greenandefficientextractionofresveratrolfrom...

Research ArticleGreen and Efficient Extraction of Resveratrol fromPeanut Roots Using Deep Eutectic Solvents

Jingnan Chen Xingxing Jiang Guolong Yang Yanlan Bi and Wei Liu

Provincal Key Laboratory for Transformation and Utilization of Cereal Resource College of Food Science and TechnologyHenan University of Technology Zhengzhou 450001 China

Correspondence should be addressed to Jingnan Chen chenjingnan813126com and Wei Liu liuwei307hotmailcom

Received 31 July 2018 Revised 11 October 2018 Accepted 14 November 2018 Published 13 December 2018

Guest Editor Mariusz Korczynski

Copyright copy 2018 Jingnan Chen et al 2is is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Deep eutectic solvents (DESs) a new group of ecofriendly solvent combined with the ultrasonic-assisted extraction (UAE)technique were first successfully used for extraction of resveratrol from peanut roots Resveratrol in the extract was analyzed andquantified using a HPLC-UV method A series of DESs consisting of choline chloride (ChCl) and 14-butanediol citric acid andethylene glycol were formulated finding ChCl14-butanediol was a most proper extraction system 2e optimal extractionparameters were obtained using a BoxndashBehnken design (BBD) test combined with response surface methodology as follows 40of water in ChCl14-butanediol (13 gg) at 55degC for 40min and solidliquid ratio of 130 gmL 2e total extraction content andextraction yield of resveratrol from peanut roots could reach 3891mgkg and 8819 respectively under such optimal conditions2e present study will provide a typical example for using DESs to extract natural bioactive compounds from plants

1 Introduction

Peanut (Arachis hypogaea Linn) belongs to the family ofRosales and is widely cultivated around the world as animportant economical crop 2is species is capable ofproducing stilbene derivatives including resveratrol (354prime-trihydroxy-stilbene) and other stilbenoids [1] Resveratrolhas attracted tremendous interest ascribing to its strongbiological activity such as antioxidant anti-inflammatoryanticancer cardiovascular protection and cardioprotectionfunctions [2ndash4] In addition it has been widely used as anactive ingredient in cosmetic medicine and health productsNaturally resveratrol is extracted from peanut [5] Japaneseknotweed [6] grape [7] and other plants Peanut roots arethe waste products in the field after peanut harvesting whichare the cheapest sources of resveratrol [8]

Extraction of resveratrol from peanut roots should bebased on green and sustainable technology Conventionallyorganic solvents such as alcohols and acetone are widely usedin extraction of bioactive components from natural productsresources in the fields of cosmetic food and pharmaceutical

industries [9 10] However the organic solvents pollution is aserious environmental issue 2erefore the green extractionby using the new and environmentally friendly solvents wasneeded urgently [11] In this connection the deep eutecticsolvents (DESs) have emerged as a better alternative toconventional solvents [12] DESs can be easily synthesizedwith quaternary ammonium salt and hydrogen bond donor(HBD) with a gentle heating temperature range (70degCndash90degC)and no further purification is required [13] Numerous DESsconsisting of different components such as choline chlorideurea organic acids polyols and sugars have been developed[14ndash16] In addition to as an green extraction solvent DESshave other advantages including the simple synthesis lowcost biodegradable chemical inertness and no toxic factors[17 18] In fact DESs have been applied in the extraction of awide variety of natural compounds including phenoliccompounds [19 20] flavonoids [21 22] sugars [23] andproteins [24 25]

With the aim of development of green extractiontechnology for separation bioactive compounds from oilprocessing by-products the present study was to establish a

HindawiJournal of ChemistryVolume 2018 Article ID 4091930 9 pageshttpsdoiorg10115520184091930

highly efficient and green extraction technology for theextraction of resveratrol from peanut roots using deep eu-tectic solvents assisted by ultrasonic extraction methods(DESs-UAE) First a series of DESs were prepared bymixingthe varying ratios of polyols organic acid and carbamidewith ChCl Second various parameters of DESs-UAE inextracting resveratrol were optimized and systematicallyevaluated using a BoxndashBehnken design

2 Materials and Methods

21 Chemicals and Reagents 14-Butanediol (gt98) andcitric acid (gt99) were obtained from Kwangfu FineChemical Industry Research Institute (Tianjin China)Ethylene glycol (gt98) was purchased from Fuyu FineChemical Co Ltd (Tianjin China) Lactic acid (gt95) waspurchased from Sinopharm Chemical Reagent Co Ltd(Shanghai China) Glycerol (gt99) 12-propylene glycol(gt99) DL-malic acid (gt99) and carbamide (gt99) werepurchased from Kermel Chemical Reagent Co Ltd(Tianjin China) Choline chloride (gt98) was obtainedfrom Macklin Biochemical Co Ltd (Shanghai China)Resveratrol (354prime-trihydroxy stilbene ge98) was obtainedfrom Sigma-Aldrich Co (St Louis MO USA) Methanol(the chromatographic grade) was purchased from VBS bi-ologic Co (USA) All samples and solutions prepared forHPLC analysis were filtered through the 045 μm nylonmembranes prior to use

Standard stock solutions resveratrol compound wasdirectly prepared in methanol 2e concentration ofresveratrol in the standard stock solutions was all 500 μgmLWorking standard solutions resveratrol compound wasobtained by diluting the stock solutions with methanol to aseries of proper concentrations2e standard stock solutionsand working standard solutions were all stored at 4degC in adark place

22 Plant Materials Peanut roots were purchased fromHebei province in China2e plant material was cleaned anddried at 40degC 2e dry plant material was ground intopowder with a blender and passed through 50mesh and thenstored in a dry place at room temperature until used

23 Preparation of DESs All of the chemicals used in DESspreparations were dried at 60degC for 24h 2e DESs wereprepared at specific ratios of choline chloride to the hydrogendonor (ie 14-butanediol glycerol and lactic acid Table 1)2e varying ratios of choline chloride with the hydrogendonor were stirred in a flask at 80degC for 5ndash10min until ahomogeneous transparent colourless liquid was formed2eseDESs samples were treated by vacuum drying prior to use

24 Deep Eutectic Solvents Ultrasonic-Assisted Extraction ofResveratrol About 050 g peanut roots powder was weighedinto an 50mL centrifuge tube and followed by addition of10mL of extraction solvent 2e mixture was then ultra-sonically treated Ultrasound-assisted extraction (UAE) was

performed using an ultrasonicator under 40 kHz and 400W(SCQ-7201B Shengyan Ultrasonic Instrument Co LtdShanghai China) 2e extraction was carried out underdifferent conditions After extraction the mixture wascentrifuged (10min 2500 rpm) with a bench-scale centri-fuge 2e supernate was separated and filtered through a045 μm membrane prior to HPLC analysis

2e extraction content was calculated according to thefollowing equation

extraction content(mgkg)

mass of resveratrol(mg)

mass of weighed peanut roots powder(kg)

(1)

2e extraction yield was defined as follows [26]

extraction yield()

mass of the resveratrol in extraction solutionsumof themass of resveratrol in sample

times 100

(2)

2e mass of resveratrol in the extraction solution (one-step extraction) was determined by HPLC-UV 2e summass of resveratrol in the sample was calculated by analysisof the total mass of resveratrol in the combined extractionsolutions afforded by continuously extracting three timeswith methanol according to the standard method

25 Experimental Design and Statistical Analysis Firstly themole ratio of hydrogen bond donors and ChCl (14-butanediol ChCl 1 1 2 1 3 1 4 1 and 5 1) thepercentage of water (10 20 30 40 50 and 100)solid-liquid ratio (1 10 1 20 1 30 1 40 and 1 50)extraction temperature (20 30 40 50 60degC and 70degC) andextraction time (20 30 40 50 and 60min) were optimizedby single-factor experiments respectively FurthermoreBoxndashBehnken experimental design (BBD) with responsesurface methodology (RSM) was used to estimate the mosteffective combination of extraction parameters accordingto the single-factors experiments A three-level (minus1 0 and+1) four-factor BoxndashBehnken design (BBD) was applied toevaluate the interaction effect of the factors the percentageof water (A) solidliquid ratio (B) extraction temperature

Table 1 Different systems of natural deep eutectic solvents (DESs)

Abbreviation Component 1 Component 2 Molarratio

DES-1 Choline chloride 14-Butanediol 1 2

DES-2 Choline chloride 12-Propyleneglycol 1 2

DES-3 Choline chloride Glycerol 1 2DES-4 Choline chloride Ethylene glycol 1 2DES-5 Choline chloride DL-Malic acid 1 2DES-6 Choline chloride Lactic acid 1 2DES-7 Choline chloride Carbamide 1 2DES-8 Choline chloride Citric acid 1 2

2 Journal of Chemistry

(C) and extraction time (D) on the extraction content ofresveratrol (Y) 29 experiments running with differentcombinations of four factors were carried out totally 2esecond-order polynomial given in the equation was appliedto correlate the interaction of each factor to the responseFor the four factors this equation is

Y β0 + 11139444

i1βiXi + 1113944

4

i1βiiX

2i + 1113944

4

i11113944

4

ji+1βijXiXj (3)

where Xi and Xj are the independent coded variables whichinfluence the response Y Y is the predicted response βi is theparameter estimated for the variable βii and βij are theparameters estimated for the interaction between variables iand i and i and j and β0 βi βii and βij are the regressioncoefficients for intercept linearity square and interactionrespectively2e variables of each factor were changed in therange of between minus1 and 1 for the appraisals while thedependent variable was the extraction content of resveratrol

All above experimental statistical analyses were analyzedusing the software Design-Expert 806 (Stat-Ease Minne-apolis MN USA) Analysis of variance (ANOVA) wascarried out to evaluate the optimal conditions for theresveratrol extraction from peanut roots using the DESs-UAE method A significance level of plt 005 was performedfor each influential factor

26 HPLC Analysis of Resveratrol 2e determination ofresveratrol content was carried out on an HPLC system(Waters e2695 USA) 2e chromatographic separation ofresveratrol was performed on Waters Symmetry C18reversed-phase column (250mm times 5mm times 46mm 5 μm)2e mobile phase consisting of 05 formic acid aqueoussolution (A) and methanol (B) was filtered through a045 μm membrane filter prior to use 2e gradient elutionwas performed as follows 0ndash6min 28 B 6ndash15min 28ndash60 B 15ndash20min 60ndash100 B and 20ndash25min 100 B 2eflow rate and injection volume were 10mLmin and 10 μLrespectively and the column temperature was set at 30degC2e resveratrol was identified by comparing the retentiontime with the standard and the quantification of resveratrolwas carried out at 306 nm 2e HPLC analysis of theresveratrol standard and peanut roots sample was shown inFigure 1

27 Statistical Analysis Experimental results were obtainedas the mean value plusmn standard deviation (SD) (n 3) 2esignificance of difference was assessed using ANOVADifferences were considered significant when the p valuewas lt005

3 Results and Discussion

31Effect ofDESsonResveratrolExtraction 2e componentsof DESs have significant influence on their physicochemicalproperties such as polarity viscosity and dissolving ca-pacity which will directly influence their extraction effi-ciency In the present study ChCl-based DESs were

synthesized by ChCl combining with different hydrogenbond donors (HBDs) including 14-butanediol citric acidethylene glycol lactic acid glycerol 12-propylene glycolDL-malic acid and carbamide 2e obtained eight DESs withdifferent physicochemical properties were used forextracting resveratrol from peanut roots 2e extractioncontents were shown in Figure 2 2e results indicated thatthe DESs type indeed strongly influenced the resveratrolextraction efficiency 2e sequence of DESs for the extrac-tion contents of resveratrol was as follows DES-1 (ChCl14-Buta) gt DES-2 (ChCl12-PG) gt DES-4 (ChClEG) gt DES-6(ChClLA) gt DES-7 (ChClCa) gt DES-3 ((ChClGly) gtDES-5 (ChClMA) gt DES-8 (ChClCA) 2e optimal DESwhich provided the highest extraction content (2644 plusmn006mgkg) of resveratrol was DES-1 (composed of ChCl14-butanediol) 2ese data indicated that the polyalcohol-based DESs had a better extraction efficiency than organicacid-based DESs except for DES-3 ((ChClglycerol) 2iswas because the strength of H-bonding interactions of theorganic acid-based DESs would be the most efficient or theenthalpy of hole formation of polyalcohol-based DESswould be better for the resveratrol extraction [27] More-over resveratrol belong to polyhydroxyphenols and thepolyalcohol-based DESs had a more suitable polarity for theresveratrol extraction In addition the much higher viscosityof glycerol-based DES also limited its extraction efficiency ofresveratrol 2erefore the following experiments wereaimed at optimizing the extraction processing of resveratrolusing choline chloride-14-butanediol (ChCl14-Buta)(DES-1) as an extraction solvent

32 Effect of Choline Chloride14-Butanediol Molar Ratio onResveratrol Extraction 2e effect of the choline chloride14-butanediol molar ratio (1 1 1 2 1 3 1 4 and 1 5) onthe resveratrol extraction was examined (Figure 3) 2eresults showed that the maximum resveratrol content(2879 plusmn 012mgkg) could be achieved at choline

140 145 150 155 160 165 170 175 180

0000

0005

0010

0015

0020

0025

0030

0035

0040

AU

Time (min)

Resveratrol

Figure 1 HPLC chromatogram of resveratrol extracted frompeanut roots (the red and black lines mean the standard ofresveratrol and sample respectively)

Journal of Chemistry 3

chloride14-butanediol molar ratio of 1 3 Further in-creasing of the proportion of 14-butanediol (gt1 3) wouldcause the decline of resveratrol content (1 4 2476 plusmn047mgkg and 1 5 2521 plusmn 030mgkg)2e increase of the14-butanediol molar ratio in DES would result in the vis-cosity of DES decreasing and the polarity increasing whichmight affect the effectiveness of mass transport and diffusionof resveratrol from peanut roots [28ndash30] 2erefore theChCl14-butanediol molar ratio of 1 3 (molmol) was se-lected for the next experiments

33 Effect ofWaterContent inDESs onResveratrol Extraction2e high viscosity of DESs not only hinders the masstransport from plant matrices to solution but also leads tohandling difficulties Polarity is another important property

of DESs since it affects the solubilizing ability of DESs 2eaddition of water to DES can decrease the viscosity of theDESs adjust the polarity and increase the solubility of thetarget compounds In this study ChCl14-Buta- (DES-1-)water mixture with water fraction ranging from 10 to 50(vv) was evaluated for the extraction of resveratrol frompeanut roots (Figure 4)

As shown in Figure 4 the extraction power of theresveratrol was significantly improved with the increasingproportion of water up to maximum (2826 plusmn 077mgkg) at30 (vv) 2is was because that the addition of water led toa decrease in the viscosity of the reaction media improvingthe mass transfer from peanut roots to solution thereforeenhancing the extraction efficiency However higher con-centration of water in ChCl14-Buta (DES-1) (40ndash100)led to the decrease in the extraction amount of resveratrol

11 12 13 14 150

10

20

30

40

50

Extr

actio

n co

nten

t (m

gkg

)

Choline chloride14-butanediol molar ratio (molmol)

Figure 3 Effect of choline chloride14-butanediol molar ratio on the extraction content of resveratrol from peanut roots UAE conditionssolidliquid ratio of 1 20 (gmL) 25degC 30min ultrasonic power of 40 kHz extraction solvent of choline chloride14-butanediol (DES-1)and 20 of water (vv)

DES-1 DES-2 DES-3 DES-4 DES-5 DES-6 DES-7 DES-80

5

10

15

20

25

30

Extra

ctio

n co

nten

t (m

gkg

)

Extraction solvent

fe

c

e

b

dc

a

Figure 2 Effect of DESs on the extraction content of resveratrol from peanut roots UAE conditions solidliquid ratio of 1 20 (gmL) 25degC30min ultrasonic power of 40 kHz and choline chloride14-butanediol molar ratio of 1 2 20 of water (vv) (DES-1 ChCl14-Buta DES-2ChCl12-PG DES-3 ChClGly DES-4 ChClEG DES-5 ChClMA DES-6 ChClLA DES-7 ChClCa and DES-8 ChClCA) (data areexpressed as the mean plusmn SD means in the group with different letters differ significantly at plt 005)


(40 2720 plusmn 084 50 2739 plusmn 014 and 100 2202 plusmn021) (mgkg) 2is was probably because higher concen-tration of water weakened the interactions betweenresveratrol and ChCl14-Buta (DES-1) and also increasedthe polarity of extraction solution Furthermore the excesswater made the ChCl14-Buta (DES-1) diluted whichmightresult in the disruption of hydrogen bonds of DESs com-ponents and the loss of the supermolecular structure con-sequently [31] Hence water content of 30 in the ChCl14-Buta was considered as the optimal ratio

34 Effect of SolidLiquid Ratio on Resveratrol Extraction2e solidliquid ratio was evaluated (Figure 5) From theresults we could find that the extraction efficiency ofresveratrol increased from 1317 plusmn 032 to 3834 plusmn 054mgkgwith the increase of the solidliquid ratio from 1 10 to 1 30(gmL) But further increase of the solidliquid ratio had noobvious effect on the extraction content of resveratrol (1 403688 plusmn 018 and 1 50 3804 plusmn 021) (mgkg) indicating thatthe target compound could be fully extracted at 1 30 gmLof the solidliquid ratio2erefore 1 30 gmL of solidliquidratio was selected for the further experiments

35 Effect of Extraction Temperature on ResveratrolExtraction 2e temperature affects the viscosity and solu-bility of solvents and therefore affects the extraction efficiencyof resveratrol As shown in Figure 6 the extraction content ofthe resveratrol increased continually with the increasingextraction temperatures from 20degC to 60degC (20degC 3566 plusmn015 and 60degC 4053 plusmn 067) (mgkg) 2e elevated tem-perature might decrease the viscosity of the DESs inducingthe full contact of the material with the extraction solvent2eextraction efficiency at 70degC (4052 plusmn 067mgkg) had nochange compared to 60degC However with further increase of

temperature the extraction content of resveratrol decreasedslightly (80degC 3746 plusmn 0002mgkg) probably because thehigher extraction temperature would make the resveratroloxidized or decomposed Comprehensively considering theextraction efficiency and energy saving 60degC was selected asthe optimal extraction temperature

36 Effect of Extraction Time on Resveratrol Extraction2e extraction time was also investigated and the resultswere shown in Figure 7 2e highest extraction content ofthe resveratrol was obtained at 30min (3915 plusmn 007mgkg)

110 120 130 140 1500

10

20

30

40

50

Extr

actio

n co

nten

t (m

gkg

)

Solidliquid ratio (gmL)

Figure 5 Effect of solidliquid ratio on the extraction content ofresveratrol from peanut roots UAE conditions 25degC 30min ul-trasonic power of 40 kHz extraction solvent of choline chloride14-butanediol (DES-1) choline chloride14-butanediol molarratio of 1 3 and 30 of water (vv)

30 40 50 60 70 8020

25

30

35

40

45

50

Extra

ctio

n co

nten

t (m

gkg

)

Extraction temperature (degC)

Figure 6 Effect of extraction temperatures on the extractioncontent of resveratrol from peanut roots UAE conditions solidliquid ratio of 1 30 (gmL) 30min ultrasonic power of 40 kHzextraction solvent of choline chloride14-butanediol (DES-1)choline chloride14-butanediol molar ratio of 13 and 30 ofwater (vv)

10 20 30 40 50 10010

20

30

40

50

Extr

actio

n co

nten

t (m

gkg

)

Water content ()

Figure 4 Effect of water content on the extraction content ofresveratrol from peanut roots UAE conditions solidliquid ratio of1 20 (gmL) 25degC 30min ultrasonic power of 40 kHz extractionsolvent of choline chloride14-butanediol (DES-1) and cholinechloride14-butanediol molar ratio of 1 3


With the prolonging of extraction time from 30 to 60minthe resveratrol content decreased slightly (40min 3765 plusmn037 50min 3741 plusmn 005 and 60min 3464 plusmn 006)(mgkg)2is trend might be because resveratrol took placeoxidation or decomposition during the long time extrac-tion process 2us 30min was chosen as the optimal ex-traction time

37 Optimization of the Extraction Process by the BBD AssayFurther optimization of DESs-UAE resveratrol extractionconditions (water content liquidsolid ratio extraction tem-perature and extraction time) was carried out by a BoxndashBehnken design (BBD) method 2e data were analyzed usingDesign-Expert 806 software for statistical analysis of variance(ANOVA) and regression analysis (Table 2) 2e regressionequation model for resveratrol extraction was obtained andshown in the following equation

Y 3758 + 252A + 481B + 147C + 174D + 085AB

minus 155ACminus 08ADminus 153BC + 057BDminus 093CD

minus 621A2 minus 948B

2 minus 182C2 minus 327D

2

(4)where Y is the extraction content of resveratrol (mgkg)and A B C and D represented water content solidliquidratio extraction temperature and extraction timerespectively

2e analysis of variance (ANOVA) was performed toevaluate the optimal extraction conditions of resveratrol(Table 3) 2e F-value of the model was 3103 (plt 00001)indicating that the afforded model was significant ldquoLack offit F-valuerdquo was 174 (p 03117) demonstrating thatthe lack of fit of the quadratic models was not significantand the experiment data fitted well to the model 2e re-gression analysis of the data showed the coefficient of the

determination (R209688) value for resveratrol was sig-nificant implying that this quadratic model was suitable todescribe the response of the experiment regarding to theresveratrol

2e effect of these factors affecting the resveratrol ex-traction was in an order of B (liquidsolid ratio) gt A (watercontent) gtD (extraction time) gt C (extraction temperature)which was determined by the absolute value of the liner termcoefficient of the regression equation 2e p value of thequadratic term of A2 and B2 was both lt00001 respectivelyimplying that water content (A) and solidliquid (B) ratioboth had significant effects on the extraction content ofresveratrol

2e effect and interaction of four factors on the ex-traction yields of resveratrol were examined by the three-dimensional response surface (Figure 8) Figure 8(a) showedthe effects of water content solidliquid ratio and theirinteraction on the extraction content of resveratrol It wasobserved that the highest extraction content was affordedwith the water content range of 15ndash55 and solidliquidratio of 25ndash38mLg When solidliquid ratio was a certainvalue the extraction content of resveratrol had the trend ofincreasing first and then decreasing with the increase ofwater contentWhen the water content was fixed the yield of

20 30 40 50 6020

25

30

35

40

45

50

Extr

actio

n co

nten

t (m

gkg

)

Extraction time (min)

Figure 7 Effect of extraction time on the extraction content ofresveratrol from peanut roots UAE conditions solidliquid ratio of1 30 (gmL) 60degC ultrasonic power of 40 kHz extraction solventof choline chloride14-butanediol (DES-1) choline chloride14-butanediol molar ratio of 1 3 and 30 of water (vv)

Table 2 Experimental data and the obtained response values withdifferent combinations of water content (A) solidliquid ratio (B gmL) extraction temperature (C degC) and extraction time (D min)used in BBD

RunFactor

Extraction content YA B C D

1 70 30 30 40 32402 40 30 55 40 38333 40 10 30 40 18174 10 30 55 10 22445 70 50 55 40 29086 40 50 55 70 30667 10 30 30 40 23548 40 30 30 10 26869 40 30 55 40 381210 40 50 55 10 278811 40 30 55 40 381912 10 30 55 70 282613 10 50 55 40 29314 70 30 55 10 289515 10 30 80 40 307616 70 30 55 70 315617 70 30 80 40 334118 40 10 55 10 210719 40 30 55 40 380320 40 30 80 10 331421 40 10 55 70 215722 40 30 30 70 333423 70 10 55 40 187724 40 30 55 40 352225 40 10 80 40 214926 40 30 80 70 358827 40 50 80 40 307328 10 10 55 40 160329 40 50 30 40 3353


Table 3 ANOVA statistics analysis of the model for the extraction of resveratrol

Source Sum of squares df Mean square F value p value probability gt F SignificanceModel 116803 14 8343 3103 lt00001 lowastlowast

A (water content ) 7610 1 7610 2831 00001 lowastlowast

B (solidliquid ratio gmL) 27752 1 27752 10322 lt00001 lowastlowast

C (extraction temperature degC) 2578 1 2578 959 00079 lowast

D (extraction time min) 3645 1 3645 1356 00025 lowast

AB 29 1 29 108 03164AC 964 1 964 358 00792AD 256 1 256 095 03458BC 938 1 938 349 00829BD 131 1 131 049 04969CD 349 1 349 130 02738A2 24987 1 24987 9224 lt00001 lowastlowast

B2 58345 1 58345 21701 lt00001 lowastlowast

C2 2144 1 2144 797 00135D2 6944 1 6944 2583 00002Residual 3764 14 269Lack of fit 3062 10 306 174 03117 Not significantPure error 702 4 176Correlation total 120567 28lowastplt 001 lowastlowastplt 0001

10Water content ()

10

152025303540

1826

3442

50Liquidsolid ratio (mLg)

Extr

actio

n co

nten

t (m

gkg

)

2540

5570

(a)

Water content ()

152025303540

Extraction temperature (ordmC)

Extr

actio

n co

nten

t (m

gkg

)

10304050

607080

2540

5570

(b)

Water content ()

152025303540


Extr

actio

n co

nten

t (m

gkg

)

101022

3446

5870

2540

5570

(c)

152025303540

1018

26 3442 50

Liquidsolid ratio (mLg)Extraction temperature (ordmC)

Extr

actio

n co

nten

t (m

gkg

)

304050

607080

(d)

Figure 8 Continued


resveratrol was increased and then kept at the stable levelwith the increase of solidliquid ratio 2e interaction effectof water content and solidliquid ratio on the resveratrolcontent was not significant 2e same trend was observed inFigures 8(b)ndash8(f ) (the interactive effects of water contentextraction time and solidliquid ratio on the extractioncontent of resveratrol) In summary the interactions inwater content extraction time extraction temperature andsolidliquid ratio on the resveratrol extraction from peanutsroots were no significant which was consistent with theanalysis result of the regression model

2e optimal conditions for the extraction of resveratrolfrom peanut roots were obtained based on the establishedmodel which were as follows water content of 40 (vv)solidliquid ratio of 130 gmL extraction temperature of55degC and extraction time of 40min Under these optimumconditions the predicted extraction content of resveratrolfrom peanut roots was 3839mgkg 2e verification ex-periment was also performed and the obtained extractioncontent of resveratrol was up to 3891mgkg which in-dicated that the established model was considered to bereliable and reasonable

4 Conclusion

In this study a new type of green and efficient solvent DESscoupled with ultrasonic-assisted extraction (UAE) andHPLC-UV was developed to extract resveratrol from peanutroots 2e optimal DES-UAE conditions were obtainedusing a BBD test combined with a response surface meth-odology as follows extraction solvent 40 of water in ChCl14-butanediol (13 gg) extraction temperature 55degC solidliquid ratio 1 30 gmL and extraction time 40min Underthe above optimum conditions the total extraction contentof resveratrol from peanut roots was up to 3891mgkg andthe extraction yield was 8946 It was concluded that thisDES-UAE-HPLC method was a fast safe and efficient ex-traction method for the preparation and determination ofresveratrol from peanut roots

Abbreviations

DESs Deep eutectic solventsUAE Ultrasonic-assisted extractionBBD BoxndashBehnken designHBD Hydrogen bond donor

Data Availability

2e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

2e authors declare that there are no conflicts of interestregarding the publication of this article

Acknowledgments

2e authors gratefully acknowledge the financial supportfrom the Funds of National Natural Science Foundation ofChina (no 31772003) Provincal Key Laboratory forTransformation and Utilization of Cereal Resource HenanUniversity of Technology (no PL2017001) Henan NaturalScience Foundation (no 162300410046) and Henan sci-entific and technical projects (no 182102110024)

References

[1] M M Hasan M Cha V K Bajpai and K H Baek ldquoPro-duction of a major stilbene phytoalexin resveratrol in peanut(Arachis hypogaea) and peanut products a mini reviewrdquoReviews in Environmental Science and BioTechnology vol 12no 3 pp 209ndash221 2012

[2] R Kolahdouz Mohammadi and T Arablou ldquoResveratrol andendometriosis in vitro and animal studies and underlyingmechanismsrdquo Biomedicine and Pharmacotherapy vol 91pp 220ndash228 2017

[3] L M Hung J K Chen S S Huang R S Lee and M J SuldquoCardioprotective effect of resveratrol a natural antioxidant

152025303540

1826

34 4250

Liquidsolid ratio (mLg)Extraction time (min)

Extr

actio

n co

nten

t (m

gkg

)

10 102234465870

(e)

152025303540

Extraction time (min)Extraction temperature (ordmC

)

Extr

actio

n co

nten

t (m

gkg

)

102234

465870

3040

50 60 7080

(f )

Figure 8 (andashf) refers to the target compounds response values under the two variables Response surfaces representations for resveratrolfrom peanut roots (a) varying water content and liquidsolid ratio (b) varying water content and temperature (c) varying water content andtime (d) varying temperature and liquidsolid ratio (e) varying liquidsolid ratio and time (f ) varying temperature and time


derived from grapesrdquo Cardiovascular Research vol 47 no 3p 549 2000

[4] D G Wanga W Y Liub and G T Chena ldquoA simple methodfor the isolation and purification of resveratrol from Polyg-onum cuspidatumrdquo Journal of Pharmaceutical Analysisvol 3 no 4 pp 241ndash247 2013

[5] Q Xiong Q Zhang D Zhang Y Shi C Jiang and X ShildquoPreliminary separation and purification of resveratrol fromextract of peanut (Arachis hypogaea) sprouts by macro-porous adsorption resinsrdquo Food Chemistry vol 145 pp 1ndash72014

[6] D Zhang X Li D Hao et al ldquoSystematic purification ofpolydatin resveratrol and anthraglycoside B from Polygonumcuspidatum Sieb et Zuccrdquo Separation and PurificationTechnology vol 66 no 2 pp 329ndash339 2009

[7] M C Pascual-Martı A Salvador A Chafer and A BernaldquoSupercritical fluid extraction of resveratrol from grape skinof Vitis vinifera and determination by HPLCrdquo Talantavol 54 no 4 pp 735ndash740 2001

[8] C D Liu Y Y Wen and J M Chiou ldquoComparative char-acterization of peanuts grown by aquatic floating cultivationand field cultivation for seed and resveratrol productionrdquoJournal of Agricultural and Food Chemistry vol 51 no 6pp 1582ndash1585 2003

[9] S Jiang Q Liu Y Xie et al ldquoSeparation of five flavonoidsfrom tartary buckwheat (Fagopyrum tataricum (L) Gaertn)grains via off-line two dimensional high-speed counter-current chromatographyrdquo Food Chemistry vol 186pp 153ndash159 2015

[10] X H Yao D Y Zhang M H Duan et al ldquoPreparation anddetermination of phenolic compounds from Pyrola incarnataFisch with a green polyols based-deep eutectic solventrdquoSeparation and Purification Technology vol 149 pp 116ndash1232015

[11] M Cvjetko Bubalo S Vidovic I Radojcic Redovnikovic andS Jokic ldquoGreen solvents for green technologiesrdquo Journal ofChemical Technology and Biotechnology vol 90 no 9pp 1631ndash1639 2015

[12] E L Smith A P Abbott and K S Ryder ldquoDeep eutecticsolvents (DESs) and their applicationsrdquo Chemical Reviewsvol 114 no 21 pp 11060ndash11082 2014

[13] A P Abbott D Boothby G Capper D L Davies andR K Rasheed ldquoDeep eutectic solvents formed betweencholine chloride and carboxylic acids versatile alternatives toionic liquidsrdquo Journal of the American Chemical Societyvol 126 no 29 pp 9142ndash9147 2004

[14] A Paiva R Craveiro I Aroso M Martins R L Reis andA R C Duarte ldquoNatural deep eutectic solvents-solvents forthe 21st centuryrdquoACS Sustainable Chemistry and Engineeringvol 2 no 5 pp 1063ndash1071 2014

[15] K Radosevic N Curko V G Srcek et al ldquoNatural deepeutectic solvents as beneficial extractants for enhancement ofplant extracts bioactivityrdquo LWT-Food Science and Technologyvol 73 pp 45ndash51 2016

[16] Y T Dai J V Spronsenb and G J Witkamp ldquoNatural deepeutectic solvents as new potential media for green tech-nologyrdquo Analytica Chimica Acta vol 766 no 5 pp 61ndash682013

[17] Z F Wei X Q Wang X Peng et al ldquoFast and green ex-traction and separation of main bioactive flavonoids fromRadix Scutellariaerdquo Industrial Crops and Products vol 63no 4 pp 175ndash181 2015

[18] B M Cvjetko N Curko M Tomasevic K K Ganic andR I Redovnikovic ldquoGreen extraction of grape skin phenolics

by using deep eutectic solventsrdquo Food Chemistry vol 200pp 159ndash166 2016

[19] K Pang Y C Hou W Z Wu W J Guo W Peng andK N Marsh ldquoEfficient separation of phenols from oils viaforming deep eutectic solventsrdquo Green Chemistry vol 14no 9 pp 2398ndash2401 2012

[20] Y T Dai G J Witkamp R Verpoorte and Y H ChoildquoNatural deep eutectic solvents as a new extraction media forphenolic metabolites in Carthamus tinctorius Lrdquo AnalyticalChemistry vol 85 no 13 pp 6272ndash6278 2013

[21] M W Nam J Zhao M S Lee J H Jeong and J LeeldquoEnhanced extraction of bioactive natural products usingtailor-made deep eutectic solvents application to flavonoidextraction from Flos sophoraerdquo Green Chemistry vol 17no 3 pp 1718ndash1727 2015

[22] W T Bi M Tian and K H Row ldquoEvaluation of alcohol-based deep eutectic solvent in extraction and determination offlavonoids with response surface methodology optimizationrdquoJournal of Chromatography A vol 1285 pp 22ndash30 2013

[23] A K Das M Sharma D Mondal and K Prasad ldquoDeepeutectic solvents as efficient solvent system for the extractionof k-carrageenan from Kappaphycus alvareziirdquo CarbohydratePolymers vol 136 pp 930ndash935 2016

[24] N Li Y Z Wang K J Xu Y H Huang Q Wen andX Q Ding ldquoDevelopment of green betaine-based deep eu-tectic solvent aqueous two-phase system for the extraction ofproteinrdquo Talanta vol 152 pp 23ndash32 2016

[25] K Xu Y Z Wang Y H Huang N Li and Q Wen ldquoA greendeep eutectic solvent-based aqueous two-phase system forprotein extractingrdquo Analytica Chimica Acta vol 864 pp 9ndash20 2015

[26] F Y Du X H Xiao and G K Li ldquoApplication of ionic liquidsin the microwave-assisted extraction of trans -resveratrolfrom Rhizma Polygoni Cuspidatirdquo Journal of Chromatogra-phy A vol 1140 no 1-2 pp 56ndash62 2007

[27] A P Abbott A Y M Al-Murshedi O A O Alshammariet al ldquo2ermodynamics of phase transfer for polar moleculesfrom alkanes to deep eutectic solventsrdquo Fluid Phase Equi-libria vol 448 pp 99ndash104 2017

[28] M Hayyan C Y Looi A Hayyan and W F Wong ldquoIn vitroand in vivo toxicity profiling of ammonium-based deep eu-tectic solventsrdquo Plos One vol 10 no 2 Article ID e01179342015

[29] A P Abbott R C Harris and K S Ryder ldquoApplication ofhole theory to define ionic liquids by their transport prop-ertiesrdquo Journal of Physical Chemistry B vol 111 no 18pp 4910ndash4913 2007

[30] C DrsquoAgostino R C Harris A P Abbott L F Gladden andM D Mantle ldquoMolecular motion and ion diffusion in cholinechloride based deep eutectic solvents studied by 1H pulsedfield gradient NMR spectroscopyrdquo Physical ChemistryChemical Physics vol 13 no 48 pp 21383ndash21391 2011

[31] Y T Dai G J Witkamp R Verpoorte and Y H ChoildquoTailoring properties of natural deep eutectic solvents withwater to facilitate their applicationsrdquo Food Chemistry vol 187pp 14ndash19 2015


TribologyAdvances in

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Analytical Methods in Chemistry

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Volume 2018

Bioinorganic Chemistry and ApplicationsHindawiwwwhindawicom Volume 2018

SpectroscopyInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

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Medicinal ChemistryInternational Journal of


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SpectroscopyAnalytical ChemistryInternational Journal of


MaterialsJournal of



BioMed Research International Electrochemistry

International Journal of


Na

nom

ate

ria

ls


Journal ofNanomaterials

Submit your manuscripts atwwwhindawicom

highly efficient and green extraction technology for theextraction of resveratrol from peanut roots using deep eu-tectic solvents assisted by ultrasonic extraction methods(DESs-UAE) First a series of DESs were prepared bymixingthe varying ratios of polyols organic acid and carbamidewith ChCl Second various parameters of DESs-UAE inextracting resveratrol were optimized and systematicallyevaluated using a BoxndashBehnken design

2 Materials and Methods

21 Chemicals and Reagents 14-Butanediol (gt98) andcitric acid (gt99) were obtained from Kwangfu FineChemical Industry Research Institute (Tianjin China)Ethylene glycol (gt98) was purchased from Fuyu FineChemical Co Ltd (Tianjin China) Lactic acid (gt95) waspurchased from Sinopharm Chemical Reagent Co Ltd(Shanghai China) Glycerol (gt99) 12-propylene glycol(gt99) DL-malic acid (gt99) and carbamide (gt99) werepurchased from Kermel Chemical Reagent Co Ltd(Tianjin China) Choline chloride (gt98) was obtainedfrom Macklin Biochemical Co Ltd (Shanghai China)Resveratrol (354prime-trihydroxy stilbene ge98) was obtainedfrom Sigma-Aldrich Co (St Louis MO USA) Methanol(the chromatographic grade) was purchased from VBS bi-ologic Co (USA) All samples and solutions prepared forHPLC analysis were filtered through the 045 μm nylonmembranes prior to use

Standard stock solutions resveratrol compound wasdirectly prepared in methanol 2e concentration ofresveratrol in the standard stock solutions was all 500 μgmLWorking standard solutions resveratrol compound wasobtained by diluting the stock solutions with methanol to aseries of proper concentrations2e standard stock solutionsand working standard solutions were all stored at 4degC in adark place

22 Plant Materials Peanut roots were purchased fromHebei province in China2e plant material was cleaned anddried at 40degC 2e dry plant material was ground intopowder with a blender and passed through 50mesh and thenstored in a dry place at room temperature until used

23 Preparation of DESs All of the chemicals used in DESspreparations were dried at 60degC for 24h 2e DESs wereprepared at specific ratios of choline chloride to the hydrogendonor (ie 14-butanediol glycerol and lactic acid Table 1)2e varying ratios of choline chloride with the hydrogendonor were stirred in a flask at 80degC for 5ndash10min until ahomogeneous transparent colourless liquid was formed2eseDESs samples were treated by vacuum drying prior to use

24 Deep Eutectic Solvents Ultrasonic-Assisted Extraction ofResveratrol About 050 g peanut roots powder was weighedinto an 50mL centrifuge tube and followed by addition of10mL of extraction solvent 2e mixture was then ultra-sonically treated Ultrasound-assisted extraction (UAE) was

performed using an ultrasonicator under 40 kHz and 400W(SCQ-7201B Shengyan Ultrasonic Instrument Co LtdShanghai China) 2e extraction was carried out underdifferent conditions After extraction the mixture wascentrifuged (10min 2500 rpm) with a bench-scale centri-fuge 2e supernate was separated and filtered through a045 μm membrane prior to HPLC analysis

2e extraction content was calculated according to thefollowing equation

extraction content(mgkg)

mass of resveratrol(mg)

mass of weighed peanut roots powder(kg)

(1)

2e extraction yield was defined as follows [26]

extraction yield()

mass of the resveratrol in extraction solutionsumof themass of resveratrol in sample

times 100

(2)

2e mass of resveratrol in the extraction solution (one-step extraction) was determined by HPLC-UV 2e summass of resveratrol in the sample was calculated by analysisof the total mass of resveratrol in the combined extractionsolutions afforded by continuously extracting three timeswith methanol according to the standard method

25 Experimental Design and Statistical Analysis Firstly themole ratio of hydrogen bond donors and ChCl (14-butanediol ChCl 1 1 2 1 3 1 4 1 and 5 1) thepercentage of water (10 20 30 40 50 and 100)solid-liquid ratio (1 10 1 20 1 30 1 40 and 1 50)extraction temperature (20 30 40 50 60degC and 70degC) andextraction time (20 30 40 50 and 60min) were optimizedby single-factor experiments respectively FurthermoreBoxndashBehnken experimental design (BBD) with responsesurface methodology (RSM) was used to estimate the mosteffective combination of extraction parameters accordingto the single-factors experiments A three-level (minus1 0 and+1) four-factor BoxndashBehnken design (BBD) was applied toevaluate the interaction effect of the factors the percentageof water (A) solidliquid ratio (B) extraction temperature

Table 1 Different systems of natural deep eutectic solvents (DESs)

Abbreviation Component 1 Component 2 Molarratio

DES-1 Choline chloride 14-Butanediol 1 2

DES-2 Choline chloride 12-Propyleneglycol 1 2

DES-3 Choline chloride Glycerol 1 2DES-4 Choline chloride Ethylene glycol 1 2DES-5 Choline chloride DL-Malic acid 1 2DES-6 Choline chloride Lactic acid 1 2DES-7 Choline chloride Carbamide 1 2DES-8 Choline chloride Citric acid 1 2



Y β0 + 11139444

i1βiXi + 1113944

4

i1βiiX

2i + 1113944

4

i11113944

4

ji+1βijXiXj (3)









140 145 150 155 160 165 170 175 180

0000

0005

0010

0015

0020

0025

0030

0035

0040

AU

Time (min)

Resveratrol







11 12 13 14 150

10

20

30

40

50

Extr

actio

n co

nten

t (m

gkg

)




5

10

15

20

25

30

Extra

ctio

n co

nten

t (m

gkg

)

Extraction solvent

fe

c

e

b

dc

a








110 120 130 140 1500

10

20

30

40

50

Extr

actio

n co

nten

t (m

gkg

)



30 40 50 60 70 8020

25

30

35

40

45

50

Extra

ctio

n co

nten

t (m

gkg

)



10 20 30 40 50 10010

20

30

40

50

Extr

actio

n co

nten

t (m

gkg

)

Water content ()





Y 3758 + 252A + 481B + 147C + 174D + 085AB




2






20 30 40 50 6020

25

30

35

40

45

50

Extr

actio

n co

nten

t (m

gkg

)




RunFactor


1 70 30 30 40 32402 40 30 55 40 38333 40 10 30 40 18174 10 30 55 10 22445 70 50 55 40 29086 40 50 55 70 30667 10 30 30 40 23548 40 30 30 10 26869 40 30 55 40 381210 40 50 55 10 278811 40 30 55 40 381912 10 30 55 70 282613 10 50 55 40 29314 70 30 55 10 289515 10 30 80 40 307616 70 30 55 70 315617 70 30 80 40 334118 40 10 55 10 210719 40 30 55 40 380320 40 30 80 10 331421 40 10 55 70 215722 40 30 30 70 333423 70 10 55 40 187724 40 30 55 40 352225 40 10 80 40 214926 40 30 80 70 358827 40 50 80 40 307328 10 10 55 40 160329 40 50 30 40 3353











10Water content ()

10

152025303540

1826

3442


Extr

actio

n co

nten

t (m

gkg

)

2540

5570

(a)

Water content ()

152025303540


Extr

actio

n co

nten

t (m

gkg

)

10304050

607080

2540

5570

(b)

Water content ()

152025303540


Extr

actio

n co

nten

t (m

gkg

)

101022

3446

5870

2540

5570

(c)

152025303540

1018

26 3442 50


Extr

actio

n co

nten

t (m

gkg

)

304050

607080

(d)

Figure 8 Continued




4 Conclusion


Abbreviations


Data Availability




Acknowledgments


References




152025303540

1826

34 4250


Extr

actio

n co

nten

t (m

gkg

)

10 102234465870

(e)

152025303540


)

Extr

actio

n co

nten

t (m

gkg

)

102234

465870

3040

50 60 7080

(f )







































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls





Y β0 + 11139444

i1βiXi + 1113944

4

i1βiiX

2i + 1113944

4

i11113944

4

ji+1βijXiXj (3)









140 145 150 155 160 165 170 175 180

0000

0005

0010

0015

0020

0025

0030

0035

0040

AU

Time (min)

Resveratrol







11 12 13 14 150

10

20

30

40

50

Extr

actio

n co

nten

t (m

gkg

)




5

10

15

20

25

30

Extra

ctio

n co

nten

t (m

gkg

)

Extraction solvent

fe

c

e

b

dc

a








110 120 130 140 1500

10

20

30

40

50

Extr

actio

n co

nten

t (m

gkg

)



30 40 50 60 70 8020

25

30

35

40

45

50

Extra

ctio

n co

nten

t (m

gkg

)



10 20 30 40 50 10010

20

30

40

50

Extr

actio

n co

nten

t (m

gkg

)

Water content ()





Y 3758 + 252A + 481B + 147C + 174D + 085AB




2






20 30 40 50 6020

25

30

35

40

45

50

Extr

actio

n co

nten

t (m

gkg

)




RunFactor


1 70 30 30 40 32402 40 30 55 40 38333 40 10 30 40 18174 10 30 55 10 22445 70 50 55 40 29086 40 50 55 70 30667 10 30 30 40 23548 40 30 30 10 26869 40 30 55 40 381210 40 50 55 10 278811 40 30 55 40 381912 10 30 55 70 282613 10 50 55 40 29314 70 30 55 10 289515 10 30 80 40 307616 70 30 55 70 315617 70 30 80 40 334118 40 10 55 10 210719 40 30 55 40 380320 40 30 80 10 331421 40 10 55 70 215722 40 30 30 70 333423 70 10 55 40 187724 40 30 55 40 352225 40 10 80 40 214926 40 30 80 70 358827 40 50 80 40 307328 10 10 55 40 160329 40 50 30 40 3353











10Water content ()

10

152025303540

1826

3442


Extr

actio

n co

nten

t (m

gkg

)

2540

5570

(a)

Water content ()

152025303540


Extr

actio

n co

nten

t (m

gkg

)

10304050

607080

2540

5570

(b)

Water content ()

152025303540


Extr

actio

n co

nten

t (m

gkg

)

101022

3446

5870

2540

5570

(c)

152025303540

1018

26 3442 50


Extr

actio

n co

nten

t (m

gkg

)

304050

607080

(d)

Figure 8 Continued




4 Conclusion


Abbreviations


Data Availability




Acknowledgments


References




152025303540

1826

34 4250


Extr

actio

n co

nten

t (m

gkg

)

10 102234465870

(e)

152025303540


)

Extr

actio

n co

nten

t (m

gkg

)

102234

465870

3040

50 60 7080

(f )







































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls








11 12 13 14 150

10

20

30

40

50

Extr

actio

n co

nten

t (m

gkg

)




5

10

15

20

25

30

Extra

ctio

n co

nten

t (m

gkg

)

Extraction solvent

fe

c

e

b

dc

a








110 120 130 140 1500

10

20

30

40

50

Extr

actio

n co

nten

t (m

gkg

)



30 40 50 60 70 8020

25

30

35

40

45

50

Extra

ctio

n co

nten

t (m

gkg

)



10 20 30 40 50 10010

20

30

40

50

Extr

actio

n co

nten

t (m

gkg

)

Water content ()





Y 3758 + 252A + 481B + 147C + 174D + 085AB




2






20 30 40 50 6020

25

30

35

40

45

50

Extr

actio

n co

nten

t (m

gkg

)




RunFactor


1 70 30 30 40 32402 40 30 55 40 38333 40 10 30 40 18174 10 30 55 10 22445 70 50 55 40 29086 40 50 55 70 30667 10 30 30 40 23548 40 30 30 10 26869 40 30 55 40 381210 40 50 55 10 278811 40 30 55 40 381912 10 30 55 70 282613 10 50 55 40 29314 70 30 55 10 289515 10 30 80 40 307616 70 30 55 70 315617 70 30 80 40 334118 40 10 55 10 210719 40 30 55 40 380320 40 30 80 10 331421 40 10 55 70 215722 40 30 30 70 333423 70 10 55 40 187724 40 30 55 40 352225 40 10 80 40 214926 40 30 80 70 358827 40 50 80 40 307328 10 10 55 40 160329 40 50 30 40 3353











10Water content ()

10

152025303540

1826

3442


Extr

actio

n co

nten

t (m

gkg

)

2540

5570

(a)

Water content ()

152025303540


Extr

actio

n co

nten

t (m

gkg

)

10304050

607080

2540

5570

(b)

Water content ()

152025303540


Extr

actio

n co

nten

t (m

gkg

)

101022

3446

5870

2540

5570

(c)

152025303540

1018

26 3442 50


Extr

actio

n co

nten

t (m

gkg

)

304050

607080

(d)

Figure 8 Continued




4 Conclusion


Abbreviations


Data Availability




Acknowledgments


References




152025303540

1826

34 4250


Extr

actio

n co

nten

t (m

gkg

)

10 102234465870

(e)

152025303540


)

Extr

actio

n co

nten

t (m

gkg

)

102234

465870

3040

50 60 7080

(f )







































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls









110 120 130 140 1500

10

20

30

40

50

Extr

actio

n co

nten

t (m

gkg

)



30 40 50 60 70 8020

25

30

35

40

45

50

Extra

ctio

n co

nten

t (m

gkg

)



10 20 30 40 50 10010

20

30

40

50

Extr

actio

n co

nten

t (m

gkg

)

Water content ()





Y 3758 + 252A + 481B + 147C + 174D + 085AB




2






20 30 40 50 6020

25

30

35

40

45

50

Extr

actio

n co

nten

t (m

gkg

)




RunFactor


1 70 30 30 40 32402 40 30 55 40 38333 40 10 30 40 18174 10 30 55 10 22445 70 50 55 40 29086 40 50 55 70 30667 10 30 30 40 23548 40 30 30 10 26869 40 30 55 40 381210 40 50 55 10 278811 40 30 55 40 381912 10 30 55 70 282613 10 50 55 40 29314 70 30 55 10 289515 10 30 80 40 307616 70 30 55 70 315617 70 30 80 40 334118 40 10 55 10 210719 40 30 55 40 380320 40 30 80 10 331421 40 10 55 70 215722 40 30 30 70 333423 70 10 55 40 187724 40 30 55 40 352225 40 10 80 40 214926 40 30 80 70 358827 40 50 80 40 307328 10 10 55 40 160329 40 50 30 40 3353











10Water content ()

10

152025303540

1826

3442


Extr

actio

n co

nten

t (m

gkg

)

2540

5570

(a)

Water content ()

152025303540


Extr

actio

n co

nten

t (m

gkg

)

10304050

607080

2540

5570

(b)

Water content ()

152025303540


Extr

actio

n co

nten

t (m

gkg

)

101022

3446

5870

2540

5570

(c)

152025303540

1018

26 3442 50


Extr

actio

n co

nten

t (m

gkg

)

304050

607080

(d)

Figure 8 Continued




4 Conclusion


Abbreviations


Data Availability




Acknowledgments


References




152025303540

1826

34 4250


Extr

actio

n co

nten

t (m

gkg

)

10 102234465870

(e)

152025303540


)

Extr

actio

n co

nten

t (m

gkg

)

102234

465870

3040

50 60 7080

(f )







































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls






Y 3758 + 252A + 481B + 147C + 174D + 085AB




2






20 30 40 50 6020

25

30

35

40

45

50

Extr

actio

n co

nten

t (m

gkg

)




RunFactor


1 70 30 30 40 32402 40 30 55 40 38333 40 10 30 40 18174 10 30 55 10 22445 70 50 55 40 29086 40 50 55 70 30667 10 30 30 40 23548 40 30 30 10 26869 40 30 55 40 381210 40 50 55 10 278811 40 30 55 40 381912 10 30 55 70 282613 10 50 55 40 29314 70 30 55 10 289515 10 30 80 40 307616 70 30 55 70 315617 70 30 80 40 334118 40 10 55 10 210719 40 30 55 40 380320 40 30 80 10 331421 40 10 55 70 215722 40 30 30 70 333423 70 10 55 40 187724 40 30 55 40 352225 40 10 80 40 214926 40 30 80 70 358827 40 50 80 40 307328 10 10 55 40 160329 40 50 30 40 3353











10Water content ()

10

152025303540

1826

3442


Extr

actio

n co

nten

t (m

gkg

)

2540

5570

(a)

Water content ()

152025303540


Extr

actio

n co

nten

t (m

gkg

)

10304050

607080

2540

5570

(b)

Water content ()

152025303540


Extr

actio

n co

nten

t (m

gkg

)

101022

3446

5870

2540

5570

(c)

152025303540

1018

26 3442 50


Extr

actio

n co

nten

t (m

gkg

)

304050

607080

(d)

Figure 8 Continued




4 Conclusion


Abbreviations


Data Availability




Acknowledgments


References




152025303540

1826

34 4250


Extr

actio

n co

nten

t (m

gkg

)

10 102234465870

(e)

152025303540


)

Extr

actio

n co

nten

t (m

gkg

)

102234

465870

3040

50 60 7080

(f )







































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls













10Water content ()

10

152025303540

1826

3442


Extr

actio

n co

nten

t (m

gkg

)

2540

5570

(a)

Water content ()

152025303540


Extr

actio

n co

nten

t (m

gkg

)

10304050

607080

2540

5570

(b)

Water content ()

152025303540


Extr

actio

n co

nten

t (m

gkg

)

101022

3446

5870

2540

5570

(c)

152025303540

1018

26 3442 50


Extr

actio

n co

nten

t (m

gkg

)

304050

607080

(d)

Figure 8 Continued




4 Conclusion


Abbreviations


Data Availability




Acknowledgments


References




152025303540

1826

34 4250


Extr

actio

n co

nten

t (m

gkg

)

10 102234465870

(e)

152025303540


)

Extr

actio

n co

nten

t (m

gkg

)

102234

465870

3040

50 60 7080

(f )







































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls






4 Conclusion


Abbreviations


Data Availability




Acknowledgments


References




152025303540

1826

34 4250


Extr

actio

n co

nten

t (m

gkg

)

10 102234465870

(e)

152025303540


)

Extr

actio

n co

nten

t (m

gkg

)

102234

465870

3040

50 60 7080

(f )







































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls








































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls




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