Genetic and chemical analysis of Carthamus
species growing in Egypt and Libya
Name of dispatched researcher（派遣研究者名）

Dalia Adel Mohamed AlMahdy

Instructor（指導教官，研究指導者）

Meselhy Ragab Meselhy

Afﬁliation of Instructor（所属）

Faculty of Pharmacy, Cairo University

Host Collaborator（受入研究者）

Katsuko Komatsu

Introduction:
Carthamus L. is a genus belonging to the tribe Cynareae (thistle), subfamily Tubuliﬂorae
and family Compositae. The genus includes about 25 species, distributed from Spain and North
Africa across the Middle East to northern India (Ashri & Knowles, 1960). Carthamus tinctorius
L. known as Safﬂower is one of the most famous species and is the only species that is widely
cultivated. Safflower (known in Egypt as Kurtum and in Japan as Beni bana) was prized inancient Egypt as a source of red-yellow and orange dyes for cotton and silk, as well as ceremonial
ointment and to anoint mummies prior to binding. Safflower seeds and packets and garlandsof ﬂorets have been found with 4000-year-old mummies. Safﬂower has been used in the Middle
East, India and Africa for purgative and alexipharmic (antidote) effects, as well as in a medicatedoil, to promote sweating and cure fevers (Weiss, 1971). Safﬂower is currently cultivated in Egypt,
mainly for its seeds which are used as source of edible oil.
The dried florets of C. tinctorius have been used extensively for over 2,500 years in
Traditional Chinese Medicine (TCM) to treat stroke, coronary heart disease and angina pectoris(Zheng, Dong, & She, 1998). In the Compendium of Materia Medica, it is described as being able
to invigorate the circulation of blood”, which suggests its potential in circulatory system (Zhu,
1998). As well, Safﬂower was also found to demonstrate a number of other biological activity e.g.
antioxidant (Jun et al., 2011), anti-inﬂammatory (Choi, Kim, & Lee, 2010) and hepatoprotective
activities (Zhang et al., 2011).
Major active constituents isolated from Safflower are the C-glycoside pigments;hydroxysafﬂor yellow A, safﬂor yellow A, tinctormine, safﬂor yellow B, carthamin and safﬂomin
A (Kazuma et al., 2000; Meselhy et al., 1993; Onodera, Obara, Osone, Maruyama, & Sato, 1981;
Takahashi et al., 1984; X. Zhou, Tang, Xu, Zhou, & Wang, 2014). All of these compounds havebeen classiﬁed into the quinochalcone family of ﬂavonoid which have a unique structure with a− 72 −

C-glycosylated cyclohexanonedienol moiety (Fig. 1). In addition several kaempferol, quercetin,
luteolin and apigenin mono-, di- and triglycosides have been reported from the ﬂorets and theleaves of C. tinctorius (Hattori et al., 1992; Lee, Chang, Kim, Park, & Choi, 2002; Lim, 2007) as
well as alkaloids (Jiang, Lu, Yang, Zhang, & Zhang, 2008) and cinnamic acid derivatives (Y.-Z.
Zhou et al., 2008).
Regarding C. lanatus L., few reported the isolation of flavonoids, phenolic acids andsesquiterpene glycosides from the aerial parts (Mikhova, Duddeck, Taskova, Mitova, & Alipieva,
2004; San Feliciano, Medarde, Del Rey, Miguel Del Corral, & Barrero, 1990; Taskova, Mitova,
Mikhova, & Duddeck, 2003). Nothing was reported concerning the chemical composition of C.
divaricatus (Beg. et Vacc.) Pamp. or C. leucocaulos Sibthorp Smith.
C. tinctorius has several varieties which are cultivated widely in the Middle East, India,
Africa, China and Japan. However, genetic and chemical diversity of Carthamus species remains
unclear, which affects therapeutic effects.
The aim of this work is to present a comparative phylogenetic study and metabolite proﬁlingof several Carthamus species from Egypt and Libya.

Fig. 1: Structures of common compounds isolated from C. tinctorius L.

Part I: Genetic Characterization based on ITS sequences
Materials and Methods:
Plant Material
Three Carthamus species were collected from Libya, and two varieties were collected from
Egypt (Table 1). Vouchers are stored at the Museum of Materia Medica, Institute of Natural
Medicine, University of Toyama, Japan.
Genomic DNA Extraction
About 20-100 mg of the samples were cut to small pieces, frozen for 30 min at -80 ˚C andground using Tissuelyser for 2 min at 30 Hz. Total DNA was then extracted by DNeasy Plant
Mini Kit (Qiagen, Germany). Gel electrophoresis was performed to determine the quality andquantity of the DNA.

− 73 −

Table 1: Sample listof Carthamus species used in genetic study
Table 1: Sample list of Carthamus species used in genetic study
Code
1

2

DA-1

DA-3

Part

Date of

Used

Collection

var. Shandaweil, Sohag district, Leaves

May, 2013

Species
Carthamus

tinctorius

SourcetypicusUpper Egypt

C. tinctorius var. inermis
intimus

Shandaweil, Sohag district, Leaves

May, 2013

Upper Egypt
3DA-5

C. lanatus L.

Barqa district “near borders Flowerof Benghazi”, Libya

4

DA-6

5

DA-7

head

C. leucocaulos Sibthorp Smith Barqa district “near borders Flowerof Benghazi”, Libyaof Benghazi”, Libya

May, 2013head

C. divaricatus (Beg. Et Vacc.) Barqa district “near borders Leaves
Pamp.

May, 2013

May, 2013

6

DA-9

C. tinctorius L.

EuropeLeavesJuly, 20147

DA-10 C. tinctorius L.

Japan

Seeds

Sep, 2014

Polymerase Chain Reaction (PCR) Amplification

Genomic DNA Extraction

Using the tDNA as a template, ITS region (Fig. 2) of all the samples was amplified bypolymerase
chain reaction
using
Cycler.
reaction
About 20-100
mg of the(PCR)
samples
wereTakara
cut to PCR
small Thermal
pieces, frozen
for The30 minat -80 mixture
˚C andcontained:
0.5 µlTissuelyser
KOD-Plus-DNA
polymerase,µl dNTPs,
1 µl
MgSO4,
13.5
dH2O, Plant
2.5 µlground usingfor 2 minat 30 Hz.2.5
Total
DNA wasthenextracted
byµlDNeasy
10 ×
Buffer,2 µl of eachprimer Gel(ITS-1F,
18S-25S-3wasR)performed
and 1 µl templateDNA.
thermalMini
Kit (Qiagen,Germany).
electrophoresis
to determine
the Thequalityandcycling
conditions
for amplification were as follows: initial denaturation at 96 °C for 4 minquantityof the DNA.
followed by 35 cycles of denaturation at 94 °C for 20 s, annealing at 55 °C for 30 s, extension at

Polymerase
Reaction(PCR)°C for 7 min. The PCR product was detected by 1.0%
68 °C
for 1 min Chainand ﬁnal
extension
at 68Amplification
agarose gel electrophoresis followed by purification using SV gel and PCR clean up system

Using the tDNA as a template, ITS region (Fig. 2) of all the samples was amplified by(Promega, U.S.A.) and the DNA concentration was determined spectrophotometrically.

polymerase chain reaction (PCR) using Takara PCR Thermal Cycler. The reaction mixture
Sequencing and Molecular analysis

contained: 0.5 µl KOD-Plus-DNA polymerase, 2.5 µl dNTPs, 1 µl MgSO4, 13.5 µl dH2O, 2.5 µl

10×Buffer,
2 µl of each primer (ITS-1F, 18S-25S-3’R) and 1 µl template DNA. The thermal
Sequencing reaction of purified PCR products was carried out using ABI PRISM Bigdye
cycling conditions
amplification
as follows:
initialU.S.A.)
denaturation
at 96°C for(ITS-1F,
4 min
Terminatorv3.1 Cyclefor
sequencing
kitswere(Applied
Biosystems,with theprimers
followed by 35
cycles of denaturation
at 94(Table°C for 20s, annealingat 55 °C cycling
for 30 s,condition
extensionwas
at2, Fig.
2). The thermal
In18S-25S-3’R,
In18S-25S-5’F,
18S-25S-3’R)
as follows: Initial denaturation at 96 °C for 1 min
4 followed by 26 cycles of denaturation at 96 °C
for 10 s, annealing and extension at 50 °C for 5 s and ﬁnal extension at 60 °C for 4 min.
The products were purified using SAM solution and X Terminator, centrifuged and thesequence was determined directly by an ABI Prism 3100-Avant Genetic Analyzer (Applied
Biosystems, U.S.A.). The obtained sequences were edited and assembled and a consensus
sequence of each sample was reached. The DNA sequences were compared and aligned using the− 74 −

program MULTALIGN (http://multalin.toulouse.inra.fr/multalin/multalin.html).
The borders of ITS1, 5.8S and ITS2 regions were determined by comparison with the known
sequences of mung bean and rice (Schiebel & Hemleben, 1989; Takaiwa, Oono, & Sugiura, 1985).
Further, the DNA sequences of the tested samples were subjected to BLAST (http:// www.

68 °C for 1 min and final extension at 68 °C for 7 min. The PCR product was detected by 1.0%

ncbi.nlm.nih.gov/blast/blast.cgi) for better identification of sequence at species level and the

agarose gel electrophoresis followed by purification using SV gel and PCR clean up system(Promega,
U.S.A.)
thecompared
DNA concentration
was determined
resulting
GenBankdata andwere
and alignedwith the spectrophotometrically.
DNA sequences of the tested
samples.
In18S-25S-5’F

ITS-1F

18S

5.8S

25S

ITS-1

ITS-2

255bp

218bp
In18S-25S-3’R

18S-25S-3’R

Fig. 2: Approach for Sequence Determination of ITS region

Fig. 2: Approach for Sequence Determination of ITS region

Table 2: Primers usedTable
for PCRand Sequencing
2: Primers
used for PCRReactionand Sequencing Reaction

Sequencing and Molecular analysis
PrimerSequence (5’→3’)

Length(bp)ForwardSequencing
primerreaction of purified PCR products was carried out using ABI PRISM Bigdye
ITS-1FTerminator v3.1 Cycle
TCC
ACT GAATAT CATTTA G U.S.A.) with the primers
22 (ITS-1F,
sequencing
kitsCCT(Applied
Biosystems,
In18S-25S-5’F
TCT CGC ATC GAT GAA GAA CG
20
In18S-25S-3’R, In18S-25S-5’F, 18S-25S-3’R) (Table 2, Fig. 2). The thermal cycling condition

Reverse primer
was as follows: Initial denaturation at 96 °C for 1 min followed by 26 cycles of denaturation at

In-18S-25S-3’R
GAC TCG ATG GTT CAC GGG ATT CT
23
96 °C for 10 s, annealing and extension at 50 °C for 5 s and final extension at 60 °C for 4 min.
18S-25S-3’R
CCA TGC TTA AAC TCA GCG GGT
21The products were purified using SAM solution and X Terminator, centrifuged and thesequence was determined directly by an ABI Prism 3100-Avant Genetic Analyzer (Applied

Results
Discussion:
Results
andandDiscussion:
Biosystems,
U.S.A.). The obtained sequences were edited and assembled and a consensus(Figs.
3-6) andSeven
plant ofsamplesfrom wasEgypt,
Libya,and Japan
werealigned
studied.
The
sequence
each sample
reached.
TheEurope
DNA sequences
werecomparedusing
thenucleotide
sequenceof internaltranscribed spacer (ITS) region of all samples were clearlyprogram
MULTALIGN(http://multalin.toulouse.inra.fr/multalin/multalin.html).

Seven plant samples from Egypt, Libya, Europe and Japan (Figs. 3-6) were studied. The

nucleotideandsequenceof internal transcribed spacer (ITS) region of all samples were clearly
determinedcompared.
Theandbordersof ITS1,
5.8S andregionswereby comparison
with
known
The sequences
differedin length
byITS2one basepair,
in determinedwhich the ITS1
region was
255thebp,
thedeterminedcompared.
C. tinctorius
5.8S TherRNA
gene region
was 163bp, and218 bpsamplessequencesdifferedin length
bytheoneITS2baseregion
pair, wasin whichtheforITS1
region was
255 bp, thesequences of mung bean and rice (Schiebel & Hemleben, 1989; Takaiwa, Oono, & Sugiura, 1985).

Further,the DNA
testedsamples
wereDA-6
subjected
to BLAST(Table (http://3). It(DA-1, DA-3
and DA-10)
and sequences
219 bp for ofthetheothersamples(DA-5,and DA-9)

5.8S rRNA gene region was 163 bp, and the ITS2 region was 218 bp for C. tinctorius samples

www.ncbi.nlm.nih.gov/blast/blast.cgi)
for betterof sequenceat speciesvar.
leveltypicus
and theC. tinctorius
is worthy
to mention that both samples from
Egypt,identification
DA-1 and DA-3,

werecomparedand alignedDA-5
with thesequencesof thesamples.
inermisGenBank
C. DNA
lanatusC.tested
leucocaulos
and var.resulting
had the datasame
sequence,
meanwhile,and DA-6(DA-1, DA-3 and DA-10) and 219 bp for the other samples (DA-5, DA-6 and DA-9) (Fig. 3). It is

worthythetosame
mentionthat both samples from Egypt, DA-1 and DA-3, C. tinctorius var. typicus andshowedsequence.
var.
inermishad theofsame
sequence,
meanwhile,
DA-5 C.Nucleotide
lanatus andDA-6 Database
C. leucocaulos
C. tinctoriusThe
ITS sequences
depositedin International
Sequence(INSD:
DDBJ/EMBI/NCBI)
showedthe same sequence.included 6 main types,5 which differed from each other by three
nucleotides at positions 464, 531 and 624 (Table 3). Type 1 (JQ230977 (1)) shows C-C-C, type 2

The ITS sequences of C. tinctorius deposited in International Nucleotide Sequence Database(HQ112166 (2)) shows C-C-Y, type 3 (HQ112178 (3)) shows C-C-T, type 4 (HQ112171 (4)) shows(INSD: DDBJ/EMBI/NCBI) included 6 main types, which differed from each other by three

Y-C-C, type 5 (GU969649 (5)) shows T-C-C and ﬁnally Type 6 (GU969651 (6)) shows T-T-C.

nucleotides at positions 464, 531 and 624 (Table 3). Type 1 (JQ230977 (1)) shows C-C-C, type 2(HQ112166 (2)) shows C-C-Y, type 3 (HQ112178 (3)) shows C-C-T, type 4 (HQ112171 (4))− 75 −

shows Y-C-C, type 5 (GU969649 (5)) shows T-C-C and finally Type 6 (GU969651 (6)) shows TT-C.

On comparing the GenBank data available for C. lanatus, C. leucocaulos and C. divaricatus,
they showed a somewhat similar sequences but which are different from that of C. tinctorius,
where C. divaricatus (GU969623) differed in 23 nucleotides positions, C. lanatus (GU969619
C. lanatus subsp. baeticus and HM921409 C. lanatus CAULA-7) in 20 positions, while C.
leucocaulos (GU969634 C. leucocaulos and GU969635 C. leucocaulos) differed in 24 nucleotide
positions.
Regarding the studied samples, they showed ITS sequences that could be separated into two
main groups, the ﬁrst group comprises the C. tinctorius samples from Egypt and Japan where
they all showed similar sequence, to that found in GenBank differing at nucleotides positions 464
and 624, where C. tinctorius var. typicus (DA-1) and C. tinctorius var. inermis (DA-3) exhibited
additive peaks of thymine and cytosine (Y). In the case of C. tinctorius sample from Japan (DA-10),
additive peaks of thymine and cytosine (Y) were as well observed at nucleotide positions 464 and531, while position 624 showed cytosine.
The second group contained sequences of C. lanatus (DA-5), C. leucocaulos (DA-6) and
Carthamus sample from Europe (DA-9). Samples DA-5 and DA-6 showed similar sequence to C.
divaricatus (GU969623), C. turkistanikus (GU969653) and C. lanatus ssp. baeticus (GU969619)
from GenBank. Their patterns differed at nucleotide positions 117, 131, 201, 204, 211, 229, 255,
387, 439, 490 and 491.
In the case of Carthamus sample from Europe, it showed close similarity with C. lanatus ssp.
baeticus (GU969619), differing only in 3 nucleotides at positions 133, 597 and 617, where DA-9
showed peaks of (Y-W-Y) instead of (T-T-C). These ﬁndings clearly show that this sample is mostprobably C. lanatus L. sample not C. tinctorius L. as previously identiﬁed.

Fig. 3: C. tin
var. typicus flower heConcerning sample DA-7, previously identiﬁed as C. divaricatus (Beg. Et Vacc.) Pamp., itclearly possessed quite a different sequence from all Carthamus samples, as well as the reported
data in GenBank. The sample also showed different morphological characters than that ofCarthamus species, where the size of the flower head was significantly smaller and showing
differences in shapes of bracts. The ITS sequence of the sample showed >99% homology to
Eryngium campestre (family Apiaceae) HE602451 from GenBank as well as similar morphologicalcharacters.
C. tinctorius var. inermis flower head
Fig.Fig.
3: C.4:tinctorius
var. typicus flower headFig. 4: C. tinctorius var. inermis flower headFig. 3: C. tinctorius var. typicus flower headFig. 4: C. tinctorius var. inermis flower headFig. 5: C
flower head

− 76 −

Fig. 5: C. lanatus
flower headFig. 6: C
leucocaulos flower

Table 3: Comparison of ITS sequence among various samples of Carthamus species
Fig. 6: C. leucocaulos flower head

8
Fig. 5: C. lanatus flower head

Table 3: Comparison of ITSITS-1
sequence among 5.8S
various samples of Carthamus
species
ITS-2
Samples
Table 3:

Table 3: Comparison
among
1 of
1 1ITS
1 1
2of 2ITS
2 sequence
2among2 2 various
3
4 various
4 4 4 samples
4of4Carthamus
4 4of 4Carthamus
4 5species
5 5 species
5 5 5 5 5 6Comparison
sequence
samples
5 9 1 3 3 3 9 0 0 1 1 2 5
8
5.8S
ITS-1
0 8 7 1 3 4 8 1 4 1 9 9 5
7
1 1 1 1 1 2 2 2 2 2 2
3
T T C C T C T T C C G C T 5.8SC
5 9 1 3 3 3ITS-1
9 0 0 1 1 2 5
8
* * 1
* * 1
* * 1
* * 2
* * 2
* * 2
*
*
3
0 8 7 1
1 3 1
4 8 2
1 4 2
1 9 2
9 5
7
*5 *9 *1 *3 *3 3
* 9
* 0
* 0
* 1
* 1
* 2
* 5
*
8*
T T C C T C T
T C
C G
C T
C
*0 *8 *7 *1 *3 4
* 8
* 1
* 4
* 1
* 9
* 9
* 5
*
7*
* * * * * * * * * * * * *
*
*T T
* C
* C
* T
* C
* T
* T
* C
* C
* G
* C
* T
*
*
C
* * * * * * * * * * * * **** ** ** ** ** ** ** ** ** ** ** ** ***** * * * * * * * * * * * *
*
* C
* * T
* * * G* * * ** T
* ** C
*
T**
C
* * T
* * * T* * * T**** * * * * * * * * * * * *** * * * * * * * * * * * *** * * * * * * * * * * * **C C * Y * T G C T * T * *
*
T*
C
* C
* T
* T
* * T
* G
* * T
* * T
* * C
*

2
2
4
C
2
*
4
2
*
2
C
*
2
*
*
C
*
**
*
**
*
*
*
*
*

3
8
4
G
3
*
4
8
*
3
G
*
8
*
*
G
*
**
*
**
*
*
*
*
*

3 5 6 7 7 8 9 9 3 5 5 5 6 7 9 9
ITS-2
9 4 4 2 6 9 0 1 1 2 5 7 5 5 0 7
4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5
G A C A G T C G
C A G A A T T T
ITS-2
3 5 6 7 7 8 9 9
3 5 5 5 6 7 9 9* 4* *4 *4 *4 *4 *4 *4 *5 *5 *5 *5 *5 *5 *5 *54
9 4 4 2 6 9 0 1 1 2 5 7 5 5 0 7* 5* *6 *7 *7 *8 *9 *9 *3 *5 *5 *5 *6 *7 *9 *93GA C
A G T C G C A G A A T T T
* 4
* Y
9
4 *2 *6 *9 *0 *1 *1 *2 *5 *7 *5 *5 *0 *7* * * * * * * * * * * * * * * *
* A
* C
T A
* *G *T C
* G
* C
* A
* G
* A
* A
* T
* T
* T
*
G
* * * * * * * * * * * * * * * *** ** *T ** ** ** ** ** *T ** ** ** ** ** ** *** * Y * * * * * * * * * * * * ** C* * * * * ** C** ** C** ** C
** T
** C
** C
** **
T
* * *T *G *C *C T
* * Y
*
*
*
*
*
*
*
*
*
*
*
*
*
* * T * * * * * T * * * * * * ** * T * * * * * * * * * * * * ** C * G C C T C * C * C T C C *T* C* *T *G *C *C T
* C
* *T *C ** *C T
* C
* C
* *

1
7
6
C
1
*6
7
*1
C
*7
*
*
C
*
**
*
**
**
*
*
*

C. tinctorius
C. tinctorius
C. tinctoriusaSamples
JQ230977(1)b
HQ112178(3)Samplesc
aHQ112166(2)d JQ230977(1)bHQ112171(4)eaHQ112178(3)JQ230977(1)cGU969649(5)fbHQ112166(2)(6)
dGU969651
HQ112178(3)HQ112171(4)C.ceHQ112166(2)divaricatus
GU969649(5)d（GU969623）
f HQ112171(4)GU969651(6)e
C. turkestanicus
C.f GU969649(5)divaricatus（GU969653）
GU969651 (6)（GU969623）
C.
ssp.
C. lanatus
divaricatus
C. turkestanicus C C T T * T G * T * T * C
T* * T C * G C C T C * C * C T C C * *C C * Y*** * * C * G C C T C * C * C T C C * *baeticus
T * T G *C *T * T * C（GU969623）（GU969653）
C.(GU969619）
turkestanicus
C. lanatus ssp.
C C * Y * T G C T * T * *** * * C * G C C T C * C * C T C C * *C. lanatus（GU969653）
baeticus** * * C * G C C T C * C * C T C C * *C C * T * T G * * * T * C
CAULA-7** * * C * G C C T C * C * C T * C A *C C * T * T G * * * T * CC.
lanatus ssp.
(GU969619）（HM921409）
baeticus** * * C * G C C T C * C * C T C C * *C C * T * T G * * * T * CC.
lanatusgC.
leucocaulos(GU969619）
CAULA-7*
T
C C * *T * T G * *T * T * C* C* * C * G C C T C * C *A C T C
* C T
A C
*
(GU969634)
C.
lanatus（HM921409）
DA-3* * * * * * * * * * * * ** *Y *G *C *C T
* C
* ** C* ** C* T* ** C* A* **CAULA-7
g DA-1,**** ** ** C
C. leucocaulos C C * T * T G * * * T * C
DA-10
*C C
* * * * T
* G
* * T
* * T
* * C
*
* C
* * C
* *Y *G *C *C T
* C
* *Y *C *A C
* T
* C
* C
* T
* C
*
*
T
（HM921409）(GU969634)
g DA-5 , DA-6
C C Y Y * T G Y Y Y T M Y
Y* * K C * G C C Y S * C * C T C C * *C.DA-1,
leucocaulos
DA-3
*C C
* * * ** T
* * ** T* * * ** C*
* * ** C* Y * *C *C T
* * ** C
* * C
* * C
* * T
* *
***
T
DA-9
C C ** *T Y
T G
G * * ** T
T * C
* C
* * C ** GG C C T C
C * C A
* C T
T C C
C W C
Y
(GU969634)* * * * * * * * * * * * *** * * * Y * * * * * Y * * * * * * * *DA-10
DA-1, DA-3
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
Y
*
*
*
*
*
*
*
*
*
*
*
*
*
G C C.
C leucocaulos,
Y S * C * C T C C * **
DA-5
DA-6
C C
Y Y DA-3:* T G
Y Y Tvar.
M inermis,Y
Y DA-5:* C.
* lanatus,
K C * DA-6:
DA-1:
C., tinctorius
var.
typicus,C. Y
tinctorius
DA-10
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
Y
*
*
*
*
* Y * * * * * * * *C * TDA-10:
Y T G* * T(Japan)* C** * * C * G C C T C * C * C T C C W Y
DA-9: DA-9
C. tinctorius C(Europe),C. *tinctorius
DA-5 , DA-6
C C Y Y * T G Y Y Y T M Y
Y* * K C * G C C Y S * C * C T C C * *
DA-9
C =C
* TSDA-3:
Y
T G*tinctorius* Tvar.
C* DA-5:* C.
* lanatus,* C * DA-6:
G C C.
C leucocaulos,
T C * C * C T C 9
C W Y
DA-1:
C. tinctorius
typicus,C.W
inermis,K = G&T;
M = A&C;var.
R
A&G;= C&G;=* A&T;Y *= C&T
DA-9:
C.
tinctorius(Europe),DA-10:
C.
tinctorius(Japan)(a): JQ230977(1): JQ230977,
tinctorius,
SBB-1162;
GU724280,
C.C.
tinctorius;
C. tinctorius, CAUTI-19;
DA-1:
C. tinctorius
var. typicus,C.
DA-3:
C. tinctorius
var. inermis,
DA-5:
lanatus, HM921410,
DA-6: C. leucocaulos,
GU969650,
tinctorius,
DAV:27591;
C.
tinctorius; GU969647, C. tinctorius; EF483946, C. tinctorius
DA-9:
C. tinctorius(Europe),DA-10:
C.GU969648,
tinctorius(Japan)K = G&T;
M =C.A&C;
R= A&G;S= C&G;W= A&T; Y= C&T
cultivar, BJ1063; EF483943, C. tinctorius cultivar, W66730; HQ407426, C. palaestinus; GU969643, C. palaestinus;(a)(1):
JQ230977,
GU724280,
C. tinctorius;
HM921410,
C. C.
tinctorius,
GU969642,
palaestinus
voucher
DAV:24847;
C. glaucus,
HW-024;
HQ112172,
glaucus,CAUTI-19;
HW-019;K :=JQ230977
G&T; M C.
= A&C;R = A&G;
SC.=tinctorius,
C&G;
W =SBB-1162;
A&T;HQ112177,
Y = C&T
HQ112154,
C. tinctorius,
glaucus, DAV:27591;
HW-001; HQ112176
C. C.glaucus,
HW-023;
GU969644,
C. persicus;
GU969639,
C.
GU969650, C.
GU969648,
tinctorius;
GU969647,
C. tinctorius;
EF483946,
C. tinctorius(a)oxyacanthus
voucher
GH:244792;
GU969641,
C. oxyacanthus;
GU969640,
oxyacanthus(Malik& Babbar, Berner: JQ230977(1):EF483943,
JQ230977,
C.tinctorius
tinctorius,
SBB-1162;
GU724280,
C. tinctorius;
HM921410,
C. tinctorius,
CAUTI-19;
cultivar,
BJ1063;C.
cultivar,
W66730;
HQ407426,
C.C.palaestinus;
GU969643,
C. palaestinus;&
McMahon C.
andpalaestinus(Bowles etDAV:27591;al) .
GU969650,
tinctorius,
GU969648,
C. tinctorius;
GU969647,
C. tinctorius;
EF483946,
C. tinctorius
GU969642,
voucher
DAV:24847;
HQ112177,
C. glaucus,
HW-024;
HQ112172,
C. glaucus,
HW-019;cultivar, BJ1063;
EF483943,
C. tinctorius
cultivar,C.W66730;
palaestinus;
C. palaestinus;
HQ112154,
C. glaucus,
HW-001;
HQ112176
glaucus,HQ407426,
HW-023; C.
GU969644,
C. GU969643,
persicus; GU969639,
C.
(b): HQ112166(2):
HQ112166,
C. glaucus,
HW-013;
EF483948,
C.
tinctorius
BJ1067;(Malik
EF483947,
C. tinctorius
oxyacanthus
voucher
GH:244792;
GU969641,
C. HQ112177,
oxyacanthus;
C.cultivar
oxyacanthus
Babbar, Berner
GU969642,
C.
palaestinus
voucher
DAV:24847;
C.GU969640,
glaucus,
HW-024;
HQ112172,
C. &glaucus,
HW-019;cultivar
BJ673
et al etand
et al).
HQ112154,
C.(Huhglaucus,
HW-001;
HQ112176
C. glaucus, HW-023; GU969644, C. persicus; GU969639, C.
&
McMahonand(Bowlesal)Chapman.
oxyacanthus voucher GH:244792; GU969641, C. oxyacanthus; GU969640, C. oxyacanthus (Malik & Babbar , Berner
(b)
(c)
(b)
HQ112178(3):HQ112178,
HW-015et al).
HQ112166(2):
HQ112166,
glaucus,HW-000;
HW-013;HQ112168,
EF483948,C.
C.glaucus,
tinctorius
cultivar(Huh
BJ1067;
EF483947, C. tinctorius& :McMahon
and(Bowles
et al)C.
.C.glaucus,
cultivar BJ673 (Huh et al and Chapman et al).
(d)
(c)
(b)
HQ112155,
C. glaucus
HW-002;: HQ112171(4):HQ112166 (2):HQ112171,
HQ112166,C.
C.glaucus
glaucus,HW-018;
HW-013;HQ112164,
EF483948,C.
C.glaucus,
tinctoriusHW-011;
cultivar BJ1067;
EF483947,
C. tinctorius(c)
EF483950,
C. tinctorius
EF483949,
C. tinctorius
cultivarHW-015
BJ2701;(HuhEF483945,: HQ112178(3):HQ112178,
C. ToziSpiny;
glaucus,etHW-000;
HQ112168,
C. glaucus,
et al). C. tinctorius cultivar
cultivar
BJ673(Huh
et alcultivar
and Chapmanal).
ENANA ; EF483944, C. tinctorius cultivar LESAF (Huh et al and Chapman et al).
(c)(d)
: HQ112178(3):et al). C. glaucus HW-002;
HQ112171(4): HQ112178,
HQ112171, C.
C. glaucus,
glaucus HW-000;
HW-018; HQ112168,
HQ112164, C. glaucus,
glaucus, HW-015
HW-011;(Huh
HQ112155,(e): GU969649
GU969649,
tinctorius; FJ539127,
al and Daniel& Knoess).
EF483950,
C. (5):
tinctorius
cultivarC.ToziSpiny;
EF483949, C.
C. tinctorius(Bowles
tinctorius cultivar et
BJ2701;
EF483945,
C. tinctorius cultivar(d)
HQ112171,
C. cultivar
glaucusLESAF
HW-018;
HQ112164,
C. glaucus,etHW-011;
HQ112155, C. glaucus HW-002;
ENANA; EF483944,
C. tinctorius(Huh
et al and Chapmanal).
(f) : HQ112171(4):: GU969651 (6): GU969651, C. tinctorius.
EF483950, C. tinctorius cultivar ToziSpiny; EF483949, C. tinctorius cultivar BJ2701; EF483945, C. tinctorius cultivar
(e)
(g)
ENANA
; EF483944,
C. tinctorius
LESAF(Huh etand Chapman et etal).al and Daniel & Knoess).
: GU969649(5):
GU969649,
C. cultivar
tinctorius;
FJ539127,
C.
tinctorius(Bowles
GU969634:
GU969634,
C. leucocaulos;
GU969635,
C.alleucocaulos.
(f)(e): GU969651 (6): GU969651, C. tinctorius.

: GU969649 (5): GU969649, C. tinctorius; FJ539127, C. tinctorius(Bowles et al and Daniel & Knoess).

(g)(f) : GU969634: GU969634, C. leucocaulos; GU969635, C. leucocaulos.

10: GU969651 (6): GU969651, C. tinctorius.

(g): GU969634: GU969634, C. leucocaulos; GU969635, C. leucocaulos.

10− 77 −
10

6 6
2 2
0 4
6 6
C
2 2
-6 T
6
0 4
-2 Y
2
C
-0 *4
- T
- *
- C
Y
-- T
*
- *
-T
- *Y
-- **
- *
T *
T
- *
T *
T *
T *
T *
T *
T *
T *
- *Y
T
- *
T
T *
- Y
T
T **
- *
- Y
T
*
- *
T *
T *
T *

Part II: Chemical Profiling
The aim of the work reported here is to deﬁne and compare the phytochemical compositionof the different parts of the Carthamus ﬂower heads, as well as the leaves for the samples underinvestigation using HPLC-DAD and HPLC-DAD-MS.
An HPLC analysis was conducted ﬁrst to determine the optimal conditions for analysis using
C. tinctorius sample provided from Kanazawa University and the samples under study.
Plant Material:
The dried ﬂower heads of the samples under investigation were separated into ﬂorets, pappus,
inner bracts and outer bracts, in addition to the leaves (Table 4, Fig. 7), they were groundseparately using an electric crusher or in a mortar using liquid nitrogen for samples of small
amounts.
Different quantities of plant material were used for extraction; 20 mg, 50 mg and 100 mg todetermine the suitable amount. The powders were then vortexed and homogenized separately
with 5 ml MeOH using an ultrasonic bath for 30 min. Extracts were centrifuged at 4000 rpm for
10 min and ﬁltered through 22 µm membrane ﬁlter.
HPLC Apparatus:
Shimadzu HPLC (pump: LC-10AD vp, Autosampler: SIL-10AD vp, Detector: SPD-M20A
multiwavelength detector)
Column: Zorbax SB-C18, 250 × 4.6 mm, 5 µm, Agilent.
Solvent: A: Acetonitrile, B: 0.1% Aqueous formic acid
Flow rate: 1 ml/min
Column temp: 25 ˚CInjection volume: 20 µl

7: Different
partsofofC.
C. tinctorius
tinctorius var.
usedin Chemical
studystudy
Fig.Fig.
7: Different
parts
var.typicus
typicususedin Chemical

Table 4: Sample list for Chemical profiling:− 78 −
Code
DA-1

SpeciesC. tinctorius var. typicus

Site
Shandaweil, Sohag district, Upper Egypt

Part Used
LeavesFig. 7: Different parts of C. tinctorius var. typicus used in Chemical study

Table 4:profiling:
Sample list for Chemical profiling:
Table 4: Sample list for Chemical
Code
DA-1
DA-2-1DA-2-2DA-2-3DA-2-4DA-3DA-4-1DA-4-2DA-4-3SpeciesC. tinctorius var. typicus
C. tinctorius var. typicus
C. tinctorius var. typicus
C. tinctorius var. typicus
C. tinctorius var. typicus
C. tinctorius var. inermis
C. tinctorius var. inermis
C. tinctorius var. inermis
C. tinctorius var. inermis

DA-5-1DA-5-2DA-5-3

C. lanatus L.
C. lanatus L.
C. lanatus L.

DA-5-4DA-6-1DA-6-2

C. lanatus L.
C. leucocaulos Sibthorp Smith
C. leucocaulos Sibthorp Smith

DA-6-3DA-6-4
DA-12
DA-13-1
DA-13-2
DA-13-3
DA-13-4

C. leucocaulos Sibthorp Smith
C. leucocaulos Sibthorp Smith
C. tinctorius L.
C. tinctorius L.
C. tinctorius L.
C. tinctorius L.
C. tinctorius L.

Site
Shandaweil, Sohag district, Upper Egypt
〃
〃
〃
〃
〃
〃
〃
〃
Barqa district “near borders of Benghazi”, Libya
〃
〃
〃
〃
〃

〃
〃
Kanazawa University〃〃〃〃

Part Used
LeavesFlorets
PappusInner bract
Outer bract
LeavesPappusInner bract
Outer bract
Florets
PappusInner bract
Outer bract
Florets
PappusInner bract
Outer bract
LeavesFlorets
PappusInner bract
Outer bract

12
Results:- Extraction:
Results of HPLC analysis of the tested samples showed that 50 mg were suitable for samplesof the leaves and ﬂorets, while 100 mg of powdered plant material was optimal for pappus, innerand outer bract samples.
- Chromatographic Conditions:
Mixtures of Acetonitrile in combination with 0.1% aqueous formic acid were tried as mobile
phases and good separation was achieved with the following conditions:
Mobile phase: A: Acetonitrile, B: 0.1% Aq. Formic acid
Program: 0-30 min, 10-30% A; 30-35 min, 30-35% A; 35-50 min, 35-70% A; 50-55 min, 70-10% A
ﬁnally 55-60 min, 10%A.
Injection volume: 20 µl, Flow rate: 1 ml/min, Column temp.: 25˚C
- Two chromatograms were examined from the DAD spectral data: the 407 nm chromatogram fordetecting quinochalcones and the 350 nm chromatogram for detecting ﬂavonols.

− 79 −

Discussion:Comparing the chromatograms of the different tested organs (Figs. 8-14), it was clear thatquinochalcones are mainly present in the samples of the ﬂorets, while all other samples (pappus,
inner bracts, outer bracts and leaves) showed peaks having characteristic UV pattern of ﬂavonols
which is in accordance with previous references (Lee et al., 2002; Yue, Tang, Li, & Duan, 2013).
It is worthy to mention that the two C. tinctorius varieties (typicus and inermis) from Egypt
exhibited similar pattern in all tested samples, which was different from those samples of C.
lanatus and C. leucocaulos from Libya (Fig. 9-14). These results are in accordance with theresults obtained from the genetic study where these samples showed identical patterns.
As well, the extracts of the inner and outer bracts showed similar patterns to each and tothat of the leaves (Fig. 8).
Up to date, there is no report dealt with chemical constituents of the different parts ofCarthamus florets viz; pappus, inner bracts and outer bracts, so it is of interest to compare
and identify chemical components using HPLC-DAD-MS. Preliminary data was obtained from
HPLC-DAD-MS analysis, and annotation of peaks and quantiﬁcation of major constituents areundergoing.

components using HPLC-DAD-MS. Preliminary data was obtained from HPLC-DAD-MS analysis, andannotation of peaks and quantification of major constituents are undergoing.

Fig 8: Chromatograms of different parts of C. tinctorius (Kanazawa Univ.) at 407 nm
Fig 8: Chromatograms of different parts of C. tinctorius (Kanazawa Univ.) at 407 nm− 80 −

Fig 8: Chromatograms of different parts of C. tinctorius (Kanazawa Univ.) at 407 nm

Fig. 9: Chromatograms of Carthamus floret samples at 350 nm
Fig. 9: Chromatograms of Carthamus floret samples at 350 nm

14

Fig.
of Carthamus
samples
at 407 nm
Fig.10:
10: Chromatograms
Chromatograms of Carthamus
floretfloret
samples
at 407 nm

Fig. 10: Chromatograms of Carthamus floret samples at 407 nm

Fig. 11: Chromatograms of Carthamus floret samples at 512 nm

Fig. 11: Chromatograms of Carthamus floret samples at 512 nm
Fig. 11: Chromatograms of Carthamus floret samples at 512 nm
15− 81 −
15

DA-5-2DA-5-2

Fig. 12: Chromatograms of Carthamus pappus samples at 350 nm
Fig. 12: Chromatograms of Carthamus pappus samples at 350 nm
Fig. 12:Chromatograms of Carthamus pappus samples at 350 nm

matograms of Carthamus pappus samples at 350 nm

Fig 13:
Fig 13:Chromatograms of Carthamu
Chromatograms of Carthamu
inner bracts
samples
at 350 nm
Fig 13:Chromatogramsof Carthamus
Fig 13: inner bracts samples at 350 nm
inner bracts samples at 350 nm
Chromatograms of Carthamus

s at 350 nm

16
16
Fig 14: Chromatograms of Carthamus outer bracts samples at 350 nm

16− 82 −

Experiences gained and Significance of the young researcher fellowship:
1-

Learn the protocol of gene sequence analysis in authentication and quality evaluation of
herbal drugs/medicinal plants, using Carthamus species collected from Egypt and Libya asa case study.

2-

Clarify the differences/similarities among Carthamus species in rDNA ITS sequence.

3-

Operate an LC-MS instrument and obtain chemical proﬁling of the Carthamus species to
explore the relation between genotypes and chemo-types.

4-

Strengthen the collaboration between ICCO in Egypt and Japan.

Acknowledgment:
I am deeply grateful to Prof. Katsuko Komatsu and Assistant Prof. Shu Zhu (Departmentof Pharmacognosy, Institute of Natural Medicine, University of Toyama, Japan) for offering
an extremely good environment for me and for this research. I am also very grateful to all thelaboratory members for their indispensable help during my stay. This work was supported by
2014 GRANT-IN-AID for general research II program, Institute of Natural Medicine, Universityof Toyama, Japan.
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04x
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− 84 −