Phenolic compounds from Carthamus tinctorius

Article abstract of DOI:10.1023/B:CONC.0000025456.09089.84

The contents of quercetin, luteolin, apigenin, isorhamnetin, umbelliferone, and daphnoretin in common safflower and of acacetin in the flowers were determined based on the physical and chemical properties of the isolated substances.

Full text of DOI:10.1023/B:CONC.0000025456.09089.84

Chemistry of Natural Compounds, Vol. 40, No. 1, 2004
PHENOLIC COMPOUNDS FROM Carthamus tinctorius
T. A. Suleimanov
UDC 547.992
The contents of quercetin, luteolin, apigenin, isorhamnetin, umbelliferone, and daphnoretin in common
safflower and of acacetin in the flowers were determined based on the physical and chemical properties of
the isolated substances.
Key words: Carthamus tinctorius, orthodihydroxy group, bathochromic shift, hydroxy groups.
Common safflower (Carthamus tinctorius L.) belongs to the Asteraceae family. The genus comprises 19 species, of
which 5 are represented in the flora of Azerbaidzhan [1]. Safflower is known from antiquity in India, Egypt, Iran and other
Asian countries, and North Africa. This plant replaces sunflower in certain regions of Russia and arid regions of Asia. It is
used to produce high-qualitymargarine; is found in dietetic products for patients with hypocholesterolemia, myocardial infarct,
and atherosclerosis; and also possesses purgative and cholagogic activity. It is applied externally to heal burns and other skin
diseases. Several medicinal preparations include safflower: safflower oil (USA), obesitol (England), Red Dragon pills
(China) [2-4].
In spite of the wide use of the oil, the chemical composition of the plant is poorly studied. It is known that the oil
content is 15-30% depending on the habitat. The non-drying oil contains up to 75% linoleic acid and up to 7% saturated fatty
acids [2, 5, 6]. The chalcone glycosides carthamine and carthamidin-5-glucoside were found in the petals [5].
The aerial part of C. lanata contained bisabolane fucoside, 3-norbisabolane fucoside, intermediol β-D-fucopyranoside,
and intermediol β-D-fucopyranoside α′,α-methylbutyrate [7, 8]. The new quinochalcone C-glycoside-Ca2+ antogonist -
tinctormine was also found [9]. The dyes carthamine (C H O ·H O) and safflower bile (C H O ) were isolated from the
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22 11
2
24 30 15
bright saffron-yellow petals [10].
We studied phenolic compounds from the herb and flowers of safflower. Ground raw material was extracted with
alcohol (70%). The extract was evaporated and chromatographed using n-butanol:acetic acid:water (4:1:2) in the first direction
and acetic acid (15%) in the second direction. Analysis of the chromatograms in the visible and UV regions used specific
reagents allow to detect in the herb and flowers up to 30 phenolic compounds. Some of them were hydroxycinnamic
acids (6 compounds) and hydroxycoumarins (4-5 compounds), the rest were flavonoids.
The evaporated aqueous extract was treated successivelywith ether, CHCl , ethylacetate, and n-butanol. The ether and
3
CHCl extracts from the herb were separated over a polyamide sorbent column with elution by CHCl and CHCl :alcohol
3
3
3
mixtures of increasing alcohol concentration. Compounds 1-4 were isolated from the ether extract; 5 and 6, from the CHCl3
extract; and 7, from the CHCl extract of flowers.
3
Compounds 1-4 and 7 are yellowcrystals that are verysoluble in ethanol, methanol, and acetone and insoluble in water
and CHCl3.
The aglycons of the studied compounds were identified using their chromatographic mobilities in
benzene:ethylacetate:acetic acid:water (50:50:1:1) and CHCl :acetic acid:water (13:6:1) and the Bryant cyanidin reaction.
3
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IR spectra of the aglycons showed that the phenolic hydroxyls appear at 3385-3275 cm ; carbonyl of the γ-pyrone ring
at 1670-1660 cm . Aromatic rings give several strong bands at 1615-1510 cm . Furthermore, an absorption band at
980 cm that is characteristic of methoxyl is found in the spectra of 4 and 7.
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-1
-1
2
Demethylation of 4 and 7 forms 1 and 3, respectively. The absorption maxima of the long-wavelength band in the UV
spectra of 1 and 4 are weaker than those of the short-wavelength one. This is characteristic of flavonols. Theyare almost equal
in 2, 3, and 7, which indicates they are flavones.
Azerbaidzhan Medical University. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 13-15, January-
February, 2004. Original article submitted October 2, 2003.
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0
009-3130/04/4001-0013 2004 Plenum Publishing Corporation
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The presence of free hydroxyls was established using ionizing and complexing reagents. Thus, addition of sodium
acetate tostarting solutions ofthe compounds produced a bathochromic shift ofthe long-wavelength maxima by15-21 nm. This
is consistent with a free hydroxyl on C-7 in the compounds. Sodium ethoxide causes a bathochromic shift of the long-
wavelength maximum in 2 (53 nm) and 3 (45 nm) and has no effect on 7. Compound 1 gives a hypsochromic shift of 50 nm
and of 13 nm with boric acid and sodium acetate. This is consistent with the presence of an orthodihydroxy group.
Compound 2 gives an analogous shift of 24 nm with boric acid and sodium acetate. Adding zirconium chloride to
starting solutions of 1-4 and 7 causes a bathochromic shift of the long-wavelength maxima by 17-91 nm. This disappears upon
adding citric acid, which is typical for a free hydroxyl on C-5. For 1 and 4, it decreases to 53 nm, which is due to the presence
in them of hydroxyls on C-3 also.
Alkaline destruction of the compounds produced fluoroglucinol and acids: for 1 and 2, 3,4-dihydroxybenzoic; for 3,
p-hydroxybenzoic; for 4, 3′-methoxy-4′-hydroxybenzoic; for 7, 4′-methoxybenzoic.
Qualitative reactions; physicochemical properties; UV, IR, and PMR spectra; alkaline destruction; constants of
demethylated derivatives; and chromatographic comparison with authentic samples established that 1 is quercetin; 2, luteolin;
3
, apigenin; 4, isorhamnetin; and 7, acacetin. The lack of melting point depressions of mixtures with authentic samples
confirmed these assignments.
Compounds 5 and 6 were coumarin derivatives based on qualitative color reactions, i.e., the presence of blue
fluorescence in UV light upon treatment of the chromatograms with alcoholic base (10%) and a yellowish-orange color in
daylight after treatment of the chromatograms with diazotized sulfanilic acid. The strong blue fluorescence of 5 and 6, which
intensifies after exposure to ammonia vapor or alcoholic base, is consistent with a free hydroxyl on C-7 in the structure.
The presence in the UV spectrum of a strong absorption maximum at 320-340 nm and in the IR spectrum of an
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absorption band at 1710-1730 cm , which is typical for stretching vibrations of a γ-pyrone carboxyl, confirm the correctness
of assigning them as coumarin derivatives [11].
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The presence in 5 of strong blue fluorescence and of an absorption band at 3200 cm in the IR spectrum is consistent
with the presence of a hydroxyl on C-7. Therefore, 5 should correspond to umbelliferone. The lack of melting-point depression
of a mixture of 5 with umbelliferone indicated that they are identical.
A comparison of the properties of known coumarins and 6 obtained by us and its acetyl (mp 240-242°C) and methyl
(mp 239-241°C) derivatives showed that it was similar to daphnoretin, which was first isolated from Daphne odora Thunb. and
also from seeds of a Coronilla species [12]. This assumption was confirmed by thermal decomposition of 6 in a stream of H2.
Chromatographyof the decomposition products detected scopoletin and umbelliferone (5). Thermal destruction confirmed that
6
isolated by us was identical to daphnoretin-6-methoxy-7-hydroxy-3,7′-dicoumarin ether.
EXPERIMENTAL
Melting points were determined on a Kofler block; UV spectra, on an SF-46 spectrophotometer; IR spectra, on a UR-20
instrument. PMR spectra were recorded on a Bruker WP-400 spectrophotometer (working frequency 400 MHz, DMSO-d6
solvent).
Isolation of Phenolic Compounds. Air-dried safflower herb (2 kg) was extracted with alcohol (70%) at a 1:10 raw
material:extractant ratio. The aqueous alcohol extract was evaporated to 2 L of aqueous solution and treated successively with
diethylether, CHCl , ethylacetate, and n-butanol. The ether extract was evaporated and separated over a polyamide sorbent
3
column (h = 60 cm, d = 4 cm) with elution by CHCl and CHCl :alcohol mixtures with increasing alcohol concentrations.
3
3
Fractions of 100-150 mL were collected. The qualitative compositions of the fractions were analyzed bypaper chromatography
using benzene:ethylacetate:acetic acid:water (50:50:1:1), CHCl :acetic acid:water (13:6:1), and acetic acid (50%). Similar
3
fractions were combined and evaporated. Dry solids were crystallized from alcohol. The ether fraction of safflower
afforded 1-4.
The extract of safflower flowers (2 kg) was isolated analogously. The CHCl extract of safflower herb afforded 5 and
3
6
by the same method; the CHCl extract of flowers, 7.
3
Quercetin (1). (3,5,7,3′,4′-pentahydroxyflavone), C H O , mp 313-315°C, UV spectrum (λmax, nm): 256, 370;
15 10 7
+
+
CH COONa 274, 385; +CH COONa + H BO 264, 383; +ZrO(NO ) 270, 461; + ZrO(NO ) + citric acid 265, 428;
3
3
3
3
3 2
3 2
C H ONa 250, 320.
2
5
1
4

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IR spectrum: 1665 (C=O), 3385-3300 (OH), 1612, 1560, 1518 cm (aromatic C=C).
Luteolin (2). (5,7,3′,4′-tetrahydroxyflavone), C H O , mp 327-329°C, UV spectrum (λmax, nm): 256, 265, 352;
15 10 6
+
+
CH COONa 272, 368; +CH COONa + H BO 265, 376; +ZrO(NO ) 280, 405; +ZrO(NO ) + citric acid 256, 352;
3
3
3
3
3 2
3 2
C H ONa 272, 405.
2
5
The IR spectrum has been reported [13].
PMR (δ, ppm, J/Hz): 6.35 (1H, d, J = 2.0, H-6), 6.50 (1H, d, J = 2.0, H-8), 6.10 (s, H-3), 6.83 (1H, d, J = 8.0, H-5′),
7
.29 (br.s, H-2′), 7.32 (1H, s, H-6′).
Apigenin(3). (5,7,4′-trihydroxyflavone), C H O , mp343-346°C, UVspectrum (λ , nm): 272, 343; +CH COONa
15
10
5
max
3
2
75, 365; +CH COONa + H BO 272, 345; +ZrO(NO ) 310, 355; +ZrO(NO ) + citric acid 270, 345; +C H ONa 280, 410.
3
3
3
3 2
3 2
2 5
The IR spectrum has been reported [13].
Isorhamnetin (4). (3,5,7,4′-tetrahydroxy-3′-methoxyflavone), C H O , mp 317-319°C, UV spectrum (λmax, nm):
16 12 7
2
55, 266, 372; +CH COONa 275, 385; +CH COONa + H BO 256, 370; +AlCl 265, 430; +C H ONa 255, 334.
3 3 3 3 3 2 5
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IR spectrum: 1658 (C=O), 3455 (OH), 2895 (CH ), 1605, 1565, 1505 cm (aromatic C=C).
3
Umbelliferone (5). (7-hydroxycoumarin), C H O , mp 228-230°C, UV spectrum (λmax, nm): 218, 251, 323;
9
6 3
+
C H ONa 231, 370.
2 5
The IR spectrum has been reported [14].
Daphnoretin(6). (6-methoxy-7-hydroxy-3,7′-dicoumaricether), C H O , mp254-256°C, UVspectrum (λmax, nm):
19 12 7
2
28, 265, 325, 345; +C H ONa 240, 280, 368, 393.
2 5
The IR spectrum has been reported [14].
Acacetin (7). (5,7-dihydroxy-4′-methoxyflavone), C H O , mp 268-270°C, UV spectrum (λmax, nm): 270, 328;
16 12 5
+
+
CH COONa 275, 380; +CH COONa + H BO 272, 338; +ZrO(NO ) 277, 245; +ZrO(NO ) + citric acid 270, 328;
3
3
3
3
3 2
3 2
C H ONa 280, 377.
2
5
The IR spectrum has been reported [13].
Demethylation of 4 and 7. Compounds 4 and 7 (10 mg each) were dissolved in acetic anhydride (5 mL), treated
gradually with HI (d = 1.7), refluxed gently for 2 h, cooled, and diluted into sodium thiosulfate solution (5 mL). Light yellow
precipitates of the demethylation products of 4 and 7 were recrystallized twice from aqueous ethanol to afford from 4 quercetin
(
1), mp 312-315°C, and from 7 apigenin (2), mp 342-345°C.
Daphnoretin Acetate. A solution of 7 (10 mg) in absolute pyridine (3-5 mL) was treated with acetic anhydride
3.5 mL), left for 3 d, and poured into a crystallizer containing cold water. The resulting crystals were recrystallized from
(
methanol to afford daphnoretin acetate, mp 240-242°C.
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