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FARMACIA, 2008, Vol.LVI, 6

QUANTITATIVE ANALYSIS OF BIO- ACTIVECOMPOUND IN HIBISCUS SABDARIFFA L.EXTRACTS. NOTE IQUANTITATIVE ANALYSIS OF FLAVONOIDS

SUHAD S. HUMADI1, VIORICA ISTUDOR 21University of Baghdad, Faculty of Pharmacy2University of Medicine and Pharmacy Carol Davila, Faculty of

Pharmacy, raian !uia ", sect# 2, $2$%&", 'omania

AbstractIn the phytochemical extraction re earche there ha !een an increa in" #eman#

to $in# a imple, rapi#, an# preci e metho# $or the %&antitati'e analy i o$ $la'onoi#($la'one , anthocyanin , an# tannin ) in plant extract . Thi paper #e cri!e the %&antitati'e

#etermination o$ $la'onoi# ($la'onol ) calc&late# a %&ercetin e%&i'alent in #i$$erent (i)iscus sa)dariffa extract analy*e# & in" #i$$erent pectrophotometric metho# + Chri tM-ller , Chan" et al an# ome mo#i$ie# metho# , to o!tain extract ith the hi"he t$la'onoi# content ho in" hypo"lycemic (pro'e# !y experiment per$orme# on aloxanin#&ce# #ia!etic rat ) an# anti oxi#ant acti'itie . The o!taine# re &lt in#icate# that the$la'onoi# percent #etermine# & in" the Chri t M&ller/ metho# ho e# the lo e t 'al&eamon" all other metho# , hile the hi"he t percent&al 'al&e a analy*e#via Chan" et almetho# & e# $or the #etermination o$ !oth the $ree a"lycone o!taine# !y hy#roly i ith 0 HCl $or 3 min&te an# "lyco ylate# $orm o$ $la'onol .

Re !"at4n cercetarea $itochimic5 a extractelor, exi t5 o cerere #in ce 6n ce mai mare #e

meto#e #e anali*5 cantitati'5 a $la'onoi#elor ($la'one, antociani, tanin&ri) care 5 $ie

imple, rapi#e, electi'e 7i preci e. 4n acea t5 l&crare a&torii a!or#ea*5 #eterminareacantitati'5 a $la'ono*i#elor #in extractele #e (i)iscus sa)dariffa 8. (Mal'aceae) &tili*9n#mai m&lte meto#e pectro$otometrice+ Chri t M-ller , Chan" 7i cola!., 7i c9te'a 'arianteale ace teia. O!:inerea ace tor extracte are ca cop e'i#en:ierea $rac:i&nii c& cel mai !&n poten:ial hipo"licemiant ('eri$icat prin experiment pe 7o!olani c& #ia!et *aharat in#&experimental prin aloxan) 7i antioxi#ant. Re*&ltatele o!:in&te arat5 c5 procent&al,con:in&t&l 6n $la'one #eterminat prin meto#a Chri t M-ller con#&ce la cele mai mici'alori, 6n timp ce prin meto#a Chan" 7i cola!. e o!:in cele mai mari 'alori.

4n concl&*ie, meto#a Chan" 7i cola!. e te cea mai impl5, rapi#5, 7i preci 5 pentr& #eterminarea con:in&t&l&i 6n $la'onoli (li!eri 7i "lico*i#a:i), prec&m 7i extrac:ia c&metanol 7i hi#roli*a hetero*i#elor prin re$l&xare c& HCl 0 , timp #e 3 min.

;la'onoi# analytical metho# (i)iscus sa)dariffa

al&mini&m chlori#e hy#roly i $la'onol

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INTRODUCTION

There ha !een con i#era!le intere t in the $la'onoi# content o$ $oo# an# plant ince the early 1otter ?1@ #emon trate# a relation hip !et een a #iet rich in $r&it an#

'e"eta!le an# a re#&ce# ri $or chronic #i ea e . Beca& e re#&ce# ri #i#not correlate ith tra#itional n&trient , attention ha $oc& e# on many nonn&trient, potentially !ioacti'e compo&n# , o$ hich the $la'onoi#con tit&te one $amily ?2@.

;la'onoi# are nat&rally occ&rrin" polyphenolic compo&n# ith aC C C !ac !one. Thi "ro&p o$ plant pi"ment hich are $o&n# in$r&it , 'e"eta!le , "rain , !ar , root , tem , $lo er , tea, an# ine can !echemically &!#i'i#e# into ix tr&ct&ral cate"orie + $la'one , $la'onol ,$la'anone , $la'anonol , $la'an ol (catechin ), an# anthocyani#in . The ecompo&n# (a"lycone ) are commonly "lyco ylate# (at one or more ite

ith a 'ariety o$ &"ar ) an# may al o !e al oxylate# or e teri$ie#. A a

re &lt, o'er 333 #i$$erent $la'onoi# ha'e !een i#enti$ie# in plant material? @.The metho# that ha'e !een reporte# $or the #etermination o$

$la'onoi# are !a e# on the al&mini&m chlori#e complex $ormation, hichi one o$ the mo t commonly & e# analytical proce#&re applie# to the$la'onoi# content #etermination in 'ario& plant ? =@. In "eneral,al&min&m chlori#e $orm aci# ta!le complexe ith the C 0 eto "ro&p an#either the C or the C hy#roxyl "ro&p o$ $la'one an# $la'onol . Ina##ition, al&min&m chlori#e $orm aci# la!ile complexe ith theortho#ihy#roxyl "ro&p in the A or B rin" o$ $la'onoi# . The analytical metho#

ere & e# either to #etermine the "lyco ylate# or the non"lyco ylate#$la'onoi# ?=,

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$or 1 ho&r, an# then $iltrate ere complete# to 2 ml !y the ame extractin"ol'ent.

A'a& s#s )+ t e +&a/)')#( c)'te't #' e,tracts.Deter"#'at#)' )+ +&a/)')#(s acc)r(#'$ t) t e C r#st-M!&&ers

"et )( ?12 10@. Thi metho# i applie# to the a"lycone partF there$ore the content

o$ total $la'onoi# i calc&late# a %&ercetin type.>roce#&re+ 23 m8 o$ acetone, 2 m8 o$ 2 HCl an# 1 m8 o$ 3.

hexamethylenetetramine ere a##e# to 2 ml extract (corre pon#in" to 2"o$ plant material) an# re$l&xe# at 3C $or 3 min&te . The extract a$iltere# an# re extracte# t ice ith 23 ml acetone $or 13 min. A$ter coolin"an# $iltration, the extract a ma#e &p to 133.3 m8 ith acetone (!a ic

ample ol&tion, BSS). 23 m8 o$ BSS a mixe# ith 23 m8 o$ ater an#then extracte# ith ethyl acetate ($ir t ith 1 m8 an# then three time ith13 m8). Lthyl acetate extract ere rin e# t ice ith ater then $iltere# an#ma#e &p to 3.3 m8 ith ethyl acetate (S1). In 13 m8 o$ S1 3. m8 o$ 3.

ol&tion o$ o#i&m citrate an# 2 m8 o$ AlCl (prepare# !y #i ol'in" 2 " o$ AlCl in 133 m8 o$ acetic aci# in methanol) ere a##e# an# then ma#e&p to 2 .3 m8 ith methanolic ol&tion o$ acetic aci# ( ample ol&tion,SS). The ame proce#&re a per$orme# ith !lan ample ol&tion !&t

itho&t AlCl. A$ter 0 min&te , yello ol&tion ere $iltere# an#a! or!ance at 02 nm a mea &re#. The content o$ total $la'onoi# ae'al&ate# &pon three in#epen#ent analy e . The yiel# a calc&late# a%&ercetin percent & in" the $ollo in" expre ion+" N A 3.PP2 !,

here A i a! or!ance an# ! repre ent the ma o$ #ry her!al material in

"ram .Deter"#'at#)' )+ +&a/)')#(s b C a'$ et a&. "et )( ?1 ,1 )@;la'onol in (i)iscus sa)dariffa extract ere expre e# a

%&ercetin e%&i'alent. E&ercetin (Si"ma, Kermany) a & e# to per$orm thecali!ration c&r'e ( tan#ar# ol&tion o$ .2 , 12. , 2 .3, 3.3, =3.3 an#133.3 Q" m81 in =3 ethanol*!+! . Sample extract (1" plant material in2 ml extract) ere all e'aporate# to #ryne an# re #i ol'e# in =3 ethanol to !e rea#y $or the analytical te t.

1 m8 o$ a ample (ethanolic ol&tion (i)iscus ) a mixe# ith m8 < ethanol*!+! ), 3.2 m8 13 al&min&m chlori#e*m+! ), 3.2 m8 o$ 1

mol 8 1

pota i&m acetate an# . m8 ater. A 'ol&me o$ 13 (m+! )al&min&m chlori#e a &! tit&te# !y the ame 'ol&me o$ #i tille# ater in

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!lan . A$ter inc&!ation at room temperat&re $or 3 min&te , the a! or!anceo$ the reaction mixt&re a mea &re# at 01 nm.

C a'$ et a&. ")(#+#e( "et )(sSe'eral mo#i$ication ere ma#e to the Chan" et al. metho# in

or#er to clari$y the acc&racy o$ thi metho#. The e mo#i$ication ere a$ollo +

M)(#+#e( "et )( AThe mo#i$ication i applie# to the ample preparation & in" the

ame tan#ar# %&ercetin c&r'e. Lach extract a hy#roly*e# ith 2 HCl(1+1) $or = ho&r , then the $la'onoi# ere extracte# ith three %&antitie o$ ethyl acetate (each o$ 1 m8), a$ter hich the ethyl acetate layer ae'aporate# to #ryne &n#er re#&ce# pre &re, then the re i#&e a re#i ol'e# & in" =3 ethanol to a 'ol&me o$ 2 m8. Thi ol&tion a & e#$or the analytical proce#&re. The $la'onoi# content a expre e# a%&ercetin e%&i'alent.

M)(#+#e( "et )( Be per$ormem the ame mo#i$ication a in metho# A except that

the hy#roly i o$ $la'onoi# i carrie# o&t & in" 0 HCl conc. $or 3min&te . The $la'onoi# content a al o expre e# a %&ercetin e%&i'alent.

Ver#+#cat#)' )+ t e C a'$ et a& "et )(>roce#&re+ 3.1" o$ %&ercetin a #i ol'e# in =3 ethanol to a

'ol&me o$ 133 m8 in a 'ol&metric $la (thi i ol. A). 3.1 " o$ %&ercetinere #i ol'e# in m8 o$ concentrate# ethanol in a 133 m8 'ol&metric $la ,

then e a##e# 2. " o$ Ma"ne i&m trip an# 1 m8 o$ concentrate# HCl,each o$ hich a a##e# tep i e. The color chan"e# $rom yello to #ar 'iolet re# color. The 'ol&me a then complete# to 133 m8 & in" =3ethanol (thi i ol. B). The 'eri$ication experiment a per$orme# & in" theori"inal Chan" et al, metho# ( itho&t mo#i$ication ) & in" the ame tan#ar#c&r'e e%&ation. In three erie o$ 13 m8 'ol&metric $la e p&t 3.P ,1, an#1.2 m8 o$ oln A in the $ir t erie F hile in the econ# erie e p&t amixin" %&antitie o$ (1m8 oln A 1m8 oln B),( 3.P oln A 1.2 m8 olnB), an# $inally (1.2 oln A 3.P m8 oln B) , the thir# erie containe#3.P , 1, an# 1.2 m8 o$ oln B. A$ter inc&!ation at room temperat&re $or 3min&te , the a! or!ance o$ the reaction mixt&re a mea &re# at 01 nm.

Al&min&m chlori#e a excle# $rom the !lan erie . ( ote that the'ol&me o$ the ample < ethanol ho&l# !e collecti'ely 0 m8).

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The $inal te t ample concentration & in" the Chan" et al metho# ithen o!taine# & in" the $ollo in" $orm&la+

(R D; V 133)here RN re &lt o!taine# $rom the tan#ar# c&r'e e%&ationF D;N

#il&tion $actorF VN 'ol&me o$ toc ol&tionF 133N $or 133 "ram #rie# plantF N ei"ht o$ plant & e# in experiment in m".

The compari on o$ the re &lt o!taine# $rom the metho# #i c& e#a!o'e i ma#e on "ram !a e i.e. the $la'onoi# content in the extracte#

ol&tion i calc&late# a " (" E&ercetin per133" o$ #rie# plant material),a repre ente# in the ta!le II.

Tab&e II;la'onoi# percent content analy*e# !y #i$$erent

pectrophotometric metho# in three extractin" ol'entLxt. o.

Mea &rement !yChri t M&ller

metho#

Mea &rement !yChan" et al

metho#

Mo#i$ie# metho#A

Mo#i$ie# metho#B

L 1 3.32< " 3.3 0 3.3 = " 3.32 3.3 " 3.32 3.3 " 3.323L 2 3.320 " 3.3 0 3.30

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Tab&e IIThe 'eri$ication proce#&re re &lt

Test s)&!t#)' T.S Abs)rba'ceAbs.

C)'ce'trat#)'C)'c."$1%22"&s)&!t#)'

Abs. D#++ere'ces

C)'c.D#++ere'ces

T.S (1.2 m8 ol. A) 3.312 3.1=P

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