Compounds from flowers of Daucus carota L. SSp. carota and their antioxidant activity

Full text of DOI:10.1007/s10600-010-9479-7

Chemistry of Natural Compounds, Vol. 45, No. 6, 2009
COMPOUNDS FROM FLOWERS OF Daucus carota L. SSP. carota
AND THEIR ANTIOXIDANT ACTIVITY
1*
2
1
Yurdanur Akgul, Lale Yildiz Aktas, and Huseyin Anil
UDC 547.972
Daucus carota L. ssp. carota, wild carrot, belongs to the family Apiaceae (Umbelliferae). It is a tall robust biennial
spiny-fruited herb that grows in dried-out fields [1]. It is indigenous to Europe and has antibacterial, stumulant [2],
antisteroidogenic [3], anti-flammatory [4], antiseptic, carminative, diuretic, and hepatoprotective properties [5]. Chemical
studies on the seed oil and the root of Daucus carota L. have revealed a variety of chemical counstituents such as daucane-type
sesquiterpenes [6–9], flavonoids [10], polyacetylenes [11], fatty oils [12], β-carotene [13], and essential oil [14].
The dichloromethane–methanol (1:1) extract of the flowers of Daucus carota L. was partitioned between water and
n-butanol. The n-butanol soluble portion was subjected to successive column chromatographic separations using silica gel to
afford compounds 2–5. The water soluble fraction give compound 1. Identification of compounds 1–5 was performed using
1
13
1D ( H, C)-2D NMR (HMBC, HMQC, COSY), LC/MS-APCI, and literature data.
Compound 1 was obtained as a white amorphous solid from the water soluble fraction. The positive LC/MS-APCI
+
spectrum of 1 showed a molecular ion peak at m/z 183.10 [M+H] corresponding to themolecular formula C H O . The main
6
14 6
+
+
1
13
fragment peaks were recorded at m/z [M–OH] 165.10 and m/z [M–CH=OH] 152.10. The H NMR and C NMR spectra
revealed signals for eight aliphatic hydrogens at δ 3.62 (2H), 3.71 (4H), and 3.77 (2H) and three carbons at δ 63.51, 69.65,
H
C
and 71.28. The melting point of compound 1 was not identified (decomposed above 149°C). The melting point of the acetylated
1
derivative 1a was found to be 121°C. The H NMR spectrum of 1a showed signals at δ 5.42 (2H, d, J = 8.0 Hz), 5.06 (2H, m),
H
1
1
4.21 (2H, dd, J = 2.8, 2.8 Hz), 4.06 (2H, dd, J = 5.2, 5.2 Hz), and 2.06, 2.04, and 2.02 (18H, s). The H– H COSY spectrum
showed a correlation between signals at δ 5.42 and 5.06, δ 5.06 and 4.21, δ 5.06 and 4.06, and δ 4.21 and 4.06. The
H
H
H
H
13
C NMR spectrum showed the presence of three carbonyl signals at δ 170.71, 170.05, and 169.85, three methyl signals at
C
δ 21.01, 20.83, and 20.76, and three carbon signals at δ 62.06, 67.69, and 68.14. Furthermore, the HMQC spectrum
C
C
showed a correlation between the carbon at δ 68.14 and the proton signals at δ 5.06; between δ 67.79 and the proton
C
H
C
signals at δ 5.42; between δ 62.02 and the proton signals at δ 4.21 and 4.06. Compound 1 was identified as D,L-iditol
H
C
H
based on NMR and LC/MS-APCI data. Also compound 1 was confirmed to be D,L-iditol by comparison with the melting
point of 1a in the literature [18]. This compound was isolated and reported for the first time from Daucus carota L. The plant
is a new source of D- and L-iditol.
CH
O
3
O
OH OH
'
1
2
H C
'
3
3
'
O
O
HO
2
4''
3''
H C
3
OH
O
'
6
1''
OH OH
H C
3
4
'
5
O
3
2''
5''
OCH
3
CH
3
1
3
(D, L-Iditol)
1) Ege University, Faculty of Science, Chemistry dept., 35100 Bornova-Izmir, Turkey, fax: + 90 232 388 82 64,
e-mail: yurdanur.akgul@ege.edu.tr; 2) Ege University, Faculty of Science, Biology dept., 35100 Bornova-Izmir, Turkey.
Published in Khimiya Prirodnykh Soedinenii, No. 6, pp. 742–744, November–December, 2009. Original article submitted
April 7, 2008.
0009-3130/09/4506-0889 ©2009 Springer Science+Business Media, Inc.
889

1
13
TABLE 1. H and C NMR Spectroscopic Data for Compound 1 (400 MHz and 125 MHz in D O) and Compound 1a (CDCl )
2
3
1
1a
C atom
H–H COSY
(J/Hz)
(J/Hz)
δH
δC
δH
δC
1
2
3
4
5
6
CH3
63.57
69.65
71.27
71.27
69.65
63.57
3.73–3.66(2H, m)
3.62–3.57 (1H)
3.81 (1H, d, J = 2.8)
3.78 (1H, d, J = 2.4)
3.62–3.57 (1H)
62.06
67.69
68.14
68.14
67.69
62.06
21.01
20.83
20.75
170.71
170.05
169.85
4.21 (2H, dd, J = 2.8, H-1a, 6a)
5.06 (1H, m)
5.42 (1H,d, J = 8.0)
5.42 (1H, d, J = 8.0)
5.06 (1H, m)
4.06 (2H, dd, J = 5.2, H-1b, 6b)
2.06 (6H, s)
2.03 (6H, s)
H1a, H1b, H2
H1a, H6a, H3
H4
H5
H6b,H1b, H4
H6a, H6b, H5
3.73–3.66 (2H, m)
2.01 (6H, s)
C=O
1
13
TABLE 2. H and C NMR Spectroscopic Data for Compound 3 (400 MHz and 125 MHz in CDCl )
3
C atom
H–H COSY
HMBC
δC
δH (J/Hz)
1
2
3
77.43
71.44
17.01
131.93
107.51
143.76
135.56
149.18
101.80
56.81
5.78 (1H, d, J = 6.4)
5.36 (1H, m)
1.11 (3H, d, J = 6.8)
H2
H1, H3
H2
H2′, H2, H3
H1, H3
H1
H1
1
′
2
′
6.56 (1H, d, J = 1.2)
H1, H6
′
OMe, H2 , H6
′
′
3
′
4
′
H2 , H6
′
′
H2 , H6 , OCH O
′
′
′
5′
2
6.58 (1H, d, J = 1.6)
3.89 (3H, s)
H2
OMe
6
′
OMe
167.35
166.86
128.04
127.70
139.13
138.31
15.99
15.88
20.73
101.87
1
2
3
4
5
H2, H5
H1, H5
′′
′′
′′
′′
′′
′′
′′
H4 , H5
′′
′′
′′
6.11 (2H, m)
1.97 (6H, m)
H4
H3
′′
′′
H4 , H5
′′
1.88 (6H, m)
5.96 (2H, s)
H3
′′
OCH2O
OCH2O
Compound 2 was isolated as a yellow powder from the n-butanol portion. It was identified by comparison with the
reported spectroscopic data [15] as kaempferol-3-O-β-glucoside [15], which was isolated and reported for the first time from
the Daucus carota L.
Compound 3 was isolated as an oil from the n-butanol soluble fraction after repeated silica gel column chromatography.
1
The H NMR spectrum of the compound showed signals due to two aromatic protons (δ 6.57, d and 6.56, d), two olefinic
H
protons (δ 6.11, m), two aliphatic protons (δ 5.77, d and 5.36, m), one methoxy group (δ 3.89, s), one methylenedioxy
H
H
H
13
group (δ 5.96, s), and three signals from five methyl groups (δ 1.97, 1.88, 1.11). The C NMR spectrum showed the
H
H
presence of six aromatic carbons, four olefinic carbons, one methyleneoxy carbon, one methoxy carbone, two aliphatic oxygen-
1
1
substituted carbons, and methyl carbons (Table 2). The H– H COSY spectrum showed a correlation between H1 (δ 5.77)
H
and H2 (δ 5.36); between H2 (δ 5.36) and H3 (δ 1.11); between H3′′ (δ 6.11 ) and H4′′, H5′′ (δ 1.97, 1.88). Compound
H
H
H
H
H
+
3 had a molecular ion peak in the LC/MS-APCI spectrum at m/z [M+H] 391.15 corresponding to the molecular
formula C H O .
21 26
7
890

RSA, %
100
80
60
40
20
0
Extract
1
2
3
4, 5 BHT Ascorbic
acid
Fig. 1. Antioxidant Activity of Isolated Compounds.
Also the mass spectrum gave a base peak at m/z 291.15 corresponding to the loss of the one angelate. The NOESY
spectrum of compound 3 indicated that there was no cross peak between H1 and H2 (J = 6.4Hz). The data indicated that 3 was
laserine [16]. The structure was confirmed by HMBC and HMQC experiments. Compound 3 was isolated and reported for the
first time from Daucus carota L.
Compounds 4 and 5 were isolated as a mixture (1:1, stigmasterol and sitosterol). Their structures were identified
1
13
easily from H, C NMR spectra, and correlated literature data [17].
The antioxidant capacity of isolated compounds was evaluated using the DPPH method [19]. Although the
dichloromethane–methanol (1:1) extract of the flowers of Daucus carota L. has significant antioxidant activity (32.57%),
isolated compounds showed slight activity. Compound 2 has higher free radical-scavenging activity (9.12%) than the other
compounds (Fig. 1).
General Experimental Procedures. Optical rotation: Perkin–Elmer 341 Polarimeter; NMR: VarianAS-400 on Mercury
plus console; LC/MS-APCI: ALIGENT 1100 MSD. All solvents used were of analytical grade (Carlo Erba).
Silicagel Merck 7734 and Silicagel Merck 13895 were used for column cromatograpy and PTLC. TLC was carried
out using Kieselgel 60 F (Merck 5554); spots were visualized by spraying with 20% H SO .
254
2
4 aq
Plant Material. Flowers of Daucus carota L. ssp. carota were collected in June 2006 from Sasali-Izmir (Turkey) and
were identified by Dr. Serdar Gokhan Senol, Department of Botany, Faculty of Science, Ege university. A voucher specimen
(EGE.HERB:40759) is preserved in the herbarium of Ege university.
Extraction and Isolation. The shade-dried flowers (898 g) were extracted exhaustively with dichloromethane–
methanol (1:1, v/v) and concentrated under reduced pressure to afford 185.10 g of extract. Twenty grams of extract was
partioned between n-butanol and water. Compound 1 was isolated from the water fraction. After removal of the water, it was
dissolved in MeOH–CH Cl (4:1 v/v). Precipitate 1 was obtained (900 mg). The n-butanol soluble fraction was chromatographed
2
2
using a silica gel column (400 g, hexane–CH Cl increasing CH Cl and 90:10:1 CH Cl –MeOH–water, CH Cl –MeOH–water
2
2
2
2
2
2
2
2
80:20:2, CH Cl –MeOH–water 70:30:3). Twenty fractions were collected (A-U). Fraction K gave compound 3 (eluting solvent:
2
2
hexane–CH Cl 40:60). After purification with PTLC (hexane–CH Cl –ethyl acetate 10:10:3), compound 3 was obtained
2
2
2
2
(20 mg). Compounds 4 and 5 were isolated from fraction R. After purification with PTLC (hexane–CH Cl –ethyl acetate
2
2
10:10:5), 6 mg mixture of compound was obtained. Compound 2 was obtained from fraction S- (elueting solvent:
CH Cl –MeOH–water 80:20:2, CH Cl –MeOH–water 70:30:3).After purification with PTLC (CH Cl –MeOH–water 70:30:3),
2
2
2
2
2
2
20 mg of compound 2 was obtained.
D,L-Iditol (1): white amorphous solid;
[α]2D5
183.10 (100), 165.10, 152.10; H and C NMR (Table 1).
Acetylation of Compound 1 (1a): 100 mg compound 1 was acetylated in pyridine with Ac O according to the usual
0° (c 0.1, MeOH); mp decomposed; LCMS/APCI m/z (rel. ints.):
1
13
2
1
13
precedure. White powder, mp 121°C [18]; H and C NMR (Table 1).
1
13
Kaempferol-3-O-β-glucoside (2): yellow amophous solid; H and C NMR [15].
[α]2D5
Laserine (3): colorless oil;
282.30 (20.5), 83.10 (45.7); 60.10 (31.4); H and C NMR (Table 2).
+48° (c 0.1, CH Cl ); LCMS/APCI m/z (rel. ints.): 391.15 (0.3), 291.15 (100),
2 2
1
13
Antioxidant Activity. The free radical scavenging activity was assayed spectrophotometrically [19]. The DPPH
(2,2-diphenyl-1-picrylhydrazyl) radical has a deep violet color due to its unpaired electron, and radical scavenging activity can
be followed spectrophotometrically by a loss of absorbance at 515 nm. An appropriate dilution series at six different
891

concentrations (0.05–1 mg sample/mL methanol or 70% ethanol–water) was prepared, and 0.1 mL of each dilution was added
–5
to 1.9 mL of a 6.0 × 10 M methanol solution of DPPH, shaken well by vortex, and allowed to react at room temperature. The
free radical scavenging activity of samples was calculated according to the formula:
DPPH radical scavenging activity (%) = [(negative control A – sample A )/negative control A )] × 100
515
515
515
–5
As a blank, 70% ethanol–water or methanol solvent (0.1 mL) was used. DPPH solution (1.9 mL, 6.0 × 10 M) and
70% ethanol–water or methanol solvent (0.1 mL) was used as a negative control. BHT and ascorbic acid were used as a
positive control. All determinations were performed in triplicate.
ACKNOWLEDGMENT
The authors thank Assoc. Prof. Stephan Thomas Astley for proofing the manuscript. Thanks are also due to
Dr. Serdar Gokhan Senol for plant identification.
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