Biocompatible Deep Eutectic Solvents Based on Choline Chloride:Characterization and Application to the Extraction of Rutin fromSophora japonicaBing-Yi Zhao,† Pei Xu,† Fu-Xi Yang,† Hong Wu,† Min-Hua Zong,*,‡ and Wen-Yong Lou*,†,‡

†Lab of Applied Biocatalysis, School of Light Industry and Food Sciences, and ‡State Key Laboratory of Pulp and Paper Engineering,South China University of Technology, Guangzhou 510640, China

*S Supporting Information

ABSTRACT: The development of novel green solvents hasbeen one of the hottest subjects in green chemistry. Deepeutectic solvents (DESs) have logically and naturally emergedin the search for more biocompatible and biodegradablesolvents. In this study, some basic physical properties,including viscosity, conductivity, and density, of 20 DESsprepared from choline chloride and various hydrogen-bonddonors were investigated systematically. In addition, thebiocompatibility of the tested DESs was qualitatively andquantitatively evaluated using two Gram-positive (Staph-ylococcus aureus and Listeria monocytogenes) and two Gram-negative (Escherichia coli and Salmonella enteritidis) bacteria. Aclosed bottle test was used to assess the biodegradability ofthese DESs. The results demonstrated that these choline chloride-based DESs were excellent solvents with extremely low toxicityand favorable biodegradability. Finally, DESs were used to extract a flavonoid (rutin) from the flower buds of Sophora japonica.An extraction efficiency of 194.17 ± 2.31 mg·g−1 was achieved using choline chloride/triethylene glycol containing 20% water.The excellent properties of DESs indicate their potential as promising green solvents for the extraction of rutin with favorableprospects for wide use in the field of green technology.

KEYWORDS: Deep eutectic solvent (DES), Choline chloride, Physical properties, Biocompatibility, Biodegradability, Rutin

■ INTRODUCTION

Deep eutectic solvents (DESs) are emerging as alternatives toconventional ionic liquids (ILs) and organic solvents, attractingattention in many fields due to their unique advantages. Aspromising solvents, DESs not only retain the excellent merits ofILs, but also overcome their shortcomings. They have themerits such as low vapor pressure, nonflammability, simplepreparation, easy purification, and low price. A DES, a eutecticmixture, is generally composed of two or three cheap and safecomponents that are capable of associating with each otherthrough hydrogen-bond interactions.1 In most cases, the DES isprepared by mixing a quaternary ammonium salt with ahydrogen-bond donor (HBD) which has the ability to form ahydrogen bond with the halide anion of the quaternaryammonium salt.Workers have combined different starting materials to

synthesize solvents with eutectic behavior, and there are alarge number of reports on their physical properties. Daiprepared DESs from a wide range of natural products andinvestigated the molecular interactions using nuclear magneticresonance spectroscopy.2 Mukhtar et al. measured the basicphysical parameters of novel phosphonium-based DESs and

found that the type of salt and HBD had a significant effect onthe studied properties.3

Despite the advantages of ILs, some aspects definitelychallenge their development. One is that some by-products(e.g., water and salt) generate in the preparation process, whichis rarely mentioned.4,5 Another is related to their biodegrad-ability and bioaccumulation. Consequently, there is increasingfocus on their potential influence on the environment.6,7 As forDESs, their ecological footprint has not yet been thoroughlyinvestigated and few relevant studies have been published.8,9

Therefore, the label “green” for DESs should be used withcaution and it is essential to determine their biodegradationpotential.DESs have been used in many fields, such as organic

reactions, electrochemical, nanoparticles, and drugs.1,10−13

However, only a few studies have focused on the use ofDESs for the extraction of bioactive compounds.14−19 Rutin is akind of flavonoid and can be used to treat hypertension andcerebral hemorrhage.20 Rutin is abundant in the flower buds of

Received: July 2, 2015Revised: September 24, 2015Published: September 27, 2015

Research Article

pubs.acs.org/journal/ascecg

© 2015 American Chemical Society 2746 DOI: 10.1021/acssuschemeng.5b00619ACS Sustainable Chem. Eng. 2015, 3, 2746−2755

Sophora japonica, and traditionally extracted by hot water,methanol, and ethanol with relatively low extraction efficiency,due to its low solubility in these solvents. To extend theapplications of DESs in the extraction of bioactive naturalproducts, it is of great interest to attempt to extract rutin usingthese novel solvents which are able to significantly damage thecell wall, release product, and enhance the solubility of rutin.In this work, our team rationally designed and prepared a

series of DESs based on choline chloride and natural, renewableproducts (Figure 1). Their physical properties, biocompati-bility, and biodegradability were studied systematically. Inaddition, utilization of the prepared DESs for the extraction of

rutin from the flower buds of Sophora japonica (Figure S1,Supporting Information), for the first time, was explored. Thesediscoveries will serve as a bridge between knowledge of theenvironmental fate of DESs and bright prospects for applicationin human health.

■ MATERIALS AND METHODSChemicals. Choline chloride (ChCl) (≥98% mass fraction purity)

was purchased from Sinopharm Chemical Reagent Co., Ltd.(Shanghai, China). Urea, acetamide, ethylene glycol, glycerol, 1,4-butanediol, triethylene glycol, xylitol, D-sorbitol, p-toluenesulfonic acid,oxalic acid, levulinic acid, malonic acid, malic acid, citric acid, tartaric

Figure 1. Structure of DESs based on choline chloride.

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acid, xylose, sucrose, fructose, glucose, and maltose (all ≥99% massfraction purity) were all purchased from Tianjin Kermel ChemicalReagent Co., Ltd. (Tianjin, China). Rutin (≥98% mass fraction purity)was purchased from Shanghai Yuanye Biotechnology Co., Ltd.(Shanghai, China). S. japonica bud was purchased from ZhanJiangYizhou Medicines Co., Ltd. (Guangdong, China). All other chemicalswere of analytical grade.Preparation of DESs. DESs were synthesized by mixing choline

chloride and HBDs at a defined molar ratio (see Table 1) and heatingat 100 °C for 2−4 h at an atmospheric pressure under constant stirringuntil a stable homogeneous liquid was formed.21 Sugar-based DESswere prepared using the same conditions but under a nitrogenatmosphere. All the prepared DESs were allowed to cool to roomtemperature and dried in a vacuum oven at 50 °C for 24 h. Thesolvents were stored in sealed laboratory vials and kept in a desiccator.Physical Properties. The water contents of the samples were

measured using a Metrohm Karl−Fischer (model 890) titrator. Theviscosities of the DESs were measured with a HAAKE RheoStress 600at 100 Hz from 25−80 °C at a rate of 5 °C min−1. The conductivity ofall samples was measured with a conductivity meter (Shanghai LeiciDDS-307A) at a preset temperature. The densities of all samples weredetermined using a 5 cm3 pycnometer calibrated with deionized waterat 30 °C. As for the determination of viscosities, conductivity, anddensities, all the DESs except sugar-based DESs were dried at 100 °Cto minimize the water content. All measurements were performed atconstant temperature. The relative standard deviation for all the testswere less than 1%.Biocompatibility. The tested strains were incubated in LB

medium consisting of nutrient broth (18g L−1) and agar (15g L−1)at 37 °C for 12 h. A 6 mm filter paper was soaked with DES andequilibrated for 12 h in a closed vessel before applying to seededplates. The plates were cultivated at 37 °C for 24 h, and then thediameters of the inhibition zones were measured and recorded. Allexperiments were performed in triplicate to ensure accuracy and thereported result is the average value with a relative standard deviation of0.6%.A WST-1 assay, a modification of the classical MTT test, was used

to quantitatively determine cellular biocompatibility of DESs.22 Afterenrichment, the concentration of the bacterial suspension wascalibrated to 0.5 McIntosh turbidity containing 1 × 108 CFU mL−1

and diluted with nutrient broth to 1 × 106 CFU mL−1. Four types ofbacteria were seeded in 96-well plates at a concentration of 5 × 105

CFU mL−1 in each well, and the tested DESs were added atconcentrations of 8−52 mM, at 2 mM intervals, with a final volume of100 μL. An inoculum without DES addition was used as a control. Theplates were lightly shaken, sealed, and incubated at 37 °C for 20 h andthen the absorbance at 600 nm was measured on a microplate reader(Tecan Infinite 200 PRO). Each test was repeated four times. Theminimal inhibitory concentration (MIC, mM) was the lowestconcentration of DES solution that prevented the growth of amicroorganism after a specified incubation period. After incubation,the bacterial suspension in the plate was cultured and observed. Theminimal bactericidal concentration (MBC, mM), the lowestconcentration of DES solution required to kill ≥99.9% of the testbacterium, was also determined.

Biodegradability. The biodegradability of the as-prepared DESswas determined according to the closed bottle test.23 The standard forthis method is 60% theoretical oxygen demand for the referencesubstance in a 14 d window within the 28 d period of the experiment.A solution of each DES (3 mg L−1) in mineral medium was inoculatedindividually into a fresh lake water sample at a concentration of 1 mL·L−1. An inoculum without DES addition was used as the control andsodium benzoate was used as the reference substance. The mineralmedium was composed of 8.50 mg L−1 KH2PO4, 21.75 mg L−1

K2HPO4, 33.40 mg L−1 Na2HPO4·2H2O, 0.5 mg L

−1 NH4Cl, 27.50 mgL−1 CaCl2, 22.50 mg L

−1 MgSO4·7H2O, and 0.25 mg L−1 FeCl3·6H2O.

The bottles were kept at 25 °C in the dark for 28 d and the biologicaloxygen demand was measured every 7 d. Each assay was performed intriplicate to ensure accuracy, and the reported result is the averagevalue with a relative standard deviation of 0.58%.

Extraction of Rutin from S. japonica. Powdered S. japonica buds(1.00 g) were mixed with 10 mL of solvent in a flask. Twenty differentDESs containing 20% water were used as the solvents with methanol/water (60%, v/v) and ethanol/water (60%, v/v) as the controls. Theflask was placed in a water bath at 55 °C with continuous stirring for20 min, and the mixture was centrifuged at 12000g for 10 min toremove the solids. The supernatant was diluted, filtered through a 0.45μm nylon membrane and analyzed by HPLC. Each extraction wasperformed in triplicate to ensure accuracy, and the reported result isthe average value with a relative standard deviation of 0.16%.

The direct separation of rutin from the extraction solution of DESwas carried out using column chromatography with a column (15 mm× 500 mm) packed with wet AB-8 macroporous resin. The bedvolume (BV) of resin was 40 mL. A 20 mL aliquot of DES extraction

Table 1. Physical Parameters of DESs

DESs salt/HBD molar ratio water content wt % viscositya Pa·s conductivitya μS·cm−1 densitya g/cm3

ChCl/urea 1:2 1.89 ± 0.01 0.214 1287 1.1879ChCl/acetamide 1:2 2.83 ± 0.02 0.127 2710 1.0852ChCl/ethylene glycol 1:2 3.79 ± 0.01 0.025 9730 1.1139ChCl/glycerol 1:2 1.68 ± 0.01 0.177 1647 1.1854ChCl/1,4-butanediol 1:4 2.87 ± 0.01 0.047 2430 1.0410ChCl/triethylene glycol 1:4 2.47 ± 0.03 0.044 1858 1.1202ChCl/xylitol 1:1 1.21 ± 0.01 3.867 172.6 1.2445ChCl/D-sorbitol 1:1 1.10 ± 0.02 13.736 63.3 1.2794ChCl/p-toluenesulfonic acidb 1:1 5.85 ± 0.01 0.183 1138 1.2074ChCl/oxalic acidc 1:1 6.68 ± 0.02 0.089 2350 1.2371ChCl/levulinic acid 1:2 2.55 ± 0.01 0.119 1422 1.1320ChCl/malonic acid 1:1 3.36 ± 0.01 0.616 732 1.2112ChCl/malic acid 1:1 1.72 ± 0.01 11.475 41.4 1.2796ChCl/citric acidb 1:1 4.06 ± 0.01 45.008 18.4 1.3313ChCl/tartaric acid 2:1 1.35 ± 0.05 66.441 14.3 1.2735ChCl/xylose/water 1:1:1 9.85 ± 0.02 0.887 1092 1.2505ChCl/sucrose/water 5:2:5 5.43 ± 0.03 3.939 147.2 1.2737ChCl/fructose/water 5:2:5 9.35 ± 0.02 0.598 1399 1.2095ChCl/glucose/water 5:2:5 9.35 ± 0.06 0.584 2820 1.2094ChCl/maltoseb/water 5:2:5 9.47 ± 0.01 3.122 421 1.2723

aDetermined at 30 °C. bMonohydrate. cDehydrate.

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solution was injected at the flow rate of 3BV/h and washed withsufficient deionized water first, and then eluted with ethanol/water(70/30,v/v) at the flow rate of 4.5BV/h. The ethanolic fraction wascollected and analyzed with HPLC. The solution was concentratedand dried under vacuum to get the solid rutin.HPLC Analysis. Samples were analyzed by RP-HPLC on a 4.6 mm

× 250 mm (5 μm) Zorbax SB-C18 column (Agilent TechnologiesIndustries Co. Ltd.) using a Waters HPLC system consisting of twoWaters 1525 pumps and a Waters 2489 UV detector set at 254 nm.The mobile phase was a mixture of methanol, water, and phosphateacid (35/65/0.3, v/v/v). The flow rate was 1.0 mL min−1 and theinjection volume was 10 μL. The peak was detected at 16.25 minaccording to the standard rutin which was observed at 16.34 min(Figure S2, Supporting Information).

■ RESULTS AND DISCUSSION

Physical Properties. A number of DESs have beenprepared in the literature, but studies on these solvents arerelatively sporadic.24−26 In this work, four types of DESs weresynthesized based on amines, polyalcohols, organic acids, andsugars at various molar ratios. All DESs obtained from solidstarting materials were optically transparent and fluid at roomtemperature. Like ILs, the formation of liquid DESs is partiallydetermined by the melting points of the components and alsoby the interactions between the cation and anion (The thermalproperties of the DESs or individual substances are available inTable S1, Supporting Information).26 The lattice energies ofthe quaternary salt and HBD, together with entropy changesduring liquid formation, play an important role in determiningthe melting point of the DES.27 A melting point of 12 °C was

obtained for a DES composed of choline chloride and urea(with melting points of 302 °C and 133 °C, respectively) in aratio of 1:2.28 The significant reduction of melting point wasdue to interactions between the HBD molecule and the halideanions of choline chloride (Figure S3, Supporting Informa-tion).29 The smaller the charge and the larger the size of theion, the less energy is required to break the bond.30 It wastherefore supposed that the HBD worked as a special agent thatinteracted with the anionic species to increase its effective size,which conversely reduced interaction with the cation, resultingin the depression of melting point.31−33

Researchers could tailor the properties of DESs to makethem ideal candidates relying on their water content, viscosity,conductivity, and density. Table 1 shows that the watercontents of many DESs were <3.8%, except where the HBDswere hydrated acids such as p-toluenesulfonic acid, oxalic acid,and citric acid, or sugar-based DESs containing water. Evenwhen the water content of DESs is high, it is beneficial for theirapplication. Water is an abundant natural substance that acts asboth hydrogen-bond donor and acceptor. It is therefore likelyto interact strongly with the components of DESs, interferingwith the coordination sphere of the ions in favor of hydrogenbond and influencing the entropic state of the final mixture. Inaddition, water absorbed by the solvent from the surroundingatmosphere is inevitable during preparation. Increasing watercontent decreases viscosity and density of the DES.The viscosity of DESs is governed by hydrogen bond, van

der Waals and electrostatic interactions. Depending on theintermolecular interactions, the viscosity of DESs is certainly

Figure 2. Effect of temperature on the viscosity of DESs: (a) amine-based DESs; (b) alcohol-based DESs; (c) acid−based DESs; (d) sugar-basedDESs.

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higher than some conventional solvents, but similar to ILs.24,26

The experimental viscosity data for the four kinds of DESs, as afunction of temperature, are depicted in Figure 2. It was notedthat the viscosities of DESs were highly sensitive totemperature. Viscosity decreased as temperature increasedbecause of the weakening of van der Waals and hydrogen bondinteractions attributed to that the molecules obtain enoughkinetic energy to overcome intermolecular forces and enablefree movement. It can be concluded from Figure 2b−d that thepresence of extra hydroxyl groups creates more hydrogenbonds, increasing the attractive forces between molecules andmaking the liquid more viscous. For example, the viscosities ofChCl/glycerol and ChCl/xylitol at 30 °C were 0.177 Pa·s and3.867 Pa·s, respectively, where xylitol has two extra hydroxylgroups compared with glycerol. As expected, the presence ofwater strongly decreases the viscosity of these fluids comparingto those of the dried DESs. This phenomenon was particularlyremarkable for sugar-based DESs. For example, the viscosity ofcholine chloride/fructose is 44 times higher than that of thewater-added sample.34

The relationship between conductivity and temperature wasalso studied. Figure 3 shows that conductivity increased

significantly when the temperature increased and was inverselyproportional to viscosity. The reason for this phenomenon isthat kinetic energy from heating increases the frequency ofcollisions between molecules resulting in weak intermolecularforces and increased conductivity.35 Figure 3 shows that liquidswith low viscosity had higher ionic conductivity, consistent withthe Walden rule describing the strong relationship betweenionic conductivity and viscosity. It was also noted that the tris-hydroxyl-HBD DES, ChCl/glycerol, had higher conductivitycompared with the poly-hydroxyl-HBD DES, ChCl/D-sorbitol.Fewer hydroxyl groups produced fewer hydrogen bonds, whichled to greater ion mobility and ionic conductivity. Theseobservations conform to the statement that ionic structure, size,and shape strongly affect ionic conductivity.36

Generally, the densities of DESs were higher than those ofwater and most conventional polar organic solvents.37 Thepresence of carboxylic group in the HBD and the varyingdegree of hydrogen bond in these systems contributed to thehigher densities as shown in Table 1. For example, the densityof ChCl/levulinic acid was much lower than other acid−basedDESs, probably due to the presence of more moles of acid inChCl/levulinic acid with a molar ratio of 1:2. When comparingChCl/glycerol to ChCl/oxalic acid, it could be concluded that

the introduction of the carboxylic group led to an increase indensity, which was consistent with Florindo’s report.21 A 3Dnetwork of hydrogen bonds was formed by interactionsbetween D-sorbitol and anions from ChCl, resulting in theincreased density of the DES. Moreover, the density alsodepended on the free volume and hole theory, which could beused to explain their viscosity, conductivity, and density indepth.31,38 It has been proposed that liquids contain mobilevacancies or holes with a random position and size. If the size ofthe hole is equal to or greater than that of its adjacent ion, theion is able to move.39 During heating, the vibrations of anionsand cations in DESs induce molecular rearrangements becauseof changes to vacancies or holes caused by weak interactionsbetween the ions. This results in decreased density andviscosity, and increased conductivity of the liquid.40

Biocompatibility. Bacterial growth inhibition is onemethod of toxicology assessment that is low cost and providesmeaningful information within a short period of time. It was ofgreat significance to qualitatively assess the antimicrobialactivity of the prepared DESs toward four types of bacteria.As shown in Table 2, amine-, alcohol- and sugar-based DESsdid not inhibit bacterial growth, while the organic acid−basedDESs had a significant inhibitory effect, indicating theirpotential application as antibacterial agents (Figure 4). Thiswas inconsistent with the hypothesis that DESs are nontoxicand biocompatible solvents.26,41,42 Hayyan et al. reported thatChCl-based DESs had no inhibitory effect on Escherichia coli orStaphylococcus aureus.43 The amine-, alcohol- and sugar-basedDESs were digested by bacteria, since these HBDs could beabsorbed by simple or facilitated diffusion as nitrogen or carbonsources. Conversely, the organic acid−based DESs inhibitedbacterial growth mainly as a result of pH change. The pHvalues of the tested DES solutions were far below the optimalpH (6.5−7.5) for bacterial growth. Actually, when the pH ofDESs were readjusted to neutral before the tests, no inhibitoryeffects on bacteria were observed (data not shown). This is inagreement with the nature of the HBD affecting the acidity orbasicity of the corresponding DES.3 Acidic HBDs couldinactivate cells by denaturing proteins located on the cellwall, resulting in cell collapse and death.44 Choline chloride isnontoxic and approved by the European Food SafetyAuthority,45 but it has an effect on the hydrogen bond thatinfluences both physical properties and chemical structure.46 Ithas been confirmed that charge delocalization as a result ofhydrogen bond made a mixture more toxic compared with itsindividual components because chemicals with delocalizedcharges are more toxic than those with localized charges. Thehydrogen bond network in acid−based DESs is dense andcompact, so delocalized charges have greater impact, especiallyin ChCl/citric acid and ChCl/tartaric acid. Hayyan also foundthat the cytotoxicity of phosphonium-based DESs was muchhigher than that of their individual component.47

To further explore their toxicity, the MIC and MBC valueswere determined for seven acid−based DESs. Hayyan examinedthe toxicity of DESs using in vitro cell lines and in vivo animalmodel and found that the cytotoxicity effect of DESs varieddepending on cell lines.48 Table 3 clearly shows that MIC andMBC values for Gram-negative bacteria (E. coli and S.enteritidis) were generally lower than those for Gram-positivebacteria (S. aureus and L. monocytogenes). Thus, Gram-negativebacteria were more sensitive than Gram-positive bacteria toacid−based DESs. A similar phenomenon was previouslyreported by Hou, who evaluated the toxicity of cholinium

Figure 3. Effect of temperature on the conductivity of DESs.

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amino acid ILs toward bacteria.23 However, this is contrary tothe traditional rule that, for pesticides, Gram-negative bacteriaare more tolerant than Gram-positive bacteria because theformer have a cell wall containing protective lipopolysacchar-ide.49 The reason may be that the mechanism of toxicity forChCl-based DESs differs from that of insecticide. Radosevic etal. used light microscopy to examine the cytotoxicity of ChCl/oxalic acid toward CCO cells cultured in DME medium

containing Ca2+.50 They assumed that one reason for theinhibitory effect of ChCl/oxalic acid was related to theformation of cell damaging calcium oxalate crystals. In ourwork, all ChCl-based DESs had much lower antimicrobialeffects compared with imidazolium- and pyridinium-basedILs.51 Some DESs even had lower toxicity than the choliniumamino acid ILs reported by Hou, where the MIC and MBCvalues for [ChCl][Tryptophan] were as high as 23.4 mM and

Table 2. Influence of DESs on the Bacteria Inhibition (cm)

entry DES E. coli S. enteritidis S. aureus L. moncytogenes

1 ChCl/urea (1:2) NIa NI NI NI2 ChCl/acetamide (1:2) NI NI NI NI3 ChCl/ethylene glycol (1:2) NI NI NI NI4 ChCl/glycerol (1:2) NI NI NI NI5 ChCl/1, 4-butanediol (1:4) NI NI NI NI6 ChCl/triethylene glycol (1:4) NI NI NI NI7 ChCl/xylitol (1:1) NI NI NI NI8 ChCl/D-sorbitol (1:1) NI NI NI NI9 ChCl/p-toluenesulfonic acid (1:1) 1.71 ± 0.09 1.20 ± 0.01 1.12 ± 0.02 0.70 ± 0.0110 ChCl/oxalic acid (1:1) 2.48 ± 0.03 1.93 ± 0.07 1.97 ± 0.07 1.50 ± 0.0111 ChCl/levulinic acid (1:2) 1.65 ± 0.05 1.60 ± 0.10 1.00 ± 0.01 0.97 ± 0.0812 ChCl/malonic acid (1:1) 1.53 ± 0.03 1.17 ± 0.03 1.32 ± 0.03 0.93 ± 0.0713 ChCl/malic acid (1:1) 1.92 ± 0.08 1.22 ± 0.03 1.50 ± 0.05 1.10 ± 0.1014 ChCl/citric acid (1:1) 1.93 ± 0.13 1.77 ± 0.03 1.58 ± 0.08 1.30 ± 0.0515 ChCl/tartaric acid (2:1) 1.76 ± 0.14 1.50 ± 0.01 1.50 ± 0.19 1.10 ± 0.0516 ChCl/xylose/water (1:1:1) NI NI NI NI17 ChCl/sucrose/water (5:2:5) NI NI NI NI18 ChCl/fructose/water (5:2:5) NI NI NI NI19 ChCl/glucose/water (5:2:5) NI NI NI NI20 ChCl/maltose/water (5:2:5) NI NI NI NI

aNI, no inhibition.

Figure 4. Inhibitory effect of different DESs on Escherichia coli with inhibition and without inhibition in a Petri dish. (A) ChCl/urea influence on theEscherichia coli. (B) ChCl/citric acid influence on the Escherichia coli.

Table 3. Toxicity of DESs toward Bacteria, Expressed as MIC and MBC

E. coli S. enteritidis S. aureus L. moncytogenes

entry DES MIC MBC MIC MBC MIC MBC MIC MBC pHa

1 ChCl/p-toluenesulfonic acid (1:1) 18b 28 26 40 18 34 30 50 4.602 ChCl/oxalic acid (1:1) 12 18 12 22 12 26 14 30 1.743 ChCl/levulinic acid (1:2) 12 16 12 26 14 22 12 36 4.424 ChCl/malonic acid (1:1) 18 20 20 34 16 30 24 48 2.105 ChCl/malic acid (1:1) 14 20 18 42 14 24 22 48 2.436 ChCl/citric acid (1:1) 12 20 16 38 12 28 20 42 4.087 ChCl/tartaric acid (2:1) 14 18 18 40 12 20 16 44 3.70

ameasured at the 10 mmol/L of DESs aqueous solution. bmmol/L.

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31.3 mM, respectively.23 The benign biocompatibility of theseChCl-based DESs is attributed to the combination of nontoxiccholine chloride with natural, renewable HBDs. It wasconfirmed by Frade et al. that viability was dependent on theconcentration of ILs, but the ChCl-based solvents are generallynontoxic or of low toxicity.52

Differences in toxicity of the various ChCl-based DESs canbe related to the different HBDs since they share the samehydrogen-bond acceptor. The higher toxicity of DESscontaining organic acids might be partly explained by thechange of pH (Table 3) because environmental pH change canalter cellular proliferation and metabolic properties. The ChCl/p-toluenesulfonic acid DES, which contained a benzene ringstructure, had the lowest toxicity. The toxicity of the acid−based DESs decreased with elongation of the carbon chain(Table 3, entries 2 and 4). However, this effect was not evidentwhen comparing entries 2, 3, and 6, which had similarinhibitory effects, possibly due to the introduction of an acetylgroup and polar carboxyl group. As shown in entries 5 and 7,the addition of an extra hydroxyl group in the HBD resulted ina slight increase in antibacterial activity. ChCl/oxalic acid,ChCl/levulinic acid, and ChCl/citric acid had the highesttoxicity, while the lowest toxicity was observed for ChCl/malonic acid and ChCl/p-toluenesulfonic acid. So the toxicityof the various ChCl-based DESs was associated with pH andthe HBA compounds.Biodegradability. The biodegradability of the DESs,

important for understanding their environmental impact andfate, was evaluated according to the closed bottle test. DESswere added to aqueous medium containing oxygen. Then themixture was inoculated with lake microorganisms, and thebiodegradation value was determined at a defined time interval(Table 4). On the basis of the Closed Bottle Test, thebiodegradability of sodium benzoate was 62.8% on the 14th dayof this research, so the method was valid. The biodegradabilityfor all the tested DESs was >69.3% after 28 d, therefore all ofthem could be considered as biodegradable green solvents. The

levels of biodegradation were as follows: amine-based DESs ≈sugar-based DESs > alcohol-based DESs > acid−based DESs.The highest and the lowest biodegradability were found to be97.1% and 69.3% for ChCl/urea and ChCl/triethylene glycol,respectively. The values for ChCl/urea and ChCl/glycerol were81.2% and 83.2% in 14 d, respectively, and the biodegradationof sugar-based DESs reached about 70%. Moreover, thebiodegradability of DESs and reference substance were fasterin the first 14 d period than in the last 14 d period.The biodegradation values for 20 different DESs were far

higher than conventional imidazole and pyridine ILs.53,54 Thecomponents of the tested DESs contributed significantly to thehigh degrees of biodegradation. Generally, biodegradablecompounds first cross the cell wall in a variety of ways, suchas free diffusion, facilitated diffusion, or active transport, and arethen oxidized enzymatically. Intermediate products aremetabolized to water and carbon dioxide or transformed intoconstituent materials of the cell. This is the primary reason forthe good biodegradability of amine- and sugar-based DESs.Long-chain substances are less amenable to transmembranetransport, resulting in the relatively low biodegradation ofChCl/triethylene glycol. There are also exclusive carriers forthe transportation of choline salt anion on the bacterial cellmembrane, so choline chloride is easily transported. Inaddition, the hydroxyl, carboxyl and amino groups of theDESs are potential sites for enzyme reactions and likely toincrease their degradation. Moreover, the acidic HBDs arereadily metabolized by microorganisms, leading to the 70%−80% biodegradation rate observed for acid-based DESs.The potential for choline amino acid ILs as biodegradable

solvents has been acknowledged previously. Hou et al. assessedthe biodegradability of 18 types of cholinium-based amino acidILs and concluded that the ILs could be denoted as “readilybiodegradable” based on their high level of biodegradability(62%−87%).23 Wen evaluated the biodegradability of 8 DESsbased on choline chloride and choline acetate, and two of themhad the biodegradability of about 80%.55 Our research may

Table 4. Biodegradation of DESs Determined by Closed Bottle Test

biodegradability (%)

entry DESs and reference substance 7 days 14 days 21 days 28 days

1 ChCl/urea (1:2) 39.7 ± 0.6 81.2 ± 0.7 90.3 ± 0.6 97.1 ± 0.72 ChCl/acetamide (1:2) 25.8 ± 0.5 62.5 ± 0.1 81.1 ± 0.6 89.5 ± 0.63 ChCl/ethylene glycol (1:2) 24.1 ± 0.5 58.2 ± 0.5 77.3 ± 0.5 81.9 ± 0.64 ChCl/glycerol (1:2) 46.3 ± 1.5 83.2 ± 0.6 90.9 ± 0.6 95.9 ± 0.75 ChCl/1, 4-butanediol (1:4) 29.4 ± 0.8 51.6 ± 1.1 62.0 ± 0.1 73.6 ± 0.96 ChCl/triethylene glycol (1:4) 10.7 ± 1.5 29.7 ± 0.5 51.4 ± 0.3 69.3 ± 0.57 ChCl/xylitol (1:1) 31.6 ± 2.4 66.0 ± 0.6 77.6 ± 0.8 84.3 ± 0.68 ChCl/D-sorbitol (1:1) 37.4 ± 1.5 63.4 ± 0.4 80.1 ± 0.6 86.2 ± 0.59 ChCl/p-toluenesulfonic acid (1:1) 32.3 ± 1.4 72.8 ± 0.4 76.3 ± 2.1 80.4 ± 0.310 ChCl/oxalic acid (1:1) 40.6 ± 0.4 61.4 ± 0.5 65.0 ± 0.4 73.4 ± 1.511 ChCl/levulinic acid (1:2) 33.9 ± 0.8 49.4 ± 1.0 67.2 ± 0.5 74.2 ± 2.212 ChCl/malonic acid (1:1) 34.6 ± 1.3 50.2 ± 0.6 60.8 ± 1.6 76.3 ± 1.313 ChCl/malic acid (1:1) 37.9 ± 0.9 62.9 ± 0.7 73.3 ± 0.6 79.4 ± 1.014 ChCl/citric acid (1:1) 39.5 ± 1.3 65.3 ± 1.6 75.0 ± 0.8 81.6 ± 0.715 ChCl/tartaric acid (2:1) 54.2 ± 1.4 76.4 ± 0.6 81.3 ± 1.0 84.6 ± 0.316 ChCl/xylose/water (1:1:1) 50.8 ± 1.3 70.6 ± 0.3 82.0 ± 1.1 89.7 ± 0.717 ChCl/sucrose/water (5:2:5) 55.6 ± 0.4 68.0 ± 1.9 87.4 ± 1.8 91.6 ± 0.318 ChCl/fructose/water (5:2:5) 48.4 ± 0.5 73.6 ± 1.3 88.2 ± 1.6 93.7 ± 1.319 ChCl/glucose/water (5:2:5) 58.6 ± 1.2 77.4 ± 1.0 89.4 ± 1.0 92.0 ± 0.420 ChCl/maltose/water (5:2:5) 53.0 ± 0.8 73.7 ± 2.0 84.6 ± 1.2 90.0 ± 0.521 sodium benzoate 57.9 ± 1.0 62.8 ± 1.1 79.0 ± 0.2 81.5 ± 0.7

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encourage a shift of attention from classical imidazolium andpyridinium ILs to DESs from natural sources such as amines,alcohols, organic acids, and sugars. It is worth noting that some“readily biodegradable” classical ILs with long alkyl chains havealso been reported to be highly toxic because of their lipophiliccharacter. Conversely, ILs with short alkyl side chains were safebut suffered from reduced biodegradability.7 However, thisconflict between toxicity and biodegradability was not apparentin the DESs tested in this study since low toxicity wasassociated with good biodegradability.Extraction of Rutin from S. japonica. In the pioneering

work of the potential application of natural deep eutecticsolvents incellular metabolism and physiology by Choi et al.,56

it was found that DESs had a strong ability to dissolve theflavonoid rutin, as well as other cellular metabolic compounds,indicating 50 to 100 times higher solubility of rutin than that inwater. In that case, however, the use of DESs for extraction ofrutin from a plant was not involved. Therefore, it was of greatinterest to investigate the extraction of the valuable rutin fromthe flower buds of Sophora japonica with the as-preparedcholine chloride-based DESs. Twenty DESs at a defined molarratio (Figure 1) were tested on the basis of previous studies.The viscosities of the DESs were generally high, whichhampered mass transfer from powder to solution. Therefore,water (20%, v/v) was added to the DES (except for sugar-basedDESs) for successful extraction of rutin. Solvents including 60%ethanol/water and 60% methanol/water were selected asreference extraction solvents.57 Clear superiority in extractionamount of rutin was observed with the evaluated DESscompared with 60% methanol and 60% ethanol.As shown in Figure 5, the amount of rutin extracted by

ChCl/triethylene glycol (194.17 ± 2.31 mg) and ChCl/

levulinic acid (197.80 ± 3.14 mg) was higher than that by othersolvents. The extraction efficiencies of amine- and alcohol-based DESs were superior to those of sugar-based DESs,because the latter had a higher viscosity. Rutin is readily solublein alkaline solvent, so the extraction efficiencies of the acid−

based DESs were lower than those with amine- or alcohol-based DESs, except for ChCl/levulinic acid and ChCl/malonicacid. In view of the high cost of levulinic acid, the optimalextraction solvent was ChCl/triethylene glycol. By using theabove-described separation method, the recovery of theextracted rutin form DES was 99.3 mg/g.In addition to the viscosity of the DES, the effect of the

structure of the HBD should also be considered. Rutin is aflavonoid, which can be considered as a HBD. Therefore, theHBDs and rutin compete for the chloride anion. If a moleculeof HBD has sufficient hydrogen-bond donor groups orbranches, it can envelope the chloride anion, resulting inconsiderable steric hindrance and preventing interactionsbetween rutin and chloride anion. This is a possible explanationfor the high extraction efficiencies of ChCl/triethylene glycoland ChCl/levulinic acid. Therefore, a favorable solvent shouldhave the correct distance between HBD groups and chlorideanion.

■ CONCLUSIONSIn this work, the physical properties (water content, viscosity,conductivity, density) of a variety of DESs were studied indetail and the hole theory was used to explain the intrinsicmicroscopic links between viscosity, conductivity, and density.The qualitative study on biocompatibility of the DESs revealedthat amine-, alcohol- and sugar-based DESs were benignsolvents, while the acid-based DESs were harmful to Gram-negative (E. coli and S. enteritidis) and Gram-positive (S. aureusand L. monocytogenes) bacteria. Quantitative research on thebiocompatibility of the acid-based DESs illustrated that eventhough they have antimicrobial activity, they might still bereferred to as “green solvents” because of their low toxicitycompared with traditional solvents and ILs. Additionally, allDESs were environmentally friendly solvents with biodegrada-tion >69%, classified as “readily biodegradable”. Moreover, itwas evident that rutin could be readily and efficiently extractedwith ChCl/triethylene glycol solution, indicating that DESwould have great potential to extract rutin from Sophorajaponica.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acssusche-meng.5b00619.

The chemical structure of rutin; HPLC analysis ofstandard rutin and extracted rutin; the interaction of aHBD with the quaternary ammonium salt cholinechloride; the thermal properties of the DESs orindividual substances (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*Tel.: +86-20-87111452. E-mail: btmhzong@scut.edu.cn.*Tel.: +86-20-22236669. Fax: +86-20-22236669. E-mail:wylou@scut.edu.cn.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe wish to thank the National Natural Science Foundation ofChina (21336002; 21222606; 21376096), the Key Program of

Figure 5. Effect of various DESs on the amount of rutin extracted fromS. japonica (solid/liquid ratio of 1 g/10 mL, 55 °C, 20 min).

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Guangdong Natural Science Foundation (S2013020013049),and the Fundamental Research Funds for the ChineseUniversities (2015PT002; 2015ZP009) for partially fundingthis work.

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