Isolation and Extraction of Total Flavonoids f rom Epimedium

Koreanum Nakai by Supercritical Fl uid Extraction

*
LIU Chun-ming
1, 2
, ZHAO Xue-liang
3
, LIU Zhi-qiang
1* *
and XING Jun-peng
1
1.
L aboratory of New Dr ug Resear ch
,
Changchun I nstit ute of App lied Chemistry
,
Chinese Academy of Sciences
,
Changchun
, 1322,
P
.
R
.
China
!
2.
Department of Chemistry
,
Changchun T eacher s

Coll ege
,
Changchun
1332,
P
.
R
.
China
!
3.
China
-
Japan nion ! ospital
,
Jili n ni" ersity
,
Changchun
1332,
P
.
R
.
China
"e#ei$ed June
1, 2%
* &upp'( )ed *+ )he Na)u(al &#ien#e and , e#hn'l'g+ -'unda)i'n '. Jilin /( '$in#e0 N'. 22132-13 .
* * ,' 4 h'm #'( (e5p'nden#e 5h'uld *e add( e55ed. 6-mail7 liu8q9 #ia#. :l. #n

I n )he p( e5en) pape( i5 ( ep'( )ed )he me)h'd .'( )he i5'la)i'n and e;)(a#) i'n '. )') al .la$'n'id5 '.
#pimedium $ or eanum N a%ai *+ mean5 '. 5upe(#( i)i#al .luid e;) (a#)i'n0 &-63 . <+ e;amining p(e55u(e, )em-
pe(a) u( e
,
am'un)5 '. m'di.ie( and e;) (a#)i'n )ime
,
)he 'p)imi8ed #'ndi)i'n '. &-6 i5 #'n.i( med a5
3
=/a
and 1 , 4 i)h 2> e)han'l a5 )he m'di.ie( . , he 5ample5 4 e(e 5)a)i#all+ e;)( a#) ed .'( 3 min, .'ll'4 ed *+
d+nami# e;)( a#)i'n .'(
12
min a) a .l'4 (a)e '.
1
mL
?
min
.
,he quan)i)a)i$e anal+5i5 '. )')al .la$'n'id5 4 a5
pe( .'( med *+ U@-@i5 5pe#) ('ph')'me)( +. C'mpa(ed 4i)h ) he #'n$en) i'nal me)h'd, )he &-6 me)h'd i5 m'( e
e..i#ien), m'(e ( apid and m'( e .( iendl+ en$i('nmen)all+.
Keywords &upe( #( i)i#al .luid e;)( a#)i'n0 &-63 , -la$'n'id5, #p imedium $ or eanum N a%a
Articl e ID 1A-B%0 2%3 -1-22-%
Introduction
,he he(*al pl an) #pimedi um $oreanum Na%ai
0 &er ber idaceae3 i5 a na) i$e he(* g('4ing in ) he
Chang*ai ='un) ain (egi'n, Jilin p('$in#e '. China,
and i5 u5ed a5 a ) 'ni#, an) i-(heuma) i# and aph('-
di5ia# in ) (adi) i'nal Chine5e medi#ine, ) 'ge)he(
4i)h . 'u( ') he( Chine5e /ha(ma#'p'#ia-(e#'(ded
5pe#ie57 6pimedium *(e$i#'(num =a;im, 6pimedi-
um 5agi)) a) um =a;im, 6pimedium pu*e5#ene5
=a;im and 6pimedium 4u5hanen5e , . &. Cing
D 1E
.
I) ha5 *een (ep'() ed ) ha) in #pimedium $ ore-
anum Na%ai ) he(e a( e )he #'mp'nen)5
.l a$'n'id5
D 2 1E
,
alFal'id5
D 11 13E
,
p'l+5a##ha-
(ide5
D 1%, 1AE
, mi#('elemen) 5
D 11, 12E
, and 5' 'n, am'ng
4hi#h . la$'n'id5 a(e ) he main #'mp'nen) 5. "e#en)
pha(ma#'l'gi#al 5) udie5 dem'n5) (a) e ) ha) ) ') al
.l a$'n'id5 in #p imedium $oreanum Na%ai 5h'4
) he an)i-in.lamma) '(+ e. .e#) 5
D 1GE
and '*$i'u5 h+-
p') en5i$e a#) i$i)+
D 1BE
. , he+ #an imp( '$e )he im-
mune . un#) i'n
D 2E
, in#(ea5e )he *'ne den5i) +, en-
han#e ) he e5) (adi'l le$el , de#( ea5e IL-1 #'n#en) (a-
)i'n in 5e(um, and p(e$en) '5) e'p'( '5i5
D 21 23E
.
Up ) ill n'4, )')al .la$'n'id5 ha$e *een mainl +
i5'l a) ed .('m #pimedi um $ oreanum Na%ai *+ e;-
)( a#) i'n 4i) h e) han'l
D 2%E
. In ) hi5 ) ime-#'n5umed
p( '#e55, la(ge am'un) 5 '. 5'l$en) 5 a(e #'n5umed
4hi#h ma+ p'llu)e )he en$i('nmen) *adl +.
Cu( (en) l+, )he &-6 ) e#hnique a. .'(d5 man+
ad$an) age5 '$e( )he m'(e #'n$en) i'nal e;) (a#)i'n
me) h'd. &upe(#(i) i#al #a(*'n di';ide i5 4idel+ u5ed
a5 an e;) (a#) i'n 5'l$en) due ) ' i) 5 l'4 #(i) i#al
p'in) , n'n-)';i#i) +, #hemi#al ine() ne55, n'n-e;pl'-
5i$ene55, l'4 $ap'( l'55, a$aila*ili)+ and ea5e '.
(em'$al . ('m ) he e;) (a#)ed 5+5) em5. -u() he(m'(e,
) he e;)( a#) d'e5 n') need ) ' *e #'n#en) (a)ed *e-
#au5e 'nl+ a li) ) le am'un) '. '(gani# 5'l$en) i5
u5ed. , he5e ad$an) age5 ha$e a) ) (a#) ed in#(ea5ing
in) e(e5) .( 'm (e5ea(#he(5
.
Al) h'ugh &-6 ha5 *een
empl '+ed .'( ) he e;) (a#) i'n '. man+ #la55e5 '.
he( *5 and na) u(al p('du#)5
D 2A 33E
,
'nl + a .e4 5) ud-
ie5 in$'l$ing ) he 5epa(a) i'n '. .la$'n'id5 ha$e *een
pu*li5hed
D 3% 32E
. , ' ) he *e5) '. 'u( Fn'4ledge, n'
(ep'() ha5 +e) appea(ed 'n ) he &-6 '. #p imedium
$ oreanum Na%ai
.
In ) hi5 4'(F
,
4e . '#u5ed 'u( a) ) en) i'n 'n ) he
e;) (a#)i'n '. ) ') al . la$'n'id5 in #p imedium $ore-
anum Na%ai *+ &-6
.
,he in. luen#e5 '. p(e55u(e
,
) empe(a) u( e
,
am'un) '. e)han'l a5 ) he m'di. ie( and
e;) (a#)i'n ) ime 4e(e e$alua) ed in '( de( ) ' ge) 5a) i5-
.a#)'(+ (e5ul) 5
.
,he ) ') al .la$'n'id5 e;) (a#) ed 4e(e
anal+8ed *+ U@-@i5 5pe#) ('me)(+, and )he (e5ul) 5
4e( e #'mpa( ed 4i) h ) h'5e '*)ained *+ ) he #'n$en-
CH6=
.
"6&
.
CHIN6&6 U
. 2%, 20 13 , 22 21
)i'nal e;) (a#) i'n me) h'd
.
Experimental
1 Plant Materi al
,he lea$e5 '. #p imedium $ or eanum Na%ai
4e( e #'l le#) ed . ('m ) he Chang*ai ='un) ain, Jilin
/('$in#e '. China in Jul+, 23, and ) he ma;imal
am'un) '. . la$'n'id5 4a5 p('du#ed and u5ed .'( all
) he e;pe(imen) 5. , he plan) ma) e(ial 4a5 d(ied in
) he 5hade . '( 5e$e(al da+5 and ) hen pul$e(i8ed.
2
Equi pment and Reagents
&-6 4a5 pe(.'(med 'n an
0
HA
221-A-13
5u-
pe(#(i)i#al .luid e;) (a#) '(
0
Nan) 'ng
,
Huaan =a-
#hine -a#) '(+
3 ,
4hi#h i5 de5igned . '( 4'(Fing
p( e55u(e5 and )empe(a)u(e5 up ) '
A
=/a and
1 ,
(e5pe#)i$el+
.
A U@
-
@i5 5pe#)( 'ph') 'me) e(
0
='del
11,
Agilen)
3
4i)h
1
#m qua() 8 a*5'( p-
)i'n #ell5 4a5 u5ed ) ' anal+8e quan) i) a) i$el+ ) he ) '-
)al . la$'n'id5 a) 22 nm. Anal+) i#al g(ade e)han'l
4a5 u5ed a5 a m'di.ie( and . '( )he #'n$en) i'nal e;-
)( a#) i'n '. ) he ) ') al . la$'n'id5. Hi5) illed 4a) e( 4a5
u5ed in all ) he e;pe(imen) 5.
!onventional Extraction
,he d(ied and pul$e(i8ed pl an) ma) e(ial '.
A. g 4a5 e;) (a#) ed 4i) h A mL '. 2> e)han'l
unde( (e.l u; di5) illa) i'n . '( 2 h. , he e;) (a#) i'n
4a5 (epea) ed .'( ) h(ee ) ime5 in '(de( ) ' #'mple) el+
e;) (a#) ) he e. .e#) i$e #'mp'nen) 5 .( 'm ) he lea$e5 '.
#p imedium $ or eanum Na%ai
.
, he #'m*ined e;-
)( a#) 5 4e(e #'n#en) (a) ed in a (')a(+ e$ap'(a)'( a) a
(edu#ed p(e55u(e un) il a $i5#'u5 (e5idue (emained
.
,he (e5idue 4a5 e;) (a#) ed 4i) h pe)( 'leum e) he(
0
*
.
p
. 1 B 3
in '(de( ) ' (em'$e #hl'('ph+ll
,
and
) hen ) he aque'u5 la+e(5 4e(e e;) (a#) ed .'( ) h( ee
) ime5 4i)h e)h+l a#e) a) e
,
.inal l+
,
) he e) h+l a#e) a) e
la+e(5 4e(e #'m*ined and #'n#en) (a) ed a) a ( edu#ed
p( e55u(e ) ' +iel d 1 mL '. a +ell'4 5'lu) i'n.
" Supercritical Fluid Extraction
,he &-6 e;pe(imen) 4a5 pe(. '(med in ) he
p( e55u(e (ange .('m 2 ) ' 3A =/a and ) he ) empe(a-
)u(e ( ange .('m % ) ' 2 . , he 5upe(#(i) i#al . luid
.l '4 ( a) e 4a5 #'n) ('lled a) 1 mL? min. In ea#h e;-
pe(imen), ) he e;) (a#) '( 4a5 pa#Fed 4i) h A. g '.
d( + #pimedi um $oreanum Na%ai p'4de( , 4hi#h
4a5 mi;ed 4i) h A g '. gla55 *ead5 ) ' in#(ea5e ) he
#'n) a#) 5u(. a#e *e) 4een ) he plan) ma) e(ial and ) he
5upe(#(i) i#al #a(*'n di';ide unde( $a(i'u5 #'ndi-
)i'n5
.
<')h ) he ) 'p and *') ) 'm pa() 5 '. ) he #ell
4e( e . illed 4i)h a la+e( '. gl a55 4''l in '( de( ) ' de-
#(ea5e )he unu5ed 5pa#e
.
-'( all ) he m'di.ie( 5) ud-
ie5
,
) he m'di.ie(5 4e(e added *+ mean5 '. a 5epa-
(a) e pump 5' a5 ) ' ge) )he m'5) a##u(a#+ and (ep('-
du#i*ili) +. , he e;) (a#) ed anal+) e5 4e(e #'lle#) ed in
3 mL '. BA> e) han'l in a 1 mL . la5F a) (''m
) empe(a) u( e.
# T$e %nalysi s of Total Fl avonoids
,he am'un) 5 '. )he ) ') al . la$'n'id5 4e(e de-
) e(mined *+ mean5 '. U@-@i5 5pe#) ('me) (+ 4i) h
I#a((in a5 5) anda(d a##'( ding )' )he me)h'd .('m
(e.e(en#e
D 12E .
,he a*5'( *an#e 4a5 mea5u(ed a-
gain5) a *lanF a)
22
nm
,
and ) he )')al . la$'n'id5
pe(#en) age 4a5 e5)ima) ed 4i)h ) he aid '. ) he . 'l-
l'4ing linea( equa) i'n *a5ed 'n ) he #ali*( a)i'n
#u($e '. I#a(( in. A I . 3GB- . A1, r I
. BBB2, 4he( e A i5 ) he a*5'(*an#e $alue, and i5
) he I#a((in ma55 #'n#en) (a) i'n in g? mL. ,he #ali-
*( a) i'n #u( $e 4a5 linea( *e)4een 12 g? mL.
Results and &iscussion
1 T$e Inf luence of Modi fier on t$e Extraction Ef'
ficiency
<e#au5e '. ) he limi) ed 5'lu*ili)+ '. p'la( '(-
gani# #'mp'und5 in 5upe(#(i) i#al #a(*'n di';ide e;-
) (a#) i'n
,
)he quan) i) a) i$e e;) (a#) i'n '. p'la( ana-
l+) e5 4i) h pu(e 5upe(#(i)i#al .l uid '. CO
2
i5 di55a) i5-
.a#)'(+. ,' imp('$e ) he e;) (a#)i'n e. .i#ien#+ '. ) '-
) al . la$'n'id5, 2> e) han'l and 1> e)han'l
4e( e u5ed a5 )he m'di. ie(. 6)han'l 4a5 #h'5en due
) ' i) 5 l'4 ) ';i#i)+ #'mpa(ed 4i) h ) he ') he( '(gani#
5'l$en) 5. A) )he $e(+ *eginning '. d+nami# e;) (a#-
) i'n, ) he m'di. ie( 4a5 added *+ mean5 '. a 5epa-
(a) e pump in '(de( ) ' '*)ain ) he highe5) a##u(a#+
and (ep('du#i*il i) +
.
,he in. luen#e5 '. di. . e(en) $'l-
ume5 '. )he m'di. ie( 'n ) he e;) (a#) i'n e. .i#ien#+ '.
) ') al . la$'n'id5 a(e 5h'4n in ,a*le
1.
Table 1 T$e influence of modif ier on extracted
ef ficiency of total flavonoids
a
='di. ie(
Addi)i'n
am'un) 5? mL
6; )(a#) i'n
e. . i#ien#+
0 > 3
b
6) han'l
3 2. 1
A 2. G
2 3. %
2> 6) han'l 3 %. 1
A %. B
2 A. %
/u(e CO
2
1. 1
a. &upe(#(i) i#al #a(*'n di';ide a) 2A =/a, A ! b. mean $al-
ue5 0 nI 33 e;p(e55ed a5 ) he pe(#en) age (e. e(ed ) ' ) he d( + 4ei gh) .
,he e;pe(imen) al (e5ul) 5 dem'n5) (a) e ) ha) ) he
addi) i'n '. e) han'l #an 5u*5) an)iall+ enhan#e ) he
+ield '. ) ') al e;) (a#) ed . la$'n'id5. A) a #e() ain
) empe(a) u( e and a p(e55u( e, ) he highe( ) he am'un)
'. ) he m'di.ie( i5, ) he highe( ) he e;) (a#)i'n +iel d
2G

CH6=
.
"6&
.
CHIN6&6 U
.
@'l
. 2
a#hie$ed i5
.
<u) 4hen )he 5ame am'un) '. ) he
m'di.ie( i5 u5ed
,
)he e;)( a#) ed +ield5 4i) h
2>
e)han'l a5 ) he m'di. ie( a(e highe( ) han ) h'5e 4i) h
pu(e e) han'l a5 ) he m'di. ie(
.
, hi5 #'uld *e e;-
plained *+ ) he . a#) ) ha) p'la( #'n5)i) uen) 5 in ) he
pl an) 4'uld *e ea5ie( )' *e e;)( a#) ed 4i)h a m'(e
p'la( 5'l $en)
D 3AE
.
A5 a (e5ul )
, 2
mL '.
2>
e)han'l 4a5 5ele#)ed in ) he 5u*5equen) e;pe(i-
men) 5
.
2
T$e Influence of Pressure on t$e Extraction Ef'
ficiency
,he p( e55u(e and ) empe(a) u(e a(e ) he m'5) im-
p'( )an) ph+5i#al pa(ame)e(5 in &-6 e;pe(imen)
,
4hi#h #an a. .e#) ) he anal+) e 5'lu*ili) + '*$i'u5l+
.
-'( e;amining )he in.luen#e '. p(e55u(e, ) he +ield5
'. ) ') al e;)(a#) ed . la$'n'id5 a) di. .e(en) p(e55u(e5
4e( e in$e5) iga) ed a) % and 4i) h 2> e) han'l a5
) he m'di.ie(. ,he e;pe(imen) al (e5ul) 5 5h'4 ) ha)
) he +iel d '. ) ') al e;)( a#) ed . la$'n'id5 in#(ea5e5
4i)h )he in#(ea5e '. ) he p(e55u(e $a(+ing . ('m 2 ) '
3 =/a, and ) hen, )he e;) (a#) i'n +ield in#(ea5e5
5ligh) l+ 4hile ) he p(e55u(e i5 in#(ea5ed .('m 3 ) '
3A =/a0 5ee -ig. 13 , 4hi#h dem'n5) (a) e5 ) ha) ) he
in#(ea5e '. ) he den5i) + '. 5upe(#(i) i#al #a(*'n di';-
ide (e5ul) 5 in ) he in#(ea5e '. ) he e;) (a#) i'n +ield.
<u) high p(e55u(e (e5ul) 5 in g(ea) e( #'5) . '( ) he e;-
)( a#) i'n 5+5)em, a5 4ell a5 )he 5) (ingen) 'pe( a) i'n
de5ign and in#( ea5ed ene( g+ demand
D 22E
. , he(e. '(e,
3 =/a i5 ) he 'p)imal p( e55u(e . '( )he e;)( a#) i'n.
Fig
. 1
T$e eff ect of pressure on t$e extraction eff iciency
of total flavonoids at #' .
T$e Influence of Temperature on t$e Extraction
Efficiency
,he e. .e#) '. )empe(a)u(e 'n ) he e;)( a#) i'n '.
) ') al .la$'n'id5 . ('m #p imedium $ oreanum Na%ai
4a5 in$e5) iga) ed 'n Feeping all ') he( e;) (a#) i'n pa-
(ame) e(5 ) ' *e . i;ed. , he e;) (a#) i'n e. .i#ien#+ '.
) ') al . la$'n'ide5 *+ 5upe(#(i)i#al . luid #a(*'n di';-
ide a) .'u( di.. e(en) ) empe(a) u(e50 %, A, 1 and
2 3 i5 5h'4n in -ig. 2. ,he e;) (a#) i'n +ield in-
#(ea5e5 al'ng 4i) h ) he )empe(a)u(e #hanging .('m
%
)'
1 ,
*u) 4i) h ) he ) empe(a) u(e in#(ea5e
. ('m
1
) '
2 ,
)he e;) (a#)i'n +ield de#(ea5e5
5l igh)l+
.
,he5e (e5ul) 5 dem'n5) (a) e ) ha) ) he in-
#(ea5e '. ) he $ap'( p(e55u( e #au5ed *+ ) he in#(ea5e
'. ) empe( a) u(e appea(5 ) ' #'mpen5a) e . '( ) he de-
#(ea5e in 5'lu*ili)+ #au5ed *+ d('pping '. 5upe(#(i) -
i#al . luid den5i) +
,
(e5ul) ing in )he e;)(a#) i'n +iel d
) ' in#(ea5e 4i) h ) he ) empe(a) u(e in#( ea5e .('m
%
) '
1 ,
*u) 4hen ) he )empe( a)u(e #'n) inue5 ) '
in#(ea5e
,
) he ( edu#) i'n '. 5upe( #(i) i#al #a(*'n di';-
ide den5i) + *e#'me5 d'mina) e in )he e;) (a#) i'n p('-
#e55
D 2B, 3%E
.
, hu5 an e;) (a#) i'n ) empe( a) u(e '.
1
4a5 5ele#) ed . '( e;)(a#) ing ) he m'5) '. ) ') al
. la$'n'id5
.
Fig. 2 T$e ef fect of temperature on t$e extraction eff i'
ciency of total f lavonoids at
2#
MPa
.
"
T$e Infl uence of Extraction Time on t$e Ex'
traction Ef ficiency
Fig. T$e ef fect of time on extraction eff iciency of
total f lavonoids at
2#
MPa and
#' .
A. )e(
3
min '. #'n5) an) 5) a)i# e;) (a#)i'n
) ime
,
) he e. .e#) '. ) he d+nami# e;) (a#)i'n ) ime
. ('m
3
) '
1A
min 'n ) he e;)( a#) i'n e. .i#ien#+ 4a5
5) udied
.
,he e;pe(imen) al (e5ul )5 dem'n5)( a) e ) ha)
) he l'nge( ) he 5upe(#( i) i#al #a(*'n di';ide i5 in #'n-
) a#) 4i) h )he ma) (i;
,
) he highe( ) he am'un) '. ) ') al
. la$'n'id5 e;) (a#)ed i5
0
5ee -ig
. 33 ,
and ) he e;) (a#-
) i'n +iel d in#(ea5e5 5'me4ha) a. ) e( )he d+nami# e;-
) (a#) i'n '.
12
min
.
&' )he #'m*ina)i'n '.
3
min
'. 5) a) i# e;) (a#)i'n and 12 min '. d+nami# e;) (a#-
N' 2B
. 1
LIU Chun
-
ming et al
.
)i'n 4a5 #h'5en . '( e;) (a#) i'n
.
A##'(ding ) ' )he (e5ul) 5 men) i'ned in ) he p(e-
$i'u5 5e#) i'n5
,
) he 'p) imal pa( ame) e(5 '. ) ') al
.l a$'n'id5 e;) (a)ed *+ &-6 a(e a5 . 'll'45
7 2
mL
'.
2>
e) han'l a5 ) he m'di. ie(
, 3
=/a
, 1
and
3
min '. 5) a)i#
? 12
min '. d+nami# e;) (a#)i'n
.
#
T$e !omparison of SFE (it$ !onventional Ex'
traction
,he #'mpa(i5'n (e5ul) 5 '. &-6 4i) h #'n$en-
)i'nal e;) (a#)i'n unde( ) he 'p) imal #'ndi) i'n5 a(e
li5) ed in , a*le
2.
,he (e5ul )5 5h'4 ) ha) ) he e;) (a#-
)i'n +ield '. &-6 i5 *e) )e( ) han ) ha) '. #'n$en) i'nal
e;) (a#) i'n
.
,hu5 )he p(e5en) &-6 me) h'd i5 u5e.ul
.'( ) he e;) (a#)i'n '. . la$'n'id5 . ('m #p imedium
$ oreanum Na%ai .
Table 2 T$e comparison of total flavonoids extracted by
t$e conventional met$od and by supercritical
carbon dioxide extraction
&ample
C'n$en) i'nal
e;) (a#) i'n
&-6
1 1. 1A> 1. 21>
2 1. 2G> 1. 23>
3 A. BA> 1. AB>
% 1. 1G> 1. 1G>
A 1. %> 1. 3%>
A$e(age $al ue5 1. 22> 1. A1>
&) anda(d de$ia) i'n . 2G> . 21>
"ela) i$e 5 ) anda(d de$ia) i'n %. 1> 3. 2>
!onclusions
,he e.. e#) 5 '. m'di. ie(, p(e55u( e, )empe(a)u(e
and e;) (a#) i'n )ime 'n ) he e;)( a#) i'n e. . i#ien#+ '.
) ') al .la$'n'id5 . ('m #p imedium $ oreanum Na%ai
ha$e *een in$e5) iga) ed. ,he &-6 me) h'd i5 #'n5id-
e( ed )' *e $e(+ e. .i#ien) .'( ) he e;) (a#)i'n '. ) ') al
.l a$'n'id5, 4hi#h i5 qui#Fe(, l e55 ) ';i# ) ' ) he en$i-
('nmen) and m'(e e. .i#ien) ) han #'n$en) i'nal me)h-
'd5.
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21

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