Green Chemistry

PAPER

Cite this: Green Chem., 2018, 20,1879

Received 22nd December 2017,Accepted 17th March 2018

DOI: 10.1039/c7gc03820h

rsc.li/greenchem

Two-phase systems developed with hydrophilicand hydrophobic deep eutectic solvents forsimultaneously extracting various bioactivecompounds with different polarities†

Jun Cao,a,b Luyao Chen,a Mohan Li,a Fuliang Cao,b Linguo Zhaob andErzheng Su *a,b,c

Two-phase systems developed with hydrophilic deep eutectic solvents (DESs) and hydrophobic DESs

were prepared in this study for the first time. Ginkgo biloba leaves containing several bioactive com-

pounds were chosen as the model plant material to evaluate the possibility of the two-phase DES

system as a designer solvent for the simultaneous extraction of the various bioactive compounds with

different polarities. After tailoring, a two-phase DES system prepared with two hydrophilic DESs and one

hydrophobic DES at a volume ratio of 35 : 5 : 40 could effectively extract flavonoids, terpene trilactones

(TTLs), procyanidine (PAC) and polyprenyl acetates (PPAs) simultaneously from the Ginkgo biloba leaves.

The extraction conditions were further optimized using the response surface methodology. When the

two-phase DES system was employed in the extraction under the optimal conditions, the PPAs moved

into the hydrophobic phase, and the flavonoids, TTLs, and PAC were enriched into the hydrophilic

phase. The first extraction rates for PAC, flavonoids, TTLs and PPAs were 86.07%, 77.72%, 93.29% and

94.63%, respectively. After extraction, the hydrophilic and hydrophobic phases containing different bio-

active compounds could be easily separated for different preparations. Extraction with the two-phase

DES system is a new application trial of DESs, which has the potential to be widely used for the extrac-

tion of bioactive compounds from plant materials or removal of different polar impurities from specific

extracts.

Introduction

There are a lot of natural bioactive compounds present inplant resources with specific physiological and chemical pro-perties, which have been widely applied in pharmaceutical,food, perfume, and cosmetics industries. Traditional processesto extract these bioactive compounds usually employ petro-chemical and volatile organic solvents. Therefore, high energyconsumption, large use of organic solvents, environment pol-lution and low extraction rates are always linked to these tra-ditional extraction processes.

With the emphasis on environmental issues, the demandfor green and natural products is progressively increasing,which attracts more and more attention towards green extrac-tion technology for bioactive compounds. According to therequirements of green chemistry, the definition of greenextraction of natural products can be modified as follows:“Green extraction is based on the discovery and design ofextraction processes that reduce energy consumption, allowthe use of alternative solvents and renewable natural products,and ensure a safe and high quality extract/product”.1 Greensolvents are considered as the alternative solvents to toxicorganic solvents due to their high solvent power, non-toxicity,biodegradability and non-inflammability. Extraction withsupercritical CO2 has been developed since the 1970s and hasreached a relatively mature stage. Ionic liquids (ILs) areanother kind of green solvents with adjustable properties, andthey have been studied systematically. However, their limit-ations are the complex preparation processes and high costs.

In recent years, deep eutectic solvents (DESs) have beenconsidered as green solvent alternatives to conventionalorganic solvents and ILs.2,3 DESs are commonly made up of

†Electronic supplementary information (ESI) available. See DOI: 10.1039/c7gc03820h

aCollege of Light Industry and Food Engineering, Nanjing Forestry University,

Nanjing 210037, China. E-mail: ezhsu@njfu.edu.cn; Fax: +86 25 85428906;

Tel: +86 25 85428906bCo-innovation Center for the Sustainable Forestry in Southern China,

College of Forestry, Nanjing Forestry University, Nanjing 210037, ChinacState Key Laboratory of Natural Medicines, China Pharmaceutical University,

Nanjing 210009, China

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two or more non-toxic, biodegradable and inexpensive com-ponents.4 When compared with ILs, DESs have the advantagesof biodegradability, low toxicity, easy preparation, and novelproperties. Due to their green character, DESs have beenemployed to replace conventional volatile organic solvents toextract various natural bioactive compounds and fragrancessuch as phenolic acids, flavonoids, astaxanthin, anthocyanins,organic acids, stilbenes, alkaloids, polygodial and vanillin.5–15

Except for hydrophilic DESs, hydrophobic DESs have alsobeen prepared to extract hydrophobic bioactive components,for example, polyprenyl acetates (PPAs), artemisinin andneonicotinoids.16–18

There are quite a few plant materials containing bothhydrophilic bioactive compounds and hydrophobic bioactivecompounds. For example, the major bioactive constituents ofthe Chinese herb Radix Salviae Miltiorrhizae can be classifiedinto hydrophilic depsides derived from caffeic acid, e.g., sal-vianolic acids and lipophilic components including diterpe-noids tanshinones.19 Ginkgo biloba leaves contain hydro-phobic PPAs and partially hydrophilic components such asflavonoids, terpene trilactones (TTLs) and procyanidine(PAC).20 The traditional extraction processes employingorganic solvents often mainly extract the hydrophilic com-ponents or lipophilic components only. The ignored lipophi-lic or hydrophilic components remaining in the extractionresidues are discarded, which leads to waste of resources andenvironmental pollution. At present, hydrophilic or hydro-phobic DESs have been designed to enhance the extractionefficiency of bioactive components with various polarities.We wondered whether there was a DES-DES two-phasesystem formed with hydrophilic DES and hydrophobic DES,which could simultaneously extract the bioactive compoundswith various polarities from plant materials. The two-phasesystem containing DES has been reported. A hydrophobicDES-water two-phase system was first developed by Van Oschand evaluated for the recovery of volatile fatty acids fromdiluted aqueous solutions.21 A DES-salt aqueous solutiontwo-phase system has also been established to extractprotein and anthraquinones.22–25 To the best of our knowl-edge, there are no studies on the DES-DES two-phase systemprepared with two or more DESs to simultaneously extractthe bioactive compounds with various polarities from plantmaterials. Our group has carried out some experiments onthe extraction of Ginkgo flavonoids, TTLs, PAC and PPAsfrom Ginkgo biloba leaves using hydrophilic or hydrophobicDESs.18,26 Based on the previous studies, Ginkgo bilobaleaves are selected as the model plant material because ofthe high contents of bioactive compounds with differentpolarities. We aim to prepare a two-phase DES system andevaluate the possibility of this two-phase system as a tunablesolvent to simultaneously extract bioactive compounds withdifferent polarities from the model plant. PPAs with hydro-phobicity and PAC, TTLs and flavonoids with certain hydro-philicities are chosen as the target bioactive compounds toinvestigate the extraction abilities of the DES-DES two-phasesystem.

Experimental sectionMaterials

Ginkgo Biloba leaves picked in October were purchased fromthe Chinese Herb Transaction Center (Bozhou, China). Theleaves were dried at 40 °C in a vacuum oven to constant weightand then pulverized to 30–40 mesh using a disintegrator. Thewater content of the Ginkgo biloba leaves was 3.42%.

The standard PAC compound present in Ginkgo bilobaleaves was donated by Zhejiang Conba Pharmaceutical Co., Ltd(Hangzhou, China). The standard rutin (≥98%) and ginkgolideA (≥98%) compunds were obtained from the National Institutefor the Control of Pharmaceutical and Biological Products(Beijing, China). Hydroxylamine hydrochloride (99%) andferric chloride (98%) were bought from Alfa Aesar ChemicalCo. Ltd (Shanghai, China). Methyl trioctyl ammonium chloride(≥99.0%) was obtained from Adams Reagent Co., Ltd(Shanghai, China). All chemicals and reagents used for theDES preparation were obtained from Aladdin IndustrialCorporation (Shanghai, China). Methanol, isopropanol andn-hexane of chromatographic grade were provided by Tedia CoInc. (Shanghai, China). Deionized water was prepared using aMilli-Q® Ultrapure Water System (Millipore, Billerica, MA). Allother chemicals and reagents used in the experiments were ofanalytical reagent grade.

Determination of bioactive compounds in Ginkgo bilobaleaves

HPLC analysis of PPAs was performed on an Elite P1201 HPLCsystem equipped with a SinoChrom ODS-BP C18 column (4.6 ×200 mm, 5 µm). The mobile phase was a mixture of isopro-panol–methanol–n-hexane–water at a ratio of 45 : 22 : 25 : 3(v/v). The column temperature was controlled at 27 °C, and theflow rate was maintained at 1.0 mL min−1. The injectionvolume was 10 µL. The absorbance was detected at 210 nm.18

The content of PPAs was calculated by means of a calibrationcurve established with the regression equation y = 1179.4x +12.746 (R2 = 0.9991, Tables S1–S3†).

The content of PAC was detected using the spectrophoto-metric method employing 4-(dimethylamino) cinnamaldehyde(DMAC).27 The detection procedure was as follows: HCL(12.5 mL) and water (12.5 mL) were added into 75 mL ofethanol to prepare the acidic ethanol solution, which wasrequired to be used immediately after it was prepared. DMAC(50 mg) was dissolved in the acidic ethanol solution to make50 mL of the DMAC solution. The DMAC solution was freshlyprepared daily. One mL of the sample was added into 3 mL ofDMAC solution and kept for 10 min at room temperature forthe color reaction. After the reaction, the mixture was detectedat 644 nm. The content of PAC was calculated by means of acalibration curve established with the regression equation y =8.8157x + 0.0114 (R2 = 0.9994, Tables S1–S3†).

Flavonoids were detected using the rutin method.28,29 Thesample (0.5 mL) was mixed with 0.3 mL of NaNO2 solution(5.0%, w/w). After reacting for 6 min at room temperature,0.3 mL of Al(NO3)3 solution (10%, w/w) was added into the

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mixture. The reaction system was kept for 6 min at room tem-perature before adding the latter reaction reagents, which were4 mL of NaOH solution (4%, w/w) and 4.5 mL of 70% (v/v)ethanol. The mixture was detected at 510 nm after 20 min. Thecontent of flavonoids was calculated by means of a calibrationcurve established using rutin as the standard. The regressionequation was y = 6.1786x − 0.0689 (R2 = 0.9989, TablesS1–S3†).

The colorimetric method was used for the determination ofTTL content using ginkgolide A as the standard.26

Hydroxylamine solution (0.4 mL) consisting of 13.9% (w/w)hydroxylamine hydrochloride solution and 3.5 mol L−1 NaOHsolution (1 : 2, v/v) was added into 1 mL of the sample, and themixture was reacted at 25 °C for 5 min. A hydrochloric acidsolution (0.4 mL, 3.0 mol L−1) and 0.2 mL of FeCl3 solution(6.0%, w/w) were added to the abovementioned reactionmixture and mixed. Then, 5.0 mL of 70% (v/v) ethanol wasadded. The mixture was detected at 517 nm. The calibrationcurve was established using ginkgolide A as the standard. Thelinear regression equation was y = 1.2243x − 0.00154 (R2 =0.9992, Tables S1–S3†).

Preparation and tailoring of two-phase DES system

Preparation of two-phase DES system. The two-phase DESsystem was prepared with hydrophobic DES and hydrophilicDES. DESs were prepared by heating different component mix-tures having a certain molar ratio to 80 °C with constant stir-ring until a homogeneous liquid was formed. MCO, which wasthe optimal DES for the extraction of PPAs,18 was chosen asthe hydrophobic phase. MCO was prepared by mixing methyltrioctyl ammonium chloride, capryl alcohol and octylic acid ata molar ratio of 1 : 2 : 3. The hydrophilic phase was selectedfrom the DESs: Ch-LA1, BE-EG and Ch-M, and they showed thehighest extraction yields for Ginkgo flavonoids, TTLs and PAC,respectively.26 Ch-LA1 was prepared from choline chloride andlevulinic acid at a molar ratio of 1 : 2 with a water content of40% (w/w). BE-EG was prepared from betaine and ethyleneglycol at a molar ratio of 1 : 3 with a water content of 40%(w/w). Ch-M was prepared from choline chloride and malonicacid at a molar ratio of 1 : 2 with a water content of 55% (w/w).The two-phase DES system was prepared by mixing the hydro-phobic DES with hydrophilic DES at a volume ratio of 1 : 1.

Tailoring of two-phase DES system. Three hydrophilic DESsCh-LA1, BE-EG and Ch-M were chosen to construct the two-phase DES system. To choose an appropriate DES, severalgroups of experiments were carried out. First, the extractionabilities for Ginkgo flavonoids, TTLs and PAC were comparedbetween Ch-LA1, BE-EG and Ch-M by changing the extractionconditions. Second, the two-phase DES system constructedwith MCO and only one hydrophilic DES (Ch-LA1 or BE-EG orCh-M) was prepared to evaluate the extraction abilities for fourdifferent kinds of polar bioactive compounds (Ginkgo flavo-noids, TTLs, PAC and PPAs). Last, the two-phase DES systemconstructed with MCO and two hydrophilic DESs (Ch-LA1 andBE-EG, Ch-LA1 and Ch-M, or BE-EG and Ch-M) was preparedto evaluate the extraction abilities for four different kinds of

polar bioactive compounds (Ginkgo flavonoids, TTLs, PAC andPPAs). The volume ratio between the two hydrophilic DESs waschanged from 10 : 0 to 0 : 10.

Extraction of bioactive compounds from Ginkgo biloba leavesusing the two-phase DES system

The hydrophobic DES MCO (2 mL) and 2 mL of hydrophilicDES or DESs mixture were mixed in a 20 mL ground-glass stop-pered flask to construct the two-phase DES system. The Ginkgobiloba leaves powder (0.2 g) was added to the two-phase DESsystem. The extraction was performed by stirring at 150 rpm at25 °C for a certain time, followed by centrifugation at 10 000rpm for 10 min. The samples were withdrawn from the hydro-philic phase for determining the contents of flavonoids, TTLsand PAC and from the hydrophobic phase for determining thecontent of PPAs.

Optimization of the extraction procedure employing the two-phase DES system

A comparison of the extraction methods. After DES tailoring,the most efficient two-phase DES system was chosen as theextraction solvent to compare the extraction efficiencies of thedifferent extraction methods. Stirring (150 rpm), air-bathshaking (150 rpm) and ultrasonic methods (50 w) were com-pared. Apart from the extraction conditions signed in thebrackets, the other extraction parameters were kept constant:the solid to solvent ratio was 1 : 20 (w/v, g mL−1), extractiontime was 30 min and extraction temperature was 50 °C.

Optimization of the extraction conditions using RSM.Single-factor experiments presented in the ESI† were con-ducted to determine the preliminary range of the extractionvariables for scientific experimental design based on theresponse surface methodology (RSM). Central compositedesign (CCD) combined with RSM was used to optimize theextraction conditions. Three parameters, namely, solid tosolvent ratio (X1), extraction temperature (X2) and extractiontime (X3) were investigated at five levels. The extraction yieldsof PPAs, flavonoids, TTLs and PAC were taken as the fourresponses. Twenty experimental runs were conducted inrandom order. The actual levels of the variables set in theexperimental design are listed in Table 1. Furthermore, asecond-order polynomial model was applied to analyze theexperimental data.

Results and discussionPreparation and tailoring of the two-phase DES system

The two-phase DES system could be prepared successfully bymixing MCO and hydrophilic DES (Fig. 1A). It could be dividedinto two-phases quickly after stirring and extracting (Fig. 1).The upper phase was MCO and the bottom phase was thehydrophilic DES (Fig. 1). It can also be seen from Fig. 1 thatthe color of the upper phase after the extraction was green,which might be due to the presence of hydrophobic chloro-

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phyll extracted from the Ginkgo biloba leaves by the hydro-phobic upper phase.

Selection of hydrophilic DES for constructing two-phase DESsystem. An ideal hydrophilic phase in the two-phase DESsystem should show high extraction yields for Ginkgo flavo-noids, TTLs and PAC at the same time. According to our pre-vious study,26 Ch-LA1, BE-EG and Ch-M showed the highestextraction yields for Ginkgo flavonoids, TTLs and PAC, respect-ively. Therefore, we wondered if one DES among the threeDESs showing high extraction efficiency for one componentcould effectively extract the other two components at the sametime. At first, the extraction abilities of Ch-LA1, BE-EG andCh-M for Ginkgo flavonoids, TTLs and PAC were separately

investigated. As the extraction time is a significant parameteraffecting the extraction yield, three extraction times (10 min,25 min and 45 min) were employed for investigation. Theresults are shown in Fig. 2. Because BE-EG interfered with theanalysis of TTLs, it was abandoned from the comparison. Ch-LA1 showed a higher extraction ability for flavonoids, whereasCh-M was more efficient in extracting TTLs and PAC. Theextraction abilities of Ch-LA1 and Ch-M for the three bioactivecompounds were different because of their polarity differ-ences. Unfortunately, we could not find out which one of Ch-LA1 and Ch-M would be more suitable as the hydrophilicphase for constructing the two-phase DES system.

The abovementioned investigation was not carried outunder the optimized conditions for the extraction of Ginkgoflavonoids, TTLs, and PAC.18,26 The extraction method andconditions are important factors influencing the extractionefficiency in the extraction process. Therefore, the extractionabilities of Ch-LA1 and Ch-M were further investigated underthe optimized extraction conditions developed in our previousstudies.18,26 Stirring at 50 °C for 15 min were the optimal con-ditions for flavonoids extraction, stirring at 65 °C for 53 minwere the optimal conditions for PAC extraction, and ultrasonic-assisted extraction (UAE) at 45 °C and 100 W for 20 min werethe optimal conditions for TLLs extraction. The weakest andstrongest extraction conditions stirring at 50 °C for 15 min and65 °C for 53 min, respectively, were chosen for comparing theextraction abilities of Ch-LA1 and Ch-M. This time, the contentof PPAs in the hydrophilic DES was also detected. The resultsare shown in Fig. 3. It could be found that the extraction yieldsof Ginkgo flavonoids, TTLs, and PAC increased along with anincrease in the extraction temperature and time. Similar to theresults obtained in Fig. 2, Ch-LA1 showed a higher extractionability for flavonoids, whereas Ch-M was more efficient inextracting TTLs and PAC. Surprisingly, PPAs could also beextracted by Ch-LA1 and Ch-M with higher extraction yields.

Table 1 The program of central composite design and the responsevalues

Run X1 X2 X3

Bioactive compounds inthe hydrophilic DES phase(mg g−1)

PPAs in the MCOphase (mg g−1)PAC Flavonoids TTLs

1 20 60 40 21.25 2.24 22.23 75.792 30 60 40 21.18 2.24 21.18 75.533 10 60 40 19.49 2.20 17.75 64.664 20 60 40 21.70 2.24 22.70 75.145 20 60 40 21.47 2.23 23.38 74.496 20 70 40 20.66 2.28 23.57 75.307 20 50 40 13.93 2.11 18.34 56.478 15 55 45 17.78 2.21 19.23 65.639 20 60 30 18.22 2.10 22.37 63.3210 25 65 45 20.72 2.22 23.82 74.7411 20 60 40 20.60 2.25 22.76 74.2812 15 65 35 20.50 2.24 22.19 74.6913 20 60 50 20.40 2.25 23.73 73.8814 15 65 45 20.81 2.33 22.55 73.3615 25 55 45 18.83 2.28 22.89 76.0516 20 60 40 20.58 2.25 22.97 74.3117 15 55 35 15.85 2.02 18.35 63.3118 20 60 40 20.70 2.25 22.97 75.7219 25 55 35 17.25 2.20 20.92 72.2120 25 65 35 21.08 2.26 22.24 75.30

Fig. 1 The two-phase DES system prepared with hydrophobic DES andhydrophilic DES. (A) The newly prepared two-phase DES system and (B)the two-phase DES system after extraction and centrifugation.

Fig. 2 The extraction yields of Ginkgo flavonoids, TTLs and PACextracted by Ch-LA1 and Ch-M. (a) The bioactive compounds extractedby Ch-LA1 and (b) the bioactive compounds extracted by Ch-M.Extraction conditions: solid to solvent ratio = 1 : 10, and extraction tem-perature = 25 °C.

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We doubted if the PPAs extracted into the hydrophilic DESsaffected the extraction abilities of the hydrophilic DESs forGinkgo flavonoids, TTLs, and PAC. A comparison of the extrac-tion abilities of Ch-LA1 and Ch-M in the true two-phase DESsystem may be more meaningful.

The extraction yields of four bioactive compounds found inGinkgo biloba leaves extracted using the MCO/Ch-LA1 andMCO/Ch-M two-phase DES systems are listed in Table 2. Asexpected, the content of PPAs in MCO increased with theextension of time for both two-phase DES systems, whereasthose in hydrophilic DESs decreased with the extension oftime until they were unable to be detected. The MCO/Ch-LA1two-phase DES system showed a higher extraction ability toPPAs. It could be concluded that the two-phase DES systemcould effectively reduce the content of hydrophobic com-ponents (PPAs) in the hydrophilic phase and was effective inenriching the bioactive compounds with different polaritiesinto the extraction phases with different polarities. That is tosay, the compounds with some hydrophilicity could beextracted into the hydrophilic DES phase, and the hydrophobiccompounds could be extracted into the hydrophobic DESphase.

Through the above three groups of experiments, we foundout that the extraction ability of Ch-LA1 and Ch-M should becompared in the true two-phase DES system. Ch-LA1 or Ch-Malone was not competent enough to be used as the hydrophilicDES phase to construct the two-phase DES system for effectivelyextracting Ginkgo flavonoids, TTLs and PAC at the same time.

Tailoring the hydrophilic DES for constructing the two-phase DES system. Ch-LA1 showed a higher extraction abilityfor flavonoids, and Ch-M was more efficient in extracting TTLsand PAC. Therefore, we wondered if we could mix Ch-LA1 withCh-M to prepare a new hydrophilic DES for constructing thetwo-phase DES system to fulfill our goal. The volume ratio ofCh-LA1 to Ch-M was modulated from 10 : 0 to 0 : 10 at an inter-val of 1. The mixture of Ch-LA1 and Ch-M was used as thehydrophilic DES phase for constructing the two-phase DESsystem for extracting the bioactive compounds from Ginkgobiloba leaves. The results are shown in Fig. 4A.

It could be found that the extraction yields of flavonoids,TTLs and PAC initially increased and then decreased with theincrease in the volume ratio of Ch-M. The addition of a certainamount of Ch-M to Ch-LA1 improved the extraction ability ofCh-LA1 for PAC and TTLs. Similarly, the addition of a certainamount of Ch-LA1 to Ch-M improved the extraction ability ofCh-M for flavonoids. It has been reported that mixing twoDESs may yield one with more suitable properties for a particu-lar application.30 A series of new hydrophilic DESs with novelproperties were prepared by mixing Ch-LA1 and Ch-M, whichwere more efficient in extracting the flavonoids, TTLs and PAC.When the volume ratio of Ch-LA1 : Ch-M was 8 : 2, the two-phase DES system with mixed Ch-LA1 and Ch-M as the hydro-philic phase showed the highest extraction yield for PAC; thisyield was 8.90% higher than that of the MCO/Ch-LA1 two-phase DES system and 7.25% higher than that of the MCO/Ch-M two-phase DES system. The two-phase DES system withmixed Ch-LA1 and Ch-M (8 : 2, v/v) as the hydrophilic phaseshowed an extraction yield for TTLs similar to the extractionyield of the MCO/Ch-M two-phase DES system; this yield was34.62% higher than that of the MCO/Ch-LA1 two-phase DESsystem. The two-phase DES system with mixed Ch-LA1 and Ch-M(9 : 1, v/v) as the hydrophilic phase showed the highest flavo-noid extraction yield; this yield was 7.44% higher than that ofthe MCO/Ch-LA1 two-phase DES system and 17.72% higherthan that of the MCO/Ch-M two-phase DES system. When the

Fig. 3 The extraction yields of four bioactive compounds found inGinkgo biloba leaves extracted using Ch-LA1 and Ch-M under the opti-mized conditions used for flavonoids and PAC extraction. Extractionconditions: solid to solvent ratio = 1 : 10.

Table 2 The extraction yields of the bioactive compounds found in Ginkgo biloba leaves extracted using the two-phase DES systema

Hydrophilic phase and extraction time

Bioactive compounds in the hydrophilic DES phase (mg g−1)

PPAs in the MCO phase (mg g−1)PAC Flavonoids TTLs PPAs

Ch-LA1 (15 min) 13.04 ± 0.87 1.81 ± 0.02 12.90 ± 0.50 1.37 ± 0.06 65.04 ± 1.13Ch-M (15 min) 14.25 ± 0.58 1.39 ± 0.01 17.09 ± 0.66 2.15 ± 0.08 38.76 ± 1.80Ch-LA1 (30 min) 15.90 ± 1.07 2.07 ± 0.05 14.45 ± 0.52 Undetected 75.54 ± 2.14Ch-M (30 min) 16.14 ± 0.89 1.76 ± 0.02 22.10 ± 0.37 Undetected 42.42 ± 1.01Ch-LA1 (45 min) 17.29 ± 1.31 2.07 ± 0.03 20.36 ± 0.74 Undetected 77.86 ± 0.47Ch-M (45 min) 17.71 ± 0.86 1.88 ± 0.03 22.81 ± 0.94 Undetected 59.01 ± 1.57

a The solid to solvent ratio = 1 : 10 and the extraction temperature = 50 °C.

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volume ratio of Ch-LA1 : Ch-M was less than 5 : 5, the extrac-tion yield of PPAs decreased with an increase in the Ch-Mvolume. Based on the abovementioned results, the volumeratio of Ch-LA1 : Ch-M was further investigated within therange from 10 : 0 to 7 : 3 at an interval of 0.25. The results aredisplayed in Fig. 4B.

It could be seen that the fine tuning of the volume ratio ofCh-LA1 : Ch-M did lead to a change in the extraction yield. Forthe Ch-LA1 : Ch-M system having a volume ratio in the range of10 : 0–7 : 3, the extraction yields of PAC and TTLs increased andthen decreased. The extraction yields of flavonoids and PPAsremained almost the same. There was not a certain ratio showinghigh extraction ability for all of the target bioactive compounds.Three volume ratios (8.75 : 1.25, 8.25 : 1.75 and 7.75 : 2.25) of Ch-LA1 : Ch-M were selected for the next optimization step.

Optimization of the extraction process employing the two-phase DES system

Selection of the extraction methods. An appropriate extrac-tion method may enhance the extraction yield, cut down con-

sumption and improve economic efficiency. The effects ofdifferent extraction methods on the extraction yields of thetwo-phase DES system for flavonoids, TTLs, PAC and PPAswere investigated (Table 3). It could be found that the stirringmethod showed the highest extraction yields for the four bio-active compounds in three kinds of two-phase DES systemsstudied, followed by the air-bath shaking method and UAEmethod. When the stirring method was used, the extractionyields of PAC and PPAs in the three kinds of the two-phaseDES systems were almost the same. However, the extractionyield of the flavonoids decreased and that of TTLs increasedwith the increase in the Ch-M volume. When the UAE or air-bath shaking method was employed, the extraction yields ofthe four bioactive compounds except TTLs decreased orremained almost unchanged. Considering the extraction yieldsobtained for the four bioactive compounds as a whole, the stir-ring method was chosen as the extraction method, and Ch-LA1 : Ch-M at a volume ratio of 8.75 : 1.25 was adopted as thehydrophilic phase to form the two-phase DES system withMCO at a volume ratio of 1 : 1.

Fig. 4 The effect of the Ch-LA1 to Ch-M volume ratio on the extraction efficiency of the two-phase DES system. (A) The volume ratio of Ch-LA1 : Ch-M varied from 10 : 0 to 0 : 10 and (B) the volume ratio of Ch-LA1 : Ch-M varied from 10 : 0 to 7 : 3. Extraction conditions: solid to solventratio = 1 : 20, extraction temperature = 50 °C and extraction time = 30 min.

Table 3 The extraction efficiency of the two-phase DES system employing different extraction methodsa

Volume ratio of Ch-LA1 : Ch-M Extraction method

Bioactive compounds in hydrophilic DES phase(mg g−1)

PPAs in MCO phase (mg g−1)PAC Flavonoids TTLs

8.75 : 1.25 Stirring 17.60 ± 0.85 2.18 ± 0.02 17.80 ± 0.87 75.74 ± 2.01UAE 12.13 ± 0.98 1.88 ± 0.05 13.03 ± 0.56 47.87 ± 1.11Air-bath shaking 14.12 ± 0.91 1.80 ± 0.05 12.50 ± 0.06 57.81 ± 1.70

8.25 : 1.75 Stirring 16.33 ± 1.10 2.05 ± 0.09 18.37 ± 0.37 74.44 ± 1.58UAE 10.64 ± 0.96 1.67 ± 0.03 15.52 ± 0.06 46.62 ± 1.83Air-bath shaking 14.44 ± 0.48 1.80 ± 0.06 14.45 ± 0.62 51.12 ± 1.74

7.75 : 2.25 Stirring 17.01 ± 0.92 2.02 ± 0.02 20.08 ± 0.50 74.89 ± 1.60UAE 8.75 ± 0.86 1.32 ± 0.05 15.92 ± 0.34 43.68 ± 0.69Air-bath shaking 14.70 ± 0.47 1.67 ± 0.03 15.07 ± 023 44.37 ± 1.01

a Solid to solvent ratio = 1 : 20, extraction temperature = 50 °C and extraction time = 30 min.

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The extraction abilities of the mixed hydrophilic DES-MCOtwo-phase system and single hydrophilic DES-MCO two-phasesystem were compared (Table 4). Except for the extraction yieldof TTLs with MCO/Ch-LA1 : Ch-M being lower than that ofMCO/Ch-M, the MCO/Ch-LA1 : Ch-M two-phase system alwaysshowed higher extraction yields for the target bioactive com-pounds found in Ginkgo biloba leaves.

Optimization of the extraction conditions using RSM. Theextraction yield of bioactive compounds from plant resourcesis affected by the extraction conditions such as extractiontemperature, solid to solvent ratio and extraction time. Aninvestigation of the effect of these extraction conditions wasconducted using RSM-based optimization, which allows theevaluation of the interaction effects between variables and vari-ables optimized in their full scope through fewer experi-ments.31 Single-factor experiments were carried out to choosethe appropriate parameter ranges for RSM optimization. Theresults are shown in Table S4 and Fig. S1, S2 found in theESI.† According to the results obtained from the single-factorexperiments, the factor ranges for RSM optimization wereselected as follows: solid to solvent ratio (X1) from 1 : 10 to1 : 30 (g mL−1), temperature (X2) from 50 to 70 °C and extrac-tion time (X3) from 40 to 50 min. The extraction yields of PAC,flavonoids, TTLs and PPAs were taken as responses and wereexpressed as Y1, Y2, Y3 and Y4, respectively. The resultsobtained from 20 experimental runs of CCD are shown inTable 1.

The second-order polynomial equation was applied toexpress the proposed model after multiple regression analysisof the experimental data. The second-order polynomialequations for the responses and variables in terms of thecoded levels are as follows:

Y1 ¼ 20:98003þ 0:394038X1 þ 1:678975X2 þ 0:488663X3

� 0:24525X1X2 � 0:12695X1X3 � 0:44395X2X3

� 0:2156X12 � 0:97511X2

2 � 0:47175X32:

Y2 ¼ 2:245034þ 0:015494X1 þ 0:042881X2 þ 0:038594X3

� 0:03996X1X2 � 0:03051X1X3 � 0:02686X2X3

� 0:00465X12 � 0:01041X2

2 � 0:01625X32:

Y3 ¼ 22:82395þ 0:9009X1 þ 1:241888X2 þ 0:4694X3

� 0:6121X1X2 þ 0:289625X1X3 � 0:11453X2X3

� 0:84675X12 � 0:47459X2

2 þ 0:049515X32:

Y4 ¼ 75:32499þ 2:690713X1 þ 3:658725X2 þ 1:587175X3

� 2:16575X1X2 þ 0:28735X1X3 � 1:0096X2X3

� 1:03146X12 � 2:08384X2

2 � 1:40494X32:

The results for ANOVA for the proposed models are shownin Tables S5–S8 in the ESI.† The significance of each coeffi-cient was checked by the F-test and p-values. If the F-value wasmore and the p-value was less than 0.05, the model was con-sidered to be significant. The p-values in the four ANOVAresults were all less than 0.05, indicating the models were sig-nificant and suitable for the optimization of the extractionconditions. The p-value was applied to verify the significanceof the linear coefficients, cross product coefficients and quad-ratic term coefficients of the parameters. In this case, thelinear coefficients (X1, X2, X3) were considered to be significantfor the extraction of the four compounds (p < 0.05). The crossproduct coefficients X1X2 had a significant effect on the extrac-tion yields of flavonoids, TTLs and PPAs (p < 0.05). The extrac-tion yield of the flavonoids was also affected by the crossproduct coefficients X1X3.

The results of the ANOVA analysis were consistent withthose of 3D response surface plots, which gave a visualgraphical representation of the effects of variables and theinteraction effects between the variables (shown in Fig. S3–S6 in the ESI†). Analysis of 3D response surface plots gavethe same results when compared with the analysis ofANOVA.

The optimal extraction conditions were also fitted out usingRSM and were as follows: solid to solvent ratio of 1 : 19.699(g mL−1), extraction temperature of 65 °C and extraction timeof 41.954 min. The predicted extraction yields of PAC, flavo-noids, TTLs and PPAs were 21.62 mg g−1, 2.28 mg g−1,23.71 mg g−1 and 76.87 mg g−1, respectively. The verificationexperiment was conducted under a solid to solvent ratio of1 : 20 (g mL−1) at 65 °C for 42 min, which gave the practicalextraction yields of 21.28 ± 0.92 mg g−1, 2.22 ± 0.05 mg g−1,22.86 ± 0.06 mg g−1 and 74.28 ± 1.94 mg g−1 for PAC, flavo-noids, TTLs and PPAs, respectively. The residue of the Ginkgobiloba leaves after the first extraction was collected for asecond extraction step under the optimal conditions to calcu-late the extraction rate. The results showed that the first extrac-tion rates of PAC, flavonoids, TTLs and PPAs were 86.07%,77.72%, 93.29% and 94.63%, respectively.

Table 4 A comparison of the extraction abilities among the three two-phase DES systems studieda

Two-phase DES system

Bioactive compounds in the hydrophilic DES phase (mg g−1)

PPAs in the MCO phase (mg g−1)Flavonoids (mg g−1) PAC (mg g−1) TTLs (mg g−1)

MCO/Ch-LA1 : Ch-M 2.18 ± 0.02 17.6 ± 0.85 17.80 ± 0.87 75.74 ± 2.01MCO/Ch-LA1 2.07 ± 0.05** 15.90 ± 1.07** 14.45 ± 0.52** 75.54 ± 2.14MCO/Ch-M 1.88 ± 0.03** 16.14 ± 0.89** 22.10 ± 0.37** 42.42 ± 1.01**

a Solid to solvent ratio = 1 : 20, extraction temperature = 50 °C, stirring extraction time = 30 min. Compared with the data of MCO/Ch-LA1 : Ch-Min the same column, ** indicated p < 0.01.

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Conclusions

This study provided a practical example showing the feasibilityof constructing a two-phase DES system and its application inthe simultaneous extraction of bioactive compounds withdifferent polarities. A two-phase DES system that was formedusing Ch-LA1/Ch-M/MCO at a volume ratio of 35 : 5 : 40showed a high extraction ability towards the bioactive com-pounds with different polarities found in the Ginkgo bilobaleaves. The extraction yields for PAC, flavonoids, TTLs andPPAs were 21.28 ± 0.92 mg g−1, 2.22 ± 0.05 mg g−1, 22.86 ±0.06 mg g−1 and 74.28 ± 1.94 mg g−1, respectively, which corre-sponded to the first extraction rates of 86.07%, 77.72%,93.29% and 94.63%. The two-phase DES system could effec-tively enrich the bioactive compounds with different polaritiesin the upper phase or bottom phase. The upper phase andbottom phase could be easily separated after the extractionprocess. Similar to general DESs, the two-phase DES systemcould also be tailored for maximal extractability of diverse bio-active compounds from plant materials. The application of thetwo-phase DES system in multi-component extraction is a newtrial in the application of DESs. This system can be widelyused for the extraction of bioactive compounds from otherplant materials or in similar situations such as impurityremoval.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This study was supported by the “Six Talent Peaks Project inJiangsu Province (2015-JY-016)”, the “China PostdoctoralScience Foundation (2016M600417 and 2017T100373)”, the“333 project of Jiangsu Province (BRA2017458)”, the “OpenProject of State Key Laboratory of Natural Medicines (No.SKLNMKF201802)”, “A Project Funded by the PriorityAcademic Program Development of Jiangsu Higher EducationInstitutions, PAPD” and the “Project of Innovative andEntrepreneurial Training Program for College Students inJiangsu Province (SPITP, 201710298040Z)”.

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1886 | Green Chem., 2018, 20, 1879–1886 This journal is © The Royal Society of Chemistry 2018

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