Vc was less than 1.5 mg/mL, the hydroxyl radical clearance rate increased rapidly. When the concentration of Vc reached 1.5 mg/mL, all of the hydroxyl radicals were basically eliminated. The IC50 of Vc, DMY, and DMY-16-acetate were 0.68 mg/mL, 1.73 mg/ mL, and 1.79 mg/mL, respectively. This result showed that the hydroxyl radical clearance ability of the product DMY-16acetate remained stable after the acetylation of DMY. What’s more, DMY and DMY-16-acetate offered better efficiency in removing hydroxyl radicals than DPPH free radical. Fentons reagent, consisting of Fe2+ and H2O2, can oxidize organic compounds to inorganic compounds.28 Therefore, it is helpful to reduce the concentration of Fe2+ in Fenton reaction in order to limit the oxidative damage in the human body. Figure 6 illustrated the Fe2+-chelating ability of the substrate DMY and the product DMY-16-acetate, with EDTA as the control. As the concentration of EDTA, DMY, and DMY-16-


S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00011. Supplementary Figure: Scanning electron micrograph of the immobilized lipase (Figure S1). The method: HPLC analysis of DMY and DMY-16-acetate; Larger-scale synthesis of DMY esters for the purification and structure determination of DMY esters; The lipid-solubilities and antioxidant abilities of DMY and DMY-16-acetate. Supplementary Tables: Effect of different DESs on the solubility of DMY and the conversion of the [email protected] acylation of DMY (Table S1). Effect of different Ch-Gly/DMSO volume ratios in Ch-Gly/ DMSO cosolvent on acetylation of DMY by [email protected] PD-MNPs (Table S2). Enzymatic acetylation of DMY by [email protected] at a DMY concentration of 300 mM (Table S3). Solubility of DMY and DMY-16-acetate in oil (Table S4) (PDF)


Corresponding Author

*Prof. Wen-Yong Lou, E-mail: [email protected]; Tel/fax: +86-20-22236669. ORCID

Shi-Lin Cao: 0000-0002-7702-5279 Jian Zhou: 0000-0002-3033-7785 Wen-Yong Lou: 0000-0003-3474-3446 Author Contributions #

S.-L.C. and X.D. contributed equally to this work.

Figure 6. Effects of antioxidant concentration on iron-chelating rate. 2087

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We wish to thank the Program of State Key Laboratory of Pulp and Paper Engineering (2017ZD05), the National Natural Science Foundation of China (21676104; 21336002; 21376096), the Key Program of Guangdong Natural Science Foundation (S2013020013049), and the Open Funding Project of the State Key Laboratory of Bioreactor Engineering for partially funding this work. We also thank SCUT Doctoral Student Short-Term Overseas Visiting Study Funding Project. Notes

The authors declare no competing financial interest.

ABBREVIATIONS USED ANL, Aspergillus niger lipase; [email protected], immobilized Aspergillus niger lipase; ChCl, choline chloride; DESs, deep eutectic solvents; DMSO, dimethyl sulfoxide; DMY, dihydromyricetin; Gly, glycerol; HDBs, hydrogen-bond donors; PDMNPs, polydopamine-coated magnetic nanoparticles; THF, tetrahydrofuran


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DOI: 10.1021/acs.jafc.7b00011 J. Agric. Food Chem. 2017, 65, 2084−2088

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