Rev. bras. farmacogn. vol.26 número5

(1)

w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

A

validated

and

densitometric

HPTLC

method

for

the

simultaneous

quantification

of

reserpine

and

ajmalicine

in

Rauvolfia

serpentina

and

Rauvolfia

tetraphylla

Devendra

Kumar

Pandey

a,∗

_,

_Radha

a

_,

_Abhijit

_Dey

b

a_Department_of_{Biotechnology,}_Lovely_Professional_University,_Phagwara,_Punjab,_India

b_Department_of_Biological_Sciences,_Presidency_University_(Erstwhile_Presidency_College),_Kolkata,_India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received8December2015

Accepted11April2016

Availableonline28May2016

Keywords:

Highperformancethinlayer

chromatography Reserpine Ajmalicine

R.serpentinaandR.tetraphylla

a

b

s

t

r

a

c

t

Highperformancethinlayerchromatographicmethod(HPTLC)hasbeendevelopedforthequantification ofreserpineandajmalicineinrootpartoftwodifferentpopulationofRauvolfiaserpentina(L.)Benth. exKurzandRauvolfiatetraphyllaL.,Apocynaceae,collectedfromPunjabandUttarakhand.HPTLCof methanolicextractofrootcontainingindolealkaloids,i.e.,reserpineandajmalicine,wasperformedon TLCSilicagel60F254 (10cm×10cm)plateswithtoluene:ethylacetate:formicacid(7:2:1),asmobile phase.Quantificationofthereserpineandajmalicinewasperformedintheabsorption–reflectionmode at268nm.Therecoveryofreserpineandajmalicinewere99.3and98.7%respectively.Thecalibration curveswerelinearforboththereserpineandajmalicine,intherangeof200–1200ng.HPTLCdensitometry hasbeenperformedfortheestimationofreserpineandajmalicineinrootpartofR.serpentinaandR. tetraphyllaforthefirsttime.Themethodissimple,rapidandcosteffectiveandcanbeusedforroutine analysisofajmalicineandreserpineindifferentRauvolfiaspeciesaswellasforqualitycontrolofherbal drugscontainingRauvolfiaspecies.

Introduction

Indole alkaloids are the bioactive compounds derived from plantswhichpossessanarrayofpharmacologicalpropertiessuch asanticancer(Wangetal.,2015;Zhu etal.,2015), antimalarial (Chierrito etal.,2014), antimicrobial(Cheenprachaet al.,2014) andcerebroprotective(Biradaretal.,2013)properties.Themajor bioactive alkaloids reported from various Rauvolfia species are reserpine,reserpiline, ajmaline,ajamalacine, rauvolfinine, serpi-nine,serpentine,serpentinine,yohimbine,vomilenine,picrinine, vinorine,norseredamine,seredamine(Stöckigtetal.,1981;Batista etal.,1996;Duezetal.,1986;DeBruynetal.,1989;Katoetal., 2002;Binduetal.,2014).Reserpineandajmalicineareconsidered astwomajorindolealkaloidsfromvariousRauvolfiaspecies. Reser-pineisacommonalkaloidknowntodepressthecentralnervous systemandtolowerbloodpressure(Faisaletal.,2005).Reserpine hasbeenreportedforantihypertensive(ShamonandPerez,2009), neuroprotective(Aryaetal.,2009)andanticancer(Abdelfatahand Efferth,2015)properties whereas ajmalicine hasbeenassessed

∗ Correspondingauthor.

E-mail:devendra.15673@lpu.co.in(D.K.Pandey).

for central depressant and adrenergic blocking (Bhargava and Borison,1957),antihypertensiveandcytotoxic(Fernández-Pérez etal.,2013)activities.

GenusRauvolfia,belongingtothe familyApocynaceae, com-prisesaround80specieswhicharedistributedintropicalclimatic conditions.Traditionally, R.serpentina(L.) Benth.ex Kurz, com-monly known as Sarpagandha, wasreported against snakebite, insomnia,melancholia,schizophreniaormoreviolentmental dis-orders,diarrhea,dysentery,choleraandcolic,scabies,malaria,eye inflammation,etc.(DeyandDe,2010,2012).R.serpentinahasan economicalimportanceandtherootpartoftheplantisusedin manyAyurvedicpolyherbalformationsi.rsarpagandhavati. Rau-volfiatetraphyllaL.popularlyknownas“devilpepper”or“bestill tree”isanendangeredwoodyshrubnativeintropicalAmericas (Faisaletal.,2013).EthnomedicinalimportanceofR.tetraphylla wasfoundintermsofitsuseagainstsnakebite,tostimulate uter-inecontractionandtofacilitatedifficultchildbirthcases(Sarma etal.,1999;DeyandDe,2012).Moreover,R.serpentinahasbeen reportedforpharmacologicalpropertiessuchasantbacterial, anti-inflammatoryandcytotoxicity(DeyandDe,2010).R.tetraphylla has also been reported to possess antipsychotic (Gupta et al., 2012a), antibacterialactivityandanti-inflammatory (GangaRao etal.,2012)properties.

http://dx.doi.org/10.1016/j.bjp.2016.04.002

(2)

EarlierRauvolfiaalkaloidswereanalyzedbyquantitativethin layer chromatography (Habib and Court, 1971; Nguyen and Nikolova,1989).HPLCandTLCidentification,estimationand sep-arationofindolealkaloidfromR.serpentinaandR.vomitoriawas alsoachieved(Cieri,1983;Klyushnichenkoetal.,1994).Reserpine andrescinnamineinR.serpentinapreparationswasdetectedby liquidchromatographywithfluorescence(Cieri,1987).Recently, HPLC-UV and GC–MS were applied to determine indole alka-loidsandrelatedcompoundsinR.verticillata(Hongetal.,2013). In addition reserpine, ajmaline, and ajmalicine in R. serpentina weredeterminedbyreversed-phaseHPLC(Srivastavaetal.,2006). HPTLC,HPLC,anddensitometrywereusedfortheseparation of differentindolealkaloids fromR.serpentina roots (Guptaet al., 2006).Quantitativedensitometricdeterminationofreserpineand ajmalineby HPTLC wasperformed in R. vomitoria (Katiˇc et al., 1980).However,thereisnoHPTLCreportonsimultaneous quan-tificationofreserpineand ajmalicineindifferentpopulationsof Rauvolfiaspecies.Altitudinalandseasonalmodulationof stigmas-terolwasdeterminedin R.serpentinafollowing HPTLCmethods (Dey and Pandey, 2014a,b). In continuation to our studies on HPTLCprofilingofmedicinalplants (Pandeyet al.,2015,2016), here we report the simultaneous HPTLC determination of two indolealkaloids,ajmalicineandreserpineintwowildpopulationof R.serpentinaandR.tetraphylla.

Materialandmethods

Chemicalsandplantmaterials

Theanalyticalgradechemicalsusedinexperimentwere pur-chased fromE. Merck, India. TheHPTLC plate Silica gel60F254 (10cm×10cm)usedintheexperimentwerepurchasedfromE. Merck(Darmstadt, Germany). The standards, reserpine(A) and ajmalicine(B)werepurchasedfromHimedia,India.

TheplantsamplesofRauvolfiaspecieswascollectedfrom differ-entareasofPhagwara(Punjab)andDehradun(Uttarakhand)inthe monthsofSeptember–October2014.Theplantmaterialcollected fromdifferentplaceswasauthenticatedonthebasisof morpholog-icalcharactersbyTaxonomistintheDepartmentofBotany,Lovely ProfessionalUniversity,Phagwara(Punjab).Avoucherspecimens (VoucherNo.121214and151214ofR.serpentina(L.)Benth.exKurz andR.tetraphyllaL.,respectively)weredepositedatthe Depart-mentofBiotechnology,LovelyProfessionalUniversityfor future reference.

Preparationofsamplesolution

Theair dried (25–30◦_C) _root_parts _of _Rauvolfia _species ₍₁_g)

were extracted thrice with 20ml of methanol for 45min by refluxmethodat70◦_C_in_temperature_controlled_water_bath._The

methanolicextractsweretransferredinconicalflaskandwere con-centratedandre-dissolvedin1mlofmethanol.

Preparationofstandardsolutions

Stocksolutionsofreserpineandajmalicine(1mgml−1₎_were preparedbydissolvingtheajmalicineandreserpineinmethanol respectively,and differentamounts(2,4,6, 8,10 and12␮l) of thesewereloadedtoaTLCplate,usinganLinomatapplicatorV forpreparingcalibrationcurves.

Chromatography

ACAMAGHPTLCscannerequippedwithanautomatic Linomat-V automatic sample applicator, TLC scanner III, and integrated

software WINCATS version:1.4.4.6337 wasused for the analy-sisofindolealkaloidsindifferentsamples.Thestationaryphase waspre-coatedsilicagel HPTLC60F254 (10cm×10cm)plateof 0.20mmlayerthicknessusedforthequantificationofreserpineand ajmalicineinRauvolfiaspecies.Theplantsamplesandthestandards wereloadedwiththehelpofLinomatapplicatorVontheTLCplate at8mmwidebandswithconstantapplicationrateof100nls−1 underaflowofN2gas.TheloadingofthesamplesontheTLCplate wasdonebykeepingspaceof15mmfromthebottomand15mm fromtheside,andthespacebetweentwospotswasmaintained 14.4mmoftheplate.

Detectionandestimationofreserpineandajmalicine

The TLC plate was kept in a Camag twin trough chamber (10cm×10cm),whichwaspre-saturatedwith25mlmobilephase withtoluene:ethylacetate:formicacid(7:2:1)for30min,atroom temperature(28±2◦_C)_and₅₅_±_5%_relative_humidity._The_length

of the chromatogram run was 80mm from the base and the TLCplatewas driedby usingan air dryer,in a wooden cham-ber.Quantitative evaluation of theplate wasperformedin the absorption–reflectionmodeat268nm,slitwidth4mm×0.30mm, dataresolution100␮mstep−1andscanningspeed20mms−1.The radiationusedintheanalysiswasdeuteriumandtungstenlamp. Eachanalysiswascarriedoutintriplicate.

ValidationofHPTLCdensitometrymethod

Linearity

Stocksolutionsofreserpineandajmalicine werepreparedin methanolanddifferentamounts(2,4,6,8,10and12␮l)ofthese were loaded onto a TLC plate, using Linomat applicator V for preparingsixpointscalibrationcurves.Theregressionequationand correlationcoefficientwerefromcalibrationcurves,forreserpine, Y=538+431.15Xand0.991andforajmalicine,Y=−108.53+370X and0.994(Table1andFig.1).

Limitsofdetectionandquantification

Thelimitofdetection(LOD)andlimitofquantification(LOQ) wasdeterminedbyloadingtheblankmethanolontheTLCplate followingthemethodasexplainedbefore.Thesignal-to-noiseratio wasdetermined as 3:1 and 10:1considered for LOD and LOQ, respectively.TheLODandLOQforthereserpinewere60ngand 180ngrespectively.TheLODandLOQfortheajmalicinewere50ng and150ngrespectively.

Accuracy

Tothepre-analyzedsample,50and100␮geachofreserpineand ajmalicinewereaddedinthe100mgrootpowderofhighyieldingR. serpentinarootsamples(reserpine180␮gandajmalicine170␮g) andthemixturewasanalyzedbytheproposedmethod.The exper-imentwasconductedintriplicatetocheckrecoveryandaccuracy

Table1

Reserpineandajmalicinecontent(ng␮l−1)foundindifferentpopulationofplant

RauvolfiaserpentinaandR.tetraphyllabyHPTLCmethod(n=3) collectedfrom

Uttarakhand(RS-1andRT-1)andPunjab(RS-2andRT-2).

Sample Averagereserpine (%drywt.)

%CV Average ajmalicine

(%drywt.)CV

RS-1 0.18 1.46 0.17 1.55 RS-2 0.17 1.83 0.16 1.68 RT-1 0.16 2.42 0.15 2.67 RT-2 0.15 2.01 0.14 3.87

(3)

100.0

[AU]

80.0

70.0

60.0

50.0

40.0

30.0

20.0

10.0

0.0

200.0 300.0 400.0 500.0 [nm] 700.0

200.0 300.0 400.0 500.0 [nm] 700.0 100.0

[AU]

80.0

70.0

60.0

50.0

40.0

30.0

20.0

10.0

0.0

100.0

[AU]

80.0

70.0

60.0

50.0

40.0

30.0

20.0

10.0

0.0

100.0

[AU]

80.0

70.0

60.0

50.0

40.0

30.0

20.0

10.0

0.0

A

B

Fig.1. (A)OverlayspectraofReserpineinallthetrack.(B)Overlayspectraof ajmalicineinallthetrack.

ofthesystem.Therecoveryforreserpinewasfoundtobe99.3%and 99.6%andajmalicinewere99.2%and99.5%,respectively.

Precision

Sixsamplesofsameconcentrationseachfromthestock solu-tionsofreserpineandajmalicineandspottedontheHPTLCsilica gel60F254plateandanalyzedwiththeproposedmethodandwere

expressedin%CVforthesystemprecision.Formethodprecisionsix samplesofsameconcentrationswereappliedonaHPTLCplateand analyzedbytheproposedmethodtodeterminevariationexpressed in%CV.Theresultsforreserpineandajmalicinewerefoundtobe 1.28%and1.56%,respectively.

Resultsanddiscussion

Variouscompositionsofmobilephasesweretestedtogetbetter resolutionofreserpineandajmalicine.Theresolutionofreserpine

1 2 3 4 5 6

Fig.3.Track:1–6representsthefingerprintingchromatogram:firsttwoare Rau-volfiaserpentina,rootsamplescollectedfrom(1)Dehradun(Uttrakhand)and(2) (Punjab),thirdandfourthrepresentsthestandardofreserpine(3)andajmalicine (4),FifthandsixthshowingR.tetraphyllarootsamplescollectedfromtwostates Dehradun(5)andJalandhar(6).

and ajmalicine, with symmetricaland reproducible peaks, was achieved by using mobile phase consisting of toluene:ethyl acetate:formic acid (7:2:1). Peaks corresponding to reserpine and ajmalicine were recorded at Rf 0.69 and 0.85, respectively

(Figs. 2 and 3). The methanolic extract of Rauvolfia species, when subjected to HPTLC, showed the presence of reserpine and ajmalicine peaksin allthesamples.Comparisonof theUV spectral characteristic of the peaks for standards of reserpine andajmalicine,revealedtheidentityofreserpineandajmalicine presentinallsamples.Thecalibrationcurveswerelinearinthe range of 200, 400, 600, 800, 1000, 1200ng for reserpine and ajmalicine.Peakpuritytestsofreserpineandajmalicinewerealso conductedbycomparingspectraofstandardwithreserpineand ajmalicineinRauvolfiaspeciessampletrack(Figs.1,3and4).The highercontents(0.17%and0.18%ofajmalicineandreserpine)in roots ofR. serpentinaand R.tetraphylla (0.16%and0.15%) were recorded in roots of plant samples collected from Dehradun (Uttarakhand) than compared withtheplant samplescollected fromJalandhar(Punjab)ascomparedtoroots(Table1).

Earlier,hexane,chloroform,methanol,andwaterextractsofR. serpentinarootswerereportedlycontainedmarkerindolealkaloids, ajmaline,ajmalicine,andreserpine.Chloroformwascitedasthe mostpotentsolventforextractionofthesealkaloidswhereas defat-tingwithhexanewasindicatedforthelossofthealkaloids(Gupta

1000 [AU] 800 700 600 500 400 300 200 100 0 0.00

0.10 0.20

0.30

0.40 _0.50 0.60

0.70 0.80

1.00 0 10 20

3040 50 60

7080

[Rf]

[mm] 100 Reserpine

Ajmalicine

(4)

500

400

300

200

100

0

400

300

200

100

0

0.00 0.20 0.40 0.60 0.80

50 100 150 200 250 300 350 100 200 300 400 500 600 0

1

2

3 4 5

6 7

9

11

2

1 3

3 2

3 4 5

6 9

Reserpine @ 268 nm

Reserpine @ 268 nm Ajmalicine @ 268

Ajmalicine @ 268

A

B

C

D

Reserpine

Ajmalicine 1

Fig.4.(A)StandardAjmalicine,(B)StandardReserpine,(C)ReserpineandAjmalicineinrootpartofRauvolfiatetraphyllacollectedfromDehradun,(D)ReserpineandAjmalicine

inrootpartofRauvolfiaserpentinacollectedfromDehradun.

etal.,2006).Extractionefficiencyofthetargetedindolealakaloids with“green”solventswasstudiedusingconventional, ultrasonica-tionandmicrowavetechniques(Guptaetal.,2012b).Inthepresent study we could observe the maximum contents of ajmalicine andreserpine in roots.Tissue specificmodulationof secondary metaboliteshasbeenreportedinvariousbotanicals(Williamsand Ellis,1989;HartmannandDierich,1998;Baranskaetal.,2006;Lim etal.,2010;Deyetal.,2016)which mightbeimplicatedtothe biosynthesisandaccumulationofspecificmetabolitesdepending onpresenceofspecificenzymes(Facchini,2001).

Conclusion

HPTLC densitometry is a rapid, reproducible, accurate, and selectivealternativetoHPLCfortheseparationoftheajmalicine andreserpineinR.serpentinaandR.tetraphylla.Further,rootsof R.serpentinaareenrichedinthedesiredconstituentsandcanbe usedforpreparationsofpolyherbalformulations.Itwasfoundthat thequantityofindolealkaloidsinRauvolfiatetraphyllawasatparto R.serpentinaandcanbeusedinthepreparationofherbal formula-tion.TheadvantageofHPTLCisthehighsamplethroughputwhich

resultsfromthesmallamountofsamplepreparationandlesseruse ofsolventinseparationofthecompoundsandthesimultaneous quantificationofseveralsamples.

Authors’contributions

Thecollectionandcultivationoftheplantsamplesweredoneby RadhawhiletheidentificationandHPTLCquantificationwasdone byDKPandAD.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

(5)

References

Arya,U.,Dwivedi,H.,Subramaniam,J.R.,2009.Reserpineamelioratesabetatoxicity

intheAlzheimer’sdiseasemodelinCaenorhabditiselegans.Exp.Gerontol.44, 462–466.

Abdelfatah,S.A.,Efferth,T.,2015. Cytotoxicityoftheindolealkaloidreserpine

fromRauwolfiaserpentinaagainstdrug-resistanttumorcells.Phytomedicine22, 308–318.

Baranska,M.,Baranski,R.,Schulz,H.,Nothnagel,T.,2006.Tissue-specific

accumula-tionofcarotenoidsincarrotroots.Planta224,1028–1037.

Batista,C.V.F.,Schripsema,J.,Verpoorte,R.,Rech,S.B.,Henriques,A.T.,1996.Indole

alkaloidsfromRauwolfiasellowii.Phytochemistry41,969–973.

Bhargava,K.P.,Borison,H.L.,1957.ComparativeeffectsofvariousRauwolfia

alka-loidsoncentrallyevokedvasopressorresponses.J.Pharmacol.Exp.Ther.119, 395–405.

Bindu,S.,Rameshkumar,K.B.,Kumar,B.,Singh,A.,Anilkumar,C.,2014.Distribution

ofreserpineinRauvolfiaspeciesfromIndia–HPTLCandLC–MSstudies.Ind. CropsProd.62,430–436.

Biradar,S.M.,Joshi,H.,Tarak,K.C.,2013.Cerebroprotectiveeffectofisolatedharmine

alkaloidsextractsofseedsofPeganumharmalaL.onsodiumnitrite-induced hypoxiaandethanol-inducedneurodegenerationinyoungmice.Pak.J.Biol.Sci. 16,1687–1697.

Chierrito,T.P.,Aguiar,A.C.,deAndrade,I.M.,Ceravolo, I.P.,Gonc¸alves,R.A.,de

Oliveira,A.J.,Krettli,A.U.,2014.Anti-malarialactivityofindolealkaloidsisolated

fromAspidospermaolivaceum.Malar.J.14,142.

Cheenpracha,S.,Raksat,A.,Ritthiwigrom,T.,Laphookhieo,S.,2014.Monoterpene

indolealkaloidsfrom thetwigsofKopsia arborea. Nat.Prod.Commun. 9, 1441–1443.

Cieri,U.R.,1983.IdentificationandestimationofthealkaloidsofRauvolfia

ser-pentinabyhigh-performanceliquidchromatography.J.Assoc.Off.Anal.Chem. 66,867–873.

Cieri,U.R.,1987.DeterminationofreserpineandrescinnamineinRauwolfia

ser-pentinapreparationsbyliquidchromatographywithfluorescencedetection. J.Assoc.Off.Anal.Chem.70,540–546.

DeBruyn,A.,Zhang,W.,Budˇeˇsinsk ´y,M.,1989.NMRstudyofthree

heteroyohim-binederivativesfromRauwolfiaserpentina:stereochemicalaspectsofthetwo isomersofreserpilinehydrochloride.Magn.Reson.Chem.27,935–940.

Dey,A.,Pandey,D.K.,2014a.HPTLCdetectionofaltitudinalvariationofthepotential

antiveninstigmasterolindifferentpopulationsofthetropicalethnicantidote Rauvolfiaserpentina.AsianPac.J.Trop.Med.7S1,S540–S545.

Dey,A.,Pandey,D.K.,2014b.HPTLCMethodforQuantitativeEvaluationofSeasonal

VariationofStigmasterolinRauvolfiaserpentina(L).Benth.exKurz.J.Biol.Act. Prod.Natl.4,254–261.

Dey,A.,Mukherjee,S.,De,A.,Pandey,D.K.,2016.Astigmasterolcontainingn-hexane

fractionofRauvolfiaserpentina(L).Benth.exKurz.(Apocynaceae)methanolic extractshowstissuespecificvariationofbiocidalactivityandantioxidation. J.HerbsSpicesMed.Plants22,81–91.

Dey,A.,De,J.N.,2010.Rauvolfiaserpentina(L).Benth.exKurz.–areview.Asian

J.PlantSci.9,285.

Dey,A.,De,J.N.,2012.Anti-snakevenombotanicalsusedbytheethnicgroupsof

PuruliaDistrict,WestBengal,India.J.HerbsSpicesMed.Plants18,152–165.

Duez,P.,Chamart,S.,Vanhaelen,M.,Vanhaelen-Fastré,R.,Hanocq, M.,Molle,

L.,1986.Comparisonbetweenhigh-performancethin-layer

chromatography-densitometryandhigh-performanceliquidchromatographyforthe determina-tionofajmaline,reserpineandrescinnamineinRauwolfiavomitoriarootbark. J.Chromatogr.A356,334–340.

Faisal,M.,Ahmad,N.,Anis,M.,2005.ShootmultiplicationinRauvolfiatetraphyllaL.

usingthidiazuron.PlantCellTiss.Org.80,187–190.

Faisal,M.,Alatar,A.A.,Hegazy,A.K.,2013.Molecularandbiochemical

characteri-zationinRauvolfiatetraphyllaplantletsgrownfromsyntheticseedsfollowing invitrocoldstorage.Appl.Biochem.Biotechnol.169,408–417.

Facchini,P.J.,2001.Alkaloidbiosynthesisinplants:biochemistry,cellbiology,

molecularregulation,andmetabolicengineeringapplications.Annu.Rev.Plant Physiol.PlantMol.Biol.52,29–66.

Fernández-Pérez,F.,Almagro,L.,Pedre ˜no,M.A.,GómezRos,L.V.,2013.Synergistic

andcytotoxicactionofindolealkaloidsproducedfromelicitedcellculturesof Catharanthusroseus.Pharm.Biol.51,304–310.

GangaRao,B.,UmamaheswaraRao,P.,SambasivaRao,E.,MallikarjunaRao,T.,

Praneeth,D.V.S.,2012.Evaluationofin-vitroantibacterialactivityand

anti-inflammatoryactivityfordifferentextractsofRauvolfiatetraphyllaL.rootbark. AsianPac.J.Trop.Biomed.2,818–821.

Gupta,S.,Khanna,V.K.,Maurya,A.,Bawankule,D.U.,Shukla,R.K.,Pal,A.,Srivastava,

S.K.,2012a.Bioactivityguidedisolationofantipsychoticconstituentsfromthe

leavesofRauwolfiatetraphyllaL.Fitoterapia83,1092–1099.

Gupta,S.,Shanker,K.,Srivastava,S.K.,2012b.HPTLCmethodforthe

simulta-neousdeterminationoffourindolealkaloidsinRauwolfiatetraphylla:astudyof organic/greensolventandcontinuous/pulsesonication.J.Pharm.Biomed.Anal. 66,33–39.

Gupta,M.,Srivastava,A.,Tripathi,A.,Misra,H.,Verma,R.,2006.UseofHPTLC,HPLC,

anddensitometryforqualitativeseparationofindolealkaloidsfromRauvolfia serpentinaroots.J.PlanarChromatogr.-Mod.TLC19,282–287.

Habib,M.S.,Court,W.E.,1971.EstimationofRauvolfiaalkaloidsbyquantitativethin

layerchromatography.J.Pharm.Pharmacol.2–3(Suppl.),2305.

Hartmann,T.,Dierich,B.,1998.Chemicaldiversityandvariationofpyrrolizidine

alkaloidsofthesenecioninetype:biologicalneedorcoincidence?Planta206, 443–451.

Hong,B.,Li,W.,Song,A.,Zhao,C.,2013.Determinationofindolealkaloidsand

highlyvolatilecompoundsinRauvolfiaverticillatabyHPLC-UVandGC–MS.J. Chromatogr.Sci.51,926–930.

Katiˇc,M.,Kuˇsan,E.,Proˇsek,M.,Bano,M.,1980.Quantitativedensitometric

determi-nationofreserpineandajmalineinRauwolfiavomitoriabyHPTLC.J.HighResolut. Chromatogr.3,149–150.

Kato,L.,Braga,R.M.,Koch,I.,Kinoshita,L.S.,2002.IndolealkaloidsfromRauvolfia

bahiensisA.DC.(Apocynaceae).Phytochemistry60,315–320.

Klyushnichenko,V.Y.,Yakimov,S.A.,Bychkova,T.P.,Syagailo,Y.V.,Kuzovkiha,I.N.,

Bulfson,A.N., Miroshnikov, A.I.,1994. HPLCand TLC separation of indole

alkaloid from Rauvolfia serpentina and R. vomitoria. Khim. Farm. Zh. 28, 58–61.

Lim,W.H.,Goodger,J.Q.,Field,A.R.,Holtum,J.A.,Woodrow,I.E.,2010.Huperzine

alkaloidsfromAustralasianandsoutheastAsian.Pharm.Biol.48,1073–1078.

Nguyen,K.C.,Nikolova,I.G.,1989.Quantitativedeterminationofalkaloidinrootbark

ofsomespeciesofRauvolfiaL.bythinlayerchromatography.Rastit.Resur.25, 594–599.

Pandey,D.K.,Parida,S.,Dey,A.,2016.ComparativeHPTLCanalysisofbioactive

markerbarbaloinfrominvitroandnaturallygrownAloevera(L.)Burm.f.Rev. Bras.Farmacogn.26,161–167.

Pandey,D.K.,Shahnawaz,Dey,A.,2015.ComparativeHPTLCanalysisofantioxidant

compoundgallicacidfrominvitroandnaturallygrownSteviarebaudiana.J.Biol. Act.Prod.Natl.5,397–405.

Shamon,S.D.,Perez,M.I.,2009.Bloodpressureloweringefficacyofreserpinefor

primaryhypertension.CochraneDatabaseSyst.Rev.7,CD007655.

Sarma,D.,Sarma,S.,Baruah,A.,1999.Micropropagationandinvitrofloweringof

Rauvolfiatetraphylla;apotentsourceofanti-hypertensiondrugs.PlantaMed. 65,277–278.

Srivastava,A.,Tripathi,A.K.,Pandey,R.,Verma,R.K.,Gupta,M.M.,2006.Quantitative

determinationofreserpine,ajmaline,andajmalicineinRauvolfiaserpentinaby reversed-phasehigh-performanceliquidchromatography.J.Chromatogr.Sci. 44,557–560.

Stöckigt,J.,Pfitzner,A.,Firl,J.,1981.Indolealkaloidsfromcellsuspensioncultures

ofRauwolfiaserpentinaBenth.PlantCellRep.1,36–39.

Wang, H.Y.,Wang, R.X.,Zhao, Y.X.,Liu, K., Wang, F.L.,Sun,J.Y., 2015. Three

newisomericindolealkaloidsfromNaucleaofficinalis.Chem.Biodivers.12, 1256–1262.

Williams,R.D.,Ellis,B.E.,1989.AgeandtissuedistributionofalkaloidsinPapaver

somniferum.Phytochemistry28,2085–2088.

Zhu,W.,Yang,B.,Komatsu,S.,Lu,X.,Li,X.,Tian,J.,2015.Binarystressinducesan