Research ArticleFlavonoids in Different Parts of Lysimachiaclethroides Duby Extracted by Ionic Liquid Analysis byHPLC and Antioxidant Activity Assay

Jin-fengWei12 Zhi-juan Zhang1 Li-li Cui1 andWen-yi Kang12

1 Institute of Chinese Materia Medica Henan University Kaifeng 475004 China2Kaifeng Key Laboratory of Functional Components in Health Food Kaifeng 475004 China

Correspondence should be addressed to Wen-yi Kang kangwenyhotmailcom

Received 14 January 2017 Accepted 4 May 2017 Published 13 June 2017

Academic Editor Yoshihiro Kudo

Copyright copy 2017 Jin-feng Wei et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

To establish methods for simultaneous determination of isoquercitrin astragalin in leaves quercetin and kaempferol in flowersof Lysimachia clethroidesDuby respectively the methods were ultrasound-assisted extraction combined with RP-HPLC and ionicliquid was used as the extraction solvent Meanwhile the antioxidant activity of the different extracts of L clethroideswas evaluatedPurospher STAR RP-C18 column (46mm times 250mm 5 120583m) was used for analysis The flow rate was 06mLsdotminminus1 and the columntemperature was 25∘CThe detection wavelength was 360 nmThemobile phases a and b consisted of acetonitrile-04 phosphoricacid (18 82 vv) methanol (A) and 04 phosphoric acid (B) respectively Linear ranges were 0068sim164 0060sim144 00080sim019 and 00077sim018 120583g for isoquercitrin astragalin quercetin and kaempferol respectively The average recoveries of the fourconstituents were 9917 9839 10068 and 9881 respectivelyThe antioxidant activity of the extracts was detected by DPPHABTS and FRAP Under the optimized conditions all the test solutions showed a certain antioxidant activity and the ionic liquidextracts were better than that of extract of methanol Ionic liquid used as the extraction solvent had the potential to extract activeingredients efficiently from L clethroides and this method improved the antioxidant activity with accurate and reliable results

1 Introduction

Lysimachia clethroides Duby belongs to the Primulaceaefamily and is abundant and widely distributed in ChinaThe root or the whole plant of L clethroides was usedas traditional Chinese herbal medicine with many healthbenefits in the treatment of edema jaundice dysenteryrheumatic fever amenorrhea bruises fractures traumaticbleeding mastitis boils and snakebite [1] Furthermore itstender stems and leaves serve as popular vegetable in someareas [2] Pharmacological research showed that L clethroideshad many pharmacological activities such as antitumor [34] antioxidant [5] hypoglycemic [6] and hepatoprotectiveeffect [7] The main chemical components of L clethroideswere flavonoids triterpenes and organic acids [8ndash11] Theflavonoids are responsible for the bioactivity of L clethroides[12 13]

Ionic liquids (ILs) are solely composed of cations andanions as liquid near room temperature [14] ILs own a largeamount of excellent properties including negligible vapourpressure good thermal stability wide liquid range good dis-solving and high electric conductivity [15] ILs are expectedto be greener alternatives to traditional volatile organic sol-vents with the characteristic of being environment-friendlyILs are promising solvents for extracting the active ingre-dients from traditional Chinese medicine which are notonly environment-friendly but also highly efficient [16 17]However it unavoidably involved some organic solventsduring the extraction ILs in traditional Chinese medicineextraction are applied in the initial stage it is necessary toconduct in-depth research

Our group had reported the content of four flavonoids inL clethroides [18] and on the basis of other studies [19ndash21]

HindawiJournal of ChemistryVolume 2017 Article ID 2080738 10 pageshttpsdoiorg10115520172080738

2 Journal of Chemistry

the contents of isoquercitrin astragalin quercetin andkaempferol in different parts of L clethroides using high-performance liquid chromatography (HPLC) were com-pared and a rapid effective and environment-friendly ionicliquid-based ultrasonic-assisted approach was established toimprove the extraction yield In addition the antioxidantactivities of the different extracts of L clethroides weremeasured in order to prove the efficient advantage of ionicliquid in vitro

2 Materials and Methods

21 Reagents and Materials Isoquercitrin astragalin quer-cetin and kaempferol of purity greater than 98 were pur-chased fromChengdu Pufei De Biotech Co Ltd Acetonitrileused for HPLC was purchased from Avantor PerformanceMaterials Inc (USA) Methanol used for HPLC was pur-chased from Tianjin Shield Fine Chemicals Co Ltd Thewater was Wahaha pure water

1-Butyl-3-methylimidazolium tetrafluoroborate([BMIM]BF4) 1-butyl-3-methylimidazolium hexafluoro-phosphate ([BMIM]PF6) 1-hexyl-3-methylimidazolium hex-afluorophosphate ([HMIM]PF6) and 1-butyl-3-methylim-idazolium bromide ([BMIM]Br) were obtained from MerckKGaA

11-Diphenyl-2-picrylhydrazyl (DPPH) was obtainedfrom Tokyo Chemical Industry Co Ltd 221015840-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was obtainedfrom Fluka 246-Tri(2-pyridinyl)-135-triazine (TPTZ)butylated hydroxytoluene (BHT) butyl-p-hydroxyanisole(BHA) and gallic acid propyl (PG) were obtained fromSigma 6-Hydroxy-2578-tetramethylchroman-2-carboxylicacid (Trolox) was obtained from Aldrich Chemical Co Ltd

Air-dried plant was collected from Huaxi DistrictGuizhou Province of China in June 2014 The plant wasidentified as L clethroides of Primulaceae family by Profes-sor Changqin Li (Henan University Kaifeng China) Thevoucher specimens were deposited in the Institute of NaturalProducts Henan University (number 20140612)

22 Preparation of Standard Solution Thefour standard solu-tions of isoquercitrin astragalin quercetin and kaempferolwere prepared in methanol at a concentration of 1365 1200160 and 154 120583gsdotmLminus1 and stored at 4∘C respectively Thestandard working solutions were prepared by appropriatedilution of the four stock standard solutions with methanolto the required concentrations Standardworking solutions (aand b) were prepared by diluting isoquercitrin and astragalinand quercetin and kaempferol stock solutions at properconcentrations respectively

23 Preparation of Test Sample Solution Leaves (050 g) andflowers (025 g) of L clethroides were mixed with 5mL of[BMIM]BF4 methanol solution (05molsdotLminus1) in glass vesselrespectivelyThe samples of leaves and flowers were extractedby ultrasonic-assisted extraction (20 and 45min 500W)and then centrifuged (4000 and 5000 rsdotminminus1 resp) Thenthe extract was filtrated through a 022 120583m microporous

membrane The subsequent filtrate was the test samplesolutions a and b (the optimum extraction solvent concen-tration of selected extraction solvent mesh solid-liquid ratioultrasonic time and power and centrifugation speed weresystematically investigated in the study)

24 HPLC Analysis LC-20AT high-performance liquidchromatography (Shimadzu) was equipped with a degassera quaternary gradient low pressure pump the CTO-20Acolumn oven a SPD-M20A UV-detector and a SIL-20AautosamplerThe datawere acquired and processed using LC-Solution chromatography data processing system

The two chromatographic separations were all performedon a Purospher STAR RP-C18 column (46mm times 250mm5 120583m) at a column temperature of 25∘C The flow rate ofmobile phase was 06mLsdotminminus1 The UV detection wave-length was set at 360 nm All injection volumes were 10 120583LMobile phase a consists of acetonitrile-04 phosphoric acid(18 82 vv) Mobile phase b consists of methanol (A) and04 phosphoric acid (B) and the gradient program wasas follows 0sim25min 52 A 25sim30min 52sim65 A 30sim46min 65 A The HPLC chromatograms of the standardsolution and the extract of sample were shown in Figure 1

25 Experimental Procedure Theschematic plot of the exper-imental procedure was shown in Figure 2

26 Antioxidant Activity In Vitro

261 Preparation of the Sample Solution According to theoptimized conditionsmethanol and ionic liquid sample solu-tions were prepared and concentrated to 1mL respectively

262 DPPH Assay The sample was diluted into a range ofconcentrations with methanol 10120583L of sample solution (ormethanol) and 175 120583L of DPPH methanol solution (200120583M)were added to 96-well microplate and mixed The solutionwas measured at 515 nm using a Multiskan GO microplatereader after 20min in the dark place with PG BHA andBHTas positive controls All the experiments were performedin triplicate The percentage of radical scavenging rate wascalculated using formula (1) IC50 value was calculated fromthe concentration-effect linear regression curve [22]

DPPH radical scavenging rate ()= [(1198600 minus 1198601)1198600 ] times 100

(1)

Note 1198600 was the absorbance of the DPPH itself 1198601 wasthe absorbance of sample and the positive control IC50represents the concentration of sample in 120583gsdotmLminus1 whichinhibits DPPH radical by 50

263 ABTS Assay According to literature [23] the ABTSradical working solution was preparedThen the sample wasdiluted into a range of concentrations withmethanol 10 120583L ofsample solution (or methanol) and 200120583L of ABTS solution

Journal of Chemistry 3

A

B

1

2

0

20000

40000

60000

80000

100000

120000

Inte

nsity

(mV

)

20 30 40 5010Time (min)

(a)

A

B

3 4

0

20000

40000

60000

80000

100000

120000

140000

160000

Inte

nsity

(mV

)

15 20 25 30 35 40 4510Time (min)

(b)

Figure 1 HPLC chromatograms of the standard solution (A) and the sample solution (B) (1) isoquercitrin (2) astragalin (3) quercetin and(4) kaempferol

Ultrasonic extraction

Centrifugation Supernatant

Filter

HPLC-UV

Sample powder

solution[BMIM]BF4

A

B

AB

3 4

20 30 4010 50Time (min)

0

20000

40000

60000

80000

100000

120000

Inte

nsity

(mV

)

1

2

020000400006000080000

100000120000140000160000

Inte

nsity

(mV

)

15 20 25 30 35 40 4510Time (min)

Figure 2 Schematic plot of the experimental procedure

4 Journal of Chemistry

Isoquercitrin Astragalin03

04

05

06

07

08Ex

trac

tion

yiel

d (m

gmiddotgminus

1)

Methanol[HMIM]PF6

[BMIM]Br

[BMIM]PF6

[BMIM]PF4

(a)

Quercetin Kaempferol001

002

003

004

005

Extr

actio

n yi

eld

(mgmiddot

gminus1)

Methanol[HMIM]PF6

[BMIM]Br

[BMIM]PF6

[BMIM]PF4

(b)

Figure 3 Effect of type of ILs on extraction yields Concentration of the four ILs in methanol solution 05molsdotLminus1 the particle size 50 meshsolid-liquid ratio 1 10 gsdotmLminus1 ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

were added to 96-well microplate and mixed The solutionwas measured at 405 nm after 20min in the dark place Allthe experiments were performed in triplicate Calculate thepercentage of radical scavenging rate and IC50 value

264 FRAP Assay According to literature [24] the TPTZworking solution was preparedThen the sample was dilutedinto a range of concentrations withmethanol 10120583L of samplesolution (or methanol) and 200120583L of TPTZ solution wereadded to 96-well microplate and mixed The solution wasmeasured at 593 nm after 20min at 37∘C All reactions werecarried out with three replications Calculate the reducingability of Fe3+ of the sample The results were expressed in120583mol Trolox equivalents (TE)g sample The standard curvewas linear when the concentration of Trolox was between 50and 1600 120583M Further dilution of the sample was needed ifthe FRAP value measured exceeded the linear range of thestandard curve

27 Statistical Analysis All the grouped data were statisticallyevaluated with SPSS 190 software All results are expressed asmean plusmn standard deviation (SD)

3 Results and Discussion

31 Optimization of Extraction Conditions The contentsof isoquercitrin astragalin quercetin and kaempferol inthe roots stems leaves and flowers of L clethroides weredetermined according to the method of literature [18] Theresults showed that the contents of the four flavonoidswere different and the contents of isoquercitrin astragalinin leaves quercetin and kaempferol in flowers were thehighest respectively Therefore the extraction conditions ofisoquercitrin astragalin in leaves quercetin and kaempferolin flowers of L clethroides were chosen to optimize theconditions of extraction

311 Type of ILs Thestructure of IL has a significant effect onits physicochemical properties including solubility in waterthe viscosity and extraction capacity which might have greatimpact on the extraction efficiency of target compounds [25]In our study four kinds of ILs were used as the extractionsolvents including water-soluble and water-insoluble typesIn addition methanol was selected as extraction solvent forevaluating the extraction yield compared with the developedmethod In Figure 3 the addition of ILs to methanol obvi-ously improved the extraction efficiency of the four flavonoidcompounds comparedwithmethanol as solventThesemightbe due to the strongermulti-interactions between the analytesand ILs including hydrogen bonding and van der Waalsinteraction energy [26] Among the four types of ILs theextraction efficiency of [BMIM]BF4 was the highest butothers did not show a certain regularity The reason for theabove phenomenon may be the fact that other ingredientsin leaves and flowers had an influence on the extraction oftarget analytes Therefore [BMIM]BF4 was chosen as thecomponent of extraction solvent in the following studies

312 [BMIM]BF4 Concentration The concentration effectof [BMIM]BF4 on the extraction was shown in Figure 4The extraction yields continued to rise when the concen-tration increased from 01 to 05molsdotLminus1 and then differentdegrees of decrease appeared with the further increase of theconcentration This was because the solubility of the ionicliquid solution increased with the increasing concentrationbut the diffusion and transfer capability of extraction solventdecreased which led to declining of the solventrsquos penetrationpower into the interior of sample matrix [27 28]

313 Particle Size The effect of the particle sizes of sampleon extraction efficiency was investigated In Figure 5 thehighest extraction yields of isoquercitrin astragalin in leavesquercetin and kaempferol in flowers were achieved at 24 and

Journal of Chemistry 5

IsoquercitrinAstragalin

02 04 06 08 10 1200

Concentration (molmiddotLminus1)

050

055

060

065

070

075

080

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(a)

QuercetinKaempferol

02 04 06 08 10 1200

Concentration (molmiddotLminus1)

0020

0025

0030

0035

0040

0045

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(b)

Figure 4 Effect of the concentration of IL on extraction yields Type of IL [BMIM]BF4 the particle size 50 mesh solid-liquid ratio1 10 gsdotmLminus1 ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

Isoquercitrin Astragalin03

04

05

06

07

08

09

10

Extr

actio

n yi

eld

(mgmiddot

gminus1)

10 mesh24 mesh40mesh

50mesh70mesh90mesh

(a)

Quercetin Kaempferol001

002

003

004

005

006

Extr

actio

n yi

eld

(mgmiddot

gminus1)

10 mesh24 mesh40mesh

50mesh70mesh90mesh

(b)

Figure 5 Effect of the particle size on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 solid-liquid ratio 1 10 gsdotmLminus1ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

40mesh respectively It might be the smaller the particle sizethe easier the extraction but the samples of leaves and flowerscan easily be conglomerated by ionic liquid when the particlesize was too small [29] In addition the different optimumparticle sizes of leaves and flowers might be caused by thedifference of organizational structures

314 Solid-Liquid Ratio Six different ratios of solid-liquid(1 5 1 10 1 20 1 30 1 50 and 1 80 gsdotmLminus1) were chosen toevaluate the effect of solid-liquid ratio on the extraction yieldThe results in Figure 6 indicated that themaximumextractionrates of isoquercitrin astragalin in leaves quercetin andkaempferol in flowers were obtained at 1 10 and 1 20 gsdotmLminus1

respectively Results showed that the target analytes in thesample had basically been extracted When the proportionof extraction agent was increased the extraction efficiencieswere constant or decreased slightly this finding might berelated to the viscosity of ionic liquid

315 Ultrasound Time To investigate the influence of ultra-sound time on the extraction efficiency six time pointsranging from 10 to 60min were chosen In Figure 7 themaximum extraction efficiencies of isoquercitrin astragalinin leaves quercetin and kaempferol in flowers were obtainedat 20 and 45min respectively Nevertheless the ultrasoundtime was further extended the extraction yields of target

6 Journal of Chemistry

IsoquercitrinAstragalin

1 101 5 1 30 1 50 1 801 20Solid-liquid ratio (gmiddotmL

minus1)

05

06

07

08

09

10Ex

trac

tion

yiel

d (m

gmiddotgminus

1)

(a)

QuercetinKaempferol

Extr

actio

n yi

eld

(mgmiddot

gminus1)

1 101 5 1 30 1 50 1 801 20Solid-liquid ratio (gmiddotmL

minus1)

002

003

004

005

006

007

(b)

Figure 6 Effect of solid-liquid ratio on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

IsoquercitrinAstragalin

10 20 30 45 605

Ultrasound time (min)

07

08

09

10

11

12

13

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(a)

QuercetinKaempferol

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

30 45 6010 205

Ultrasound time (min)

(b)

Figure 7 Effect of ultrasound time on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound power 500W centrifugation speed 5000 rsdotminminus1

analytes had a certain decrease the reason for the abovephenomenon may be the fact that abundant sonication heatdestroys the structure of the analytes [30]

316 Ultrasonic Power The ultrasonic power had a sig-nificant effect on the extraction of the analytes In ourstudy the effect of different ultrasonic powers on extractionefficiency was investigated from 200 to 500W (in Figure 8)Results showed that the extraction efficiencies were increasedwith the increase of ultrasonic power Therefore 500W waschosen as ultrasonic power in this study

317 Centrifugation Speed The influence of centrifugationspeed on the extraction yield of the target analyte was

investigated The centrifugation time was set at 5min whichwas a reasonable time for productivity of the method [31]In Figure 9 results showed that the maximum extrac-tion yields of isoquercitrin astragalin in leaves quercetinand kaempferol in flowers were obtained at 4000 and5000 rsdotminminus1 respectively The reason might be that thehigher centrifugal speed affects the solubility of the analytesso that the solubility of the analytes is reduced

32 Method Validation

321 System Suitability Test (SST) System suitability test(SST) of HPLC includes the theoretical plate number (n) theresolution (Rs) the tailing factor (T) and the repeatability

Journal of Chemistry 7

IsoquercitrinAstragalin

500400300200

Ultrasound power (W)

07

08

09

10

11

12

13Ex

trac

tion

yiel

d (m

gmiddotgminus

1)

(a)

QuercetinKaempferol

002

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

300 400 500200

Ultrasound power (W)

(b)

Figure 8 Effect of ultrasound power on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound time 20min (a) and 45min (b) centrifugation speed5000 rsdotminminus1

IsoquercitrinAstragalin

4000 5000 6000 7000 80003000

Centrifugation speed (rmiddotminminus1)

04

06

08

10

12

14

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(a)

QuercetinKaempferol

002

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

4000 5000 6000 7000 80003000

Centrifugation speed (rmiddotminminus1)

(b)

Figure 9 Effect of centrifugation speed on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh(a) and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound time 20min (a) and 45min (b) ultrasound power500W

The repeatability was represented by the relative standarddeviation of the peak area (RSD) The results of SST wereshown in Table 1 which shows that the chromatographicsystems had good suitability

322 Linearity The stock standard solutions a and b wereaccurately injected 05 1 2 4 6 8 10 and 12 120583L to chro-matographic instrument for the construction of calibrationcurves respectively The calibration curves were constructedby plotting the peak areas versus the injection volume of eachcompoundThe results were demonstrated in Table 2 119903 valueswere in the range from 09996 to 10000 which indicated thatthe methods had good linearity

323 Precision The stock standard solutions a and b wereaccurately injected 10 120583L to chromatographic instrumentrespectively And they were analyzed in six replicates withinone day for determining the precision of the developedassay The RSDs of peak areas for isoquercitrin astragalinquercetin and kaempferol were 046 083 023 and024 respectively The results indicated that the methodswere precise for quantitative analysis of isoquercitrin astra-galin quercetin and kaempferol

324 Stability The sample solutions were prepared underthe optimum extraction conditions and placed at roomtemperature and thenwere injected 10120583L to chromatographic

8 Journal of Chemistry

Table 1 System suitability test (SST) of HPLC

Compound Theoretical plate number Resolution Tailing factor RepeatabilityIsoquercitrin 8392 152 131 087Astragalin 8834 1095 118 064Quercetin 7071 550 125 113Kaempferol 13956 854 135 102

Table 2 Regression equations and linear ranges for four compounds

Compound Regressive equation 119903 Linear range120583gIsoquercitrin 119884 = 319 times 106119883 minus 292 times 104 09996 0068sim164Astragalin 119884 = 300 times 106119883 minus 232 times 104 09997 0060sim144Quercetin 119884 = 742 times 106119883 minus 958 times 103 10000 00080sim019Kaempferol 119884 = 890 times 106119883 minus 149 times 104 10000 00077sim018119884 peak area119883 injection amount (120583g)

Table 3 Antioxidant activity of the different extracts of L clethroides

Sample DPPH ABTS FRAPRadical scavenging rate IC50120583gsdotmLminus1 Radical scavenging rate IC50120583gsdotmLminus1 TEAC120583molsdotgminus1

Leaf rsquos methanol extract 9391 1403 plusmn 80 9701 144 plusmn 5 3858 plusmn 072Leaf rsquos ionic liquid extract 8848 638 plusmn 60 9323 50 plusmn 2 10579 plusmn 702Flowerrsquos methanol extract 9392 1004 plusmn 12 9804 151 plusmn 1 5069 plusmn 110Flowerrsquos ionic liquid extract 8977 460 plusmn 36 9636 51 plusmn 2 10583 plusmn 125[BMIM]BF4 minus443 NT 003 NT NTBHA 7600 15 plusmn 146 9994 206 plusmn 008 591911 plusmn 1410BHT 7306 12 plusmn 048 9942 656 plusmn 010 58920 plusmn 5920PG 9561 401 plusmn 008 9580 069 plusmn 005 944133 plusmn 6927Note NT means not available because of low activity BHA BHT and PG are positive controls

instrument at 0 3 6 9 12 and 24 h respectively The RSDsof peak areas for isoquercitrin astragalin quercetin andkaempferol were 067 083 165 and 174 The resultsindicated that the sample solutionwas basically stable at roomtemperature within 24 h

325 Repeatability Six test sample solutions a and b wereprocessed under the optimumextraction conditions and theninjected 10 120583L to chromatographic instrument for analysisThe RSDs of peak areas for the four compounds were203 184 115 and 140 indicating that the analyticalmethods had an acceptable level of repeatability

326 Recovery Nine batches of leaves and flowers samplesof L clethroideswere prepared and divided into three groupsrespectively Then the four standard substances at threedifferent amounts were added to the leaves and flowerssamples The spiked samples were prepared according to theoptimum extraction conditions All the calculated recoveryvalues of the analytes ranged from 9839 to 10068 and theRSDs were less than 220The results demonstrated that themethods were reasonable and feasible

33 Antioxidant Activity of the Different Extracts of Lclethroides In Vitro The antioxidant activities of methanol

and ionic liquid sample solutions were evaluated by theDPPH ABTS and FRAP assay comprehensively and theresults were shown in Table 3 Results showed that the ionicliquid extract had stronger antioxidant activity than thatof the methanol extract and the ionic liquid itself had noantioxidant activity Results indicated that the ionic liquidused as extraction solvent hadmore advantages for extractingingredients

34 Sample Analysis Ionic liquid was successfully appliedfor the determination of isoquercitrin astragalin in leavesquercetin and kaempferol in flowers of L clethroides bythe optimized conditions respectively The mass fractions ofisoquercitrin astragalin in leaves quercetin and kaempferolin flowers were 1262 1219 0061 and 0046mgsdotgminus1 (119899 = 3)respectively

4 Conclusion

Themethod ionic liquid-based ultrasonic-assisted extractionhad been developed for the extraction of isoquercitrinastragalin in leaves quercetin and kaempferol in flowers of Lclethroides Compared with the conventional extraction withmethanol the proposed approaches provided higher extrac-tion efficiencies Results demonstrated that the methods

Journal of Chemistry 9

were simple rapid green and effective ILs can be recycledby some methods such as vacuum distillation membranefiltration salting out and liquid-liquid extraction [32] Con-sidering the unique properties of ILs the methods have agreat promising prospect in sample preparation of Chineseherbal medicine

Conflicts of Interest

The authors declare that there are no conflicts of interest

Authorsrsquo Contributions

Wen-yi Kang and Jin-feng Wei conceived the research ideaZhi-juan Zhang and Li-li Cui conducted the experimentscollected the plant specimens analyzed and interpreted thedata and prepared the first draft Wen-yi Kang Zhi-juanZhang and Jin-fengWei critically read and revised the paperAll the authors read and approved the paper before its finalsubmission

Acknowledgments

This work was supported by Henan Province UniversityScience and Technology Innovation Team (16IRTSTHN019)Science and Technology Innovation Team of Kaifeng City(2016-124) Kaifeng City Science and Technology InnovationTalent (1509010) and Key project in Science and TechnologyAgency of Kaifeng (1603111)

References

[1] X M Hu W K Zhang and Q S Zhu The Chinese MateriaMedica Shanghai Shanghai Science and Technology Press1999

[2] Z-J Zhang Z-Y Xia J-MWang X-T Song J-FWei andW-Y Kang ldquoEffects of flavonoids in Lysimachia clethroides dubyon the activities of cytochrome P450 CYP2E1 and CYP3A4 inrat liver microsomesrdquo Molecules vol 21 no 6 article no 7382016

[3] L H Tang X Y Xu B G You W Zhang and Y Q WangldquoAnti-tumor effect and its mechanism of total flavones ofLysimachia clethroides Dubyrdquo Shanghai Journal of TraditionalChinese Medicine vol 41 no 5 pp 74ndash76 2007

[4] Q-M Xu Y-L Liu Y-L Feng L-H Tang and S-L YangldquoA new e-ring 120574-lactone pentacyclic triterpene from lysimachiaclethroides and its cytotoxic activitiesrdquo Chemistry of NaturalCompounds vol 48 no 4 pp 597ndash600 2012

[5] C F Li Y L Song Y X Liu and W Y Kang ldquoAntioxidantactivity of extracts from Lysimachia clethroidesrdquo Fine Chemicalsvol 25 pp 1191ndash1193 2008

[6] J F Wei ldquoStudy on Chemical Constitutens Hepatoprotectiveand Antihyperglycemic Effect of Lysimachia paridiformis varstenophylla and Lysimachia clethroidesrdquo MSD thesis 2012Henan University Kaifeng

[7] D Liang ldquoStudy on the chemical composition and biologicalactivity of Lysimachia clethroides and chemical conversion ofsyntheticrdquo MSD thesis 2012 Peking Union Medical CollegeBeijing

[8] S M Yue B Q Chen P F Yuan W H Cui and W YKang ldquoChemical Constituents of Lysimachia clethroides DubyrdquoChinese Phramaceutical Journal vol 46 pp 341ndash343 2011

[9] L F Ding Y D Guo X D Wu and Y H Ma ldquoChemicalconstituents of flavonoids in Lysimachia clethroidesrdquo ChineseTraditional Patent Medicine vol 32 no 05 pp 827ndash830 2010

[10] Q M Xu L H Tang X Li L L Hao X R Li and S LYang ldquoIsolation and Identification of 3-O-p-CoumaroyloxylPentacyclic Triterpenoids from Lysimachia clethroides DubyrdquoChin Pharm J vol 45 pp 825ndash828 2010

[11] H Y Zou and P F Tu ldquoStudies on the chemical constituents ofLysimachia clethroides Dubyrdquo Chin Tradit Herb Drugs vol 40pp 704ndash708 2009

[12] L H Tang X Y Xu B G You Y Q Wang and W ZhangldquoEffect of flavones from Lysimachia clethroides on gene expre-sion of HL-60 cellrdquo Pharm Clin Res vol 15 pp 21ndash24 2007

[13] Y Q Wang L H Tang Z Q Liang and Z L Gu ldquoPrimarystudies on the anti-uterine cervix cancer effects of the extractZE4 from Lysimachia clethroides Dubyrdquo Chin Pharmacol Bullvol 23 pp 925ndash929 2007

[14] A A Aerov A R Khokhlov and I I Potemkin ldquoWhy ionicliquids can possess extra solvent powerrdquo Journal of PhysicalChemistry B vol 110 no 33 pp 16205ndash16207 2006

[15] J F Wei P R Cao J M Wang and W Y Kang ldquoAnalysis oftilianin and acacetin in Agastache rugosa by high-performanceliquid chromatography with ionic liquids-ultrasound basedextractionrdquo Chemistry Central Journal vol 10 p 76 2016

[16] W Y Ma Y B Lu R L Hu J H Chen Z Z Zhang and YJ Pan ldquoApplication of ionic liquids based microwave-assistedextraction of three alkaloids N-nornuciferine O-nornuciferineand nuciferine from lotus leafrdquo Talanta vol 80 no 3 pp 1292ndash1297 2010

[17] W Liu D D Li H S Yang et al ldquoDetermination of oleanic acidand paeoniflorin in Paeonia lactiflora by ultrasound-assistedionic liquid-reversed phase liquid chromatographyrdquo China JChin Mater Med vol 40 pp 443ndash449 2015

[18] J F Wei Z J Zhang D D Li W Liu andW Y Kang ldquoAnalysisof four flavonoids in Lysimachia clethroides using ionic liquid-assisted extractionrdquo China J Chin Mater Med vol 40 pp 1305ndash1310 2015

[19] J F Wei Z H Yin and F D Shang ldquoVolatiles in the Lysimachiaclethroides Duby by head space solid phase microextractioncoupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS)rdquo African Journal of Pharmacy and Pharmacol-ogy vol 6 pp 2484ndash2487 2012

[20] J F Wei Y Y Li Z H Yin F Gong and F D ShangldquoAntioxidant activities in vitroand hepatoprotective effects ofLysimachia clethroidesDuby on CCl4-induced acute liver injuryin micerdquo Afr J Pharm Pharmacol vol 6 pp 743ndash750 2012

[21] J F Wei X Chang W Wang and W Y Kang ldquoChemicalconstituents from Lysimachia circaeoidesrdquo Journal of ChineseMedicine Material vol 36 pp 1441ndash1443 2013

[22] W-Y Kang Y-L Song and L Zhang ldquo120572-Glucosidaseinhibitory and antioxidant properties and antidiabetic activityof Hypericum ascyron Lrdquo Medicinal Chemistry Research vol20 no 7 pp 809ndash816 2011

[23] W-Y Kang C-F Li and Y-X Liu ldquoAntioxidant phenoliccompounds and flavonoids of Mitragyna rotundifolia (Roxb)Kuntze in vitrordquo Medicinal Chemistry Research vol 19 no 9pp 1222ndash1232 2010

10 Journal of Chemistry

[24] W Kang and J Wang ldquoIn vitro antioxidant properties andin vivo lowering blood lipid of Forsythia suspense leavesrdquoMedicinal Chemistry Research vol 19 no 7 pp 617ndash628 2010

[25] C F Poole and S K Poole ldquoExtraction of organic compoundswith room temperature ionic liquidsrdquo Journal of Chromatogra-phy A vol 1217 no 16 pp 2268ndash2286 2010

[26] Z Guo B Lue K Thomasen A S Meyer and X Xu ldquoPre-dictions of flavonoid solubility in ionic liquids by COSMO-RSexperimental verification structural elucidation and solvationcharacterizationrdquo Green Chemistry vol 9 no 12 pp 1362ndash13732007

[27] S Y Dong Q Hu J Li Y L Fu Y Q Xing and T L HuangldquoIonic liquid-based microwave-assisted extraction followedhigh-performance liquid chromatography for the simultaneousdetermination of five organic oxygen pesticides in soil samplesrdquoChinese Journal of Analysis Laboratory vol 32 pp 48ndash52 2013

[28] H Wu M Chen Y Fan F Elsebaei and Y Zhu ldquoDeter-mination of rutin and quercetin in Chinese herbal medicineby ionic liquid-based pressurized liquid extraction-liquidchromatography- chemiluminescence detectionrdquo Talanta vol88 pp 222ndash229 2012

[29] R Jin L Fan and X An ldquoIonic liquid-assisted extraction ofpaeonol from cynanchum paniculatumrdquo Chromatographia vol73 no 7-8 pp 787ndash792 2011

[30] P Liang F Wang and Q Wan ldquoIonic liquid-based ultrasound-assisted emulsification microextraction coupled with high per-formance liquid chromatography for the determination of fourfungicides in environmental water samplesrdquo Talanta vol 105pp 57ndash62 2013

[31] C K Zacharis C Christophoridis and K Fytianos ldquoVortex-assisted liquid-liquid microextraction combined with gaschromatography-mass spectrometry for the determination oforganophosphate pesticides in environmental water samplesand winesrdquo Journal of Separation Science vol 35 no 18 pp2422ndash2429 2012

[32] H Liu X Y Wei J H Li T Li and F Wang ldquoReview of ionicliquids recyclingrdquo Journal of Cellulose Science Technology vol21 pp 63ndash69 2013

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal ofInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Medicinal ChemistryInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 3

A

B

1

2

0

20000

40000

60000

80000

100000

120000

Inte

nsity

(mV

)

20 30 40 5010Time (min)

(a)

A

B

3 4

0

20000

40000

60000

80000

100000

120000

140000

160000

Inte

nsity

(mV

)

15 20 25 30 35 40 4510Time (min)

(b)

Figure 1 HPLC chromatograms of the standard solution (A) and the sample solution (B) (1) isoquercitrin (2) astragalin (3) quercetin and(4) kaempferol

Ultrasonic extraction

Centrifugation Supernatant

Filter

HPLC-UV

Sample powder

solution[BMIM]BF4

A

B

AB

3 4

20 30 4010 50Time (min)

0

20000

40000

60000

80000

100000

120000

Inte

nsity

(mV

)

1

2

020000400006000080000

100000120000140000160000

Inte

nsity

(mV

)

15 20 25 30 35 40 4510Time (min)

Figure 2 Schematic plot of the experimental procedure

4 Journal of Chemistry

Isoquercitrin Astragalin03

04

05

06

07

08Ex

trac

tion

yiel

d (m

gmiddotgminus

1)

Methanol[HMIM]PF6

[BMIM]Br

[BMIM]PF6

[BMIM]PF4

(a)

Quercetin Kaempferol001

002

003

004

005

Extr

actio

n yi

eld

(mgmiddot

gminus1)

Methanol[HMIM]PF6

[BMIM]Br

[BMIM]PF6

[BMIM]PF4

(b)

Figure 3 Effect of type of ILs on extraction yields Concentration of the four ILs in methanol solution 05molsdotLminus1 the particle size 50 meshsolid-liquid ratio 1 10 gsdotmLminus1 ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

were added to 96-well microplate and mixed The solutionwas measured at 405 nm after 20min in the dark place Allthe experiments were performed in triplicate Calculate thepercentage of radical scavenging rate and IC50 value

264 FRAP Assay According to literature [24] the TPTZworking solution was preparedThen the sample was dilutedinto a range of concentrations withmethanol 10120583L of samplesolution (or methanol) and 200120583L of TPTZ solution wereadded to 96-well microplate and mixed The solution wasmeasured at 593 nm after 20min at 37∘C All reactions werecarried out with three replications Calculate the reducingability of Fe3+ of the sample The results were expressed in120583mol Trolox equivalents (TE)g sample The standard curvewas linear when the concentration of Trolox was between 50and 1600 120583M Further dilution of the sample was needed ifthe FRAP value measured exceeded the linear range of thestandard curve

27 Statistical Analysis All the grouped data were statisticallyevaluated with SPSS 190 software All results are expressed asmean plusmn standard deviation (SD)

3 Results and Discussion

31 Optimization of Extraction Conditions The contentsof isoquercitrin astragalin quercetin and kaempferol inthe roots stems leaves and flowers of L clethroides weredetermined according to the method of literature [18] Theresults showed that the contents of the four flavonoidswere different and the contents of isoquercitrin astragalinin leaves quercetin and kaempferol in flowers were thehighest respectively Therefore the extraction conditions ofisoquercitrin astragalin in leaves quercetin and kaempferolin flowers of L clethroides were chosen to optimize theconditions of extraction

311 Type of ILs Thestructure of IL has a significant effect onits physicochemical properties including solubility in waterthe viscosity and extraction capacity which might have greatimpact on the extraction efficiency of target compounds [25]In our study four kinds of ILs were used as the extractionsolvents including water-soluble and water-insoluble typesIn addition methanol was selected as extraction solvent forevaluating the extraction yield compared with the developedmethod In Figure 3 the addition of ILs to methanol obvi-ously improved the extraction efficiency of the four flavonoidcompounds comparedwithmethanol as solventThesemightbe due to the strongermulti-interactions between the analytesand ILs including hydrogen bonding and van der Waalsinteraction energy [26] Among the four types of ILs theextraction efficiency of [BMIM]BF4 was the highest butothers did not show a certain regularity The reason for theabove phenomenon may be the fact that other ingredientsin leaves and flowers had an influence on the extraction oftarget analytes Therefore [BMIM]BF4 was chosen as thecomponent of extraction solvent in the following studies

312 [BMIM]BF4 Concentration The concentration effectof [BMIM]BF4 on the extraction was shown in Figure 4The extraction yields continued to rise when the concen-tration increased from 01 to 05molsdotLminus1 and then differentdegrees of decrease appeared with the further increase of theconcentration This was because the solubility of the ionicliquid solution increased with the increasing concentrationbut the diffusion and transfer capability of extraction solventdecreased which led to declining of the solventrsquos penetrationpower into the interior of sample matrix [27 28]

313 Particle Size The effect of the particle sizes of sampleon extraction efficiency was investigated In Figure 5 thehighest extraction yields of isoquercitrin astragalin in leavesquercetin and kaempferol in flowers were achieved at 24 and

Journal of Chemistry 5

IsoquercitrinAstragalin

02 04 06 08 10 1200

Concentration (molmiddotLminus1)

050

055

060

065

070

075

080

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(a)

QuercetinKaempferol

02 04 06 08 10 1200

Concentration (molmiddotLminus1)

0020

0025

0030

0035

0040

0045

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(b)

Figure 4 Effect of the concentration of IL on extraction yields Type of IL [BMIM]BF4 the particle size 50 mesh solid-liquid ratio1 10 gsdotmLminus1 ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

Isoquercitrin Astragalin03

04

05

06

07

08

09

10

Extr

actio

n yi

eld

(mgmiddot

gminus1)

10 mesh24 mesh40mesh

50mesh70mesh90mesh

(a)

Quercetin Kaempferol001

002

003

004

005

006

Extr

actio

n yi

eld

(mgmiddot

gminus1)

10 mesh24 mesh40mesh

50mesh70mesh90mesh

(b)

Figure 5 Effect of the particle size on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 solid-liquid ratio 1 10 gsdotmLminus1ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

40mesh respectively It might be the smaller the particle sizethe easier the extraction but the samples of leaves and flowerscan easily be conglomerated by ionic liquid when the particlesize was too small [29] In addition the different optimumparticle sizes of leaves and flowers might be caused by thedifference of organizational structures

314 Solid-Liquid Ratio Six different ratios of solid-liquid(1 5 1 10 1 20 1 30 1 50 and 1 80 gsdotmLminus1) were chosen toevaluate the effect of solid-liquid ratio on the extraction yieldThe results in Figure 6 indicated that themaximumextractionrates of isoquercitrin astragalin in leaves quercetin andkaempferol in flowers were obtained at 1 10 and 1 20 gsdotmLminus1

respectively Results showed that the target analytes in thesample had basically been extracted When the proportionof extraction agent was increased the extraction efficiencieswere constant or decreased slightly this finding might berelated to the viscosity of ionic liquid

315 Ultrasound Time To investigate the influence of ultra-sound time on the extraction efficiency six time pointsranging from 10 to 60min were chosen In Figure 7 themaximum extraction efficiencies of isoquercitrin astragalinin leaves quercetin and kaempferol in flowers were obtainedat 20 and 45min respectively Nevertheless the ultrasoundtime was further extended the extraction yields of target

6 Journal of Chemistry

IsoquercitrinAstragalin

1 101 5 1 30 1 50 1 801 20Solid-liquid ratio (gmiddotmL

minus1)

05

06

07

08

09

10Ex

trac

tion

yiel

d (m

gmiddotgminus

1)

(a)

QuercetinKaempferol

Extr

actio

n yi

eld

(mgmiddot

gminus1)

1 101 5 1 30 1 50 1 801 20Solid-liquid ratio (gmiddotmL

minus1)

002

003

004

005

006

007

(b)

Figure 6 Effect of solid-liquid ratio on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

IsoquercitrinAstragalin

10 20 30 45 605

Ultrasound time (min)

07

08

09

10

11

12

13

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(a)

QuercetinKaempferol

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

30 45 6010 205

Ultrasound time (min)

(b)

Figure 7 Effect of ultrasound time on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound power 500W centrifugation speed 5000 rsdotminminus1

analytes had a certain decrease the reason for the abovephenomenon may be the fact that abundant sonication heatdestroys the structure of the analytes [30]

316 Ultrasonic Power The ultrasonic power had a sig-nificant effect on the extraction of the analytes In ourstudy the effect of different ultrasonic powers on extractionefficiency was investigated from 200 to 500W (in Figure 8)Results showed that the extraction efficiencies were increasedwith the increase of ultrasonic power Therefore 500W waschosen as ultrasonic power in this study

317 Centrifugation Speed The influence of centrifugationspeed on the extraction yield of the target analyte was

investigated The centrifugation time was set at 5min whichwas a reasonable time for productivity of the method [31]In Figure 9 results showed that the maximum extrac-tion yields of isoquercitrin astragalin in leaves quercetinand kaempferol in flowers were obtained at 4000 and5000 rsdotminminus1 respectively The reason might be that thehigher centrifugal speed affects the solubility of the analytesso that the solubility of the analytes is reduced

32 Method Validation

321 System Suitability Test (SST) System suitability test(SST) of HPLC includes the theoretical plate number (n) theresolution (Rs) the tailing factor (T) and the repeatability

Journal of Chemistry 7

IsoquercitrinAstragalin

500400300200

Ultrasound power (W)

07

08

09

10

11

12

13Ex

trac

tion

yiel

d (m

gmiddotgminus

1)

(a)

QuercetinKaempferol

002

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

300 400 500200

Ultrasound power (W)

(b)

Figure 8 Effect of ultrasound power on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound time 20min (a) and 45min (b) centrifugation speed5000 rsdotminminus1

IsoquercitrinAstragalin

4000 5000 6000 7000 80003000

Centrifugation speed (rmiddotminminus1)

04

06

08

10

12

14

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(a)

QuercetinKaempferol

002

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

4000 5000 6000 7000 80003000

Centrifugation speed (rmiddotminminus1)

(b)

Figure 9 Effect of centrifugation speed on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh(a) and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound time 20min (a) and 45min (b) ultrasound power500W

The repeatability was represented by the relative standarddeviation of the peak area (RSD) The results of SST wereshown in Table 1 which shows that the chromatographicsystems had good suitability

322 Linearity The stock standard solutions a and b wereaccurately injected 05 1 2 4 6 8 10 and 12 120583L to chro-matographic instrument for the construction of calibrationcurves respectively The calibration curves were constructedby plotting the peak areas versus the injection volume of eachcompoundThe results were demonstrated in Table 2 119903 valueswere in the range from 09996 to 10000 which indicated thatthe methods had good linearity

323 Precision The stock standard solutions a and b wereaccurately injected 10 120583L to chromatographic instrumentrespectively And they were analyzed in six replicates withinone day for determining the precision of the developedassay The RSDs of peak areas for isoquercitrin astragalinquercetin and kaempferol were 046 083 023 and024 respectively The results indicated that the methodswere precise for quantitative analysis of isoquercitrin astra-galin quercetin and kaempferol

324 Stability The sample solutions were prepared underthe optimum extraction conditions and placed at roomtemperature and thenwere injected 10120583L to chromatographic

8 Journal of Chemistry

Table 1 System suitability test (SST) of HPLC

Compound Theoretical plate number Resolution Tailing factor RepeatabilityIsoquercitrin 8392 152 131 087Astragalin 8834 1095 118 064Quercetin 7071 550 125 113Kaempferol 13956 854 135 102

Table 2 Regression equations and linear ranges for four compounds

Compound Regressive equation 119903 Linear range120583gIsoquercitrin 119884 = 319 times 106119883 minus 292 times 104 09996 0068sim164Astragalin 119884 = 300 times 106119883 minus 232 times 104 09997 0060sim144Quercetin 119884 = 742 times 106119883 minus 958 times 103 10000 00080sim019Kaempferol 119884 = 890 times 106119883 minus 149 times 104 10000 00077sim018119884 peak area119883 injection amount (120583g)

Table 3 Antioxidant activity of the different extracts of L clethroides

Sample DPPH ABTS FRAPRadical scavenging rate IC50120583gsdotmLminus1 Radical scavenging rate IC50120583gsdotmLminus1 TEAC120583molsdotgminus1

Leaf rsquos methanol extract 9391 1403 plusmn 80 9701 144 plusmn 5 3858 plusmn 072Leaf rsquos ionic liquid extract 8848 638 plusmn 60 9323 50 plusmn 2 10579 plusmn 702Flowerrsquos methanol extract 9392 1004 plusmn 12 9804 151 plusmn 1 5069 plusmn 110Flowerrsquos ionic liquid extract 8977 460 plusmn 36 9636 51 plusmn 2 10583 plusmn 125[BMIM]BF4 minus443 NT 003 NT NTBHA 7600 15 plusmn 146 9994 206 plusmn 008 591911 plusmn 1410BHT 7306 12 plusmn 048 9942 656 plusmn 010 58920 plusmn 5920PG 9561 401 plusmn 008 9580 069 plusmn 005 944133 plusmn 6927Note NT means not available because of low activity BHA BHT and PG are positive controls

instrument at 0 3 6 9 12 and 24 h respectively The RSDsof peak areas for isoquercitrin astragalin quercetin andkaempferol were 067 083 165 and 174 The resultsindicated that the sample solutionwas basically stable at roomtemperature within 24 h

325 Repeatability Six test sample solutions a and b wereprocessed under the optimumextraction conditions and theninjected 10 120583L to chromatographic instrument for analysisThe RSDs of peak areas for the four compounds were203 184 115 and 140 indicating that the analyticalmethods had an acceptable level of repeatability

326 Recovery Nine batches of leaves and flowers samplesof L clethroideswere prepared and divided into three groupsrespectively Then the four standard substances at threedifferent amounts were added to the leaves and flowerssamples The spiked samples were prepared according to theoptimum extraction conditions All the calculated recoveryvalues of the analytes ranged from 9839 to 10068 and theRSDs were less than 220The results demonstrated that themethods were reasonable and feasible

33 Antioxidant Activity of the Different Extracts of Lclethroides In Vitro The antioxidant activities of methanol

and ionic liquid sample solutions were evaluated by theDPPH ABTS and FRAP assay comprehensively and theresults were shown in Table 3 Results showed that the ionicliquid extract had stronger antioxidant activity than thatof the methanol extract and the ionic liquid itself had noantioxidant activity Results indicated that the ionic liquidused as extraction solvent hadmore advantages for extractingingredients

34 Sample Analysis Ionic liquid was successfully appliedfor the determination of isoquercitrin astragalin in leavesquercetin and kaempferol in flowers of L clethroides bythe optimized conditions respectively The mass fractions ofisoquercitrin astragalin in leaves quercetin and kaempferolin flowers were 1262 1219 0061 and 0046mgsdotgminus1 (119899 = 3)respectively

4 Conclusion

Themethod ionic liquid-based ultrasonic-assisted extractionhad been developed for the extraction of isoquercitrinastragalin in leaves quercetin and kaempferol in flowers of Lclethroides Compared with the conventional extraction withmethanol the proposed approaches provided higher extrac-tion efficiencies Results demonstrated that the methods

Journal of Chemistry 9

were simple rapid green and effective ILs can be recycledby some methods such as vacuum distillation membranefiltration salting out and liquid-liquid extraction [32] Con-sidering the unique properties of ILs the methods have agreat promising prospect in sample preparation of Chineseherbal medicine

Conflicts of Interest

The authors declare that there are no conflicts of interest

Authorsrsquo Contributions

Wen-yi Kang and Jin-feng Wei conceived the research ideaZhi-juan Zhang and Li-li Cui conducted the experimentscollected the plant specimens analyzed and interpreted thedata and prepared the first draft Wen-yi Kang Zhi-juanZhang and Jin-fengWei critically read and revised the paperAll the authors read and approved the paper before its finalsubmission

Acknowledgments

This work was supported by Henan Province UniversityScience and Technology Innovation Team (16IRTSTHN019)Science and Technology Innovation Team of Kaifeng City(2016-124) Kaifeng City Science and Technology InnovationTalent (1509010) and Key project in Science and TechnologyAgency of Kaifeng (1603111)

References

[1] X M Hu W K Zhang and Q S Zhu The Chinese MateriaMedica Shanghai Shanghai Science and Technology Press1999

[2] Z-J Zhang Z-Y Xia J-MWang X-T Song J-FWei andW-Y Kang ldquoEffects of flavonoids in Lysimachia clethroides dubyon the activities of cytochrome P450 CYP2E1 and CYP3A4 inrat liver microsomesrdquo Molecules vol 21 no 6 article no 7382016

[3] L H Tang X Y Xu B G You W Zhang and Y Q WangldquoAnti-tumor effect and its mechanism of total flavones ofLysimachia clethroides Dubyrdquo Shanghai Journal of TraditionalChinese Medicine vol 41 no 5 pp 74ndash76 2007

[4] Q-M Xu Y-L Liu Y-L Feng L-H Tang and S-L YangldquoA new e-ring 120574-lactone pentacyclic triterpene from lysimachiaclethroides and its cytotoxic activitiesrdquo Chemistry of NaturalCompounds vol 48 no 4 pp 597ndash600 2012

[5] C F Li Y L Song Y X Liu and W Y Kang ldquoAntioxidantactivity of extracts from Lysimachia clethroidesrdquo Fine Chemicalsvol 25 pp 1191ndash1193 2008

[6] J F Wei ldquoStudy on Chemical Constitutens Hepatoprotectiveand Antihyperglycemic Effect of Lysimachia paridiformis varstenophylla and Lysimachia clethroidesrdquo MSD thesis 2012Henan University Kaifeng

[7] D Liang ldquoStudy on the chemical composition and biologicalactivity of Lysimachia clethroides and chemical conversion ofsyntheticrdquo MSD thesis 2012 Peking Union Medical CollegeBeijing

[8] S M Yue B Q Chen P F Yuan W H Cui and W YKang ldquoChemical Constituents of Lysimachia clethroides DubyrdquoChinese Phramaceutical Journal vol 46 pp 341ndash343 2011

[9] L F Ding Y D Guo X D Wu and Y H Ma ldquoChemicalconstituents of flavonoids in Lysimachia clethroidesrdquo ChineseTraditional Patent Medicine vol 32 no 05 pp 827ndash830 2010

[10] Q M Xu L H Tang X Li L L Hao X R Li and S LYang ldquoIsolation and Identification of 3-O-p-CoumaroyloxylPentacyclic Triterpenoids from Lysimachia clethroides DubyrdquoChin Pharm J vol 45 pp 825ndash828 2010

[11] H Y Zou and P F Tu ldquoStudies on the chemical constituents ofLysimachia clethroides Dubyrdquo Chin Tradit Herb Drugs vol 40pp 704ndash708 2009

[12] L H Tang X Y Xu B G You Y Q Wang and W ZhangldquoEffect of flavones from Lysimachia clethroides on gene expre-sion of HL-60 cellrdquo Pharm Clin Res vol 15 pp 21ndash24 2007

[13] Y Q Wang L H Tang Z Q Liang and Z L Gu ldquoPrimarystudies on the anti-uterine cervix cancer effects of the extractZE4 from Lysimachia clethroides Dubyrdquo Chin Pharmacol Bullvol 23 pp 925ndash929 2007

[14] A A Aerov A R Khokhlov and I I Potemkin ldquoWhy ionicliquids can possess extra solvent powerrdquo Journal of PhysicalChemistry B vol 110 no 33 pp 16205ndash16207 2006

[15] J F Wei P R Cao J M Wang and W Y Kang ldquoAnalysis oftilianin and acacetin in Agastache rugosa by high-performanceliquid chromatography with ionic liquids-ultrasound basedextractionrdquo Chemistry Central Journal vol 10 p 76 2016

[16] W Y Ma Y B Lu R L Hu J H Chen Z Z Zhang and YJ Pan ldquoApplication of ionic liquids based microwave-assistedextraction of three alkaloids N-nornuciferine O-nornuciferineand nuciferine from lotus leafrdquo Talanta vol 80 no 3 pp 1292ndash1297 2010

[17] W Liu D D Li H S Yang et al ldquoDetermination of oleanic acidand paeoniflorin in Paeonia lactiflora by ultrasound-assistedionic liquid-reversed phase liquid chromatographyrdquo China JChin Mater Med vol 40 pp 443ndash449 2015

[18] J F Wei Z J Zhang D D Li W Liu andW Y Kang ldquoAnalysisof four flavonoids in Lysimachia clethroides using ionic liquid-assisted extractionrdquo China J Chin Mater Med vol 40 pp 1305ndash1310 2015

[19] J F Wei Z H Yin and F D Shang ldquoVolatiles in the Lysimachiaclethroides Duby by head space solid phase microextractioncoupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS)rdquo African Journal of Pharmacy and Pharmacol-ogy vol 6 pp 2484ndash2487 2012

[20] J F Wei Y Y Li Z H Yin F Gong and F D ShangldquoAntioxidant activities in vitroand hepatoprotective effects ofLysimachia clethroidesDuby on CCl4-induced acute liver injuryin micerdquo Afr J Pharm Pharmacol vol 6 pp 743ndash750 2012

[21] J F Wei X Chang W Wang and W Y Kang ldquoChemicalconstituents from Lysimachia circaeoidesrdquo Journal of ChineseMedicine Material vol 36 pp 1441ndash1443 2013

[22] W-Y Kang Y-L Song and L Zhang ldquo120572-Glucosidaseinhibitory and antioxidant properties and antidiabetic activityof Hypericum ascyron Lrdquo Medicinal Chemistry Research vol20 no 7 pp 809ndash816 2011

[23] W-Y Kang C-F Li and Y-X Liu ldquoAntioxidant phenoliccompounds and flavonoids of Mitragyna rotundifolia (Roxb)Kuntze in vitrordquo Medicinal Chemistry Research vol 19 no 9pp 1222ndash1232 2010

10 Journal of Chemistry

[24] W Kang and J Wang ldquoIn vitro antioxidant properties andin vivo lowering blood lipid of Forsythia suspense leavesrdquoMedicinal Chemistry Research vol 19 no 7 pp 617ndash628 2010

[25] C F Poole and S K Poole ldquoExtraction of organic compoundswith room temperature ionic liquidsrdquo Journal of Chromatogra-phy A vol 1217 no 16 pp 2268ndash2286 2010

[26] Z Guo B Lue K Thomasen A S Meyer and X Xu ldquoPre-dictions of flavonoid solubility in ionic liquids by COSMO-RSexperimental verification structural elucidation and solvationcharacterizationrdquo Green Chemistry vol 9 no 12 pp 1362ndash13732007

[27] S Y Dong Q Hu J Li Y L Fu Y Q Xing and T L HuangldquoIonic liquid-based microwave-assisted extraction followedhigh-performance liquid chromatography for the simultaneousdetermination of five organic oxygen pesticides in soil samplesrdquoChinese Journal of Analysis Laboratory vol 32 pp 48ndash52 2013

[28] H Wu M Chen Y Fan F Elsebaei and Y Zhu ldquoDeter-mination of rutin and quercetin in Chinese herbal medicineby ionic liquid-based pressurized liquid extraction-liquidchromatography- chemiluminescence detectionrdquo Talanta vol88 pp 222ndash229 2012

[29] R Jin L Fan and X An ldquoIonic liquid-assisted extraction ofpaeonol from cynanchum paniculatumrdquo Chromatographia vol73 no 7-8 pp 787ndash792 2011

[30] P Liang F Wang and Q Wan ldquoIonic liquid-based ultrasound-assisted emulsification microextraction coupled with high per-formance liquid chromatography for the determination of fourfungicides in environmental water samplesrdquo Talanta vol 105pp 57ndash62 2013

[31] C K Zacharis C Christophoridis and K Fytianos ldquoVortex-assisted liquid-liquid microextraction combined with gaschromatography-mass spectrometry for the determination oforganophosphate pesticides in environmental water samplesand winesrdquo Journal of Separation Science vol 35 no 18 pp2422ndash2429 2012

[32] H Liu X Y Wei J H Li T Li and F Wang ldquoReview of ionicliquids recyclingrdquo Journal of Cellulose Science Technology vol21 pp 63ndash69 2013

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

International Journal ofInternational Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal ofInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

4 Journal of Chemistry

Isoquercitrin Astragalin03

04

05

06

07

08Ex

trac

tion

yiel

d (m

gmiddotgminus

1)

Methanol[HMIM]PF6

[BMIM]Br

[BMIM]PF6

[BMIM]PF4

(a)

Quercetin Kaempferol001

002

003

004

005

Extr

actio

n yi

eld

(mgmiddot

gminus1)

Methanol[HMIM]PF6

[BMIM]Br

[BMIM]PF6

[BMIM]PF4

(b)

Figure 3 Effect of type of ILs on extraction yields Concentration of the four ILs in methanol solution 05molsdotLminus1 the particle size 50 meshsolid-liquid ratio 1 10 gsdotmLminus1 ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

were added to 96-well microplate and mixed The solutionwas measured at 405 nm after 20min in the dark place Allthe experiments were performed in triplicate Calculate thepercentage of radical scavenging rate and IC50 value

264 FRAP Assay According to literature [24] the TPTZworking solution was preparedThen the sample was dilutedinto a range of concentrations withmethanol 10120583L of samplesolution (or methanol) and 200120583L of TPTZ solution wereadded to 96-well microplate and mixed The solution wasmeasured at 593 nm after 20min at 37∘C All reactions werecarried out with three replications Calculate the reducingability of Fe3+ of the sample The results were expressed in120583mol Trolox equivalents (TE)g sample The standard curvewas linear when the concentration of Trolox was between 50and 1600 120583M Further dilution of the sample was needed ifthe FRAP value measured exceeded the linear range of thestandard curve

27 Statistical Analysis All the grouped data were statisticallyevaluated with SPSS 190 software All results are expressed asmean plusmn standard deviation (SD)

3 Results and Discussion

31 Optimization of Extraction Conditions The contentsof isoquercitrin astragalin quercetin and kaempferol inthe roots stems leaves and flowers of L clethroides weredetermined according to the method of literature [18] Theresults showed that the contents of the four flavonoidswere different and the contents of isoquercitrin astragalinin leaves quercetin and kaempferol in flowers were thehighest respectively Therefore the extraction conditions ofisoquercitrin astragalin in leaves quercetin and kaempferolin flowers of L clethroides were chosen to optimize theconditions of extraction

311 Type of ILs Thestructure of IL has a significant effect onits physicochemical properties including solubility in waterthe viscosity and extraction capacity which might have greatimpact on the extraction efficiency of target compounds [25]In our study four kinds of ILs were used as the extractionsolvents including water-soluble and water-insoluble typesIn addition methanol was selected as extraction solvent forevaluating the extraction yield compared with the developedmethod In Figure 3 the addition of ILs to methanol obvi-ously improved the extraction efficiency of the four flavonoidcompounds comparedwithmethanol as solventThesemightbe due to the strongermulti-interactions between the analytesand ILs including hydrogen bonding and van der Waalsinteraction energy [26] Among the four types of ILs theextraction efficiency of [BMIM]BF4 was the highest butothers did not show a certain regularity The reason for theabove phenomenon may be the fact that other ingredientsin leaves and flowers had an influence on the extraction oftarget analytes Therefore [BMIM]BF4 was chosen as thecomponent of extraction solvent in the following studies

312 [BMIM]BF4 Concentration The concentration effectof [BMIM]BF4 on the extraction was shown in Figure 4The extraction yields continued to rise when the concen-tration increased from 01 to 05molsdotLminus1 and then differentdegrees of decrease appeared with the further increase of theconcentration This was because the solubility of the ionicliquid solution increased with the increasing concentrationbut the diffusion and transfer capability of extraction solventdecreased which led to declining of the solventrsquos penetrationpower into the interior of sample matrix [27 28]

313 Particle Size The effect of the particle sizes of sampleon extraction efficiency was investigated In Figure 5 thehighest extraction yields of isoquercitrin astragalin in leavesquercetin and kaempferol in flowers were achieved at 24 and

Journal of Chemistry 5

IsoquercitrinAstragalin

02 04 06 08 10 1200

Concentration (molmiddotLminus1)

050

055

060

065

070

075

080

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(a)

QuercetinKaempferol

02 04 06 08 10 1200

Concentration (molmiddotLminus1)

0020

0025

0030

0035

0040

0045

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(b)

Figure 4 Effect of the concentration of IL on extraction yields Type of IL [BMIM]BF4 the particle size 50 mesh solid-liquid ratio1 10 gsdotmLminus1 ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

Isoquercitrin Astragalin03

04

05

06

07

08

09

10

Extr

actio

n yi

eld

(mgmiddot

gminus1)

10 mesh24 mesh40mesh

50mesh70mesh90mesh

(a)

Quercetin Kaempferol001

002

003

004

005

006

Extr

actio

n yi

eld

(mgmiddot

gminus1)

10 mesh24 mesh40mesh

50mesh70mesh90mesh

(b)

Figure 5 Effect of the particle size on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 solid-liquid ratio 1 10 gsdotmLminus1ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

40mesh respectively It might be the smaller the particle sizethe easier the extraction but the samples of leaves and flowerscan easily be conglomerated by ionic liquid when the particlesize was too small [29] In addition the different optimumparticle sizes of leaves and flowers might be caused by thedifference of organizational structures

314 Solid-Liquid Ratio Six different ratios of solid-liquid(1 5 1 10 1 20 1 30 1 50 and 1 80 gsdotmLminus1) were chosen toevaluate the effect of solid-liquid ratio on the extraction yieldThe results in Figure 6 indicated that themaximumextractionrates of isoquercitrin astragalin in leaves quercetin andkaempferol in flowers were obtained at 1 10 and 1 20 gsdotmLminus1

respectively Results showed that the target analytes in thesample had basically been extracted When the proportionof extraction agent was increased the extraction efficiencieswere constant or decreased slightly this finding might berelated to the viscosity of ionic liquid

315 Ultrasound Time To investigate the influence of ultra-sound time on the extraction efficiency six time pointsranging from 10 to 60min were chosen In Figure 7 themaximum extraction efficiencies of isoquercitrin astragalinin leaves quercetin and kaempferol in flowers were obtainedat 20 and 45min respectively Nevertheless the ultrasoundtime was further extended the extraction yields of target

6 Journal of Chemistry

IsoquercitrinAstragalin

1 101 5 1 30 1 50 1 801 20Solid-liquid ratio (gmiddotmL

minus1)

05

06

07

08

09

10Ex

trac

tion

yiel

d (m

gmiddotgminus

1)

(a)

QuercetinKaempferol

Extr

actio

n yi

eld

(mgmiddot

gminus1)

1 101 5 1 30 1 50 1 801 20Solid-liquid ratio (gmiddotmL

minus1)

002

003

004

005

006

007

(b)

Figure 6 Effect of solid-liquid ratio on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

IsoquercitrinAstragalin

10 20 30 45 605

Ultrasound time (min)

07

08

09

10

11

12

13

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(a)

QuercetinKaempferol

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

30 45 6010 205

Ultrasound time (min)

(b)

Figure 7 Effect of ultrasound time on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound power 500W centrifugation speed 5000 rsdotminminus1

analytes had a certain decrease the reason for the abovephenomenon may be the fact that abundant sonication heatdestroys the structure of the analytes [30]

316 Ultrasonic Power The ultrasonic power had a sig-nificant effect on the extraction of the analytes In ourstudy the effect of different ultrasonic powers on extractionefficiency was investigated from 200 to 500W (in Figure 8)Results showed that the extraction efficiencies were increasedwith the increase of ultrasonic power Therefore 500W waschosen as ultrasonic power in this study

317 Centrifugation Speed The influence of centrifugationspeed on the extraction yield of the target analyte was

investigated The centrifugation time was set at 5min whichwas a reasonable time for productivity of the method [31]In Figure 9 results showed that the maximum extrac-tion yields of isoquercitrin astragalin in leaves quercetinand kaempferol in flowers were obtained at 4000 and5000 rsdotminminus1 respectively The reason might be that thehigher centrifugal speed affects the solubility of the analytesso that the solubility of the analytes is reduced

32 Method Validation

321 System Suitability Test (SST) System suitability test(SST) of HPLC includes the theoretical plate number (n) theresolution (Rs) the tailing factor (T) and the repeatability

Journal of Chemistry 7

IsoquercitrinAstragalin

500400300200

Ultrasound power (W)

07

08

09

10

11

12

13Ex

trac

tion

yiel

d (m

gmiddotgminus

1)

(a)

QuercetinKaempferol

002

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

300 400 500200

Ultrasound power (W)

(b)

Figure 8 Effect of ultrasound power on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound time 20min (a) and 45min (b) centrifugation speed5000 rsdotminminus1

IsoquercitrinAstragalin

4000 5000 6000 7000 80003000

Centrifugation speed (rmiddotminminus1)

04

06

08

10

12

14

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(a)

QuercetinKaempferol

002

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

4000 5000 6000 7000 80003000

Centrifugation speed (rmiddotminminus1)

(b)

Figure 9 Effect of centrifugation speed on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh(a) and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound time 20min (a) and 45min (b) ultrasound power500W

The repeatability was represented by the relative standarddeviation of the peak area (RSD) The results of SST wereshown in Table 1 which shows that the chromatographicsystems had good suitability

322 Linearity The stock standard solutions a and b wereaccurately injected 05 1 2 4 6 8 10 and 12 120583L to chro-matographic instrument for the construction of calibrationcurves respectively The calibration curves were constructedby plotting the peak areas versus the injection volume of eachcompoundThe results were demonstrated in Table 2 119903 valueswere in the range from 09996 to 10000 which indicated thatthe methods had good linearity

323 Precision The stock standard solutions a and b wereaccurately injected 10 120583L to chromatographic instrumentrespectively And they were analyzed in six replicates withinone day for determining the precision of the developedassay The RSDs of peak areas for isoquercitrin astragalinquercetin and kaempferol were 046 083 023 and024 respectively The results indicated that the methodswere precise for quantitative analysis of isoquercitrin astra-galin quercetin and kaempferol

324 Stability The sample solutions were prepared underthe optimum extraction conditions and placed at roomtemperature and thenwere injected 10120583L to chromatographic

8 Journal of Chemistry

Table 1 System suitability test (SST) of HPLC

Compound Theoretical plate number Resolution Tailing factor RepeatabilityIsoquercitrin 8392 152 131 087Astragalin 8834 1095 118 064Quercetin 7071 550 125 113Kaempferol 13956 854 135 102

Table 2 Regression equations and linear ranges for four compounds

Compound Regressive equation 119903 Linear range120583gIsoquercitrin 119884 = 319 times 106119883 minus 292 times 104 09996 0068sim164Astragalin 119884 = 300 times 106119883 minus 232 times 104 09997 0060sim144Quercetin 119884 = 742 times 106119883 minus 958 times 103 10000 00080sim019Kaempferol 119884 = 890 times 106119883 minus 149 times 104 10000 00077sim018119884 peak area119883 injection amount (120583g)

Table 3 Antioxidant activity of the different extracts of L clethroides

Sample DPPH ABTS FRAPRadical scavenging rate IC50120583gsdotmLminus1 Radical scavenging rate IC50120583gsdotmLminus1 TEAC120583molsdotgminus1

Leaf rsquos methanol extract 9391 1403 plusmn 80 9701 144 plusmn 5 3858 plusmn 072Leaf rsquos ionic liquid extract 8848 638 plusmn 60 9323 50 plusmn 2 10579 plusmn 702Flowerrsquos methanol extract 9392 1004 plusmn 12 9804 151 plusmn 1 5069 plusmn 110Flowerrsquos ionic liquid extract 8977 460 plusmn 36 9636 51 plusmn 2 10583 plusmn 125[BMIM]BF4 minus443 NT 003 NT NTBHA 7600 15 plusmn 146 9994 206 plusmn 008 591911 plusmn 1410BHT 7306 12 plusmn 048 9942 656 plusmn 010 58920 plusmn 5920PG 9561 401 plusmn 008 9580 069 plusmn 005 944133 plusmn 6927Note NT means not available because of low activity BHA BHT and PG are positive controls

instrument at 0 3 6 9 12 and 24 h respectively The RSDsof peak areas for isoquercitrin astragalin quercetin andkaempferol were 067 083 165 and 174 The resultsindicated that the sample solutionwas basically stable at roomtemperature within 24 h

325 Repeatability Six test sample solutions a and b wereprocessed under the optimumextraction conditions and theninjected 10 120583L to chromatographic instrument for analysisThe RSDs of peak areas for the four compounds were203 184 115 and 140 indicating that the analyticalmethods had an acceptable level of repeatability

326 Recovery Nine batches of leaves and flowers samplesof L clethroideswere prepared and divided into three groupsrespectively Then the four standard substances at threedifferent amounts were added to the leaves and flowerssamples The spiked samples were prepared according to theoptimum extraction conditions All the calculated recoveryvalues of the analytes ranged from 9839 to 10068 and theRSDs were less than 220The results demonstrated that themethods were reasonable and feasible

33 Antioxidant Activity of the Different Extracts of Lclethroides In Vitro The antioxidant activities of methanol

and ionic liquid sample solutions were evaluated by theDPPH ABTS and FRAP assay comprehensively and theresults were shown in Table 3 Results showed that the ionicliquid extract had stronger antioxidant activity than thatof the methanol extract and the ionic liquid itself had noantioxidant activity Results indicated that the ionic liquidused as extraction solvent hadmore advantages for extractingingredients

34 Sample Analysis Ionic liquid was successfully appliedfor the determination of isoquercitrin astragalin in leavesquercetin and kaempferol in flowers of L clethroides bythe optimized conditions respectively The mass fractions ofisoquercitrin astragalin in leaves quercetin and kaempferolin flowers were 1262 1219 0061 and 0046mgsdotgminus1 (119899 = 3)respectively

4 Conclusion

Themethod ionic liquid-based ultrasonic-assisted extractionhad been developed for the extraction of isoquercitrinastragalin in leaves quercetin and kaempferol in flowers of Lclethroides Compared with the conventional extraction withmethanol the proposed approaches provided higher extrac-tion efficiencies Results demonstrated that the methods

Journal of Chemistry 9

were simple rapid green and effective ILs can be recycledby some methods such as vacuum distillation membranefiltration salting out and liquid-liquid extraction [32] Con-sidering the unique properties of ILs the methods have agreat promising prospect in sample preparation of Chineseherbal medicine

Conflicts of Interest

The authors declare that there are no conflicts of interest

Authorsrsquo Contributions

Wen-yi Kang and Jin-feng Wei conceived the research ideaZhi-juan Zhang and Li-li Cui conducted the experimentscollected the plant specimens analyzed and interpreted thedata and prepared the first draft Wen-yi Kang Zhi-juanZhang and Jin-fengWei critically read and revised the paperAll the authors read and approved the paper before its finalsubmission

Acknowledgments

This work was supported by Henan Province UniversityScience and Technology Innovation Team (16IRTSTHN019)Science and Technology Innovation Team of Kaifeng City(2016-124) Kaifeng City Science and Technology InnovationTalent (1509010) and Key project in Science and TechnologyAgency of Kaifeng (1603111)

References

[1] X M Hu W K Zhang and Q S Zhu The Chinese MateriaMedica Shanghai Shanghai Science and Technology Press1999

[2] Z-J Zhang Z-Y Xia J-MWang X-T Song J-FWei andW-Y Kang ldquoEffects of flavonoids in Lysimachia clethroides dubyon the activities of cytochrome P450 CYP2E1 and CYP3A4 inrat liver microsomesrdquo Molecules vol 21 no 6 article no 7382016

[3] L H Tang X Y Xu B G You W Zhang and Y Q WangldquoAnti-tumor effect and its mechanism of total flavones ofLysimachia clethroides Dubyrdquo Shanghai Journal of TraditionalChinese Medicine vol 41 no 5 pp 74ndash76 2007

[4] Q-M Xu Y-L Liu Y-L Feng L-H Tang and S-L YangldquoA new e-ring 120574-lactone pentacyclic triterpene from lysimachiaclethroides and its cytotoxic activitiesrdquo Chemistry of NaturalCompounds vol 48 no 4 pp 597ndash600 2012

[5] C F Li Y L Song Y X Liu and W Y Kang ldquoAntioxidantactivity of extracts from Lysimachia clethroidesrdquo Fine Chemicalsvol 25 pp 1191ndash1193 2008

[6] J F Wei ldquoStudy on Chemical Constitutens Hepatoprotectiveand Antihyperglycemic Effect of Lysimachia paridiformis varstenophylla and Lysimachia clethroidesrdquo MSD thesis 2012Henan University Kaifeng

[7] D Liang ldquoStudy on the chemical composition and biologicalactivity of Lysimachia clethroides and chemical conversion ofsyntheticrdquo MSD thesis 2012 Peking Union Medical CollegeBeijing

[8] S M Yue B Q Chen P F Yuan W H Cui and W YKang ldquoChemical Constituents of Lysimachia clethroides DubyrdquoChinese Phramaceutical Journal vol 46 pp 341ndash343 2011

[9] L F Ding Y D Guo X D Wu and Y H Ma ldquoChemicalconstituents of flavonoids in Lysimachia clethroidesrdquo ChineseTraditional Patent Medicine vol 32 no 05 pp 827ndash830 2010

[10] Q M Xu L H Tang X Li L L Hao X R Li and S LYang ldquoIsolation and Identification of 3-O-p-CoumaroyloxylPentacyclic Triterpenoids from Lysimachia clethroides DubyrdquoChin Pharm J vol 45 pp 825ndash828 2010

[11] H Y Zou and P F Tu ldquoStudies on the chemical constituents ofLysimachia clethroides Dubyrdquo Chin Tradit Herb Drugs vol 40pp 704ndash708 2009

[12] L H Tang X Y Xu B G You Y Q Wang and W ZhangldquoEffect of flavones from Lysimachia clethroides on gene expre-sion of HL-60 cellrdquo Pharm Clin Res vol 15 pp 21ndash24 2007

[13] Y Q Wang L H Tang Z Q Liang and Z L Gu ldquoPrimarystudies on the anti-uterine cervix cancer effects of the extractZE4 from Lysimachia clethroides Dubyrdquo Chin Pharmacol Bullvol 23 pp 925ndash929 2007

[14] A A Aerov A R Khokhlov and I I Potemkin ldquoWhy ionicliquids can possess extra solvent powerrdquo Journal of PhysicalChemistry B vol 110 no 33 pp 16205ndash16207 2006

[15] J F Wei P R Cao J M Wang and W Y Kang ldquoAnalysis oftilianin and acacetin in Agastache rugosa by high-performanceliquid chromatography with ionic liquids-ultrasound basedextractionrdquo Chemistry Central Journal vol 10 p 76 2016

[16] W Y Ma Y B Lu R L Hu J H Chen Z Z Zhang and YJ Pan ldquoApplication of ionic liquids based microwave-assistedextraction of three alkaloids N-nornuciferine O-nornuciferineand nuciferine from lotus leafrdquo Talanta vol 80 no 3 pp 1292ndash1297 2010

[17] W Liu D D Li H S Yang et al ldquoDetermination of oleanic acidand paeoniflorin in Paeonia lactiflora by ultrasound-assistedionic liquid-reversed phase liquid chromatographyrdquo China JChin Mater Med vol 40 pp 443ndash449 2015

[18] J F Wei Z J Zhang D D Li W Liu andW Y Kang ldquoAnalysisof four flavonoids in Lysimachia clethroides using ionic liquid-assisted extractionrdquo China J Chin Mater Med vol 40 pp 1305ndash1310 2015

[19] J F Wei Z H Yin and F D Shang ldquoVolatiles in the Lysimachiaclethroides Duby by head space solid phase microextractioncoupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS)rdquo African Journal of Pharmacy and Pharmacol-ogy vol 6 pp 2484ndash2487 2012

[20] J F Wei Y Y Li Z H Yin F Gong and F D ShangldquoAntioxidant activities in vitroand hepatoprotective effects ofLysimachia clethroidesDuby on CCl4-induced acute liver injuryin micerdquo Afr J Pharm Pharmacol vol 6 pp 743ndash750 2012

[21] J F Wei X Chang W Wang and W Y Kang ldquoChemicalconstituents from Lysimachia circaeoidesrdquo Journal of ChineseMedicine Material vol 36 pp 1441ndash1443 2013

[22] W-Y Kang Y-L Song and L Zhang ldquo120572-Glucosidaseinhibitory and antioxidant properties and antidiabetic activityof Hypericum ascyron Lrdquo Medicinal Chemistry Research vol20 no 7 pp 809ndash816 2011

[23] W-Y Kang C-F Li and Y-X Liu ldquoAntioxidant phenoliccompounds and flavonoids of Mitragyna rotundifolia (Roxb)Kuntze in vitrordquo Medicinal Chemistry Research vol 19 no 9pp 1222ndash1232 2010

10 Journal of Chemistry

[24] W Kang and J Wang ldquoIn vitro antioxidant properties andin vivo lowering blood lipid of Forsythia suspense leavesrdquoMedicinal Chemistry Research vol 19 no 7 pp 617ndash628 2010

[25] C F Poole and S K Poole ldquoExtraction of organic compoundswith room temperature ionic liquidsrdquo Journal of Chromatogra-phy A vol 1217 no 16 pp 2268ndash2286 2010

[26] Z Guo B Lue K Thomasen A S Meyer and X Xu ldquoPre-dictions of flavonoid solubility in ionic liquids by COSMO-RSexperimental verification structural elucidation and solvationcharacterizationrdquo Green Chemistry vol 9 no 12 pp 1362ndash13732007

[27] S Y Dong Q Hu J Li Y L Fu Y Q Xing and T L HuangldquoIonic liquid-based microwave-assisted extraction followedhigh-performance liquid chromatography for the simultaneousdetermination of five organic oxygen pesticides in soil samplesrdquoChinese Journal of Analysis Laboratory vol 32 pp 48ndash52 2013

[28] H Wu M Chen Y Fan F Elsebaei and Y Zhu ldquoDeter-mination of rutin and quercetin in Chinese herbal medicineby ionic liquid-based pressurized liquid extraction-liquidchromatography- chemiluminescence detectionrdquo Talanta vol88 pp 222ndash229 2012

[29] R Jin L Fan and X An ldquoIonic liquid-assisted extraction ofpaeonol from cynanchum paniculatumrdquo Chromatographia vol73 no 7-8 pp 787ndash792 2011

[30] P Liang F Wang and Q Wan ldquoIonic liquid-based ultrasound-assisted emulsification microextraction coupled with high per-formance liquid chromatography for the determination of fourfungicides in environmental water samplesrdquo Talanta vol 105pp 57ndash62 2013

[31] C K Zacharis C Christophoridis and K Fytianos ldquoVortex-assisted liquid-liquid microextraction combined with gaschromatography-mass spectrometry for the determination oforganophosphate pesticides in environmental water samplesand winesrdquo Journal of Separation Science vol 35 no 18 pp2422ndash2429 2012

[32] H Liu X Y Wei J H Li T Li and F Wang ldquoReview of ionicliquids recyclingrdquo Journal of Cellulose Science Technology vol21 pp 63ndash69 2013

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

International Journal ofInternational Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal ofInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 5

IsoquercitrinAstragalin

02 04 06 08 10 1200

Concentration (molmiddotLminus1)

050

055

060

065

070

075

080

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(a)

QuercetinKaempferol

02 04 06 08 10 1200

Concentration (molmiddotLminus1)

0020

0025

0030

0035

0040

0045

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(b)

Figure 4 Effect of the concentration of IL on extraction yields Type of IL [BMIM]BF4 the particle size 50 mesh solid-liquid ratio1 10 gsdotmLminus1 ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

Isoquercitrin Astragalin03

04

05

06

07

08

09

10

Extr

actio

n yi

eld

(mgmiddot

gminus1)

10 mesh24 mesh40mesh

50mesh70mesh90mesh

(a)

Quercetin Kaempferol001

002

003

004

005

006

Extr

actio

n yi

eld

(mgmiddot

gminus1)

10 mesh24 mesh40mesh

50mesh70mesh90mesh

(b)

Figure 5 Effect of the particle size on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 solid-liquid ratio 1 10 gsdotmLminus1ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

40mesh respectively It might be the smaller the particle sizethe easier the extraction but the samples of leaves and flowerscan easily be conglomerated by ionic liquid when the particlesize was too small [29] In addition the different optimumparticle sizes of leaves and flowers might be caused by thedifference of organizational structures

314 Solid-Liquid Ratio Six different ratios of solid-liquid(1 5 1 10 1 20 1 30 1 50 and 1 80 gsdotmLminus1) were chosen toevaluate the effect of solid-liquid ratio on the extraction yieldThe results in Figure 6 indicated that themaximumextractionrates of isoquercitrin astragalin in leaves quercetin andkaempferol in flowers were obtained at 1 10 and 1 20 gsdotmLminus1

respectively Results showed that the target analytes in thesample had basically been extracted When the proportionof extraction agent was increased the extraction efficiencieswere constant or decreased slightly this finding might berelated to the viscosity of ionic liquid

315 Ultrasound Time To investigate the influence of ultra-sound time on the extraction efficiency six time pointsranging from 10 to 60min were chosen In Figure 7 themaximum extraction efficiencies of isoquercitrin astragalinin leaves quercetin and kaempferol in flowers were obtainedat 20 and 45min respectively Nevertheless the ultrasoundtime was further extended the extraction yields of target

6 Journal of Chemistry

IsoquercitrinAstragalin

1 101 5 1 30 1 50 1 801 20Solid-liquid ratio (gmiddotmL

minus1)

05

06

07

08

09

10Ex

trac

tion

yiel

d (m

gmiddotgminus

1)

(a)

QuercetinKaempferol

Extr

actio

n yi

eld

(mgmiddot

gminus1)

1 101 5 1 30 1 50 1 801 20Solid-liquid ratio (gmiddotmL

minus1)

002

003

004

005

006

007

(b)

Figure 6 Effect of solid-liquid ratio on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

IsoquercitrinAstragalin

10 20 30 45 605

Ultrasound time (min)

07

08

09

10

11

12

13

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(a)

QuercetinKaempferol

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

30 45 6010 205

Ultrasound time (min)

(b)

Figure 7 Effect of ultrasound time on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound power 500W centrifugation speed 5000 rsdotminminus1

analytes had a certain decrease the reason for the abovephenomenon may be the fact that abundant sonication heatdestroys the structure of the analytes [30]

316 Ultrasonic Power The ultrasonic power had a sig-nificant effect on the extraction of the analytes In ourstudy the effect of different ultrasonic powers on extractionefficiency was investigated from 200 to 500W (in Figure 8)Results showed that the extraction efficiencies were increasedwith the increase of ultrasonic power Therefore 500W waschosen as ultrasonic power in this study

317 Centrifugation Speed The influence of centrifugationspeed on the extraction yield of the target analyte was

investigated The centrifugation time was set at 5min whichwas a reasonable time for productivity of the method [31]In Figure 9 results showed that the maximum extrac-tion yields of isoquercitrin astragalin in leaves quercetinand kaempferol in flowers were obtained at 4000 and5000 rsdotminminus1 respectively The reason might be that thehigher centrifugal speed affects the solubility of the analytesso that the solubility of the analytes is reduced

32 Method Validation

321 System Suitability Test (SST) System suitability test(SST) of HPLC includes the theoretical plate number (n) theresolution (Rs) the tailing factor (T) and the repeatability

Journal of Chemistry 7

IsoquercitrinAstragalin

500400300200

Ultrasound power (W)

07

08

09

10

11

12

13Ex

trac

tion

yiel

d (m

gmiddotgminus

1)

(a)

QuercetinKaempferol

002

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

300 400 500200

Ultrasound power (W)

(b)

Figure 8 Effect of ultrasound power on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound time 20min (a) and 45min (b) centrifugation speed5000 rsdotminminus1

IsoquercitrinAstragalin

4000 5000 6000 7000 80003000

Centrifugation speed (rmiddotminminus1)

04

06

08

10

12

14

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(a)

QuercetinKaempferol

002

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

4000 5000 6000 7000 80003000

Centrifugation speed (rmiddotminminus1)

(b)

Figure 9 Effect of centrifugation speed on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh(a) and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound time 20min (a) and 45min (b) ultrasound power500W

The repeatability was represented by the relative standarddeviation of the peak area (RSD) The results of SST wereshown in Table 1 which shows that the chromatographicsystems had good suitability

322 Linearity The stock standard solutions a and b wereaccurately injected 05 1 2 4 6 8 10 and 12 120583L to chro-matographic instrument for the construction of calibrationcurves respectively The calibration curves were constructedby plotting the peak areas versus the injection volume of eachcompoundThe results were demonstrated in Table 2 119903 valueswere in the range from 09996 to 10000 which indicated thatthe methods had good linearity

323 Precision The stock standard solutions a and b wereaccurately injected 10 120583L to chromatographic instrumentrespectively And they were analyzed in six replicates withinone day for determining the precision of the developedassay The RSDs of peak areas for isoquercitrin astragalinquercetin and kaempferol were 046 083 023 and024 respectively The results indicated that the methodswere precise for quantitative analysis of isoquercitrin astra-galin quercetin and kaempferol

324 Stability The sample solutions were prepared underthe optimum extraction conditions and placed at roomtemperature and thenwere injected 10120583L to chromatographic

8 Journal of Chemistry

Table 1 System suitability test (SST) of HPLC

Compound Theoretical plate number Resolution Tailing factor RepeatabilityIsoquercitrin 8392 152 131 087Astragalin 8834 1095 118 064Quercetin 7071 550 125 113Kaempferol 13956 854 135 102

Table 2 Regression equations and linear ranges for four compounds

Compound Regressive equation 119903 Linear range120583gIsoquercitrin 119884 = 319 times 106119883 minus 292 times 104 09996 0068sim164Astragalin 119884 = 300 times 106119883 minus 232 times 104 09997 0060sim144Quercetin 119884 = 742 times 106119883 minus 958 times 103 10000 00080sim019Kaempferol 119884 = 890 times 106119883 minus 149 times 104 10000 00077sim018119884 peak area119883 injection amount (120583g)

Table 3 Antioxidant activity of the different extracts of L clethroides

Sample DPPH ABTS FRAPRadical scavenging rate IC50120583gsdotmLminus1 Radical scavenging rate IC50120583gsdotmLminus1 TEAC120583molsdotgminus1

Leaf rsquos methanol extract 9391 1403 plusmn 80 9701 144 plusmn 5 3858 plusmn 072Leaf rsquos ionic liquid extract 8848 638 plusmn 60 9323 50 plusmn 2 10579 plusmn 702Flowerrsquos methanol extract 9392 1004 plusmn 12 9804 151 plusmn 1 5069 plusmn 110Flowerrsquos ionic liquid extract 8977 460 plusmn 36 9636 51 plusmn 2 10583 plusmn 125[BMIM]BF4 minus443 NT 003 NT NTBHA 7600 15 plusmn 146 9994 206 plusmn 008 591911 plusmn 1410BHT 7306 12 plusmn 048 9942 656 plusmn 010 58920 plusmn 5920PG 9561 401 plusmn 008 9580 069 plusmn 005 944133 plusmn 6927Note NT means not available because of low activity BHA BHT and PG are positive controls

instrument at 0 3 6 9 12 and 24 h respectively The RSDsof peak areas for isoquercitrin astragalin quercetin andkaempferol were 067 083 165 and 174 The resultsindicated that the sample solutionwas basically stable at roomtemperature within 24 h

325 Repeatability Six test sample solutions a and b wereprocessed under the optimumextraction conditions and theninjected 10 120583L to chromatographic instrument for analysisThe RSDs of peak areas for the four compounds were203 184 115 and 140 indicating that the analyticalmethods had an acceptable level of repeatability

326 Recovery Nine batches of leaves and flowers samplesof L clethroideswere prepared and divided into three groupsrespectively Then the four standard substances at threedifferent amounts were added to the leaves and flowerssamples The spiked samples were prepared according to theoptimum extraction conditions All the calculated recoveryvalues of the analytes ranged from 9839 to 10068 and theRSDs were less than 220The results demonstrated that themethods were reasonable and feasible

33 Antioxidant Activity of the Different Extracts of Lclethroides In Vitro The antioxidant activities of methanol

and ionic liquid sample solutions were evaluated by theDPPH ABTS and FRAP assay comprehensively and theresults were shown in Table 3 Results showed that the ionicliquid extract had stronger antioxidant activity than thatof the methanol extract and the ionic liquid itself had noantioxidant activity Results indicated that the ionic liquidused as extraction solvent hadmore advantages for extractingingredients

34 Sample Analysis Ionic liquid was successfully appliedfor the determination of isoquercitrin astragalin in leavesquercetin and kaempferol in flowers of L clethroides bythe optimized conditions respectively The mass fractions ofisoquercitrin astragalin in leaves quercetin and kaempferolin flowers were 1262 1219 0061 and 0046mgsdotgminus1 (119899 = 3)respectively

4 Conclusion

Themethod ionic liquid-based ultrasonic-assisted extractionhad been developed for the extraction of isoquercitrinastragalin in leaves quercetin and kaempferol in flowers of Lclethroides Compared with the conventional extraction withmethanol the proposed approaches provided higher extrac-tion efficiencies Results demonstrated that the methods

Journal of Chemistry 9

were simple rapid green and effective ILs can be recycledby some methods such as vacuum distillation membranefiltration salting out and liquid-liquid extraction [32] Con-sidering the unique properties of ILs the methods have agreat promising prospect in sample preparation of Chineseherbal medicine

Conflicts of Interest

The authors declare that there are no conflicts of interest

Authorsrsquo Contributions

Wen-yi Kang and Jin-feng Wei conceived the research ideaZhi-juan Zhang and Li-li Cui conducted the experimentscollected the plant specimens analyzed and interpreted thedata and prepared the first draft Wen-yi Kang Zhi-juanZhang and Jin-fengWei critically read and revised the paperAll the authors read and approved the paper before its finalsubmission

Acknowledgments

This work was supported by Henan Province UniversityScience and Technology Innovation Team (16IRTSTHN019)Science and Technology Innovation Team of Kaifeng City(2016-124) Kaifeng City Science and Technology InnovationTalent (1509010) and Key project in Science and TechnologyAgency of Kaifeng (1603111)

References

[1] X M Hu W K Zhang and Q S Zhu The Chinese MateriaMedica Shanghai Shanghai Science and Technology Press1999

[2] Z-J Zhang Z-Y Xia J-MWang X-T Song J-FWei andW-Y Kang ldquoEffects of flavonoids in Lysimachia clethroides dubyon the activities of cytochrome P450 CYP2E1 and CYP3A4 inrat liver microsomesrdquo Molecules vol 21 no 6 article no 7382016

[3] L H Tang X Y Xu B G You W Zhang and Y Q WangldquoAnti-tumor effect and its mechanism of total flavones ofLysimachia clethroides Dubyrdquo Shanghai Journal of TraditionalChinese Medicine vol 41 no 5 pp 74ndash76 2007

[4] Q-M Xu Y-L Liu Y-L Feng L-H Tang and S-L YangldquoA new e-ring 120574-lactone pentacyclic triterpene from lysimachiaclethroides and its cytotoxic activitiesrdquo Chemistry of NaturalCompounds vol 48 no 4 pp 597ndash600 2012

[5] C F Li Y L Song Y X Liu and W Y Kang ldquoAntioxidantactivity of extracts from Lysimachia clethroidesrdquo Fine Chemicalsvol 25 pp 1191ndash1193 2008

[6] J F Wei ldquoStudy on Chemical Constitutens Hepatoprotectiveand Antihyperglycemic Effect of Lysimachia paridiformis varstenophylla and Lysimachia clethroidesrdquo MSD thesis 2012Henan University Kaifeng

[7] D Liang ldquoStudy on the chemical composition and biologicalactivity of Lysimachia clethroides and chemical conversion ofsyntheticrdquo MSD thesis 2012 Peking Union Medical CollegeBeijing

[8] S M Yue B Q Chen P F Yuan W H Cui and W YKang ldquoChemical Constituents of Lysimachia clethroides DubyrdquoChinese Phramaceutical Journal vol 46 pp 341ndash343 2011

[9] L F Ding Y D Guo X D Wu and Y H Ma ldquoChemicalconstituents of flavonoids in Lysimachia clethroidesrdquo ChineseTraditional Patent Medicine vol 32 no 05 pp 827ndash830 2010

[10] Q M Xu L H Tang X Li L L Hao X R Li and S LYang ldquoIsolation and Identification of 3-O-p-CoumaroyloxylPentacyclic Triterpenoids from Lysimachia clethroides DubyrdquoChin Pharm J vol 45 pp 825ndash828 2010

[11] H Y Zou and P F Tu ldquoStudies on the chemical constituents ofLysimachia clethroides Dubyrdquo Chin Tradit Herb Drugs vol 40pp 704ndash708 2009

[12] L H Tang X Y Xu B G You Y Q Wang and W ZhangldquoEffect of flavones from Lysimachia clethroides on gene expre-sion of HL-60 cellrdquo Pharm Clin Res vol 15 pp 21ndash24 2007

[13] Y Q Wang L H Tang Z Q Liang and Z L Gu ldquoPrimarystudies on the anti-uterine cervix cancer effects of the extractZE4 from Lysimachia clethroides Dubyrdquo Chin Pharmacol Bullvol 23 pp 925ndash929 2007

[14] A A Aerov A R Khokhlov and I I Potemkin ldquoWhy ionicliquids can possess extra solvent powerrdquo Journal of PhysicalChemistry B vol 110 no 33 pp 16205ndash16207 2006

[15] J F Wei P R Cao J M Wang and W Y Kang ldquoAnalysis oftilianin and acacetin in Agastache rugosa by high-performanceliquid chromatography with ionic liquids-ultrasound basedextractionrdquo Chemistry Central Journal vol 10 p 76 2016

[16] W Y Ma Y B Lu R L Hu J H Chen Z Z Zhang and YJ Pan ldquoApplication of ionic liquids based microwave-assistedextraction of three alkaloids N-nornuciferine O-nornuciferineand nuciferine from lotus leafrdquo Talanta vol 80 no 3 pp 1292ndash1297 2010

[17] W Liu D D Li H S Yang et al ldquoDetermination of oleanic acidand paeoniflorin in Paeonia lactiflora by ultrasound-assistedionic liquid-reversed phase liquid chromatographyrdquo China JChin Mater Med vol 40 pp 443ndash449 2015

[18] J F Wei Z J Zhang D D Li W Liu andW Y Kang ldquoAnalysisof four flavonoids in Lysimachia clethroides using ionic liquid-assisted extractionrdquo China J Chin Mater Med vol 40 pp 1305ndash1310 2015

[19] J F Wei Z H Yin and F D Shang ldquoVolatiles in the Lysimachiaclethroides Duby by head space solid phase microextractioncoupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS)rdquo African Journal of Pharmacy and Pharmacol-ogy vol 6 pp 2484ndash2487 2012

[20] J F Wei Y Y Li Z H Yin F Gong and F D ShangldquoAntioxidant activities in vitroand hepatoprotective effects ofLysimachia clethroidesDuby on CCl4-induced acute liver injuryin micerdquo Afr J Pharm Pharmacol vol 6 pp 743ndash750 2012

[21] J F Wei X Chang W Wang and W Y Kang ldquoChemicalconstituents from Lysimachia circaeoidesrdquo Journal of ChineseMedicine Material vol 36 pp 1441ndash1443 2013

[22] W-Y Kang Y-L Song and L Zhang ldquo120572-Glucosidaseinhibitory and antioxidant properties and antidiabetic activityof Hypericum ascyron Lrdquo Medicinal Chemistry Research vol20 no 7 pp 809ndash816 2011

[23] W-Y Kang C-F Li and Y-X Liu ldquoAntioxidant phenoliccompounds and flavonoids of Mitragyna rotundifolia (Roxb)Kuntze in vitrordquo Medicinal Chemistry Research vol 19 no 9pp 1222ndash1232 2010

10 Journal of Chemistry

[24] W Kang and J Wang ldquoIn vitro antioxidant properties andin vivo lowering blood lipid of Forsythia suspense leavesrdquoMedicinal Chemistry Research vol 19 no 7 pp 617ndash628 2010

[25] C F Poole and S K Poole ldquoExtraction of organic compoundswith room temperature ionic liquidsrdquo Journal of Chromatogra-phy A vol 1217 no 16 pp 2268ndash2286 2010

[26] Z Guo B Lue K Thomasen A S Meyer and X Xu ldquoPre-dictions of flavonoid solubility in ionic liquids by COSMO-RSexperimental verification structural elucidation and solvationcharacterizationrdquo Green Chemistry vol 9 no 12 pp 1362ndash13732007

[27] S Y Dong Q Hu J Li Y L Fu Y Q Xing and T L HuangldquoIonic liquid-based microwave-assisted extraction followedhigh-performance liquid chromatography for the simultaneousdetermination of five organic oxygen pesticides in soil samplesrdquoChinese Journal of Analysis Laboratory vol 32 pp 48ndash52 2013

[28] H Wu M Chen Y Fan F Elsebaei and Y Zhu ldquoDeter-mination of rutin and quercetin in Chinese herbal medicineby ionic liquid-based pressurized liquid extraction-liquidchromatography- chemiluminescence detectionrdquo Talanta vol88 pp 222ndash229 2012

[29] R Jin L Fan and X An ldquoIonic liquid-assisted extraction ofpaeonol from cynanchum paniculatumrdquo Chromatographia vol73 no 7-8 pp 787ndash792 2011

[30] P Liang F Wang and Q Wan ldquoIonic liquid-based ultrasound-assisted emulsification microextraction coupled with high per-formance liquid chromatography for the determination of fourfungicides in environmental water samplesrdquo Talanta vol 105pp 57ndash62 2013

[31] C K Zacharis C Christophoridis and K Fytianos ldquoVortex-assisted liquid-liquid microextraction combined with gaschromatography-mass spectrometry for the determination oforganophosphate pesticides in environmental water samplesand winesrdquo Journal of Separation Science vol 35 no 18 pp2422ndash2429 2012

[32] H Liu X Y Wei J H Li T Li and F Wang ldquoReview of ionicliquids recyclingrdquo Journal of Cellulose Science Technology vol21 pp 63ndash69 2013

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

International Journal ofInternational Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal ofInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

6 Journal of Chemistry

IsoquercitrinAstragalin

1 101 5 1 30 1 50 1 801 20Solid-liquid ratio (gmiddotmL

minus1)

05

06

07

08

09

10Ex

trac

tion

yiel

d (m

gmiddotgminus

1)

(a)

QuercetinKaempferol

Extr

actio

n yi

eld

(mgmiddot

gminus1)

1 101 5 1 30 1 50 1 801 20Solid-liquid ratio (gmiddotmL

minus1)

002

003

004

005

006

007

(b)

Figure 6 Effect of solid-liquid ratio on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) ultrasound time 30min ultrasound power 500W centrifugation speed 5000 rsdotminminus1

IsoquercitrinAstragalin

10 20 30 45 605

Ultrasound time (min)

07

08

09

10

11

12

13

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(a)

QuercetinKaempferol

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

30 45 6010 205

Ultrasound time (min)

(b)

Figure 7 Effect of ultrasound time on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound power 500W centrifugation speed 5000 rsdotminminus1

analytes had a certain decrease the reason for the abovephenomenon may be the fact that abundant sonication heatdestroys the structure of the analytes [30]

316 Ultrasonic Power The ultrasonic power had a sig-nificant effect on the extraction of the analytes In ourstudy the effect of different ultrasonic powers on extractionefficiency was investigated from 200 to 500W (in Figure 8)Results showed that the extraction efficiencies were increasedwith the increase of ultrasonic power Therefore 500W waschosen as ultrasonic power in this study

317 Centrifugation Speed The influence of centrifugationspeed on the extraction yield of the target analyte was

investigated The centrifugation time was set at 5min whichwas a reasonable time for productivity of the method [31]In Figure 9 results showed that the maximum extrac-tion yields of isoquercitrin astragalin in leaves quercetinand kaempferol in flowers were obtained at 4000 and5000 rsdotminminus1 respectively The reason might be that thehigher centrifugal speed affects the solubility of the analytesso that the solubility of the analytes is reduced

32 Method Validation

321 System Suitability Test (SST) System suitability test(SST) of HPLC includes the theoretical plate number (n) theresolution (Rs) the tailing factor (T) and the repeatability

Journal of Chemistry 7

IsoquercitrinAstragalin

500400300200

Ultrasound power (W)

07

08

09

10

11

12

13Ex

trac

tion

yiel

d (m

gmiddotgminus

1)

(a)

QuercetinKaempferol

002

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

300 400 500200

Ultrasound power (W)

(b)

Figure 8 Effect of ultrasound power on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound time 20min (a) and 45min (b) centrifugation speed5000 rsdotminminus1

IsoquercitrinAstragalin

4000 5000 6000 7000 80003000

Centrifugation speed (rmiddotminminus1)

04

06

08

10

12

14

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(a)

QuercetinKaempferol

002

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

4000 5000 6000 7000 80003000

Centrifugation speed (rmiddotminminus1)

(b)

Figure 9 Effect of centrifugation speed on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh(a) and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound time 20min (a) and 45min (b) ultrasound power500W

The repeatability was represented by the relative standarddeviation of the peak area (RSD) The results of SST wereshown in Table 1 which shows that the chromatographicsystems had good suitability

322 Linearity The stock standard solutions a and b wereaccurately injected 05 1 2 4 6 8 10 and 12 120583L to chro-matographic instrument for the construction of calibrationcurves respectively The calibration curves were constructedby plotting the peak areas versus the injection volume of eachcompoundThe results were demonstrated in Table 2 119903 valueswere in the range from 09996 to 10000 which indicated thatthe methods had good linearity

323 Precision The stock standard solutions a and b wereaccurately injected 10 120583L to chromatographic instrumentrespectively And they were analyzed in six replicates withinone day for determining the precision of the developedassay The RSDs of peak areas for isoquercitrin astragalinquercetin and kaempferol were 046 083 023 and024 respectively The results indicated that the methodswere precise for quantitative analysis of isoquercitrin astra-galin quercetin and kaempferol

324 Stability The sample solutions were prepared underthe optimum extraction conditions and placed at roomtemperature and thenwere injected 10120583L to chromatographic

8 Journal of Chemistry

Table 1 System suitability test (SST) of HPLC

Compound Theoretical plate number Resolution Tailing factor RepeatabilityIsoquercitrin 8392 152 131 087Astragalin 8834 1095 118 064Quercetin 7071 550 125 113Kaempferol 13956 854 135 102

Table 2 Regression equations and linear ranges for four compounds

Compound Regressive equation 119903 Linear range120583gIsoquercitrin 119884 = 319 times 106119883 minus 292 times 104 09996 0068sim164Astragalin 119884 = 300 times 106119883 minus 232 times 104 09997 0060sim144Quercetin 119884 = 742 times 106119883 minus 958 times 103 10000 00080sim019Kaempferol 119884 = 890 times 106119883 minus 149 times 104 10000 00077sim018119884 peak area119883 injection amount (120583g)

Table 3 Antioxidant activity of the different extracts of L clethroides

Sample DPPH ABTS FRAPRadical scavenging rate IC50120583gsdotmLminus1 Radical scavenging rate IC50120583gsdotmLminus1 TEAC120583molsdotgminus1

Leaf rsquos methanol extract 9391 1403 plusmn 80 9701 144 plusmn 5 3858 plusmn 072Leaf rsquos ionic liquid extract 8848 638 plusmn 60 9323 50 plusmn 2 10579 plusmn 702Flowerrsquos methanol extract 9392 1004 plusmn 12 9804 151 plusmn 1 5069 plusmn 110Flowerrsquos ionic liquid extract 8977 460 plusmn 36 9636 51 plusmn 2 10583 plusmn 125[BMIM]BF4 minus443 NT 003 NT NTBHA 7600 15 plusmn 146 9994 206 plusmn 008 591911 plusmn 1410BHT 7306 12 plusmn 048 9942 656 plusmn 010 58920 plusmn 5920PG 9561 401 plusmn 008 9580 069 plusmn 005 944133 plusmn 6927Note NT means not available because of low activity BHA BHT and PG are positive controls

instrument at 0 3 6 9 12 and 24 h respectively The RSDsof peak areas for isoquercitrin astragalin quercetin andkaempferol were 067 083 165 and 174 The resultsindicated that the sample solutionwas basically stable at roomtemperature within 24 h

325 Repeatability Six test sample solutions a and b wereprocessed under the optimumextraction conditions and theninjected 10 120583L to chromatographic instrument for analysisThe RSDs of peak areas for the four compounds were203 184 115 and 140 indicating that the analyticalmethods had an acceptable level of repeatability

326 Recovery Nine batches of leaves and flowers samplesof L clethroideswere prepared and divided into three groupsrespectively Then the four standard substances at threedifferent amounts were added to the leaves and flowerssamples The spiked samples were prepared according to theoptimum extraction conditions All the calculated recoveryvalues of the analytes ranged from 9839 to 10068 and theRSDs were less than 220The results demonstrated that themethods were reasonable and feasible

33 Antioxidant Activity of the Different Extracts of Lclethroides In Vitro The antioxidant activities of methanol

and ionic liquid sample solutions were evaluated by theDPPH ABTS and FRAP assay comprehensively and theresults were shown in Table 3 Results showed that the ionicliquid extract had stronger antioxidant activity than thatof the methanol extract and the ionic liquid itself had noantioxidant activity Results indicated that the ionic liquidused as extraction solvent hadmore advantages for extractingingredients

34 Sample Analysis Ionic liquid was successfully appliedfor the determination of isoquercitrin astragalin in leavesquercetin and kaempferol in flowers of L clethroides bythe optimized conditions respectively The mass fractions ofisoquercitrin astragalin in leaves quercetin and kaempferolin flowers were 1262 1219 0061 and 0046mgsdotgminus1 (119899 = 3)respectively

4 Conclusion

Themethod ionic liquid-based ultrasonic-assisted extractionhad been developed for the extraction of isoquercitrinastragalin in leaves quercetin and kaempferol in flowers of Lclethroides Compared with the conventional extraction withmethanol the proposed approaches provided higher extrac-tion efficiencies Results demonstrated that the methods

Journal of Chemistry 9

were simple rapid green and effective ILs can be recycledby some methods such as vacuum distillation membranefiltration salting out and liquid-liquid extraction [32] Con-sidering the unique properties of ILs the methods have agreat promising prospect in sample preparation of Chineseherbal medicine

Conflicts of Interest

The authors declare that there are no conflicts of interest

Authorsrsquo Contributions

Wen-yi Kang and Jin-feng Wei conceived the research ideaZhi-juan Zhang and Li-li Cui conducted the experimentscollected the plant specimens analyzed and interpreted thedata and prepared the first draft Wen-yi Kang Zhi-juanZhang and Jin-fengWei critically read and revised the paperAll the authors read and approved the paper before its finalsubmission

Acknowledgments

This work was supported by Henan Province UniversityScience and Technology Innovation Team (16IRTSTHN019)Science and Technology Innovation Team of Kaifeng City(2016-124) Kaifeng City Science and Technology InnovationTalent (1509010) and Key project in Science and TechnologyAgency of Kaifeng (1603111)

References

[1] X M Hu W K Zhang and Q S Zhu The Chinese MateriaMedica Shanghai Shanghai Science and Technology Press1999

[2] Z-J Zhang Z-Y Xia J-MWang X-T Song J-FWei andW-Y Kang ldquoEffects of flavonoids in Lysimachia clethroides dubyon the activities of cytochrome P450 CYP2E1 and CYP3A4 inrat liver microsomesrdquo Molecules vol 21 no 6 article no 7382016

[3] L H Tang X Y Xu B G You W Zhang and Y Q WangldquoAnti-tumor effect and its mechanism of total flavones ofLysimachia clethroides Dubyrdquo Shanghai Journal of TraditionalChinese Medicine vol 41 no 5 pp 74ndash76 2007

[4] Q-M Xu Y-L Liu Y-L Feng L-H Tang and S-L YangldquoA new e-ring 120574-lactone pentacyclic triterpene from lysimachiaclethroides and its cytotoxic activitiesrdquo Chemistry of NaturalCompounds vol 48 no 4 pp 597ndash600 2012

[5] C F Li Y L Song Y X Liu and W Y Kang ldquoAntioxidantactivity of extracts from Lysimachia clethroidesrdquo Fine Chemicalsvol 25 pp 1191ndash1193 2008

[6] J F Wei ldquoStudy on Chemical Constitutens Hepatoprotectiveand Antihyperglycemic Effect of Lysimachia paridiformis varstenophylla and Lysimachia clethroidesrdquo MSD thesis 2012Henan University Kaifeng

[7] D Liang ldquoStudy on the chemical composition and biologicalactivity of Lysimachia clethroides and chemical conversion ofsyntheticrdquo MSD thesis 2012 Peking Union Medical CollegeBeijing

[8] S M Yue B Q Chen P F Yuan W H Cui and W YKang ldquoChemical Constituents of Lysimachia clethroides DubyrdquoChinese Phramaceutical Journal vol 46 pp 341ndash343 2011

[9] L F Ding Y D Guo X D Wu and Y H Ma ldquoChemicalconstituents of flavonoids in Lysimachia clethroidesrdquo ChineseTraditional Patent Medicine vol 32 no 05 pp 827ndash830 2010

[10] Q M Xu L H Tang X Li L L Hao X R Li and S LYang ldquoIsolation and Identification of 3-O-p-CoumaroyloxylPentacyclic Triterpenoids from Lysimachia clethroides DubyrdquoChin Pharm J vol 45 pp 825ndash828 2010

[11] H Y Zou and P F Tu ldquoStudies on the chemical constituents ofLysimachia clethroides Dubyrdquo Chin Tradit Herb Drugs vol 40pp 704ndash708 2009

[12] L H Tang X Y Xu B G You Y Q Wang and W ZhangldquoEffect of flavones from Lysimachia clethroides on gene expre-sion of HL-60 cellrdquo Pharm Clin Res vol 15 pp 21ndash24 2007

[13] Y Q Wang L H Tang Z Q Liang and Z L Gu ldquoPrimarystudies on the anti-uterine cervix cancer effects of the extractZE4 from Lysimachia clethroides Dubyrdquo Chin Pharmacol Bullvol 23 pp 925ndash929 2007

[14] A A Aerov A R Khokhlov and I I Potemkin ldquoWhy ionicliquids can possess extra solvent powerrdquo Journal of PhysicalChemistry B vol 110 no 33 pp 16205ndash16207 2006

[15] J F Wei P R Cao J M Wang and W Y Kang ldquoAnalysis oftilianin and acacetin in Agastache rugosa by high-performanceliquid chromatography with ionic liquids-ultrasound basedextractionrdquo Chemistry Central Journal vol 10 p 76 2016

[16] W Y Ma Y B Lu R L Hu J H Chen Z Z Zhang and YJ Pan ldquoApplication of ionic liquids based microwave-assistedextraction of three alkaloids N-nornuciferine O-nornuciferineand nuciferine from lotus leafrdquo Talanta vol 80 no 3 pp 1292ndash1297 2010

[17] W Liu D D Li H S Yang et al ldquoDetermination of oleanic acidand paeoniflorin in Paeonia lactiflora by ultrasound-assistedionic liquid-reversed phase liquid chromatographyrdquo China JChin Mater Med vol 40 pp 443ndash449 2015

[18] J F Wei Z J Zhang D D Li W Liu andW Y Kang ldquoAnalysisof four flavonoids in Lysimachia clethroides using ionic liquid-assisted extractionrdquo China J Chin Mater Med vol 40 pp 1305ndash1310 2015

[19] J F Wei Z H Yin and F D Shang ldquoVolatiles in the Lysimachiaclethroides Duby by head space solid phase microextractioncoupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS)rdquo African Journal of Pharmacy and Pharmacol-ogy vol 6 pp 2484ndash2487 2012

[20] J F Wei Y Y Li Z H Yin F Gong and F D ShangldquoAntioxidant activities in vitroand hepatoprotective effects ofLysimachia clethroidesDuby on CCl4-induced acute liver injuryin micerdquo Afr J Pharm Pharmacol vol 6 pp 743ndash750 2012

[21] J F Wei X Chang W Wang and W Y Kang ldquoChemicalconstituents from Lysimachia circaeoidesrdquo Journal of ChineseMedicine Material vol 36 pp 1441ndash1443 2013

[22] W-Y Kang Y-L Song and L Zhang ldquo120572-Glucosidaseinhibitory and antioxidant properties and antidiabetic activityof Hypericum ascyron Lrdquo Medicinal Chemistry Research vol20 no 7 pp 809ndash816 2011

[23] W-Y Kang C-F Li and Y-X Liu ldquoAntioxidant phenoliccompounds and flavonoids of Mitragyna rotundifolia (Roxb)Kuntze in vitrordquo Medicinal Chemistry Research vol 19 no 9pp 1222ndash1232 2010

10 Journal of Chemistry

[24] W Kang and J Wang ldquoIn vitro antioxidant properties andin vivo lowering blood lipid of Forsythia suspense leavesrdquoMedicinal Chemistry Research vol 19 no 7 pp 617ndash628 2010

[25] C F Poole and S K Poole ldquoExtraction of organic compoundswith room temperature ionic liquidsrdquo Journal of Chromatogra-phy A vol 1217 no 16 pp 2268ndash2286 2010

[26] Z Guo B Lue K Thomasen A S Meyer and X Xu ldquoPre-dictions of flavonoid solubility in ionic liquids by COSMO-RSexperimental verification structural elucidation and solvationcharacterizationrdquo Green Chemistry vol 9 no 12 pp 1362ndash13732007

[27] S Y Dong Q Hu J Li Y L Fu Y Q Xing and T L HuangldquoIonic liquid-based microwave-assisted extraction followedhigh-performance liquid chromatography for the simultaneousdetermination of five organic oxygen pesticides in soil samplesrdquoChinese Journal of Analysis Laboratory vol 32 pp 48ndash52 2013

[28] H Wu M Chen Y Fan F Elsebaei and Y Zhu ldquoDeter-mination of rutin and quercetin in Chinese herbal medicineby ionic liquid-based pressurized liquid extraction-liquidchromatography- chemiluminescence detectionrdquo Talanta vol88 pp 222ndash229 2012

[29] R Jin L Fan and X An ldquoIonic liquid-assisted extraction ofpaeonol from cynanchum paniculatumrdquo Chromatographia vol73 no 7-8 pp 787ndash792 2011

[30] P Liang F Wang and Q Wan ldquoIonic liquid-based ultrasound-assisted emulsification microextraction coupled with high per-formance liquid chromatography for the determination of fourfungicides in environmental water samplesrdquo Talanta vol 105pp 57ndash62 2013

[31] C K Zacharis C Christophoridis and K Fytianos ldquoVortex-assisted liquid-liquid microextraction combined with gaschromatography-mass spectrometry for the determination oforganophosphate pesticides in environmental water samplesand winesrdquo Journal of Separation Science vol 35 no 18 pp2422ndash2429 2012

[32] H Liu X Y Wei J H Li T Li and F Wang ldquoReview of ionicliquids recyclingrdquo Journal of Cellulose Science Technology vol21 pp 63ndash69 2013

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

International Journal ofInternational Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal ofInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 7

IsoquercitrinAstragalin

500400300200

Ultrasound power (W)

07

08

09

10

11

12

13Ex

trac

tion

yiel

d (m

gmiddotgminus

1)

(a)

QuercetinKaempferol

002

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

300 400 500200

Ultrasound power (W)

(b)

Figure 8 Effect of ultrasound power on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh (a)and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound time 20min (a) and 45min (b) centrifugation speed5000 rsdotminminus1

IsoquercitrinAstragalin

4000 5000 6000 7000 80003000

Centrifugation speed (rmiddotminminus1)

04

06

08

10

12

14

Extr

actio

n yi

eld

(mgmiddot

gminus1)

(a)

QuercetinKaempferol

002

003

004

005

006

007

Extr

actio

n yi

eld

(mgmiddot

gminus1)

4000 5000 6000 7000 80003000

Centrifugation speed (rmiddotminminus1)

(b)

Figure 9 Effect of centrifugation speed on extraction yields Type of IL [BMIM]BF4 concentration 05molsdotLminus1 the particle size 24 mesh(a) and 40 mesh (b) solid-liquid ratio 1 10 gsdotmLminus1 (a) and 1 20 gsdotmLminus1 (b) ultrasound time 20min (a) and 45min (b) ultrasound power500W

The repeatability was represented by the relative standarddeviation of the peak area (RSD) The results of SST wereshown in Table 1 which shows that the chromatographicsystems had good suitability

322 Linearity The stock standard solutions a and b wereaccurately injected 05 1 2 4 6 8 10 and 12 120583L to chro-matographic instrument for the construction of calibrationcurves respectively The calibration curves were constructedby plotting the peak areas versus the injection volume of eachcompoundThe results were demonstrated in Table 2 119903 valueswere in the range from 09996 to 10000 which indicated thatthe methods had good linearity

323 Precision The stock standard solutions a and b wereaccurately injected 10 120583L to chromatographic instrumentrespectively And they were analyzed in six replicates withinone day for determining the precision of the developedassay The RSDs of peak areas for isoquercitrin astragalinquercetin and kaempferol were 046 083 023 and024 respectively The results indicated that the methodswere precise for quantitative analysis of isoquercitrin astra-galin quercetin and kaempferol

324 Stability The sample solutions were prepared underthe optimum extraction conditions and placed at roomtemperature and thenwere injected 10120583L to chromatographic

8 Journal of Chemistry

Table 1 System suitability test (SST) of HPLC

Compound Theoretical plate number Resolution Tailing factor RepeatabilityIsoquercitrin 8392 152 131 087Astragalin 8834 1095 118 064Quercetin 7071 550 125 113Kaempferol 13956 854 135 102

Table 2 Regression equations and linear ranges for four compounds

Compound Regressive equation 119903 Linear range120583gIsoquercitrin 119884 = 319 times 106119883 minus 292 times 104 09996 0068sim164Astragalin 119884 = 300 times 106119883 minus 232 times 104 09997 0060sim144Quercetin 119884 = 742 times 106119883 minus 958 times 103 10000 00080sim019Kaempferol 119884 = 890 times 106119883 minus 149 times 104 10000 00077sim018119884 peak area119883 injection amount (120583g)

Table 3 Antioxidant activity of the different extracts of L clethroides

Sample DPPH ABTS FRAPRadical scavenging rate IC50120583gsdotmLminus1 Radical scavenging rate IC50120583gsdotmLminus1 TEAC120583molsdotgminus1

Leaf rsquos methanol extract 9391 1403 plusmn 80 9701 144 plusmn 5 3858 plusmn 072Leaf rsquos ionic liquid extract 8848 638 plusmn 60 9323 50 plusmn 2 10579 plusmn 702Flowerrsquos methanol extract 9392 1004 plusmn 12 9804 151 plusmn 1 5069 plusmn 110Flowerrsquos ionic liquid extract 8977 460 plusmn 36 9636 51 plusmn 2 10583 plusmn 125[BMIM]BF4 minus443 NT 003 NT NTBHA 7600 15 plusmn 146 9994 206 plusmn 008 591911 plusmn 1410BHT 7306 12 plusmn 048 9942 656 plusmn 010 58920 plusmn 5920PG 9561 401 plusmn 008 9580 069 plusmn 005 944133 plusmn 6927Note NT means not available because of low activity BHA BHT and PG are positive controls

instrument at 0 3 6 9 12 and 24 h respectively The RSDsof peak areas for isoquercitrin astragalin quercetin andkaempferol were 067 083 165 and 174 The resultsindicated that the sample solutionwas basically stable at roomtemperature within 24 h

325 Repeatability Six test sample solutions a and b wereprocessed under the optimumextraction conditions and theninjected 10 120583L to chromatographic instrument for analysisThe RSDs of peak areas for the four compounds were203 184 115 and 140 indicating that the analyticalmethods had an acceptable level of repeatability

326 Recovery Nine batches of leaves and flowers samplesof L clethroideswere prepared and divided into three groupsrespectively Then the four standard substances at threedifferent amounts were added to the leaves and flowerssamples The spiked samples were prepared according to theoptimum extraction conditions All the calculated recoveryvalues of the analytes ranged from 9839 to 10068 and theRSDs were less than 220The results demonstrated that themethods were reasonable and feasible

33 Antioxidant Activity of the Different Extracts of Lclethroides In Vitro The antioxidant activities of methanol

and ionic liquid sample solutions were evaluated by theDPPH ABTS and FRAP assay comprehensively and theresults were shown in Table 3 Results showed that the ionicliquid extract had stronger antioxidant activity than thatof the methanol extract and the ionic liquid itself had noantioxidant activity Results indicated that the ionic liquidused as extraction solvent hadmore advantages for extractingingredients

34 Sample Analysis Ionic liquid was successfully appliedfor the determination of isoquercitrin astragalin in leavesquercetin and kaempferol in flowers of L clethroides bythe optimized conditions respectively The mass fractions ofisoquercitrin astragalin in leaves quercetin and kaempferolin flowers were 1262 1219 0061 and 0046mgsdotgminus1 (119899 = 3)respectively

4 Conclusion

Themethod ionic liquid-based ultrasonic-assisted extractionhad been developed for the extraction of isoquercitrinastragalin in leaves quercetin and kaempferol in flowers of Lclethroides Compared with the conventional extraction withmethanol the proposed approaches provided higher extrac-tion efficiencies Results demonstrated that the methods

Journal of Chemistry 9

were simple rapid green and effective ILs can be recycledby some methods such as vacuum distillation membranefiltration salting out and liquid-liquid extraction [32] Con-sidering the unique properties of ILs the methods have agreat promising prospect in sample preparation of Chineseherbal medicine

Conflicts of Interest

The authors declare that there are no conflicts of interest

Authorsrsquo Contributions

Wen-yi Kang and Jin-feng Wei conceived the research ideaZhi-juan Zhang and Li-li Cui conducted the experimentscollected the plant specimens analyzed and interpreted thedata and prepared the first draft Wen-yi Kang Zhi-juanZhang and Jin-fengWei critically read and revised the paperAll the authors read and approved the paper before its finalsubmission

Acknowledgments

This work was supported by Henan Province UniversityScience and Technology Innovation Team (16IRTSTHN019)Science and Technology Innovation Team of Kaifeng City(2016-124) Kaifeng City Science and Technology InnovationTalent (1509010) and Key project in Science and TechnologyAgency of Kaifeng (1603111)

References

[1] X M Hu W K Zhang and Q S Zhu The Chinese MateriaMedica Shanghai Shanghai Science and Technology Press1999

[2] Z-J Zhang Z-Y Xia J-MWang X-T Song J-FWei andW-Y Kang ldquoEffects of flavonoids in Lysimachia clethroides dubyon the activities of cytochrome P450 CYP2E1 and CYP3A4 inrat liver microsomesrdquo Molecules vol 21 no 6 article no 7382016

[3] L H Tang X Y Xu B G You W Zhang and Y Q WangldquoAnti-tumor effect and its mechanism of total flavones ofLysimachia clethroides Dubyrdquo Shanghai Journal of TraditionalChinese Medicine vol 41 no 5 pp 74ndash76 2007

[4] Q-M Xu Y-L Liu Y-L Feng L-H Tang and S-L YangldquoA new e-ring 120574-lactone pentacyclic triterpene from lysimachiaclethroides and its cytotoxic activitiesrdquo Chemistry of NaturalCompounds vol 48 no 4 pp 597ndash600 2012

[5] C F Li Y L Song Y X Liu and W Y Kang ldquoAntioxidantactivity of extracts from Lysimachia clethroidesrdquo Fine Chemicalsvol 25 pp 1191ndash1193 2008

[6] J F Wei ldquoStudy on Chemical Constitutens Hepatoprotectiveand Antihyperglycemic Effect of Lysimachia paridiformis varstenophylla and Lysimachia clethroidesrdquo MSD thesis 2012Henan University Kaifeng

[7] D Liang ldquoStudy on the chemical composition and biologicalactivity of Lysimachia clethroides and chemical conversion ofsyntheticrdquo MSD thesis 2012 Peking Union Medical CollegeBeijing

[8] S M Yue B Q Chen P F Yuan W H Cui and W YKang ldquoChemical Constituents of Lysimachia clethroides DubyrdquoChinese Phramaceutical Journal vol 46 pp 341ndash343 2011

[9] L F Ding Y D Guo X D Wu and Y H Ma ldquoChemicalconstituents of flavonoids in Lysimachia clethroidesrdquo ChineseTraditional Patent Medicine vol 32 no 05 pp 827ndash830 2010

[10] Q M Xu L H Tang X Li L L Hao X R Li and S LYang ldquoIsolation and Identification of 3-O-p-CoumaroyloxylPentacyclic Triterpenoids from Lysimachia clethroides DubyrdquoChin Pharm J vol 45 pp 825ndash828 2010

[11] H Y Zou and P F Tu ldquoStudies on the chemical constituents ofLysimachia clethroides Dubyrdquo Chin Tradit Herb Drugs vol 40pp 704ndash708 2009

[12] L H Tang X Y Xu B G You Y Q Wang and W ZhangldquoEffect of flavones from Lysimachia clethroides on gene expre-sion of HL-60 cellrdquo Pharm Clin Res vol 15 pp 21ndash24 2007

[13] Y Q Wang L H Tang Z Q Liang and Z L Gu ldquoPrimarystudies on the anti-uterine cervix cancer effects of the extractZE4 from Lysimachia clethroides Dubyrdquo Chin Pharmacol Bullvol 23 pp 925ndash929 2007

[14] A A Aerov A R Khokhlov and I I Potemkin ldquoWhy ionicliquids can possess extra solvent powerrdquo Journal of PhysicalChemistry B vol 110 no 33 pp 16205ndash16207 2006

[15] J F Wei P R Cao J M Wang and W Y Kang ldquoAnalysis oftilianin and acacetin in Agastache rugosa by high-performanceliquid chromatography with ionic liquids-ultrasound basedextractionrdquo Chemistry Central Journal vol 10 p 76 2016

[16] W Y Ma Y B Lu R L Hu J H Chen Z Z Zhang and YJ Pan ldquoApplication of ionic liquids based microwave-assistedextraction of three alkaloids N-nornuciferine O-nornuciferineand nuciferine from lotus leafrdquo Talanta vol 80 no 3 pp 1292ndash1297 2010

[17] W Liu D D Li H S Yang et al ldquoDetermination of oleanic acidand paeoniflorin in Paeonia lactiflora by ultrasound-assistedionic liquid-reversed phase liquid chromatographyrdquo China JChin Mater Med vol 40 pp 443ndash449 2015

[18] J F Wei Z J Zhang D D Li W Liu andW Y Kang ldquoAnalysisof four flavonoids in Lysimachia clethroides using ionic liquid-assisted extractionrdquo China J Chin Mater Med vol 40 pp 1305ndash1310 2015

[19] J F Wei Z H Yin and F D Shang ldquoVolatiles in the Lysimachiaclethroides Duby by head space solid phase microextractioncoupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS)rdquo African Journal of Pharmacy and Pharmacol-ogy vol 6 pp 2484ndash2487 2012

[20] J F Wei Y Y Li Z H Yin F Gong and F D ShangldquoAntioxidant activities in vitroand hepatoprotective effects ofLysimachia clethroidesDuby on CCl4-induced acute liver injuryin micerdquo Afr J Pharm Pharmacol vol 6 pp 743ndash750 2012

[21] J F Wei X Chang W Wang and W Y Kang ldquoChemicalconstituents from Lysimachia circaeoidesrdquo Journal of ChineseMedicine Material vol 36 pp 1441ndash1443 2013

[22] W-Y Kang Y-L Song and L Zhang ldquo120572-Glucosidaseinhibitory and antioxidant properties and antidiabetic activityof Hypericum ascyron Lrdquo Medicinal Chemistry Research vol20 no 7 pp 809ndash816 2011

[23] W-Y Kang C-F Li and Y-X Liu ldquoAntioxidant phenoliccompounds and flavonoids of Mitragyna rotundifolia (Roxb)Kuntze in vitrordquo Medicinal Chemistry Research vol 19 no 9pp 1222ndash1232 2010

10 Journal of Chemistry

[24] W Kang and J Wang ldquoIn vitro antioxidant properties andin vivo lowering blood lipid of Forsythia suspense leavesrdquoMedicinal Chemistry Research vol 19 no 7 pp 617ndash628 2010

[25] C F Poole and S K Poole ldquoExtraction of organic compoundswith room temperature ionic liquidsrdquo Journal of Chromatogra-phy A vol 1217 no 16 pp 2268ndash2286 2010

[26] Z Guo B Lue K Thomasen A S Meyer and X Xu ldquoPre-dictions of flavonoid solubility in ionic liquids by COSMO-RSexperimental verification structural elucidation and solvationcharacterizationrdquo Green Chemistry vol 9 no 12 pp 1362ndash13732007

[27] S Y Dong Q Hu J Li Y L Fu Y Q Xing and T L HuangldquoIonic liquid-based microwave-assisted extraction followedhigh-performance liquid chromatography for the simultaneousdetermination of five organic oxygen pesticides in soil samplesrdquoChinese Journal of Analysis Laboratory vol 32 pp 48ndash52 2013

[28] H Wu M Chen Y Fan F Elsebaei and Y Zhu ldquoDeter-mination of rutin and quercetin in Chinese herbal medicineby ionic liquid-based pressurized liquid extraction-liquidchromatography- chemiluminescence detectionrdquo Talanta vol88 pp 222ndash229 2012

[29] R Jin L Fan and X An ldquoIonic liquid-assisted extraction ofpaeonol from cynanchum paniculatumrdquo Chromatographia vol73 no 7-8 pp 787ndash792 2011

[30] P Liang F Wang and Q Wan ldquoIonic liquid-based ultrasound-assisted emulsification microextraction coupled with high per-formance liquid chromatography for the determination of fourfungicides in environmental water samplesrdquo Talanta vol 105pp 57ndash62 2013

[31] C K Zacharis C Christophoridis and K Fytianos ldquoVortex-assisted liquid-liquid microextraction combined with gaschromatography-mass spectrometry for the determination oforganophosphate pesticides in environmental water samplesand winesrdquo Journal of Separation Science vol 35 no 18 pp2422ndash2429 2012

[32] H Liu X Y Wei J H Li T Li and F Wang ldquoReview of ionicliquids recyclingrdquo Journal of Cellulose Science Technology vol21 pp 63ndash69 2013

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

International Journal ofInternational Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal ofInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

8 Journal of Chemistry

Table 1 System suitability test (SST) of HPLC

Compound Theoretical plate number Resolution Tailing factor RepeatabilityIsoquercitrin 8392 152 131 087Astragalin 8834 1095 118 064Quercetin 7071 550 125 113Kaempferol 13956 854 135 102

Table 2 Regression equations and linear ranges for four compounds

Compound Regressive equation 119903 Linear range120583gIsoquercitrin 119884 = 319 times 106119883 minus 292 times 104 09996 0068sim164Astragalin 119884 = 300 times 106119883 minus 232 times 104 09997 0060sim144Quercetin 119884 = 742 times 106119883 minus 958 times 103 10000 00080sim019Kaempferol 119884 = 890 times 106119883 minus 149 times 104 10000 00077sim018119884 peak area119883 injection amount (120583g)

Table 3 Antioxidant activity of the different extracts of L clethroides

Sample DPPH ABTS FRAPRadical scavenging rate IC50120583gsdotmLminus1 Radical scavenging rate IC50120583gsdotmLminus1 TEAC120583molsdotgminus1

Leaf rsquos methanol extract 9391 1403 plusmn 80 9701 144 plusmn 5 3858 plusmn 072Leaf rsquos ionic liquid extract 8848 638 plusmn 60 9323 50 plusmn 2 10579 plusmn 702Flowerrsquos methanol extract 9392 1004 plusmn 12 9804 151 plusmn 1 5069 plusmn 110Flowerrsquos ionic liquid extract 8977 460 plusmn 36 9636 51 plusmn 2 10583 plusmn 125[BMIM]BF4 minus443 NT 003 NT NTBHA 7600 15 plusmn 146 9994 206 plusmn 008 591911 plusmn 1410BHT 7306 12 plusmn 048 9942 656 plusmn 010 58920 plusmn 5920PG 9561 401 plusmn 008 9580 069 plusmn 005 944133 plusmn 6927Note NT means not available because of low activity BHA BHT and PG are positive controls

instrument at 0 3 6 9 12 and 24 h respectively The RSDsof peak areas for isoquercitrin astragalin quercetin andkaempferol were 067 083 165 and 174 The resultsindicated that the sample solutionwas basically stable at roomtemperature within 24 h

325 Repeatability Six test sample solutions a and b wereprocessed under the optimumextraction conditions and theninjected 10 120583L to chromatographic instrument for analysisThe RSDs of peak areas for the four compounds were203 184 115 and 140 indicating that the analyticalmethods had an acceptable level of repeatability

326 Recovery Nine batches of leaves and flowers samplesof L clethroideswere prepared and divided into three groupsrespectively Then the four standard substances at threedifferent amounts were added to the leaves and flowerssamples The spiked samples were prepared according to theoptimum extraction conditions All the calculated recoveryvalues of the analytes ranged from 9839 to 10068 and theRSDs were less than 220The results demonstrated that themethods were reasonable and feasible

33 Antioxidant Activity of the Different Extracts of Lclethroides In Vitro The antioxidant activities of methanol

and ionic liquid sample solutions were evaluated by theDPPH ABTS and FRAP assay comprehensively and theresults were shown in Table 3 Results showed that the ionicliquid extract had stronger antioxidant activity than thatof the methanol extract and the ionic liquid itself had noantioxidant activity Results indicated that the ionic liquidused as extraction solvent hadmore advantages for extractingingredients

34 Sample Analysis Ionic liquid was successfully appliedfor the determination of isoquercitrin astragalin in leavesquercetin and kaempferol in flowers of L clethroides bythe optimized conditions respectively The mass fractions ofisoquercitrin astragalin in leaves quercetin and kaempferolin flowers were 1262 1219 0061 and 0046mgsdotgminus1 (119899 = 3)respectively

4 Conclusion

Themethod ionic liquid-based ultrasonic-assisted extractionhad been developed for the extraction of isoquercitrinastragalin in leaves quercetin and kaempferol in flowers of Lclethroides Compared with the conventional extraction withmethanol the proposed approaches provided higher extrac-tion efficiencies Results demonstrated that the methods

Journal of Chemistry 9

were simple rapid green and effective ILs can be recycledby some methods such as vacuum distillation membranefiltration salting out and liquid-liquid extraction [32] Con-sidering the unique properties of ILs the methods have agreat promising prospect in sample preparation of Chineseherbal medicine

Conflicts of Interest

The authors declare that there are no conflicts of interest

Authorsrsquo Contributions

Wen-yi Kang and Jin-feng Wei conceived the research ideaZhi-juan Zhang and Li-li Cui conducted the experimentscollected the plant specimens analyzed and interpreted thedata and prepared the first draft Wen-yi Kang Zhi-juanZhang and Jin-fengWei critically read and revised the paperAll the authors read and approved the paper before its finalsubmission

Acknowledgments

This work was supported by Henan Province UniversityScience and Technology Innovation Team (16IRTSTHN019)Science and Technology Innovation Team of Kaifeng City(2016-124) Kaifeng City Science and Technology InnovationTalent (1509010) and Key project in Science and TechnologyAgency of Kaifeng (1603111)

References

[1] X M Hu W K Zhang and Q S Zhu The Chinese MateriaMedica Shanghai Shanghai Science and Technology Press1999

[2] Z-J Zhang Z-Y Xia J-MWang X-T Song J-FWei andW-Y Kang ldquoEffects of flavonoids in Lysimachia clethroides dubyon the activities of cytochrome P450 CYP2E1 and CYP3A4 inrat liver microsomesrdquo Molecules vol 21 no 6 article no 7382016

[3] L H Tang X Y Xu B G You W Zhang and Y Q WangldquoAnti-tumor effect and its mechanism of total flavones ofLysimachia clethroides Dubyrdquo Shanghai Journal of TraditionalChinese Medicine vol 41 no 5 pp 74ndash76 2007

[4] Q-M Xu Y-L Liu Y-L Feng L-H Tang and S-L YangldquoA new e-ring 120574-lactone pentacyclic triterpene from lysimachiaclethroides and its cytotoxic activitiesrdquo Chemistry of NaturalCompounds vol 48 no 4 pp 597ndash600 2012

[5] C F Li Y L Song Y X Liu and W Y Kang ldquoAntioxidantactivity of extracts from Lysimachia clethroidesrdquo Fine Chemicalsvol 25 pp 1191ndash1193 2008

[6] J F Wei ldquoStudy on Chemical Constitutens Hepatoprotectiveand Antihyperglycemic Effect of Lysimachia paridiformis varstenophylla and Lysimachia clethroidesrdquo MSD thesis 2012Henan University Kaifeng

[7] D Liang ldquoStudy on the chemical composition and biologicalactivity of Lysimachia clethroides and chemical conversion ofsyntheticrdquo MSD thesis 2012 Peking Union Medical CollegeBeijing

[8] S M Yue B Q Chen P F Yuan W H Cui and W YKang ldquoChemical Constituents of Lysimachia clethroides DubyrdquoChinese Phramaceutical Journal vol 46 pp 341ndash343 2011

[9] L F Ding Y D Guo X D Wu and Y H Ma ldquoChemicalconstituents of flavonoids in Lysimachia clethroidesrdquo ChineseTraditional Patent Medicine vol 32 no 05 pp 827ndash830 2010

[10] Q M Xu L H Tang X Li L L Hao X R Li and S LYang ldquoIsolation and Identification of 3-O-p-CoumaroyloxylPentacyclic Triterpenoids from Lysimachia clethroides DubyrdquoChin Pharm J vol 45 pp 825ndash828 2010

[11] H Y Zou and P F Tu ldquoStudies on the chemical constituents ofLysimachia clethroides Dubyrdquo Chin Tradit Herb Drugs vol 40pp 704ndash708 2009

[12] L H Tang X Y Xu B G You Y Q Wang and W ZhangldquoEffect of flavones from Lysimachia clethroides on gene expre-sion of HL-60 cellrdquo Pharm Clin Res vol 15 pp 21ndash24 2007

[13] Y Q Wang L H Tang Z Q Liang and Z L Gu ldquoPrimarystudies on the anti-uterine cervix cancer effects of the extractZE4 from Lysimachia clethroides Dubyrdquo Chin Pharmacol Bullvol 23 pp 925ndash929 2007

[14] A A Aerov A R Khokhlov and I I Potemkin ldquoWhy ionicliquids can possess extra solvent powerrdquo Journal of PhysicalChemistry B vol 110 no 33 pp 16205ndash16207 2006

[15] J F Wei P R Cao J M Wang and W Y Kang ldquoAnalysis oftilianin and acacetin in Agastache rugosa by high-performanceliquid chromatography with ionic liquids-ultrasound basedextractionrdquo Chemistry Central Journal vol 10 p 76 2016

[16] W Y Ma Y B Lu R L Hu J H Chen Z Z Zhang and YJ Pan ldquoApplication of ionic liquids based microwave-assistedextraction of three alkaloids N-nornuciferine O-nornuciferineand nuciferine from lotus leafrdquo Talanta vol 80 no 3 pp 1292ndash1297 2010

[17] W Liu D D Li H S Yang et al ldquoDetermination of oleanic acidand paeoniflorin in Paeonia lactiflora by ultrasound-assistedionic liquid-reversed phase liquid chromatographyrdquo China JChin Mater Med vol 40 pp 443ndash449 2015

[18] J F Wei Z J Zhang D D Li W Liu andW Y Kang ldquoAnalysisof four flavonoids in Lysimachia clethroides using ionic liquid-assisted extractionrdquo China J Chin Mater Med vol 40 pp 1305ndash1310 2015

[19] J F Wei Z H Yin and F D Shang ldquoVolatiles in the Lysimachiaclethroides Duby by head space solid phase microextractioncoupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS)rdquo African Journal of Pharmacy and Pharmacol-ogy vol 6 pp 2484ndash2487 2012

[20] J F Wei Y Y Li Z H Yin F Gong and F D ShangldquoAntioxidant activities in vitroand hepatoprotective effects ofLysimachia clethroidesDuby on CCl4-induced acute liver injuryin micerdquo Afr J Pharm Pharmacol vol 6 pp 743ndash750 2012

[21] J F Wei X Chang W Wang and W Y Kang ldquoChemicalconstituents from Lysimachia circaeoidesrdquo Journal of ChineseMedicine Material vol 36 pp 1441ndash1443 2013

[22] W-Y Kang Y-L Song and L Zhang ldquo120572-Glucosidaseinhibitory and antioxidant properties and antidiabetic activityof Hypericum ascyron Lrdquo Medicinal Chemistry Research vol20 no 7 pp 809ndash816 2011

[23] W-Y Kang C-F Li and Y-X Liu ldquoAntioxidant phenoliccompounds and flavonoids of Mitragyna rotundifolia (Roxb)Kuntze in vitrordquo Medicinal Chemistry Research vol 19 no 9pp 1222ndash1232 2010

10 Journal of Chemistry

[24] W Kang and J Wang ldquoIn vitro antioxidant properties andin vivo lowering blood lipid of Forsythia suspense leavesrdquoMedicinal Chemistry Research vol 19 no 7 pp 617ndash628 2010

[25] C F Poole and S K Poole ldquoExtraction of organic compoundswith room temperature ionic liquidsrdquo Journal of Chromatogra-phy A vol 1217 no 16 pp 2268ndash2286 2010

[26] Z Guo B Lue K Thomasen A S Meyer and X Xu ldquoPre-dictions of flavonoid solubility in ionic liquids by COSMO-RSexperimental verification structural elucidation and solvationcharacterizationrdquo Green Chemistry vol 9 no 12 pp 1362ndash13732007

[27] S Y Dong Q Hu J Li Y L Fu Y Q Xing and T L HuangldquoIonic liquid-based microwave-assisted extraction followedhigh-performance liquid chromatography for the simultaneousdetermination of five organic oxygen pesticides in soil samplesrdquoChinese Journal of Analysis Laboratory vol 32 pp 48ndash52 2013

[28] H Wu M Chen Y Fan F Elsebaei and Y Zhu ldquoDeter-mination of rutin and quercetin in Chinese herbal medicineby ionic liquid-based pressurized liquid extraction-liquidchromatography- chemiluminescence detectionrdquo Talanta vol88 pp 222ndash229 2012

[29] R Jin L Fan and X An ldquoIonic liquid-assisted extraction ofpaeonol from cynanchum paniculatumrdquo Chromatographia vol73 no 7-8 pp 787ndash792 2011

[30] P Liang F Wang and Q Wan ldquoIonic liquid-based ultrasound-assisted emulsification microextraction coupled with high per-formance liquid chromatography for the determination of fourfungicides in environmental water samplesrdquo Talanta vol 105pp 57ndash62 2013

[31] C K Zacharis C Christophoridis and K Fytianos ldquoVortex-assisted liquid-liquid microextraction combined with gaschromatography-mass spectrometry for the determination oforganophosphate pesticides in environmental water samplesand winesrdquo Journal of Separation Science vol 35 no 18 pp2422ndash2429 2012

[32] H Liu X Y Wei J H Li T Li and F Wang ldquoReview of ionicliquids recyclingrdquo Journal of Cellulose Science Technology vol21 pp 63ndash69 2013

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

International Journal ofInternational Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal ofInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 9

were simple rapid green and effective ILs can be recycledby some methods such as vacuum distillation membranefiltration salting out and liquid-liquid extraction [32] Con-sidering the unique properties of ILs the methods have agreat promising prospect in sample preparation of Chineseherbal medicine

Conflicts of Interest

The authors declare that there are no conflicts of interest

Authorsrsquo Contributions

Wen-yi Kang and Jin-feng Wei conceived the research ideaZhi-juan Zhang and Li-li Cui conducted the experimentscollected the plant specimens analyzed and interpreted thedata and prepared the first draft Wen-yi Kang Zhi-juanZhang and Jin-fengWei critically read and revised the paperAll the authors read and approved the paper before its finalsubmission

Acknowledgments

This work was supported by Henan Province UniversityScience and Technology Innovation Team (16IRTSTHN019)Science and Technology Innovation Team of Kaifeng City(2016-124) Kaifeng City Science and Technology InnovationTalent (1509010) and Key project in Science and TechnologyAgency of Kaifeng (1603111)

References

[1] X M Hu W K Zhang and Q S Zhu The Chinese MateriaMedica Shanghai Shanghai Science and Technology Press1999

[2] Z-J Zhang Z-Y Xia J-MWang X-T Song J-FWei andW-Y Kang ldquoEffects of flavonoids in Lysimachia clethroides dubyon the activities of cytochrome P450 CYP2E1 and CYP3A4 inrat liver microsomesrdquo Molecules vol 21 no 6 article no 7382016

[3] L H Tang X Y Xu B G You W Zhang and Y Q WangldquoAnti-tumor effect and its mechanism of total flavones ofLysimachia clethroides Dubyrdquo Shanghai Journal of TraditionalChinese Medicine vol 41 no 5 pp 74ndash76 2007

[4] Q-M Xu Y-L Liu Y-L Feng L-H Tang and S-L YangldquoA new e-ring 120574-lactone pentacyclic triterpene from lysimachiaclethroides and its cytotoxic activitiesrdquo Chemistry of NaturalCompounds vol 48 no 4 pp 597ndash600 2012

[5] C F Li Y L Song Y X Liu and W Y Kang ldquoAntioxidantactivity of extracts from Lysimachia clethroidesrdquo Fine Chemicalsvol 25 pp 1191ndash1193 2008

[6] J F Wei ldquoStudy on Chemical Constitutens Hepatoprotectiveand Antihyperglycemic Effect of Lysimachia paridiformis varstenophylla and Lysimachia clethroidesrdquo MSD thesis 2012Henan University Kaifeng

[7] D Liang ldquoStudy on the chemical composition and biologicalactivity of Lysimachia clethroides and chemical conversion ofsyntheticrdquo MSD thesis 2012 Peking Union Medical CollegeBeijing

[8] S M Yue B Q Chen P F Yuan W H Cui and W YKang ldquoChemical Constituents of Lysimachia clethroides DubyrdquoChinese Phramaceutical Journal vol 46 pp 341ndash343 2011

[9] L F Ding Y D Guo X D Wu and Y H Ma ldquoChemicalconstituents of flavonoids in Lysimachia clethroidesrdquo ChineseTraditional Patent Medicine vol 32 no 05 pp 827ndash830 2010

[10] Q M Xu L H Tang X Li L L Hao X R Li and S LYang ldquoIsolation and Identification of 3-O-p-CoumaroyloxylPentacyclic Triterpenoids from Lysimachia clethroides DubyrdquoChin Pharm J vol 45 pp 825ndash828 2010

[11] H Y Zou and P F Tu ldquoStudies on the chemical constituents ofLysimachia clethroides Dubyrdquo Chin Tradit Herb Drugs vol 40pp 704ndash708 2009

[12] L H Tang X Y Xu B G You Y Q Wang and W ZhangldquoEffect of flavones from Lysimachia clethroides on gene expre-sion of HL-60 cellrdquo Pharm Clin Res vol 15 pp 21ndash24 2007

[13] Y Q Wang L H Tang Z Q Liang and Z L Gu ldquoPrimarystudies on the anti-uterine cervix cancer effects of the extractZE4 from Lysimachia clethroides Dubyrdquo Chin Pharmacol Bullvol 23 pp 925ndash929 2007

[14] A A Aerov A R Khokhlov and I I Potemkin ldquoWhy ionicliquids can possess extra solvent powerrdquo Journal of PhysicalChemistry B vol 110 no 33 pp 16205ndash16207 2006

[15] J F Wei P R Cao J M Wang and W Y Kang ldquoAnalysis oftilianin and acacetin in Agastache rugosa by high-performanceliquid chromatography with ionic liquids-ultrasound basedextractionrdquo Chemistry Central Journal vol 10 p 76 2016

[16] W Y Ma Y B Lu R L Hu J H Chen Z Z Zhang and YJ Pan ldquoApplication of ionic liquids based microwave-assistedextraction of three alkaloids N-nornuciferine O-nornuciferineand nuciferine from lotus leafrdquo Talanta vol 80 no 3 pp 1292ndash1297 2010

[17] W Liu D D Li H S Yang et al ldquoDetermination of oleanic acidand paeoniflorin in Paeonia lactiflora by ultrasound-assistedionic liquid-reversed phase liquid chromatographyrdquo China JChin Mater Med vol 40 pp 443ndash449 2015

[18] J F Wei Z J Zhang D D Li W Liu andW Y Kang ldquoAnalysisof four flavonoids in Lysimachia clethroides using ionic liquid-assisted extractionrdquo China J Chin Mater Med vol 40 pp 1305ndash1310 2015

[19] J F Wei Z H Yin and F D Shang ldquoVolatiles in the Lysimachiaclethroides Duby by head space solid phase microextractioncoupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS)rdquo African Journal of Pharmacy and Pharmacol-ogy vol 6 pp 2484ndash2487 2012

[20] J F Wei Y Y Li Z H Yin F Gong and F D ShangldquoAntioxidant activities in vitroand hepatoprotective effects ofLysimachia clethroidesDuby on CCl4-induced acute liver injuryin micerdquo Afr J Pharm Pharmacol vol 6 pp 743ndash750 2012

[21] J F Wei X Chang W Wang and W Y Kang ldquoChemicalconstituents from Lysimachia circaeoidesrdquo Journal of ChineseMedicine Material vol 36 pp 1441ndash1443 2013

[22] W-Y Kang Y-L Song and L Zhang ldquo120572-Glucosidaseinhibitory and antioxidant properties and antidiabetic activityof Hypericum ascyron Lrdquo Medicinal Chemistry Research vol20 no 7 pp 809ndash816 2011

[23] W-Y Kang C-F Li and Y-X Liu ldquoAntioxidant phenoliccompounds and flavonoids of Mitragyna rotundifolia (Roxb)Kuntze in vitrordquo Medicinal Chemistry Research vol 19 no 9pp 1222ndash1232 2010

10 Journal of Chemistry

[24] W Kang and J Wang ldquoIn vitro antioxidant properties andin vivo lowering blood lipid of Forsythia suspense leavesrdquoMedicinal Chemistry Research vol 19 no 7 pp 617ndash628 2010

[25] C F Poole and S K Poole ldquoExtraction of organic compoundswith room temperature ionic liquidsrdquo Journal of Chromatogra-phy A vol 1217 no 16 pp 2268ndash2286 2010

[26] Z Guo B Lue K Thomasen A S Meyer and X Xu ldquoPre-dictions of flavonoid solubility in ionic liquids by COSMO-RSexperimental verification structural elucidation and solvationcharacterizationrdquo Green Chemistry vol 9 no 12 pp 1362ndash13732007

[27] S Y Dong Q Hu J Li Y L Fu Y Q Xing and T L HuangldquoIonic liquid-based microwave-assisted extraction followedhigh-performance liquid chromatography for the simultaneousdetermination of five organic oxygen pesticides in soil samplesrdquoChinese Journal of Analysis Laboratory vol 32 pp 48ndash52 2013

[28] H Wu M Chen Y Fan F Elsebaei and Y Zhu ldquoDeter-mination of rutin and quercetin in Chinese herbal medicineby ionic liquid-based pressurized liquid extraction-liquidchromatography- chemiluminescence detectionrdquo Talanta vol88 pp 222ndash229 2012

[29] R Jin L Fan and X An ldquoIonic liquid-assisted extraction ofpaeonol from cynanchum paniculatumrdquo Chromatographia vol73 no 7-8 pp 787ndash792 2011

[30] P Liang F Wang and Q Wan ldquoIonic liquid-based ultrasound-assisted emulsification microextraction coupled with high per-formance liquid chromatography for the determination of fourfungicides in environmental water samplesrdquo Talanta vol 105pp 57ndash62 2013

[31] C K Zacharis C Christophoridis and K Fytianos ldquoVortex-assisted liquid-liquid microextraction combined with gaschromatography-mass spectrometry for the determination oforganophosphate pesticides in environmental water samplesand winesrdquo Journal of Separation Science vol 35 no 18 pp2422ndash2429 2012

[32] H Liu X Y Wei J H Li T Li and F Wang ldquoReview of ionicliquids recyclingrdquo Journal of Cellulose Science Technology vol21 pp 63ndash69 2013

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

International Journal ofInternational Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal ofInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

10 Journal of Chemistry

[24] W Kang and J Wang ldquoIn vitro antioxidant properties andin vivo lowering blood lipid of Forsythia suspense leavesrdquoMedicinal Chemistry Research vol 19 no 7 pp 617ndash628 2010

[25] C F Poole and S K Poole ldquoExtraction of organic compoundswith room temperature ionic liquidsrdquo Journal of Chromatogra-phy A vol 1217 no 16 pp 2268ndash2286 2010

[26] Z Guo B Lue K Thomasen A S Meyer and X Xu ldquoPre-dictions of flavonoid solubility in ionic liquids by COSMO-RSexperimental verification structural elucidation and solvationcharacterizationrdquo Green Chemistry vol 9 no 12 pp 1362ndash13732007

[27] S Y Dong Q Hu J Li Y L Fu Y Q Xing and T L HuangldquoIonic liquid-based microwave-assisted extraction followedhigh-performance liquid chromatography for the simultaneousdetermination of five organic oxygen pesticides in soil samplesrdquoChinese Journal of Analysis Laboratory vol 32 pp 48ndash52 2013

[28] H Wu M Chen Y Fan F Elsebaei and Y Zhu ldquoDeter-mination of rutin and quercetin in Chinese herbal medicineby ionic liquid-based pressurized liquid extraction-liquidchromatography- chemiluminescence detectionrdquo Talanta vol88 pp 222ndash229 2012

[29] R Jin L Fan and X An ldquoIonic liquid-assisted extraction ofpaeonol from cynanchum paniculatumrdquo Chromatographia vol73 no 7-8 pp 787ndash792 2011

[30] P Liang F Wang and Q Wan ldquoIonic liquid-based ultrasound-assisted emulsification microextraction coupled with high per-formance liquid chromatography for the determination of fourfungicides in environmental water samplesrdquo Talanta vol 105pp 57ndash62 2013

[31] C K Zacharis C Christophoridis and K Fytianos ldquoVortex-assisted liquid-liquid microextraction combined with gaschromatography-mass spectrometry for the determination oforganophosphate pesticides in environmental water samplesand winesrdquo Journal of Separation Science vol 35 no 18 pp2422ndash2429 2012

[32] H Liu X Y Wei J H Li T Li and F Wang ldquoReview of ionicliquids recyclingrdquo Journal of Cellulose Science Technology vol21 pp 63ndash69 2013

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

International Journal ofInternational Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal ofInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

International Journal ofInternational Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal ofInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of