Evaluation of ISO Method in Saffron Qualification

F. Hadizadeh , M. Mahdavi, S.A.

Emami, Z. Khashayarmanesh, M.
Hassanzadeh, J. Asili
School of Pharmacy
Mashhad University of Medical Sci.
Mashhad
ran
M. Seifi, H. !assirli
"iotechnolo#y $ Pharmace%tical
Sciences &esearch 'enter
Mashhad University of Medical Sci.
Mashhad
ran
A. Shariatimo#hadam &. !oor(a)hsh
!ovin Saffron
P.*."o+ ,-./01-/02
Mashhad
ran
Standard nstit%te
Mashhad
ran
Keywords: crocin, HP3', S*, 4icrocrocin, safranal
Abstract
The factors which are very important in saffron are crocin color!"
picrocrocin flavor! and safranal aroma!# $i%her amount of these compounds
in saffron provide hi%her &uality of saffron# ISO the international or%ani'ation
for standardi'ation! has set a classification of saffron based on minimum
re&uirements of each &uality# Accordin% to ISO picrocrocin" safranal and crocin
are e(pressed as direct readin% of the absorbance of ) * a&ueous solution of
dried saffron at +,-" ../ and 00/ nm respectively# There was some doubt about
the accuracy of the safranal result usin% ISO method# Therefore it was decided
to evaluate the amounts of safranal and crocin in saffron accordin% to ISO
method and compare the results with the amounts of these compounds when
usin% hi%h performance li&uid chromato%raphy $123!# In $123 method in an
isocratic run a ./ cm O4S column were used# Mobile phase was mi(ture of
acetonitrile and water -5 * v6v!# A 78 detector at wavelen%ths of ./9nm for
safranal and 00/ nm for crocin was also employed# :esults indicated that
standard crocin had si%nificant absorption at ./9nm# ;e concluded that present
ISO method is not an accurate method to measure the amount of safranal in
saffron# Therefore it is su%%ested to evaluate this method and meanwhile usin% a
$123 method to evaluate the amount of safranal and crocin in saffron samples#
I<T:O473TIO<
Saffron is a food s4ice o(tained from the flo5er sti#mas of Crocus sativus 3.,
a mem(er of the ridaceae family. t 4lays an im4ortant role in the c%linary c%lt%re of
different re#ions of the 5orld, 5here it is %sed as a food additive, since it 4ossesses
4o5erf%l colorin# and flavorin# 4ro4erties d%e to its #lycosidic constit%ents
6'a(allero1*rte#a et al., 7889 : &;os et al., -,,2:<. =he most im4ortant com4o%nds in
saffron are crocin , 4icocrocin and safranal 6Fi#. -<, res4onsi(le for saffron color ,
flavor and aroma res4ectively 6Fern>ndez, 7889! 6Fi#. -<. =he amo%nts of these main
com4o%nds are %sed to e+4ress the ?%ality of saffron 6=arvand, 7880<. =he hi#her the
amo%nts of these com4o%nds in saffron, means hi#her ?%ality of saffron. S* 6the
international or#anization for standardization< is a 5orld5ide federation of national
standard (odies 6S* mem(er (odies<, they have set a classification of saffron (ased
on minim%m re?%irements of each ?%ality, and this is called S* /2/7. Accordin# to
this classification, they esta(lished 9 cate#ories 6=a(le -<. Saffron 5ith the (est
?%ality (elon#s to cate#ory , 5hich means direct readin# of the a(sor(ance at a(o%t
998nm for crocin is #rater than -,8. 6=arvand, 7880<.
Safranal is the com4o%nd mainly res4onsi(le for the aroma of

saffron s4ice
and can (e %sed as a meas%re of saffron ?%ality. Saffron sam4les are eval%ated (y a
@safranal val%eA (y means of a s4ectro4hotometric method (ased on S*

/2/7 6S*,
-,,/, S&<. Safranal val%e is e+4ressed as direct readin# of the a(sor(ance at a(o%t
//8 nm 6Alonso, 788-<# *n the other hand color stren#th of saffron 5hich is related to
crocin is e+4ressed as direct readin# at 988 nm accordin# to S* /2/7 6S*, -,,/,
S&<.
t has (een 4revio%sly re4orted that crocins have a(sor4tion at 70. and //8nm
6cis1crocin< in addition to their main 4ea) at aro%nd 998nm 6Alonso, et al., -,,B:
=arantilis, et al., -,,9<. So, e+4ression of safranal and 4icrocrocin val%e as direct
meas%rement at 70. and //8 nm accordin# to S* may (e misleadin# and sho%ld (e
revised 6=rantillis et al., -,,0: Alonso et al., 788-<. n addition sol%(ility of saffron
constit%ents in 5ater is not 4erfect. =herefore, it 5as decided to eval%ate the amo%nts
of safranal and crocin in saffron sti#mata from different cities in ran, 5ith UC
s4ectro4hotometer and com4are the res%lts 5ith the amo%nts of these com4o%nds
5hen %sin# hi#h 4erformance li?%id chromato#ra4hy 6HP3'<. n this research the
effects of e+traction 5ith ethanol 08 D 6vEv< and sonication rather than %s%al sha)in#
e+amined as 5ell as standard S* /2/7 e+traction 4roced%re.
MATE:IA2S A<4 MET$O4S
Source of Saffron and :ea%ents
A%thentic sam4les of safranal and crocin 5ere o(tained from Fl%)a 6'at !o.
-./82 $ -./89<. Saffron sam4les 5ere o(tained from different cities of Khorasan
4rovince, incl%din# =or(at1Heidarieh, Sa(zevar, Kashmar, Mashhad and !eisha4oor.
Sam4les 5ere dried in an oven at 28 F' 5ith dry silica #el %ntil constant 5ei#ht
64ercent h%midity calc%lated<. After #rindin#, the sam4les 5ere 4assed thro%#h a 8.0
mm mesh and stored in the dar) at 9 F' %ntil they 5ere %sed. =he methanol and
acetronitrile %sed 5ere 4%rchased from Merc). All of the rea#ents 5ere of HP3'
#rade.
E(traction procedure
A -88 m# mass of dry sti#mata 5as e+tracted 5ith 0ml of cold 08 D 6vEv<
ethanol in a 4estle and mortar, it 5as then transferred to a scre51ca44ed vial and a
total vol%me of 78ml 08 D 6vEv< ethanol 5as added to it. Cials 5ere sonicated for 78
min and then centrif%#ed at 9888 r4m for -8 min and 5ashed t5ice 5ith 0ml each of
the same solvent. =he s%4ernatant 5as %sed for analysis (y s4ectro4hotometric
4roced%res. An e+traction 4roced%re 5ith cold 5ater accordin# to S* /2/7 5as also
4erformed.
Spectrophotometery
=he s%4ernatant 6-ml< 5as dil%ted to 0ml 5ith B8 D 6vEv< ethanol for analysis
%sin# a Shimadz% 6=o)yo, Ja4an< UC -28 A, UC1visi(le recordin#
s4ectro4hotometer. =he a(sor4tion ma+ima for a%thentic crocin and safranal 5ere 99/
and /8B nm res4ectively as it has (een re4orted 4revio%sly 6S%Gata et al., -,,7<. A
standard c%rve 5as 4re4ared (y meas%rin# the a(sor4tion of crocin at 99/ nm.
Sample 1reparation and 3hromato%raphic 3onditions
=o se4arate crocin and safranal and meas%re amo%nts of those in a saffron
e+traction a sli#htly modified 4%(lished method 5as %sed 6S%Gata et. al., -,,7<. =he
e+tract of a%thentic sti#mata in B8 D 6vEv< ethanol 5as 4assed thro%#h a 0.8 m
Milli4ore filter 6Milli4ore, "edford, MA, U.S.A< and el%ted 5ith -88 D acetonitrile.
A Shimadz% HP3' 3'1-8AHv4 system inte#rated 5ith a Shimadz% S'31-8Av4
system controller and a SPH1-8Av4 UC1visi(le s4ectro4hotometric detector 5as %sed
for the analysis of the all com4o%nds. A Shimadz% 70cm + 9.2 mm .H. Shim14ac)
CP1*HS col%mn 5ith a -7.8

-.8 nm 4ore size and 9.2

8./ Im 4article size 5as
%sed for the se4aration and identification of com4o%nds. A &heodyne Shimadz%
Model ..70i inGector 5as %sed to inGect 78 l of the sam4le from a 70 l Hamilton
strai#ht1ed#e needle syrin#e onto the col%mn. All data 5ere recorded and analyzed on
chromato#ra4hy 5or)station Shimadz% 'lass1CP
=M
2.-8 soft5are. 'rocin 6
ma+J
99/<
and safranal 6
ma+J
/8B< 5ere se4arated on an isocratic r%n 5ith mo(ile 4hase consist
of .2 D acetonitrile in 5ater at a flo5 rate of -.7 mlEmin. n crocin chromato#rams a
4ea) at /8B 66
ma+
of safranal< 5as also a44eared.
:ES72TS A<4 4IS37SSIO<
=he amo%nt of crocin content of saffron 5hich has (een re4orted 4revio%sly is
7, m#E# 63i et al., -,,,<, 90.,, m#E# 6'a(allero1*rte#a et al., 7889< for ranian
saffron and 2../ m#E# 6S%Gata et al., -,,7< for ndian sam4les.
All direct readin# E
-D
of e+tracted saffron of sam4les in distilled 5ater at 998
nm 5ere a(ove -,8. =his indicates all the saffron sam4les (elon# to cate#ory
accordin# to S* classification 6see =a(le -<. Amo%nts of crocin in the analyzed
saffron sam4les 5ere fairly close 5hen they 5ere analyzed either (y HP3' or UC1
Cisi(le s4ectro4hotometry. As it has (een re4orted 4revio%sly 6S%Gata et al., -,,7<
there 5as a #ood correlation (et5een the crocin res%lts o(tained from the t5o
different methods 6=a(le 7<. =he e+traction method of crocin accordin# to S* /2/7
5as modified in order to im4rove the e+traction 4roced%re. n this modification
instead of 4%re distilled 5ater for e+traction, mi+t%re of distilled 5ater and ethanol
608K08< 5as %sed. Also sonication 5as also %sed to increase the yield of e+traction of
safranal and crocins. 'rocin content of one of the saffron sam4les 6Mashhad sam4le<
e+tracted %sin# classical S* method and modified 4roced%re 5ere /7./ and /2./ m#E
# res4ectively 5hen meas%red (y UC1Cisi(le s4ectro4hotometry. =he amo%nt of
crocins meas%red (y HP3' for the same sam4le 5as fo%nd to (e /B./ m#E# 6=a(le 7<.
=he amo%nts of safranal in this sam4le 5ere also increased 5hen UC1Cisi(le
s4ectro4hotometry and modified e+traction 4roced%re 5ere %sed. =hey 5ere 0.2 and
..2 m#E# for classical S* method and modified 4roced%re res4ectively. As
4revio%sly mentioned crocin has a(sor4tion at safranal a(sor4tion ma+ima 6
ma+
J
//8< , therefore, the res%lts of meas%rin# safranal (y UC1Cisi(le s4ectro4hotometry
sho5s hi#her than the real amo%nt in the saffron sam4le 6Alonso et al., -,,B:
=arantilis et al., -,,9<. For Mashhad saffron sam4le, safranal content 5as 0.2m#E#.
=he amo%nt of safranal in the same sam4le saffron sam4le 5as 8.-. m#E# 5hen
meas%red (y HP3'. Hirect readin# of this sam4le at //8 nm 5as 97.9 5hich is
(et5een 78 108 accordin#ly. As it has (een indicated (y others 6Alonso et al., 788-<
these res%lts also sho5s that the direct readin# of safranal at //8 nm 5hich is a
criteria for safranal content 6accordin# to S* /2/7< can not (e relia(le. A%thentic
crocin had also a(sor4tion at safranal ma+ima: therefore 4re4arin# a standard c%rve
for safranal at /8Bnm seemed im4ractical. *%r res%lts indicated that the hi#hest
amo%nt of crocin amon# the analyzed sam4les from different cities 5as fo%nd in
Sa(zevar 698.7 m#E#< and the lo5er one 5as in !eisha(oor 6/7.2 m#E#< 6=a(le 7<.
Accordin# to the HP3' method, the hi#hest amo%nt of safranal 5as fo%nd in
!eisha4oor sam4le 68.7, m#E#< and the lo5est amo%nt 5as in Kashmar sam4le 68.82
m#E#<. =he amo%nt of safranal from Keshmir, ndia re4orted 4revio%sly is B./ m#E#
%sin# HP3' method 6S%Gata et al., -,,7<. =his 5as fo%nd to (e -..,m#E# for ranian
sam4le 5hen meas%red (y thermal desor4tion1#as chromato#ra4hy method 6=H1L'<
6Alonso et al., 788-<. =he direct readin# of this sam4le 5as also re4orted to (e 99.,.
=he latter one is in a#reement 5ith o%r res%lts 697.9 for Mashhad sam4le<. =he
difference (et5een the content of safranal in ranian saffron in this research
68.8218.7, m#E#< 6=a(le 7< and the 4revio%sly re4orted one 6-.., m#E#< 6Alonso et al.,
788-< may (e d%e to different 4rocessin# methods and vario%s stora#e conditions in
different locations.
A3K<O;2E4=EME<TS
=his st%dy 5as s%44orted (y #rant from the Mashhad University of Medical
Sciences. Me sho%ld also than) Ms. Panahi 5ho hel4ed %s d%rin# the 4roGect.
2iterature 3ited
Alonso, L.3., Salinas, M.&. and LariGo, J. -,,B. Methods to determination
a%thenticity of aroma of saffron 6Crocus sativus 3.<. J. Food Prot. 2-K-07-1-0/8.
Alonso, L.3., Salinas, M.&. S>nchez1Fern>ndez M.A. and LariGo, J. 788-. Safranal
'ontent in S4anish Saffron. Food Sci. =ech. nt. .K770N77,.
'a(allero1*rte#a, H., Pereda1Miranda, &., &iverOn1!e#rete, 3., Hern>ndez, J.M.,
MedPci#o1&;os, M., 'astillo1Cillan%eva, A. and A(d%llae, F.. 7889. 'hemical
'om4osition of Saffron 6Crocus sativus 3.< from fo%r 'o%ntries. Acta Hort.
208K/7-1/72.
'%)o, 3., Mitso4o%lo%, =. and =simido%, M.Z. 7889. A ra4id 4roced%re for the
eval%ation of saffron colorin# stren#th %sin# tristim%l%s colorimetry. Acta Hort
208K 7B717BB.
Fern>ndez , J.A. 7889. "iolo#y, (iotechnolo#y and (iomedicine of saffron. &ecent
&es. Hevel. Plant Sci. 7K-7.1-0,.
S& 70,17. 7882. Saffron 6Crocus sativus 3<. ranian Standard $ nd%sterial
&esearch nstit%te. htt4KEE555.isiri.or#EstdE70,17.htm.
S* /2/71-$7. -,,/. Saffron 6Crocus sativus 3.<. =he nternational *r#anisation for
Standardisation 6First Edition<. S5itzerland. 55.iso.chEisoEenE.
3i, !., 3in L., K5an, M.Q., Min, Z.H. -,,,. Sim%ltaneo%s ?%antification of five
maGor (iolo#ically active in#redients of saffron (y hi#h14erformance li?%id
chromato#ra4hy. J. 'hrom. B9,K/9,1/00.
&ios, J.3., &ecio, M.'., Liner, &.M. and MaRez, S. -,,2. An %4date revie5 of saffron
and its active com4o%nds. Phytother. &es. -8K-B,1-,/.
S%Gata, C., &avishan)ar, L.A. and Cen)ataraman, 3.C. -,,7. Methods for the
analysis of the saffron meta(olites crocin, crocetins, 4icrocrocin and safranal for
the determination of the ?%ality of the s4ice %sin# thin1layer chromato#ra4hy,
hi#h14erformance li?%id chromato#ra4hy and #as chromato#ra4hy. J. 'hromato#r.
279K9,.1087.
=arantilis, P.A., Polissio%, M. and Manfait, M. -,,9. Se4aration of 4icrocrocin, cis1
transcrocins and safranal of saffron %sin# hi#h14erformance li?%id
chromato#ra4hy 5ith 4hotodiode1array detection. J. 'hromato#r. 229K0012-.
=arantilis, P.A., =so%4ras, L. and Polissio%, M. -,,0. Hetermination of saffron
6Crocus sativus 3.< com4onents in cr%de 4lant e+tract %sin# hi#h14erformance
li?%id chromato#ra4hy1UC1visi(le 4hotodiode1array detection1mass s4ectrometry.
J. 'hromato#r. 2,,K-8.1--B.
=arvand. 7882. Saffron classification, 555.tarvandsaffron.comEclassification.htm.
=a(le -. S* criteria for saffron ?%ality 6=arvand, 7880<
3haracteristic
:e&uirement
Saffron in
filaments
Saffron in 1owder
form
Test
method
"itterness, e+4ressed
as direct readin# of
the a(sor(ance
of 4icrocrocin at
a(o%t
70. nm, on dry (asis,
min.
'ate#ory
'ate#ory
'ate#ory
'ate#ory C
.8
00
98
/8
.8
00
98
/8
S*
/2/717,
cla%se -/
Safranal, e+4ressed
as
direct readin#
of the a(sor(ance
at a(o%t //8 nm, on
dry
cate#ories
min.
ma+.
78
08
78
08
S*
/2/717,
cla%se -/
'olorin# stren#th,
e+4ressed as direct
readin#
of the a(sor(ance of
crocin at a(o%t 998
nm,
on dry (asis, min.
'ate#ory
'ate#ory
'ate#ory
'ate#ory C
-,8
-08
--8
B8
-,8
-08
--8
B8
S*
/2/717,
cla%se -/
=otal nitro#en, D 6mE
m<, on dry (asis,
ma+.-<
/.8 /.8 S* -B.-
'r%(e fi(re, D 6mEm<,
on dry (asis, ma+.-<
2 2 S* 09,B

=a(le 7. Hetermination of crocin and safranal in vario%s saffron sam4les
%sin# HP3' and UC1visi(le s4ectro4hotometry
3ocation
Saffron 'rocin
6m#E#<
Safranal
6m#E#<
Hirect readin#
-
UC1visi(le
S*
e+t.
7
Modifie
d e+t.
/
S*
e+t.
7
Modifie
d e+t.
/
HP3'
Modifie
d e+t.
/
HP3'
Modified
e+t.
/
=or(atGam 7,- /7/ //., /..2 98.7 8.77
=or(at /88 /// /9.B /B.B 9-./ 8.72
Sa(zevar 7,B //8 /2./ 98.7 97., 8.8.
Kashmar 7.. /8B /7.2 /2.7 /B2 8.82
Mashhad 7., /-- /7./ /2.- /B./ 8.-.
!eisha(oor 722 9,0 /-.7 /7.2 /2., 8.7,
6-< E
-D
6-cm, 998 nm<: 67< e+traction accordin# to S* /2/7
method 5ith 5ater 6/< e+traction 5ith ethanol and 5ater 608K08< $
sonication
Fi#. -. Saffron main com4onents str%ct%res