)JOEBXJ1VCMJTIJOH$PSQPSBUJPO ...Artemisia annua L., which belongs to the family Asteraceae...

Research ArticleExtraction of Artemisinin an Active AntimalarialPhytopharmaceutical from Dried Leaves of Artemisia annua LUsing Microwaves and a Validated HPTLC-Visible Method forIts Quantitative Determination

Himanshu Misra12 Darshana Mehta1 Bhupendra Kumar Mehta1

and Dharam Chand Jain2

1 Natural Products Research Laboratory School of Studies in Chemistry and Biochemistry Vikram UniversityUjjain Madhya Pradesh 456 010 India

2 Green Technology Department Ipca Laboratories Limited Ratlam Madhya Pradesh 457 002 India

Correspondence should be addressed to Himanshu Misra himanshumisra1rediffmailcomand Dharam Chand Jain dc 52rediffmailcom

Received 23 May 2014 Accepted 7 September 2014 Published 1 October 2014

Academic Editor Qizhen Du

Copyright copy 2014 Himanshu Misra et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A simple rapid precise and accurate high-performance thin-layer chromatographic method coupled with visible densitometricdetection of artemisinin is developed and validated Samples of the dried Artemisia annua leaves were extracted via microwavesusing different solventsThis method shows the advantage of shorter extraction time of artemisinin from leaves under the influenceof electromagnetic radiations Results obtained from microwave-assisted extraction (MAE) were compared with hot soxhletextraction Chromatographic separation of artemisinin from plant extract was performed over silica gel 60 F

254HPTLC plate

using n-hexane ethyl acetate as mobile phase in the ratio of 75 25 vv The plate was developed at room temperature 25plusmn 20∘CArtemisinin separation over thin-layer plate was visualized after postchromatographic derivatization with anisaldehyde-sulphuricacid reagent HPTLC plate was scanned in a CAMAGrsquos TLC scanner 3 at 540 nm Artemisinin responses were found to be linearover a range of 400ndash2800 ng spotminus1 with a correlation coefficient 099754 Limits of detection and quantification were 40 and 80 ngspotminus1 respectively The HPTLC method was validated in terms of system suitability precision accuracy sensitivity (LOD andLOQ) and robustness Additionally calculation of plate efficiency and flow constant were included as components of validationExtracts prepared from different parts of the plant (leaves branches main stem and roots) were analyzed for artemisinin contentin which artemisinin content was found higher in the leaf extract with respect to branches and main stem extracts however noartemisinin was detected in root extractThe developedHPTLC-visiblemethod of artemisinin determination will be very useful forpharmaceutical industries which are involved in monitoring of artemisinin content during different growth stages (in vitro and invivo) of A annua for qualitative and quantitative assessment of final produce prior to commercial-scale processing for assessmentof cost-benefit ratio

1 Introduction

Malaria is a vector-borne infectious disease which affectsapproximately 400 million people every year especially inAfricaThe parasite responsible for fatal malarial infections isPlasmodium falciparum The first effective antimalarial drugto treat this dangerous infection was quinine since thenmalaria has been treated with quinoline-based drugs like

chloroquine mefloquine and pyrimethamine but malariaparasite developed resistance to these drugs [1ndash3]Artemisinin (Figure 1) is a natural product obtained froman aromatic annual herb of Asian and East European originArtemisia annua L which belongs to the family Asteraceae(Compositae) Due to the problem of resistance artemisininand its semisynthetic derivatives artemether arteether andartesunate are considered to be the most effective for the

Hindawi Publishing CorporationChromatography Research InternationalVolume 2014 Article ID 361405 11 pageshttpdxdoiorg1011552014361405

2 Chromatography Research International

H

H H

O

OO

O

O

Figure 1 Structure of artemisinin

treatment of uncomplicated malaria due to Plasmodiumfalciparum (through ACTs) and complicated cerebral malaria(through monotherapy) Central Institute of Medicinal andAromatic Plants (CSIR-CIMAP) Lucknow introducedArtemisia annua in India and developed its agrotechnologyfor commercialized production of active antimalarial drugartemisinin [4ndash8] Artemisinin has received much attentionin the treatment of drug-resistantmalaria in recent years andit is now considered a potential candidate to reduce coccidialinfection in chickens [9 10] Besides antimalarial activityartemisinin and its derivatives also possess several bioact-ivities [11] including antiviral [12] antitumor [13] and anti-cancer [14]

Presently most common commercial sources of artem-isinin are field-grown leaves and flowering tops of A annuathat are subject to seasonal and somatic variation Todaythe search for increased productivity follows two trends thesearch for new species with better yield and a further devel-opment and application of methods for genetic improvement[15] Mannan et al in 2008 [16] described that chemicalsynthesis of artemisinin is known to be tedious and expensiveTherefore search for other Artemisia species containingartemisinin and the plant source such as callus [17] shoot[18] and hairy root cultures [19 20] are also attractive alter-natives Although presence of artemisinin is supposed to bein Artemisia genus there are reports describing presence orproduction of artemisinin in species other than A annua [1016 21ndash27] Presence of artemisinin has been reported in Acina (reported from Indonesia) [21]A sieberi (reported fromIran) [10] A absinthium (reported from Pakistan) [22] Adubia andA indica (reported fromPakistan) [16] andA api-acea and A lancea [23 24] Mannan et al (2010) collected 17different Artemisia species from different hilly locationsin northern Pakistan and screened for the presence ofartemisinin results of this study revealed that artemisininwaspresent in 16 species (A moorcroftiana A vestita A vulgarisA indica A sieversiana A roxburghiana var roxburghianaA roxburghiana var gratae A parviflora A dracunculus vardracunculus A dracunculus var persica A aff TanguticaA annua A absinthium A bushriences A japonica and

A dubia) and in one species (A desertorum) artemisinin wasnot present [25] Suresh et al (2011) detected artemisinin inA abrotanum and A pallens by LCMS method [26] On theother hand Namdeo et al in 2006 [27] stated that the genusArtemisia includes ca 400 species many of these species havebeen screened for the presence of artemisinin but only Aannua and to a lower extentA apiaceawere found to produceartemisinin A annua and A apiacea were known to theChinese in antiquity and since they were easily confused witheach other Recently El-Naggar et al (2013) reported extre-mely high artemisinin concentration (485ndash490 ww) intheir study performed in the Egyptian desert for the firsttime as a new promising cultivating area for pharmaceuticalproduction of artemisinin [28]

A number of analytical methods have been developed fordetection and quantification of artemisinin like thin-layerchromatography (TLC) [29 30] TLC with visible light den-sitometric detection (TLC) [31ndash34] high-performance liquidchromatography with UV detection (HPLC-UV) [35ndash38]HPLCwith electrochemical detection (HPLC-ECD) [39 40]HPLC with evaporative light scattering detection (HPLC-ELSD) [31ndash44] HPLC with peroxyoxalate chemilumines-cence detection (HPLC-PO-CL) [45] HPLCtandem massspectrometric (LCMSMS) method [46] liquid chromatog-raphymass spectrometry (LCMS) [47] high-performancecapillary electrophoresis using self-designed conductivitydetection system [48] gas chromatography with massspectrometric detection (GC-MS) [49] GC with flameionization detection (GC-FID) [50] and enzyme-linkedimmunosorbent assay (ELISA) [51] Recently spectrophoto-metric determination of artemisinin and dihydroartemisininwas reported from India [52]

The TLC method developed by Gupta et al in 1996 [31]is really good but validation parameters were not discussedin detail Later on Bhandari et al in 2005 [34] developed RP-TLC method for simultaneous determination of artemisininartemisinic acid and arteannuin-BThismethod is very goodand well validated but costlier due to higher cost of RP-TLCplates Additionally as per the densitogram figure the base-line separation between artemisinic acid and artemisininwas not achieved So it cannot be utilized for the routineanalysis of artemisinin in industrial scale batch processingAdditionally both of the methods [31 34] are suffering fromlonger sample preparation time

During the active plant ingredient determination prepa-ration of samples is the most time-consuming step(s) forthe sample under test In some cases traditional solid-liquid(soxhlet) extraction of the herb sample performed which isvery time-consuming step and varies from a few hours tosometimes many hours Several methods have been reportedfor the measurement of artemisinin in herbal samples [31ndash34] howevermost of them are either not sufficiently sensitiveand do not offer reliable results or are difficult to applyin routine analysis Therefore advanced methods for extrac-tion and determination of these compounds such as super-critical fluid extraction and chromatography pressurizedsolvent extraction microwave-assisted extraction high-per-formance liquid chromatography coupled to mass spec-trometry or evaporative light scattering detection and its

Chromatography Research International 3

applications to plant material pharmaceutical formulationsand biological fluids have been reviewed by Christen andVeuthey (2001) [53] Recently Hao et al (2002) [54] pre-sented microwave-assisted extraction study of artemisininfromA annua leaves but in this studymicrowave irradiationtime was 12 minutes which is more Additionally number 6extraction solvent oil showed good results but it is notcommonly available solvent for extraction Although study byHao et al is good applications of many commonly availablesolvents were not performed for artemisinin extraction undermicrowave irradiation

Herein we have developed a safe effective low costand fast microwave-assisted extraction (MAE) procedure forartemisinin from dried leaves of Artemisia annua togetherwith an HPTLC method for its rapid and precise analysisunder visible light detection at 540 nm Data obtained frommicrowave-assisted extraction has been compared with thedata obtained from hot soxhlet extractionThis study reportsa faster sample preparation method with a validated proce-dure for determination of artemisinin by HPTLC methodFlow constant and number of theoretical plates (plate effi-ciency) were evaluated and included as parts of validation

2 Material and Methods

21 Plant Material Plant material was grown in our exper-imental field at Ipca Laboratories Limited Ratlam (MPIndia) after transfer of agrotechnology of high yielding Aannua variety ldquoJeevanrakshardquo [55 56] from CIMAP Luc-know IndiaThe leaves of the plant were taken air-dried andpulverized via mechanical grinder to a fine powder

22 Chemicals and Reagents All solvents used in this studywere of analytical grade Precoated silica gel 60 F

254HPTLC

plates were purchased from EMerck (Darmstadt Germany)Artemisinin (control code 103222) was purchased fromWHO Centre for Chemical Reference Substances Stock-holm Sweden (HPLC purity of artemisinin = 996)Anisaldehyde-sulphuric acid reagent was prepared bymixing5mL of anisaldehyde in 500mL of glacial acetic acid in abeaker and stirred continuously over a magnetic stirrer after5 minutes 10mL of concentrated sulphuric acid (98) wasadded slowly to the above solution through side-wall ofbeaker and was stirred continuously for 10 minutes aftercomplete addition of acid Now this reagent is ready forpostchromatographic derivatization of developedTLCplates

23 Apparatus A microwave apparatus with operatingpower 160ndash800 watts was used for MAE Minimal possibleoperation time and power were 10 seconds at 160W (20power) A computerized TLC scanner 3 withwinCATS onlinePlanar Chromatography Manager version 134 (CAMAGSwitzerland) was used for quantitative chromatographic eval-uation of test spots CAMAGrsquos Linomat 5 was utilized fornitrogen gas-assisted and controlled application of samplespots onto HPTLC plate Drying and concentration stepswere performed using rotatory evaporator (Buchi Switzer-land) model number R-205 equipped with an auto-vacuum

16000

14000

12000

10000

8000

6000

4000

2000

000 50000 10000 15000 20000 25000 30000

(AU

)

(ng)

Substance artemisinin 540nm Regression mode linear

r = 099754 sdv = 366Y = 1029962 + 4749 times X

Figure 2 Calibration curve of artemisinin

controller (model number V-800) Ultrasonicator (EnertechMumbai India) was used for homogenizing of test andstandard solutions

24 Preparation of Artemisinin Reference Standard Solutionand Calibration Curve 10mg of artemisinin reference stan-dard was dissolved in 10mL of methanol A portion (5mL)of this solution was taken in a 25mL volumetric flask anddiluted up to mark with methanol to get a standard solutionof 020mg mLminus1 concentration for quantification purpose Acalibration curve was plotted between increasing amounts ofartemisinin per spot and their peak area response A straightline was obtained between 400 and 2800 ng spotminus1 (Figure 2)

25 Stability of Artemisinin Reference Standard Solutionat Room Temperature To study the stability behavior ofartemisinin inworking standard solution it was kept at roomtemperature (room temperature ranges from 21 to 30∘C) fortwo months and this solution was analyzed after 1 month and2 months It was observed that due to degradation arte-misininwas reduced by 393 and 454 after onemonth andtwo months respectively During rainy season artemisinindeteriorates even up to 90 after 2months of storage at roomtemperature (Figure 3)

26 Soxhlet Extraction and Test Sample Preparation 100mgof fine powder was placed into an extraction thimble andextractedwith 170mL of solvent (n-hexane benzene toluenechloroform methylene dichloride ethyl acetate acetonemethanol and acetonitrile) via hot soxhlet extractionmethodfor 6 hours over a water bath The extract was evaporated invacuo and redissolved in 5mL methanol 10 120583L of these testsolutions was used for quantification purpose

27 Microwave-Assisted Extraction and Test Sample Prepa-ration 100mg of fine powder was extracted under the


Track 7 ID STD 10mgmL600

500

400

300

200

100

0minus004 016 036 056 076 096

(AU

)

Peak

1

2

Startposition

Startheight

Maxposition

Maxheight Max

() ()

Endposition

Endheight Area Area Assigned

Artemisinindegradationproduct

013

020

017

026

020

030

07

227

645

3892

226

02

16506

157870

947

9053

Artemisinin degradation

Artemisinin standard

1422

8578


Artemisinin

Rf

RfRfRf (AU) (AU) (AU)(AU) substance

product

Figure 3 Chromatogram showing degradation in artemisininstandard (prepared in methanol) after two months of storage atroom temperature during rainy season

influence of microwave energy using n-hexane benzenetoluene chloroform methylene dichloride ethyl acetateacetone methanol and acetonitrile Extraction parameters(160 watts 120 s 10mL per extraction cycle two extractioncycles and cleanup with 2mL of corresponding solvent at theend of second cycle of extraction) for microwave-assistedextraction (MAE) were the same for every solvent Theextract thus obtained was evaporated in vacuo and redis-solved in 5mL methanol 10 120583L of these test solutions wasused for quantification purpose

28 Optimization of Mobile Phase Mobile phase for thehigh-performance thin-layer chromatographic separation ofartemisinin present in plant extract was optimized usingdifferent binary mixtures (except for methylene dichloride)of few solvents Five different thin-layer plates of size 20 times100mm were taken and parameters remain common forevery plate for example application position and solventfront were 10mm and 95mm above from the base of thin-layer plate respectively Height of mobile phase was fixed to3mm in all applications since we used the same volume inevery case for the calculation of flow constant Other thin-layer parameters were also calculated for every case such as119877119891 time of thin-layer development flow constant and

number of theoretical plates (Table 1) Room temperaturewas250 plusmn 20

∘C at the time of optimization Eluted artemisininspots in all the five cases were subjected to densitometricevaluation to find out base-line separation and peak purity

500

450

400

350

300

250

200

150

100

50

0minus012

(AU

)

008 028 048 068 088

Artemisinin

Rf

Figure 4 HPTLC chromatogram showing artemisinin (119877119891= 028)

separation

status Densitometric evaluation of artemisinin separationover TLC through n-hexane ethyl acetate (75 25 vv) and n-hexane diethyl ether (50 50 vv)was showing peak purity ofup and down slopes not less than 0999 and 0998 respec-tively which is indicative of better separation with nointerfering neighboring peaks (Table 1) We therefore hadselected n-hexane ethyl acetate (75 25 vv) for furtherdevelopment Alternatively as per the peak purity data n-hexane diethyl ether (50 50 vv) may also be used for thepurpose

29 Chromatographic Analysis Thin-layer chromatographywas performed on aluminum backed HPTLC plates (60 F

254

E Merck Germany 200 times 200mm) 10 120583L of test andstandard sample spots were applied via CAMAGrsquos Linomat 5as 60mmwide bands at the height of 10mm from base spotswere simultaneously dried with N

2gas supply onto HPTLC

plates Plates were developed in a CAMAG twin troughchamber of size more than 200 times 200mm at 95mm heightfrom the base using n-hexane ethyl acetate (75 25 vv) asmobile phase Room temperature and relative humidity atthe time of development were 250 plusmn 2∘C and 45 plusmn 2respectively Plates were air-dried for complete evaporation ofmobile phase and derivatized with anisaldehyde-sulphuricacid reagent followed by heating to 110∘C for 10ndash15minutes tovisualize pink-colored spots of artemisinin This plate wasstabilized at room temperature for 30 minutes and scannedusing CAMAGrsquos TLC scanner 3 equipped with winCATSsoftware in absorption-reflection detection mode at 540 nmusing tungsten lamp HPTLC chromatogram of artemisininseparation has been shown in Figure 4 Additionally a TLCphotograph of commercial samples analysis is attached toshow background color and spots of artemisinin after post-chromatographic derivatization (Figure 5)


Table1Screeningof

different

mob

ileph

ases

foro

ptim

izationof

bette

rchrom

atograph

icseparatio

nbetweenartemisininandrelated

impu

rities

Mob

ileph

ase[sin

gleo

rbinary

solventm

ixtures

(vv)]

Timeto

travelto

solventfront

(secon

ds)

Flow

orvelocity

constant

(mm

2sminus1)

Thin-la

yerc

hrom

atograph

icdata

Thin-la

yerp

lated

ata

Densitogram

data

Specificityof

separatio

n(spo

tspectrum

purityie

peak

puritydata)

119877119891

Num

bero

ftheoretic

alplates

(119873)

119877119891

Num

bero

ftheoretic

alplates

(119873)

Correlationvalues

(119903)

Upslo

pea

Dow

nslo

peb

119899-H

exanediethylether

5050

60424

1112

80028

355111

028

76613

0999053

0998898

Methylene

dichlorid

e(100)

75537

89015

019

249333

019

7299

60998846

0997223

119899-H

exaneethylacetate

7525

6995

796

116

028

355111

027

8075

40999333

0999111

119899-H

exaneaceton

e80

20

65539

102595

028

355111

028

77072

0999075

0985880

Chloroform

methano

l95

596022

70025

060

566204

060

98015

0999280

0993213

a Spo

tstartto

spot

middle

b spo

tmiddletospot

end


Figure 5 TLC image of commercial sample analysis for artemisinindetermination

1000

900

800

700

600

500

400

300

200

100

00

1000

900

800

700

600

500

400

300

200

100

00

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000


Spectra comparison purity

(AU

)

(AU

)

Artemisinin intest sample

Wave length (nm)

Figure 6 Overlay ultraviolet absorption spectra (up and downslopes of standard and test artemisinin spots eluted onto TLC) ofartemisinin showing peak purity and 120582max at 540 nm

210 Method Validation

2101 System Suitability The system suitability test is used toensure reproducibility of the equipment The test was carriedout by applying 2120583L of the standard solution of artemisinin(10mgmLminus1) and 10 120583L of the standard solution of artem-isinin (020mg mLminus1) six times each The RSD was found tobe less than 2

2102 Specificity ThedevelopedHPTLC-visiblemethodwasfound to be specific as no interfering peak(s) was foundduring detection of artemisinin Peaks of artemisinin elutedon to HPTLC plate were found to be pure which was alsoevidenced by overlapping ultraviolet absorption spectra of upanddown slopes of the peak as shown in Figure 6 Correlationcoefficients of peak [(start middle) and (middle end)] werenot less than 099 and 099 respectively

2103 Limits of Detection and Quantitation (LOD and LOQ)Limits of detection and quantitation were determined byspotting increasing amounts (10ndash140 ng 119899 = 2) of standardartemisinin solution of concentration 10120583gmLminus1 that is

10 ng 120583Lminus1 (1mg of artemisinin per 100mL) until the averageresponses were 3 and 10 times of noise for LOD and LOQrespectively LOD and LOQ were found to be 40 and80 ng spotminus1 respectively

2104 Linearity Range The linearity of the artemisinin cali-bration plot was evaluated on seven-point scale by spottingincreasing amounts of the artemisinin working standardsolution of 200120583g mLminus1 starting from 400 to 2800 ng spotminus1The method showed good linearity in the given range with acorrelation coefficient of 099754 (1199032 = 099509) and thelinear regression equation was 119884 = 4749119883 + 1030 (119904119889V =366) (Figure 2)

2105 Precision The precision of the method was deter-mined by three replications of each sample The precision(RSD) of the replications was in between 015 and 328which is indicative of a precise method (Tables 3 and 4)

2106 Accuracy (Recovery Study) Accuracy of the methodwas studied using the method of standard addition Stan-dard artemisinin solutions were added to the extract ofthe leaves of A annua and the percentage recovery wasdetermined at three different levels For recovery study a testextract with known artemisinin content was taken now 2mLof this extract was pipetted out in three different test tubesNow 4mL 3mL and 3mL of freshly prepared stan-dard artemisinin solutions of concentrations 10mgmLminus115mgmLminus1 and 20mgmLminus1 were added to test tubes 1 2and 3 respectively The mixed solutions thus prepared werethen evaluated chromatographically The artemisinin con-tents were determined and the percent recoveries were cal-culated The results of recovery analysis are shown inTable 2

2107 Robustness Robustness of themethodwas determinedby performing small variations in mobile phase ratio heightof plate development and TLC tank saturation time Theresults indicated insignificant differences in assay and werethus indicative of a robust method

2108 Calculation of Flow Constant [57ndash59] The flow orvelocity constant (119896) is a measure of the migration rate of thesolvent front It is an important parameter for TLC usersand can be used to calculate for example development timeswith different separation distances provided that the sorbentsolvent system chamber type and temperature remain con-stant The flow constant is given by the following equation

119896 =

1198852

119865

119905

(1)

where 119896 is flow constant (mm2s) 119885119865is distance between the

solvent front and the solvent level (mm) and 119905 is the devel-opment time (seconds) The flow constant as calculated fordifferent mobile phases has been shown in Table 1


Table 2 Recovery study data of artemisinin

Artemisinin presentin test solution(120583gmLminus1)

Amount and volumeof artemisininstandard added

(120583gmLminus1)

Amount ofartemisinin in mixed

solution(120583gmLminus1)

Amount ofartemisinin detected

(120583gmLminus1)Recovery ()

63497 (2mL) 100000 (4mL) 87832 91768 1044863497 (2mL) 150000 (3mL) 115398 113853 986663497 (2mL) 200000 (3mL) 145398 108169 7439

Table 3 Screening of solvents for better extraction of artemisinin through application of microwaves

Extractionsolvent

Soxhlet extractiona Microwave-assisted extraction (MAE)b

mean contentof artemisinin(dry weight

basis)

plusmnSDc RSDd


basis)

plusmnSDc RSDd

119899-Hexane 0772 plusmn0010 130 0637 plusmn0012 188Benzene 0633 plusmn0009 142 0734 plusmn0007 095Toluene 0398 plusmn0002 050 0747 plusmn0005 067Chloroform 0614 plusmn0013 212 0742 plusmn0016 216Methylenedichloride 0580 plusmn0007 121 0671 plusmn0001 015

Ethyl acetate 0425 plusmn0007 165 0693 plusmn0011 159Acetone 0622 plusmn0002 032 0694 plusmn0007 101Methanol 0693 plusmn0007 101 0672 plusmn0012 179Acetonitrile 0488 plusmn0016 328 0698 plusmn0019 272aSoxhlet extractions were performed over a water bathbMAE conditions 100mg 14 mesh 160 watts 120 seconds 10mL times 2 cycles of extraction and cleanup with 2mLcStandard deviationdRelative standard deviation

2109 Calculation of Plate Efficiency (119873) Plate efficiencyalso known as number of theoretical plates was calculatedfor the described method by the following equation [58ndash61]

119873 =16 times 119897 times 119911

1199082 (2)

where 119897 is the position of solvent front from spot applicationposition (inmm) 119911 is the distance traveled by the analyte ontoplate (inmm) and119908 is thewidth of spot (inmm) to the direc-tion of mobile phase The plate efficiency for artemisinin isshown in Table 1

3 Results and Discussion

31 Screening of Solvents Solvents from low polar to highpolar (n-hexane benzene toluene methylene dichloridechloroform ethyl acetate acetone methanol and acetoni-trile) were used for the screening of artemisinin extractionusing hot soxhlet extraction (solvent volume 170mL solventextraction time 6 h) and microwave irradiation-assistedextraction (MAE conditions 160 watts 120 s 10mL perextraction cycle two extraction cycles and cleanupwith 2mLof corresponding solvent at the end of second cycle of extrac-tion) Results of hot soxhlet and microwave-assisted extrac-tions are summarized in Table 3

Soxhlet extraction showed good recovery of artemisininwithn-hexane (artemisinin content 0772) in comparison tochloroformmethylene dichloride ethyl acetate and acetonewe therefore have selected soxhlet extraction with n-hexaneas control for this study and further optimization results willbe compared with control value of artemisinin content thatis 0772 while other high boiling solvents could not yieldthatmuch artemisinin content due to lesser numbers of leach-ing which occurred during 6 h In contrast MAE showedgood recovery of artemisinin with high boiling solventslike benzene (0734) toluene (0747) and acetonitrile(0698) although chloroform also yielded good artemisinincontent (0742) with MAE which is in accordance with thestudy by Hao et al [54] Ethyl acetate acetone and acetoni-trile produced almost similar results with MAE while MAEwith methanol was difficult due to frequent bumping andtherefore a big volume and tall-form vial selected for error-free extraction with methanol through MAE Methanolyielded 0672 artemisininwithMAE and 0693with soxh-let extraction Pulverized leaves float over chloroform dueto lesser bulk density therefore we felt difficulty in transfer ofchloroform extract from extraction vessel to test tube and inrecollection of leaves in extraction vial for second cycle ofextraction and similarly for cleanup step Due to this reasonchloroform was not preferred


Table 4 Finalization of solvent and extraction conditions for microwave-assisted extraction of artemisinin

Extraction solvent Microwave-assisted extraction (MAE)a

mean content of artemisinin (dry wt basis) plusmnStandard deviation RSDb

Benzene 0809 plusmn0017 210Toluene 0816 plusmn0016 196aMAE conditions 100mg 14 mesh 160 watts 120 seconds 10mL times 3 cycles of extraction and cleanup with 2mLbRelative standard deviation

Table 5 Artemisinin content in different parts of whole plant

Plant part mean artemisinin content (dry wt basis) plusmnStandard deviation RSDdagger

Leaves 0776 0008 1031Main stem 0030 0002 6667Branches (excluding main stem) 0421 0008 1900Main stem + side branches (mixed sample) 0239 0002 0837Roots nd mdash mdashdaggerRelative standard deviation ndnot detected

As high boiling solvents like benzene toluene and low-boiling chloroform have given similar results these wereselected for further optimization of MAE conditions but asper previous reports without stabilization chloroform deg-rades to form small amounts of free radicals hydrochloricacid and phosgene which are extremely toxic [62ndash64]

CHCl3+O2997888rarr Co(Cl)

2

Phosgene (3)

The Bhopal tragedy in December 1984 wherein leakageof methyl isocyanate (CH

3ndashN=C=O) and phosgene from

Union Carbide pesticide plant took an unprecedented toll ofover 2000 human lives and thousands of animals is asolemn reminder of the duty of industry and government inprotecting the population from atmospheric pollution Chlo-roform was also banned by Food and Drug Administrationof America in 1976 Thus we have omitted chloroform andchosen benzene and toluene for further optimization ofMAEconditions as both of the solvents yielded the highest artem-isinin content in comparison with other solvents

Hao et al [54] had not applied few commonly availablesolvents like benzene toluene methylene dichloride ethylacetate acetone methanol and acetonitrile whereas we haveapplied all these solvents for the extraction of artemisininunder microwave irradiation (Tables 3 and 4)

32 Finalization of Solvent for Extraction For further opti-mization of MAE conditions we had taken three extractioncycles instead of two with selected solvents after screeningthat is benzene and toluene On increasing number of extrac-tion cycles from two to three benzene and toluene recovered0809 and 0816 of artemisinin respectively (Table 4)This is 479 and 569 more than that of our selectedcontrol artemisinin content (ie 0772) However Hao et al[54] described the best conditions for microwave-assistedextraction of artemisinin as follows number 6 extractionsolvent oil diameter of raw materials less than 0125mmand 12 minutes of microwave irradiation But they recov-ered only 0237 of artemisinin ( recovery of artemisinin

= 02370282 times 100 = 8404) than that present in controlsample (0282 artemisinin) which is approximately 1596less than that present in raw material

So our optimizedMAE conditions were 100mg 14mesh160watts 120 seconds 10mL times 3 cycles of extraction andcleanup with 2mL (Table 4) As shown in Table 3 tolueneextracted the highest artemisinin content from dried Aannua leaves under microwave irradiation Extraction ofartemisinin using benzene as solvent also produces almostsimilar results so as per availability any of the solvents (iebenzene or toluene) may be utilized for the extractionpurpose although our recommendation is toluene which issafer than benzene in terms of carcinogenicity

33 Artemisinin Content in Different Parts of Plant Artem-isinin content in the different parts (leaves branches mainstem and roots) of the plant was determined using soxhletextraction with n-hexane Artemisinin content was found tobe present in the decreasing order of artemisinin (Table 5)

leaves gt side branches gt main stem (4)

However no artemisinin was detected in root extractor artemisinin may be below the detection limit in rootextract of A annua These results are in accordance with theprevious studies [25 65]The leaves from the same plant mayhave different artemisinin contents according to their local-ization along the stem upper leaves contain significantlymore artemisinin thanmiddle and lower ones [56] whichwasalso verified by us using fresh green leaves from top middleand base of the single plant Artemisinin content was thenevaluated using the procedure of fresh leaves extractionreported previously [31] The artemisinin content in plantalso varies during the season Furthermore the genetic basisand environmental factors such as temperature or nutrientavailability further influence the artemisinin content in theplant [66]


4 Conclusion

The developed microwave-assisted extraction and HPTLCmethod are not only rapid but also reliable for analysis ofartemisinin in Artemisia annua This method will be usefulfor monitoring of artemisinin during different stages of plantgrowth and thereby determination of time of harvest in plantvariety development through selection of plants with higherartemisinin content and for routine industrial batch analysisfor evaluation of the commercial value of plant material Themethod also includes calculation of flow constant and thenumber of theoretical plates as components of validationDifferent parts of the plant (leaves branches main stem androots) were analyzed for the artemisinin content but theartemisinin content was found higher in the leaves withrespect to branches and the main stem however artemisininwas not detected in roots

Disclaimer

Readers are advised to use only chemical-safe microwaveapparatus and never to use domestickitchenmicrowave ovenfor any type of chemical processing The authors would notbe responsible for any loss that occurred by the use of themethod described or equipment used in this study

Conflict of Interests

This research paper is the part of the PhD degree of oneof the authors Himanshu Misra and is not intended forany financial gains Additionally no competing interests existamong CAMAG Buchi or any other scientific equipmentcompanies

Acknowledgment

Authors are very thankful to the management of Ipca Labo-ratories Limited for valuable support and facilities during thecourse of work

References

[1] W H Wernsdorfer ldquoEpidemiology of drug resistance inmalariardquo Acta Tropica vol 56 no 2-3 pp 143ndash156 1994

[2] P J De Vries and T K Dien ldquoClinical pharmacology and ther-apeutic potential of artemisinin and its derivatives in the treat-ment of malariardquo Drugs vol 52 no 6 pp 818ndash836 1996

[3] E Gkrania-Klotsas and M L Lever ldquoAn update on malariaprevention diagnosis and treatment for the returning travellerrdquoBlood Reviews vol 21 no 2 pp 73ndash87 2007

[4] A Singh V K Kaul V PMahajan A Singh L NMisra and RS Thakur ldquoIntroduction of Artemisia annua in India and iso-lation of artemisinin a promising antimalarial drugrdquo IndianJournal of Pharmaceutical Sciences vol 48 no 5 pp 137ndash1381986

[5] CIMAP ldquoDevelopment of Agro-technologies for Artemisiaannua for antimalarial drug artemisininrdquo Annual ProjectReport 1986-87 Central Institute for Medicinal and AromaticPlants Lucknow India


[7] S K Gupta P Singh P Bajpai et al ldquoMorphogenetic variationfor artemisinin and volatile oil in Artemisia annuardquo IndustrialCrops and Products vol 16 no 3 pp 217ndash224 2002

[8] S Kumar S K Gupta M M Gupta et al ldquoMethod for max-imization of artemisinin production of the plant Artemisiaannua Lrdquo Indian PatentNoNF-1222000 US 09538 892 20006393763 2002

[9] PCAllen J Lydon andHDDanforth ldquoEffects of componentsofArtemisia annua onCoccidia infections in Chickensrdquo PoultryScience vol 76 no 8 pp 1156ndash1163 1997

[10] H A Arab S Rahbari A Rassouli M H Moslemi and FKhosravirad ldquoDetermination of artemisinin inArtemisia sieberiand anticoccidial effects of the plant extract in broiler chickensrdquoTropical Animal Health and Production vol 38 no 6 pp 497ndash503 2006

[11] R S Bhakuni D C Jain R P Sharma and S Kumar ldquoSec-ondary metabolites of Artemisia annua and their biologicalactivityrdquo Current Science vol 80 no 1 pp 35ndash48 2001

[12] T Efferth M R Romero D G Wolf T Stamminger J J GMarin andMMarschall ldquoThe antiviral activities of artemisininand artesunaterdquo Clinical Infectious Diseases vol 47 no 6 pp804ndash811 2008

[13] A C Beekman P KWierenga H JWoerdenbag et al ldquoArtem-isinin-derived sesquiterpene lactones as potential antitumourcompounds cytotoxic action against bone marrow and tumourcellsrdquo Planta Medica vol 64 no 7 pp 615ndash619 1998

[14] S Oh B J KimN P SinghH Lai and T Sasaki ldquoSynthesis andanti-cancer activity of covalent conjugates of artemisinin and atransferrin-receptor targeting peptiderdquo Cancer Letters vol 274no 1 pp 33ndash39 2009

[15] J A Levy L F Marins and A Sanchez ldquoGene transfer technol-ogy in aquaculturerdquo Hydrobiologia vol 420 no 1ndash3 pp 91ndash942000

[16] A Mannan N Shaheen W Arshad R A Qureshi M Zia andB Mirza ldquoHairy roots induction and artemisinin analysis inArtemisia dubia and Artemisia indicardquo African Journal of Bio-technology vol 7 no 18 pp 3288ndash3292 2008

[17] X C He M Y Zeng G F Li and Z Liang ldquoCallus inductionand regeneration of plantlets fromArtemisia annua and changesof Qinghaosu contentsrdquo Acta Botanica Sinica vol 25 no 1 pp87ndash90 1983

[18] D P Fulzele A T Sipahimalani and M R Heble ldquoTissue cul-tures of Artemisia annua organogenesis and artemisinin pro-ductionrdquo Phytotherapy Research vol 5 no 4 pp 149ndash153 1991

[19] M B Qin G Z Li H C Ye and G F Li ldquoInduction of hairyroot fromArtemisia annuawith Agrobacterium rhizogenes andits culture in vitrordquo Acta Botanica Sinica vol 36 pp 165ndash1701994

[20] A Giri S T Ravindra V Dhingra andM L Narasu ldquoInfluenceof different strains of Agrobacterium rhizogenes on inductionof hairy roots and artemisinin production in Artemisia annuardquoCurrent Science vol 81 no 4 pp 378ndash382 2001

[21] B M Aryanti T M Ermayanti and I Mariska ldquoProduction ofantileukemic agent in untransformed and transformed rootcultures of Artemisia cinardquo Annales Bogorienses vol 8 pp 11ndash16 2001


[22] M Zia and M F Chaudhary ldquoEffect of growth regulators andamino acids on artemisinin production in the callus of Artem-isia absinthiumrdquo Pakistan Journal of Botany vol 39 no 3 pp799ndash805 2007

[23] R X Tan W F Zheng and H Q Tang ldquoBiologically activesubstances from the genus Artemisiardquo Planta Medica vol 64no 4 pp 295ndash302 1998

[24] E Hsu ldquoThe history of qing hao in the Chinese materialmedicardquo Transactions of the Royal Society of Tropical Medicineand Hygiene vol 100 no 6 pp 505ndash508 2006

[25] A Mannan I Ahmed W Arshad et al ldquoSurvey of artemisininproduction by diverse Artemisia species in northern PakistanrdquoMalaria Journal vol 9 no 1 article 310 2010

[26] J Suresh K Mruthunjaya N Paramakrishnan and M NNaganandhini ldquoDetermination of artemisinin in Artemisiaabrotanum and Artemisia pallens by LCMS methodrdquo Interna-tional Journal of Current Pharmaceutical Research vol 3 no 1pp 49ndash52 2011

[27] A G Namdeo K R Mahadik and S S Kadam ldquoAntimalarialdrug-Artemisia annuardquo Pharmacognosy Magazine vol 2 no 6pp 106ndash111 2006

[28] E-M B El-Naggar M Azazi E Svajdlenka and M ZemlickaldquoArtemisinin from minor to major ingredient in Artemisiaannua cultivated in Egyptrdquo Journal of Applied PharmaceuticalScience vol 3 no 8 pp 116ndash123 2013

[29] D L KlaymanA J LinNActon et al ldquoIsolation of artemisinin(qinghaosu) from Artemisia annua growing in the UnitedStatesrdquo Journal of Natural Products vol 47 no 4 pp 715ndash7171984

[30] M Gabriels and J Plaizier-Vercammen ldquoDevelopment of areversed-phase thin-layer chromatographic method for artem-isinin and its derivativesrdquo Journal of Chromatographic Sciencevol 42 no 7 pp 341ndash347 2004

[31] M M Gupta D C Jain R K Verma and A P Gupta ldquoA rapidanalyticalmethod for the estimation of artemisinin inArtemisiaannuardquo Journal ofMedicinal and Aromatic plant sciences vol 18no 1 pp 5ndash6 1996

[32] J A Marchese V L G Rehder and A Sartoratto ldquoQuantifi-cation of artemisinin in Artemisia annua L A comparison ofthin layer chromatography with densitometric detection andhigh performance liquid chromatography with UV detectionrdquoRevista Brasileira de Plantas Medicinais vol 4 pp 81ndash87 2001

[33] MGabriels and J A Plaizier-Vercammen ldquoDensitometric thin-layer chromatographic determination of artemisinin and itslipophilic derivatives artemether and arteetherrdquo Journal ofChromatographic Science vol 41 no 7 pp 359ndash366 2003

[34] P Bhandari A P Gupta B Singh andVK Kaul ldquoSimultaneousdensitometric determination of artemisinin artemisinic acidand arteannuin-B inArtemisia annua using reversed-phase thinlayer chromatographyrdquo Journal of Separation Science vol 28 no17 pp 2288ndash2292 2005

[35] H N ElSohly E M Croom and M A ElSohly ldquoAnalysis ofthe antimalarial sesquiterpene artemisinin in Artemisia annuaby high-performance liquid chromatography (HPLC) withpostcolumn derivatization and ultraviolet detectionrdquo Pharma-ceutical Research vol 4 no 3 pp 258ndash260 1987

[36] B L Singh D V Singh R K Verma M M Gupta D CJain and S Kumar ldquoSimultaneous determination of anti-malarial drugs using reversed phase high-performance liquidchromatography diode-array detectionrdquo Journal of Medicinaland Aromatic Plant Sciences vol 22-23 no 4A-1A pp 17ndash202000

[37] G-P Qian Y-W Yang and Q-L Ren ldquoDetermination ofartemisinin in Artemisia annua L by reversed phase HPLCrdquoJournal of Liquid Chromatography amp Related Technologies vol28 no 5 pp 705ndash712 2005

[38] N Erdemoglu I Orhan M Kartal N Adyguzel and B BanildquoDetermination of artemisinin in selected Artemisia L speciesof Turkey by reversed phase HPLCrdquo Records of Natural Prod-ucts vol 1 no 2-3 pp 36ndash43 2007

[39] N Acton D L Klayman and I J Rollman ldquoReductive elec-trochemical HPLC assay for artemisinin (Qinghaosu)rdquo PlantaMedica vol 51 no 5 pp 445ndash446 1985

[40] J F S Ferreira D J Charles KWood J Janick and J E SimonldquoA comparison of gas chromatography and high performanceliquid chromatography for artemisinin analysesrdquoPhytochemicalAnalysis vol 5 no 3 pp 116ndash120 1994

[41] B A Avery K K Venkatesh and M A Avery ldquoRapid deter-mination of artemisinin and related analogues using high-performance liquid chromatography and an evaporative lightscattering detectorrdquo Journal of Chromatography B BiomedicalSciences and Applications vol 730 no 1 pp 71ndash80 1999

[42] X-R Hu and F-H She ldquoDetermination of artemisinin con-tent in Artemisia annua from different regions by HPLC-evaporative light scattering detectionrdquoXiandai ShipinYuYaopinZazhi vol 16 pp 34ndash36 2006

[43] C A Peng J F S Ferreira and A J Wood ldquoDirect analysis ofartemisinin from Artemisia annua L using high-performanceliquid chromatography with evaporative light scattering detec-tor and gas chromatography with flame ionization detectorrdquoJournal of Chromatography A vol 1133 no 1-2 pp 254ndash2582006

[44] C-Z Liu H-Y Zhou and Y Zhao ldquoAn effective method forfast determination of artemisinin in Artemisia annua L byhigh performance liquid chromatographywith evaporative lightscattering detectionrdquoAnalytica Chimica Acta vol 581 no 2 pp298ndash302 2007

[45] A Amponsaa-Karikari N Kishikawa Y Ohba K NakashimandN Kuroda ldquoDetermination of artemisinin in human serumby high-performance liquid chromatography with on-line UVirradiation and peroxyoxalate chemiluminescence detectionrdquoBiomedical Chromatography vol 20 no 11 pp 1157ndash1162 2006

[46] J Xing H Yan S Zhang G Ren and Y Gao ldquoA high-performance liquid chromatographytandem mass spectrome-try method for the determination of artemisinin in rat plasmardquoRapid Communications in Mass Spectrometry vol 20 no 9 pp1463ndash1468 2006

[47] M Wang C Park Q Wu and J E Simon ldquoAnalysis ofartemisinin in Artemisia annua L by LC-MS with selected ionmonitoringrdquo Journal of Agricultural and FoodChemistry vol 53no 18 pp 7010ndash7013 2005

[48] B Huang and C Yao ldquoDetermination of artemisinin by cap-illary electrophoresis with conductivity detectionrdquo Fenxi CeshiXuebao vol 25 pp 109ndash111 2006

[49] H J Woerdenbag N Pras R Bos J F Visser H Hendriks andT M Malingre ldquoAnalysis of artemisinin and related sesqu-iterpenoids from Artemisia annua by combined gas chrom-atography-mass spectrometryrdquo Phytochemical Analysis vol 2no 5 pp 215ndash219 1991

[50] A T Sipahimalani D P Fulzele and M R Heble ldquoRapidmethod for the detection and determination of artemisinin bygas chromatographyrdquo Journal of ChromatographyA vol 538 no2 pp 452ndash455 1991


[51] J F S Ferreira and J Janick ldquoImmunoquantitative analysis ofartemisinin fromArtemisia annua using polyclonal antibodiesrdquoPhytochemistry vol 41 no 1 pp 97ndash104 1996

[52] T V Sreevidya and B Narayana ldquoSpectrophotometric determi-nation of artemisinin and dihydroartemisininrdquo Indian Journalof Chemical Technology vol 15 no 1 pp 59ndash62 2008

[53] P Christen and J-L Veuthey ldquoNew trends in extraction iden-tification and quantification of artemisinin and its derivativesrdquoCurrent Medicinal Chemistry vol 8 no 15 pp 1827ndash1839 2001

[54] J-Y Hao W Han S-D Huang B-Y Xue and X DengldquoMicrowave-assisted extraction of artemisinin from Artemisiaannua Lrdquo Separation and Purification Technology vol 28 no 3pp 191ndash196 2002

[55] S Kumar S Banerjee S Dwivedi et al ldquoRegistration of Jee-vanraksha and suraksha varieties of the antimalarial medicinalplantArtemisia annuardquo Journal ofMedicinal andAromatic PlantSciences vol 21 no 1 pp 47ndash48 1999

[56] Per Diemer (FAO consultant) WHO and EcoPort version byPeter Griffee (FAO) and Contributor Peter Griffee QA andTEM ldquoArtemisia annua the plant production and processingand medicinal applicationsrdquo 2013 httpwwwmmvorgsitesdefaultfilesuploadsdocsartemisinin2007 event12 Diemer-Griffee Artemisia annuapaperpdf

[57] E Hahn-Deinstrop Applied Thin-Layer Chromatography BestPractice and Avoidance of Mistakes Wiley-VCH Verlag GmbHamp Co KgaA Weinheim Germany 2nd edition 2007

[58] H Misra B K Mehta and D C Jain ldquoComparison of extrac-tion conditions and HPTLCndashUV method for determination ofquinine in different extracts of Cinchona Species barkrdquo Recordsof Natural Products vol 2 no 4 pp 107ndash115 2008

[59] HMisra DMehta B KMehta M Soni and D C Jain ldquoStudyof extraction and HPTLC - UV method for estimation of caf-feine inmarketed tea (Camellia sinensis) granulesrdquo InternationalJournal of Green Pharmacy vol 3 no 1 pp 47ndash51 2009

[60] T Kowalska K Kaczmarski and W Prus ldquoHandbook of thin-layer chromatographyrdquo inTheory and Mechanism ofThin-LayerChromatography J Sherma and B Fried Eds chapter 2 pp 47ndash80 Marcel Dekker New York NY USA 3rd edition 2003

[61] T Halkina and J Sherma ldquoComparative evaluation of theperformance of silica gel TLCplates and irregular and spherical-particle HPTLC platesrdquoActa Chromatographica no 17 pp 261ndash271 2006

[62] Martindale The Extra Pharmacopoeia The PharmaceuticalPress London UK 30th edition 1993

[63] E Turk ldquoPhosgene from chloroformrdquo Chemical amp EngineeringNews vol 76 no 9 p 6 1998

[64] K E Maudens S M R Wille and W E Lambert ldquoTraces ofphosgene in chloroform consequences for extraction of anthra-cyclinesrdquo Journal of Chromatography B vol 848 no 2 pp 384ndash390 2007

[65] X Jiang H ZhangMWang and L Zhang ldquoComparison anal-ysis of different parts and geographical origins from southwest-ern China on artemisinin content of Artemisia annua Lrdquo Cur-rent Trends in Technology and Science vol 2 no 4 pp 293ndash2972013

[66] N Delabays X Simonnet and M Gaudin ldquoThe genetics ofartemisinin content in Artemisia annua L and the breeding ofhigh yielding cultivarsrdquoCurrentMedicinal Chemistry vol 8 no15 pp 1795ndash1801 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


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


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


H

H H

O

OO

O

O

Figure 1 Structure of artemisinin

treatment of uncomplicated malaria due to Plasmodiumfalciparum (through ACTs) and complicated cerebral malaria(through monotherapy) Central Institute of Medicinal andAromatic Plants (CSIR-CIMAP) Lucknow introducedArtemisia annua in India and developed its agrotechnologyfor commercialized production of active antimalarial drugartemisinin [4ndash8] Artemisinin has received much attentionin the treatment of drug-resistantmalaria in recent years andit is now considered a potential candidate to reduce coccidialinfection in chickens [9 10] Besides antimalarial activityartemisinin and its derivatives also possess several bioact-ivities [11] including antiviral [12] antitumor [13] and anti-cancer [14]

Presently most common commercial sources of artem-isinin are field-grown leaves and flowering tops of A annuathat are subject to seasonal and somatic variation Todaythe search for increased productivity follows two trends thesearch for new species with better yield and a further devel-opment and application of methods for genetic improvement[15] Mannan et al in 2008 [16] described that chemicalsynthesis of artemisinin is known to be tedious and expensiveTherefore search for other Artemisia species containingartemisinin and the plant source such as callus [17] shoot[18] and hairy root cultures [19 20] are also attractive alter-natives Although presence of artemisinin is supposed to bein Artemisia genus there are reports describing presence orproduction of artemisinin in species other than A annua [1016 21ndash27] Presence of artemisinin has been reported in Acina (reported from Indonesia) [21]A sieberi (reported fromIran) [10] A absinthium (reported from Pakistan) [22] Adubia andA indica (reported fromPakistan) [16] andA api-acea and A lancea [23 24] Mannan et al (2010) collected 17different Artemisia species from different hilly locationsin northern Pakistan and screened for the presence ofartemisinin results of this study revealed that artemisininwaspresent in 16 species (A moorcroftiana A vestita A vulgarisA indica A sieversiana A roxburghiana var roxburghianaA roxburghiana var gratae A parviflora A dracunculus vardracunculus A dracunculus var persica A aff TanguticaA annua A absinthium A bushriences A japonica and

A dubia) and in one species (A desertorum) artemisinin wasnot present [25] Suresh et al (2011) detected artemisinin inA abrotanum and A pallens by LCMS method [26] On theother hand Namdeo et al in 2006 [27] stated that the genusArtemisia includes ca 400 species many of these species havebeen screened for the presence of artemisinin but only Aannua and to a lower extentA apiaceawere found to produceartemisinin A annua and A apiacea were known to theChinese in antiquity and since they were easily confused witheach other Recently El-Naggar et al (2013) reported extre-mely high artemisinin concentration (485ndash490 ww) intheir study performed in the Egyptian desert for the firsttime as a new promising cultivating area for pharmaceuticalproduction of artemisinin [28]

A number of analytical methods have been developed fordetection and quantification of artemisinin like thin-layerchromatography (TLC) [29 30] TLC with visible light den-sitometric detection (TLC) [31ndash34] high-performance liquidchromatography with UV detection (HPLC-UV) [35ndash38]HPLCwith electrochemical detection (HPLC-ECD) [39 40]HPLC with evaporative light scattering detection (HPLC-ELSD) [31ndash44] HPLC with peroxyoxalate chemilumines-cence detection (HPLC-PO-CL) [45] HPLCtandem massspectrometric (LCMSMS) method [46] liquid chromatog-raphymass spectrometry (LCMS) [47] high-performancecapillary electrophoresis using self-designed conductivitydetection system [48] gas chromatography with massspectrometric detection (GC-MS) [49] GC with flameionization detection (GC-FID) [50] and enzyme-linkedimmunosorbent assay (ELISA) [51] Recently spectrophoto-metric determination of artemisinin and dihydroartemisininwas reported from India [52]

The TLC method developed by Gupta et al in 1996 [31]is really good but validation parameters were not discussedin detail Later on Bhandari et al in 2005 [34] developed RP-TLC method for simultaneous determination of artemisininartemisinic acid and arteannuin-BThismethod is very goodand well validated but costlier due to higher cost of RP-TLCplates Additionally as per the densitogram figure the base-line separation between artemisinic acid and artemisininwas not achieved So it cannot be utilized for the routineanalysis of artemisinin in industrial scale batch processingAdditionally both of the methods [31 34] are suffering fromlonger sample preparation time

During the active plant ingredient determination prepa-ration of samples is the most time-consuming step(s) forthe sample under test In some cases traditional solid-liquid(soxhlet) extraction of the herb sample performed which isvery time-consuming step and varies from a few hours tosometimes many hours Several methods have been reportedfor the measurement of artemisinin in herbal samples [31ndash34] howevermost of them are either not sufficiently sensitiveand do not offer reliable results or are difficult to applyin routine analysis Therefore advanced methods for extrac-tion and determination of these compounds such as super-critical fluid extraction and chromatography pressurizedsolvent extraction microwave-assisted extraction high-per-formance liquid chromatography coupled to mass spec-trometry or evaporative light scattering detection and its







254HPTLC



16000

14000

12000

10000

8000

6000

4000

2000

000 50000 10000 15000 20000 25000 30000

(AU

)

(ng)


r = 099754 sdv = 366Y = 1029962 + 4749 times X









500

400

300

200

100

0minus004 016 036 056 076 096

(AU

)

Peak

1

2

Startposition

Startheight

Maxposition

Maxheight Max

() ()

Endposition



013

020

017

026

020

030

07

227

645

3892

226

02

16506

157870

947

9053



1422

8578


Artemisinin

Rf


product






500

450

400

350

300

250

200

150

100

50

0minus012

(AU

)

008 028 048 068 088

Artemisinin

Rf


separation



254





Table1Screeningof

different

mob

ileph

ases

foro

ptim

izationof

bette

rchrom

atograph

icseparatio


impu

rities

Mob

ileph

ase[sin

gleo

rbinary

solventm

ixtures

(vv)]

Timeto

travelto

solventfront

(secon

ds)

Flow

orvelocity

constant

(mm

2sminus1)

Thin-la

yerc

hrom

atograph

icdata

Thin-la

yerp

lated

ata

Densitogram

data

Specificityof

separatio

n(spo

tspectrum

purityie

peak

puritydata)

119877119891

Num

bero

ftheoretic

alplates

(119873)

119877119891

Num

bero

ftheoretic

alplates

(119873)

Correlationvalues

(119903)

Upslo

pea

Dow

nslo

peb

119899-H

exanediethylether

5050

60424

1112

80028

355111

028

76613

0999053

0998898

Methylene

dichlorid

e(100)

75537

89015

019

249333

019

7299

60998846

0997223

119899-H

exaneethylacetate

7525

6995

796

116

028

355111

027

8075

40999333

0999111

119899-H

exaneaceton

e80

20

65539

102595

028

355111

028

77072

0999075

0985880

Chloroform

methano

l95

596022

70025

060

566204

060

98015

0999280

0993213

a Spo

tstartto

spot

middle

b spo

tmiddletospot

end



1000

900

800

700

600

500

400

300

200

100

00

1000

900

800

700

600

500

400

300

200

100

00

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000



(AU

)

(AU

)


Wave length (nm)












119896 =

1198852

119865

119905

(1)







(120583gmLminus1)





63497 (2mL) 100000 (4mL) 87832 91768 1044863497 (2mL) 150000 (3mL) 115398 113853 986663497 (2mL) 200000 (3mL) 145398 108169 7439


Extractionsolvent



basis)

plusmnSDc RSDd


basis)

plusmnSDc RSDd




119873 =16 times 119897 times 119911

1199082 (2)















2

Phosgene (3)












4 Conclusion


Disclaimer




Acknowledgment


References














































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of







254HPTLC



16000

14000

12000

10000

8000

6000

4000

2000

000 50000 10000 15000 20000 25000 30000

(AU

)

(ng)


r = 099754 sdv = 366Y = 1029962 + 4749 times X









500

400

300

200

100

0minus004 016 036 056 076 096

(AU

)

Peak

1

2

Startposition

Startheight

Maxposition

Maxheight Max

() ()

Endposition



013

020

017

026

020

030

07

227

645

3892

226

02

16506

157870

947

9053



1422

8578


Artemisinin

Rf


product






500

450

400

350

300

250

200

150

100

50

0minus012

(AU

)

008 028 048 068 088

Artemisinin

Rf


separation



254





Table1Screeningof

different

mob

ileph

ases

foro

ptim

izationof

bette

rchrom

atograph

icseparatio


impu

rities

Mob

ileph

ase[sin

gleo

rbinary

solventm

ixtures

(vv)]

Timeto

travelto

solventfront

(secon

ds)

Flow

orvelocity

constant

(mm

2sminus1)

Thin-la

yerc

hrom

atograph

icdata

Thin-la

yerp

lated

ata

Densitogram

data

Specificityof

separatio

n(spo

tspectrum

purityie

peak

puritydata)

119877119891

Num

bero

ftheoretic

alplates

(119873)

119877119891

Num

bero

ftheoretic

alplates

(119873)

Correlationvalues

(119903)

Upslo

pea

Dow

nslo

peb

119899-H

exanediethylether

5050

60424

1112

80028

355111

028

76613

0999053

0998898

Methylene

dichlorid

e(100)

75537

89015

019

249333

019

7299

60998846

0997223

119899-H

exaneethylacetate

7525

6995

796

116

028

355111

027

8075

40999333

0999111

119899-H

exaneaceton

e80

20

65539

102595

028

355111

028

77072

0999075

0985880

Chloroform

methano

l95

596022

70025

060

566204

060

98015

0999280

0993213

a Spo

tstartto

spot

middle

b spo

tmiddletospot

end



1000

900

800

700

600

500

400

300

200

100

00

1000

900

800

700

600

500

400

300

200

100

00

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000



(AU

)

(AU

)


Wave length (nm)












119896 =

1198852

119865

119905

(1)







(120583gmLminus1)





63497 (2mL) 100000 (4mL) 87832 91768 1044863497 (2mL) 150000 (3mL) 115398 113853 986663497 (2mL) 200000 (3mL) 145398 108169 7439


Extractionsolvent



basis)

plusmnSDc RSDd


basis)

plusmnSDc RSDd




119873 =16 times 119897 times 119911

1199082 (2)















2

Phosgene (3)












4 Conclusion


Disclaimer




Acknowledgment


References














































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



500

400

300

200

100

0minus004 016 036 056 076 096

(AU

)

Peak

1

2

Startposition

Startheight

Maxposition

Maxheight Max

() ()

Endposition



013

020

017

026

020

030

07

227

645

3892

226

02

16506

157870

947

9053



1422

8578


Artemisinin

Rf


product






500

450

400

350

300

250

200

150

100

50

0minus012

(AU

)

008 028 048 068 088

Artemisinin

Rf


separation



254





Table1Screeningof

different

mob

ileph

ases

foro

ptim

izationof

bette

rchrom

atograph

icseparatio


impu

rities

Mob

ileph

ase[sin

gleo

rbinary

solventm

ixtures

(vv)]

Timeto

travelto

solventfront

(secon

ds)

Flow

orvelocity

constant

(mm

2sminus1)

Thin-la

yerc

hrom

atograph

icdata

Thin-la

yerp

lated

ata

Densitogram

data

Specificityof

separatio

n(spo

tspectrum

purityie

peak

puritydata)

119877119891

Num

bero

ftheoretic

alplates

(119873)

119877119891

Num

bero

ftheoretic

alplates

(119873)

Correlationvalues

(119903)

Upslo

pea

Dow

nslo

peb

119899-H

exanediethylether

5050

60424

1112

80028

355111

028

76613

0999053

0998898

Methylene

dichlorid

e(100)

75537

89015

019

249333

019

7299

60998846

0997223

119899-H

exaneethylacetate

7525

6995

796

116

028

355111

027

8075

40999333

0999111

119899-H

exaneaceton

e80

20

65539

102595

028

355111

028

77072

0999075

0985880

Chloroform

methano

l95

596022

70025

060

566204

060

98015

0999280

0993213

a Spo

tstartto

spot

middle

b spo

tmiddletospot

end



1000

900

800

700

600

500

400

300

200

100

00

1000

900

800

700

600

500

400

300

200

100

00

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000



(AU

)

(AU

)


Wave length (nm)












119896 =

1198852

119865

119905

(1)







(120583gmLminus1)





63497 (2mL) 100000 (4mL) 87832 91768 1044863497 (2mL) 150000 (3mL) 115398 113853 986663497 (2mL) 200000 (3mL) 145398 108169 7439


Extractionsolvent



basis)

plusmnSDc RSDd


basis)

plusmnSDc RSDd




119873 =16 times 119897 times 119911

1199082 (2)















2

Phosgene (3)












4 Conclusion


Disclaimer




Acknowledgment


References














































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Table1Screeningof

different

mob

ileph

ases

foro

ptim

izationof

bette

rchrom

atograph

icseparatio


impu

rities

Mob

ileph

ase[sin

gleo

rbinary

solventm

ixtures

(vv)]

Timeto

travelto

solventfront

(secon

ds)

Flow

orvelocity

constant

(mm

2sminus1)

Thin-la

yerc

hrom

atograph

icdata

Thin-la

yerp

lated

ata

Densitogram

data

Specificityof

separatio

n(spo

tspectrum

purityie

peak

puritydata)

119877119891

Num

bero

ftheoretic

alplates

(119873)

119877119891

Num

bero

ftheoretic

alplates

(119873)

Correlationvalues

(119903)

Upslo

pea

Dow

nslo

peb

119899-H

exanediethylether

5050

60424

1112

80028

355111

028

76613

0999053

0998898

Methylene

dichlorid

e(100)

75537

89015

019

249333

019

7299

60998846

0997223

119899-H

exaneethylacetate

7525

6995

796

116

028

355111

027

8075

40999333

0999111

119899-H

exaneaceton

e80

20

65539

102595

028

355111

028

77072

0999075

0985880

Chloroform

methano

l95

596022

70025

060

566204

060

98015

0999280

0993213

a Spo

tstartto

spot

middle

b spo

tmiddletospot

end



1000

900

800

700

600

500

400

300

200

100

00

1000

900

800

700

600

500

400

300

200

100

00

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000



(AU

)

(AU

)


Wave length (nm)












119896 =

1198852

119865

119905

(1)







(120583gmLminus1)





63497 (2mL) 100000 (4mL) 87832 91768 1044863497 (2mL) 150000 (3mL) 115398 113853 986663497 (2mL) 200000 (3mL) 145398 108169 7439


Extractionsolvent



basis)

plusmnSDc RSDd


basis)

plusmnSDc RSDd




119873 =16 times 119897 times 119911

1199082 (2)















2

Phosgene (3)












4 Conclusion


Disclaimer




Acknowledgment


References














































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



1000

900

800

700

600

500

400

300

200

100

00

1000

900

800

700

600

500

400

300

200

100

00

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000



(AU

)

(AU

)


Wave length (nm)












119896 =

1198852

119865

119905

(1)







(120583gmLminus1)





63497 (2mL) 100000 (4mL) 87832 91768 1044863497 (2mL) 150000 (3mL) 115398 113853 986663497 (2mL) 200000 (3mL) 145398 108169 7439


Extractionsolvent



basis)

plusmnSDc RSDd


basis)

plusmnSDc RSDd




119873 =16 times 119897 times 119911

1199082 (2)















2

Phosgene (3)












4 Conclusion


Disclaimer




Acknowledgment


References














































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





(120583gmLminus1)





63497 (2mL) 100000 (4mL) 87832 91768 1044863497 (2mL) 150000 (3mL) 115398 113853 986663497 (2mL) 200000 (3mL) 145398 108169 7439


Extractionsolvent



basis)

plusmnSDc RSDd


basis)

plusmnSDc RSDd




119873 =16 times 119897 times 119911

1199082 (2)















2

Phosgene (3)












4 Conclusion


Disclaimer




Acknowledgment


References














































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of











2

Phosgene (3)












4 Conclusion


Disclaimer




Acknowledgment


References














































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


4 Conclusion


Disclaimer




Acknowledgment


References














































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

























































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

)JOEBXJ1VCMJTIJOH$PSQPSBUJPO ...Artemisia annua L., which belongs to the family Asteraceae...

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