Roum. Biotechnol. Lett., Vol. 6, No.

6, 2001, pp 477-484 Copyright 2001 Bucharest University, Center for Research in Enzymology An Biotechnology, Roumanian !ociety of Biological !ciences "rinte in Romania# All right reserve # ORIGINAL PAPERS

Qualitative and Quantitative Determination of the Caffeic Acid and Chlorogenic Acid from Three Chemovarietie of Chrysanthemum balsamita L
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Receive 0 21th !eptem2er, 20013 Accepte 0 14th $ovem2er, 2001

A! tract
9hi p#per pre ent the tu/ie per)orme/ on three chemov#rietie o) the pecie 5hr! #nthemum '#l #mit# L )or the i/enti)ic#tion #n/ /o #$e o) the phen!l prop#ne /eriv#tive uch # c#))eic #ci/ #n/ chloro$enic #ci/ :hich con)er to the e pecie rem#r8#'le ph#rm#ceutic#l propertie ;hep#toprotective, chol#$o$ < choleretic=. U in$ the techni>ue o) thin l#!er chrom#to$r#ph!, /en itometr! #n/ the UV-V1& pectr# :e h#ve )oun/ th#t the three chemov#rietie o) 5hr! #nthemum '#l #mit# cont#in, in the her' pl#nt, c#))eic #ci/ #n/ chloro$enic #ci/ r#n$in$ 'et:een 0.64 up to 1.61? #n/ re pectivel! 0.-8 up to 0.4?. "e#$ord % caffeic aci , 5uantitative an 5ualitative etermination, chr! #nthenum '#l #mit#

Introduction
6he 5hr! #nthemum '#l #mit# L species 7Asteraceae8, costmary, is a her2aceous, perennial plant an it is 9ell :no9n for its chemota;onomic variety# 6he active principle is the volatile oil containe 2y the flo9ers an leaves# 6he volatile oil iffers from one chemovariety to another 2y its main chemical composition component# 69o chemical ta;ons are :no9n in Romania0 5h.'#l #mit# chemov#r c#rvon# 79hich contains a2out <0= carvona in the volatile oil8 an 5h.'#l #mit# chemov#r c#mphor# 79hich contains a2out >1= camphor in the volatile oil8# 6he thir chemical ta;on 2rought from 6he Botanical %ar ens of /ishinev has alrea y 2een acclimatize at the 'e icinal "lants &a2oratory in Brasov ?1,2@# +f 9e are to ta:e into account the main component present in the volatile oil of this chemovariety 9e can call it 7using the similarity 9ith the others8 5h.'#l #mit# chemov#r. thu6on# 79hich contains a2out A0= thuBona8#

E&'erimental
Chemical and (aterial 6he her2a of the 5hr! #nthemum '#l #mit# v#r. c#m)or# 7C2 camf#83 5hr! #nthemum '#l #mit# v#r. c#rvon# 7C2 carv#8 an 5hr! #nthemum '#l #mit# v#r. thu6on# 7C2 thuB#8 9ere o2taine from the 'e icinal "lants Research &a2oratory Brasov#
A44

A$%E&A '(RCU&E!CU, *A$+E&A ,A$%A$U, -&A, $E&+ . /+$%A

6he analytical purity methanol 9as use for e;traction an as solvent for stan ar solutions# 6he caffeic aci 9as provi e 2y Roth 7/arlsruhe, %ermany8 an the chlorogenic aci 2y Clu:a 7!9iss8# 6he thin . layer chromatography 9as performe on precoate silicagel <0 C21A plates# 6he solvents use to prepare the mo2ile phase 9ere ethyl acetate from Carlo Er2a 7+taly8 an formic aci from Roth 7/arlsruhe, %ermany8# 6he $euD"E% reagent 9as use as spray reagent 7 1D = methanolic solution of iphenyl2orate of aminoethanol an 1 = methanolic solution of polyethylenglicol A008, Roth 7/arlsruhe, %ermany8 supplie the reactives# A''aratu 6he thin . layer chromatography 9as performe 9ith *esaga A! E0 automatic applicator, normal chromatographic cham2er, an Reprostar ++ Camag apparatus# 6he ensitometry an the Fin situG UHDH+! spectra 9as performe 9ith a *esaga C* <0 ensitometer E&'erimental condition D D D 6he 6&C evelopment0 plate0 silicagel <0 C21A3 samples0 the methanolic e;tracts 9ere ma e 2y maceration in 2A hour# a# methanolic e;tract from C2 camf# 71,012< g her2a in 10 ml methanol8 2# methanolic e;tract from C2 carv# 70,IE44 g her2a in 10 ml methanol8 c# methanolic e;tract from C2 thuB# 70,I>1I g her2a in 10 ml methanol8 stan ar s0 caffeic aci 1,01 mgJml in methanol chlorogenic aci 1,01 mgJml in methanol evelopment istance0 4 cm# mo2ile phase0 ethyl acetate . formic aci . 9ater 7>0 0 10 0 10, vJv8# 6he ensitograms 9ere o2taine at 21A nm in reflection# 6he UH.H+! spectra 9ere performe Fin situG one the plate, 2et9een 200 an 400 nm#

D D D D D

Re ult and Di cu ion

6he chromatograms of the samples 9ere visualize in UH light at 21A nm# 6he plate 9as spraye 9ith $euD"E% reagent an the chromatograms 9ere visualize in UH light at E<1 nm# 6he chromatograms of the samples sho9 the presence of the spots 9ith same color an at the same Rf values as the stan ar s# )igure * an + sho9 the chromatograms of the samples an the stan ar s at 21A nm an E<1 nm spraye 9ith $euD"E% reagent#

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Kualitative an Kuantitative *etermination of the Caffeic Aci an Chlorogenic Aci from 6hree Chemovarieties of 5hr! #nthemum '#l #mit# L

)igure *, 6he chromatograms of the samples an the stan ar s in UH light at 21A nm

)igure +, 6he chromatograms of the samples an the stan ar s 9ith $euD"E% reagent, in UH light at E<1 nm

)igure -./ sho9 the ensitograms of the samples 9ith the stan ar s# +t can 2e o2serve the presence of the pea:s in the samples ensitograms, at the same R f values, as the pea:s of the stan ar s# )igure 0 sho9s the ensitograms of all three samples, 9hich prove the presence of a fe9 ifferent components in these samples#

1D chlorogenic aci

2D caffeic aci

)igure -, 6he ensitograms of the 5'. c#m) an the stan ar s at 21A nm

Roum# Biotechnol# &ett#, Hol# <, $o# <, A44DA>A 720018

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A$%E&A '(RCU&E!CU, *A$+E&A ,A$%A$U, -&A, $E&+ . /+$%A

1D chlorogenic aci

2D caffeic aci

)igure 1, 6he ensitograms of the 5'.c#rv# an the stan ar s at 21A nm

1D chlorogenic aci

2D caffeic aci

)igure /, 6he ensitograms of the 5' thu6. an the stan ar s at 21A nm

1D chlorogenic aci

2D caffeic aci

)igure 0, 6he ensitograms of the three chemovarieties samples 7 5'. 5#m)., 5'.c#rv#, 5' thu6.8

6he presence of the caffeic aci an the chlorogenic aci in the samples 9as proven 2y comparison of the UHDH+! spectra of the stan ar s 9ith the UHDH+! spectra of the separate components from the samples# )igure 2.*1 sho9 the Fin situG UHDH+! spectra#

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Roum# Biotechnol# &ett#, Hol# <, $o# <, A44DA>A 720018

Kualitative an Kuantitative *etermination of the Caffeic Aci an Chlorogenic Aci from 6hree Chemovarieties of 5hr! #nthemum '#l #mit# L

)igure 2, 6he UHDH+! spectra of the caffeic aci

stan ar

)igure 3, 6he UHDH+! spectra of the caffeic aci separate from C2 camf

)igure 4, 6he UHDH+! spectra of the caffeic aci separate from C2 carv

)igure *5, 6he UHDH+! spectra of the caffeic aci separate from C2 thuB

)igure **, 6he UHDH+! spectra of the stan ar chlorogenic aci

)igure *+, 6he UHDH+! spectra of the chlorogenic aci separate from C2 camf

6he calculate Rf values 7Ta!le *8 for the stan ar s an the separate components from the samples emonstrates also the presence of the caffeic aci an the chlorogenic aci in this three chemovarieties of 5h. '#l #mit# L. Roum# Biotechnol# &ett#, Hol# <, $o# <, A44DA>A 720018
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)igure *-, 6he UHDH+! spectra of the chlorogenic aci separate from C2 carv

)igure *-, 6he UHDH+! spectra of the chlorogenic aci separate from C2 thuB

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Ta!le *, 6he calculate Rf for the stan ar s an for the separate spots from samples !amples !tan ar s C2 camf# C2 carv# C2 thuB# Rf for caffeic aci 0,<2 0,<2 0,<2 0,<2 Rf for chlorogenic aci 0,1A 0,1A 0,1A 0,1A

6he 5uantitative etermination 9as performe 2y 6&C . ensitometry using the cali2ration curve metho # )igure */6 *0 sho9 the cali2ration curves o2taine for the caffeic aci respectively the chlorogenic aci 9ith photo ensitometer *esaga C* <0# 6he cali2ration curves 9ere performe 2y !li e Computer "rogram# 6he e5uations of this curve are0 D for caffeic aci 0 A L E0I11,0EME4IA,>>H D for chlorogenic aci 0A L 4<I,04M2<1,4<H 9here A is the pea: area an H is the applie volume in a spot# 6he concentration 9as o2taine 9ith the formula0 C= gJg L HeCetJ20m 9here C= 7gJg8 is the concentration3 H e is the corespon ing volume from the stan ar , C et is the concentration of the stan ar solution, 20 is the 5uantity of samples in Nl, an m is the 9eight of the plant use for e;traction# Ta!le +, 6he concentration o2taine from the caffeic aci an the chlorogenic aci from the Chrysanthemum 2alsamita chemovar camphora, Chrysanthemum 2alsamita chemovar# carvona an Chrysanthemum 2alsamita chemovar thuBona is0 !amples 5h.'#l .c#mphor# 5h.'#l .c#rvon# 5h.'#l . thu6on# Caffeic aci = 0,I4 = M 0,2E 1,<1 = M 0,E1 0,<A = M 0,1< Chlorogenic aci = 0,E> = M 0,0E 0,A0 = M 0,0E 0,38 % + 0,01

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Roum# Biotechnol# &ett#, Hol# <, $o# <, A44DA>A 720018

Kualitative an Kuantitative *etermination of the Caffeic Aci an Chlorogenic Aci from 6hree Chemovarieties of 5hr! #nthemum '#l #mit# L

+t can 2e o2serve that the concentration of the caffeic aci is ifferent in these three samples, 2ut the concentration of the chlorogenic aci is nearly e5ual# 6he hepatoprotective an cholagogDcholeretic action of the species ?1@ is Bustifie 2y the presence of the phenyl propanic

erivatives#

Area 76ho usan s8

Area

Holume Holume N&

N&

Holume N&

)igure */ 6he cali2ration curve for caffeic aci

)igure *0 6he cali2ration curve for chlorogenic aci 224

Roum# Biotechnol# &ett#, Hol# <, $o# <, A44DA>A 720018

A$%E&A '(RCU&E!CU, *A$+E&A ,A$%A$U, -&A, $E&+ . /+$%A

Conclu ion
6he caffeic aci an the chlorogenic aci 9ere etermine 5ualitatively an 5uantitatively in the three chemovarieties of 5hr! #nthemum '#l #mit# L#, using chromatographic an spectral metho s# 6he caffeic aci an the chlorogenic aci , responsi2le of the hepatoprotective an cholagogDcholeretic action, are the most important phenyl propane erivatives components foun in the chemovarieties of the 5h. '#l #mit# species#

Reference
1# '# 6A'A!, %# $EA'6U, A# 'ARCU&E!CU, 0l#nte me/icin#le 5hr! #nthemum '#l #mit# L., E # &u; li2ris Brasov, 1II<# 2# A# 'ARCU&E!CU, R# -"REA$, '# B-*RU%, *# B-B+6 , *ct# 0h!tother#pic# Rom#nic#, 7I 71D28, 22D2E 720008# E# C# 6# ,A&AOE+!,, *# O# 6A&&A'P, Li>. 5hrom#to$r., *0 728, AI4 71IIE8# A# ,# C,- ', K# C,E', C# '# -/"-*U, O# "# B-!!, L5 < @5, *5 7<8, A<A 71II28# 1# '# 6A'A!, A# '# RU!U, $# BUCUR, A# !+$CRA+A$U, *# RU!U, "atent no# 10E4EI 0roce/eu /e o'tinere # unui e7tr#ct /in 5hr! #nthemum '#l #mit# L, 71II18# i #rom#tice-

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Roum# Biotechnol# &ett#, Hol# <, $o# <, A44DA>A 720018