Two new acetylated flavonoid glycosides from Centaurium spicatum L

Article abstract of DOI:10.1007/s11418-011-0594-y

Two new acetylated flavonol glycosides, quercetin 3-O-[(2,4-diacetyl- α-L-rhamnopyranosyl)-(1→6)]- 2,4-diacetyl-β-D-galactopyranoside (1) and quercetin 3-O-[(2,4-diacetyl-α-L-rhamnopyranosyl)-(1→6)]-3,4- diacetyl-β-D-galactopyranoside (2), in addition to

Full text of DOI:10.1007/s11418-011-0594-y

J Nat Med (2012) 66:388–393
DOI 10.1007/s11418-011-0594-y
NOTE
Two new acetylated flavonoid glycosides from Centaurium
spicatum L
•
Ahmed E. Allam Mohamed A. El-Shanawany
•
•
Enaam Y. Backheet Alaa M. Nafady
•
•
Fumihide Takano Tomihisa Ohta
Received: 23 July 2011 / Accepted: 4 September 2011 / Published online: 12 October 2011
Ó The Japanese Society of Pharmacognosy and Springer 2011
Abstract Two new acetylated flavonol glycosides, quer-
cetin 3-O-[(2,4-diacetyl-a-L-rhamnopyranosyl)-(1?6)]-
Keywords Acetylated flavonoid glycosides ꢀ
1
Centaurium spicatum ꢀ H-NMR ꢀ 13C-NMR ꢀ 2D-NMR
2,4-diacetyl-b-D-galactopyranoside (1) and quercetin
3-O-[(2,4-diacetyl-a-L-rhamnopyranosyl)-(1?6)]-3,4-
diacetyl-b-D-galactopyranoside (2), in addition to two
known acetylated quercetin glycosides quercetin 3-O-[(2,3,
4-triacetyl-a-L-rhamnopyranosyl)-(1?6)-b-D-galactopy-
ranoside (3) and quercetin 3-O-[(2,3,4-triacetyl-a-L-rham-
nopyranosyl)-(1?6)-3-acetyl-b-D-galactopyranoside (4),
were isolated from the aerial part of Centaurium spicatum
(L.) Fritsch (Gentianaceae). Structure elucidation, espe-
cially the localization of the acetyl groups, and complete
1H and 13C NMR assignments of these biologically active
compounds were carried out using one- and two-dimen-
Introduction
Centaurium spicatum (L.) Fritsch (Gentianaceae) is an
erect small branched annual herb, widespread in Southern
Europe and North Africa, where it is used together with
other Centaurium species such as C. pulchellum in tradi-
tional medicine for the treatment of abdominal pain,
hypertension, gallstones, kidney and ureter stones, renal
colic, wounds, and diabetes [1]. The Arabic name is
‘Kantaryoun’. A survey of the current literature revealed
the isolation and identification of secoiridoids (sweroside,
swertiamarin and gentiopicrin) and polyoxygenated xant-
hones from the plant [1–4]. Alkaloids of the pyridine type
(e.g. gentianine), spicatine and the series of amides derived
from the secoiridoid glucoside swertiamarin, and kantaurin
were also shown to be present [3]. In the present work we
report the isolation and structure elucidation of two new
acetylated flavonol glycosides 1 and 2, together with two
other known compunds 3 and 4. The general methodology
used for the structure elucidation and the spectral assign-
ments of the two new acetylated flavonol glycosides from
C. spicatum is discussed in this paper.
1
sional NMR measurements, including H- and 13C-NMR,
DEPT-135, H–H COSY, HMQC and HMBC, in addition to
HR-FAB/MS experiments.
Electronic supplementary material The online version of this
article (doi:10.1007/s11418-011-0594-y) contains supplementary
material, which is available to authorized users.
A. E. Allam ꢀ F. Takano (&) ꢀ T. Ohta (&)
Pharmacognosy and Chemistry of Natural Products,
School of Pharmaceutical Sciences, Kanazawa University,
Kanazawa 920-1192, Japan
e-mail: takano@p.kanazawa-u.ac.jp
T. Ohta
e-mail: ohta@p.kanazawa-u.ac.jp
Results and discussion
A. E. Allam ꢀ A. M. Nafady
Compound 1 was obtained as yellowish green amorphous
powder (2.0 mg), soluble in methanol with [a]2D9.1 -39°
(c = 0.333, MeOH). The UV spectrum (in MeOH)
exhibited absorption maxima at 253 nm (band-II) and
356 nm (band-I), indicating a flavonol type. In addition, a
Department of Pharmacognosy, Faculty of Pharmacy,
Al-Azhar University, Assiut 71524, Egypt
M. A. El-Shanawany ꢀ E. Y. Backheet
Department of Pharmacognosy, Faculty of Pharmacy,
Assiut University, Assiut 71526, Egypt
123

J Nat Med (2012) 66:388–393
389
bathochromic shift (58 nm) with a high intensity of band-I
was observed in the NaOMe spectrum which indicated the
presence of a free OH group at position C-40. The presence
of a free OH group at position C-5 was proved through the
AlCl3 spectrum where there is a bathochromic shift
(47 nm) in band-I relative to MeOH spectrum. The pres-
ence of an ortho-dihydroxy system in ring B was confirmed
from the hypsochromic shift when HCl was added to the
AlCl3. The presence of a bathochromic shift (14 nm) in
band-II with NaOAc indicated the presence of a free OH
group at C-7. The IR spectrum of 1 (in CHCl3) indicated
the presence of hydroxyl (3,440 cm-1), carbonyl
(1,780 cm-1), and phenyl (2,980, 1,640, 1,530 cm-1). The
1H- and 13C-NMR spectra of 1 indicated the presence of a
quercetin moiety, two sugar moieties (hexoses) and four
the long-range correlation between C-1 of rhamnose (dC
99.2) and both H-6 hydrogens of galactose (dH 3.58 and
3.30, as assigned by their one-bond C–H correlation,
observed in an HMQC experiment, with C-6 of galactose at
1
dC 68.0). All H- and 13C-NMR signals of the quercetin
moiety and both sugar residues could be assigned by ana-
lyzing the H–H COSY, HMQC and HMBC spectra
(Table 1). Long-range correlations observed between the
carbonyl signal at dC 171.4 and H-2 of galactose (dH 4.99),
at dC 172.6 and H-4 of galactose (dH 5.26), at dC 171.5 and
H-2 of rhamnose (dH 5.05) and at dC 171.8 and H-4 of
rhamnose (dH 4.84) unequivocally allowed us to locate the
acetyl groups at positions C-2, C-4 of galactose and at C-2,
C-4 of the terminal rhamnose residue. This was confirmed by
four-bond correlations observed between the corresponding
carbons of galactose (C-2 at dC 70.7 and C-4 at dC 71.9 and
the methyl signals at dH 1.87 and dH 2.18, respectively) as
well as the four-bond correlations observed between the
corresponding carbons of rhamnose (C-2 at dC 70.6 and C-4
at dC 71.8 and the methyl signals at dH 1.97 and dH 2.05,
respectively). The molecular weight of 778 with a molecular
formula C35H38O20, which was evident from an [M?H]?
peak at m/z 779 in the positive ion FAB mass spectrum, and
also from [HR-FAB]?/mass at 779.2031, was in agreement
with a tetracetylated quercetin rhamnosyl galactoside.
Hence 1 could unequivocally be identified as quercetin
3-O-[(2,4-diacetyl-a-L-rhamnopyranosyl)-(1?6)]-2,4-dia-
cetyl-b-D-galactopyranoside, a new compound.
1
acetyl groups. The H-NMR spectrum showed a pair of
doublets at dH 6.21 (H-6) and dH 6.42 (H-8), and a three-
spin system with the typical coupling pattern of a 10,30,40-
trisubstituted benzene ring [dH 7.63 (H-20), 6.86 (H-50) and
7.67 (H-60)], which are two features characteristic of a
flavonol with phenolic hydroxyl groups at positions 5, 7, 30
and 40. The 13C-NMR and DEPT spectra (showing the
multiplicities of the carbon atoms) were in agreement with
a 3-substituted quercetin moiety. Substitution of quercetin
in position C-3 was evident from the chemical shift of C-2
(dC 159.5), whereas in flavonols with an unsubstituted
hydroxyl functionality at this position C-2 is expected
around dC 147 [5]. The typical doublet of the C-6 sec-
ondary methyl group of rhamnose was found at dH 0.96
Compound 2 was obtained as yellowish green amor-
phous powder (1.5 mg), soluble in methanol with [a]D29.8
-
1
(1H, d, J = 6.4 Hz) and dC 17.5 in the H- and 13C-NMR
spectra, respectively. The anomeric hydrogens showed
characteristic doublets in 1H-NMR spectrum at dH 5.30 for
galactose, with a coupling constant of 7.7 Hz, indicating a
b-configuration, and at dH 4.53 for rhamnose, with a
coupling constant of 1.7 Hz, indicating a-configuration [6].
In addition, optical rotation values of both D-galactopyra-
nose and L-rhamnopyranose tetrabenzoate derivatives of
the acid hydrolysis products of 1 were [a]3D1.5 ?54.5
34.6° (c = 0.333, MeOH). The UV spectrum (in MeOH)
exhibited absorption maxima at 256 nm (band-II) and
358 nm (band-I), indicating a flavonol type. In addition, a
bathochromic shift (54 nm) with a high intensity of band-I
was observed in the NaOMe spectrum, which indicated the
presence of a free OH group at position C-40. The presence
of a free OH group at position C-5 was proved through the
AlCl3 spectrum where there was a bathochromic shift
(43 nm) in band-I relative to MeOH spectrum. The pres-
ence of an ortho-dihydroxy system in ring B was confirmed
from the hypsochromic shift when HCl was added to the
AlCl3. The presence of a bathochromic shift (12 nm) in
band-II with NaOAc indicated the presence of a free OH
group at C-7. The IR spectrum of 2 (in CHCl3) indicated
the presence of hydroxyl (3026 cm-1), carbonyl
(1747 cm-1), and phenyl (2927, 1610, 1550 cm-1). The
1H- and 13C-NMR spectra of 2 looked very similar to those
of 1, as it has a molecular weight of 778 with the same
molecular formula, which was evident from an [M?H]?
peak at m/z 779 in the positive ion FAB mass spectrum, and
also from [HR-FAB]?/mass at 779.2031 but with a small
difference in the distribution of the acetyl groups. Based on
the assignments, a long-range C–H correlation was
(c = 0.019, CHCl3) for b-D-galactopyranose tetrabenzoate
31.8
and [a]D
?76.9 (c = 0.002, CHCl3) for a-L-rhamno-
pyranose tetrabezoate, which were identical with the
optical rotation of the synthetic models of both [7, 8]. A
long-range correlation, observed in the HMBC experiment,
between C-3 of quercetin (dC 135.1) and the anomeric
proton of galactose (dH 5.30) confirmed that this was the
site of glycosylation, and that galactose was the first sugar.
The only methylene functionality of the molecule, C-6 of
galactose, was found at dC 68.0 in the 13C NMR spectrum,
after establishing its multiplicity in the DEPT-135 experi-
ment. This chemical shift was comparable to the value
observed for C-6 of glucose in rutin, a rhamnosyl-(1–6)-
glucoside, and therefore indicated substitution of galactose
in position C-6 with rhamnose [5]. This was obvious from
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J Nat Med (2012) 66:388–393
1
Table 1 13C- and H-NMR assignments for compounds 1 and 2 recorded in CD3OD
Position
1
2
13C-NMR (d, mult)
1H-NMR [d, mult, J (Hz)]
13C-NMR (d, mult)
1H-NMR [d, mult, J (Hz)]
2
159.5,s
135.1,s
179.4,s
163
158.5,s
135.1,s
179.3,s
163.8,s
99.3,d
3
4
5
6
99.9,d
6.19,d, 2.2
6.36,d, 2.2
6.21,d, 2.4
6.42,d, 2.4
7
165.9,s
94.8,d
166.2,s
94.9,d
8
9
158.5,s
105.7,s
123.2,s
117.5,d
145.9,s
149.7,s
115.9,d
123.4,d
158.7,s
105.6,s
122.6,s
117.8,d
145.8,s
150.1,s
116.2,d
122.9,d
10
10
20
30
40
50
60
7.63,d, 2.2
7.89,d, 2.4
6.86,d, 8.7
6.86,d, 8.5
7.67,dd, 8.7, 2.2
7.64,dd, 8.5, 2.4
Galactopyranosyl
100
200
300
400
500
600
104.3,d
5.30,d, 7.7
105.6,d
70.8,d
77.4,d
72.0,d
75.6,d
68.0,d
5.23,d, 7.9
70.7,d
73.6,da
71.9,d
73.2,da
68.0,t
4.99, overlapped
3.82,dd, 10.0, 3.4
5.26,d, 3.4
4.01,dd, 10.3, 7.9
4.82,dd, 10.3, 3.4
4.05,d, 3.4
3.86, overlapped
3.58, overlapped
3.30, overlapped
3.86, overlapped
3.58, overlapped
3.30, overlapped
2-Acetyl
C=O
3-Acetyl
C=O
171.4,s
20.6,q
171.4,s
20.6,q
–CH3
1.87,s
2.18,s
–CH3
1.87,s
2.18,s
4-Acetyl
C=O
4-Acetyl
C=O
172.6,s
21.0,q
172.4,s
21.0,q
–CH3
–CH3
Rhamnopyranosyl
1
99.2,d
4.53,d, 1.7
100.0,d
70.6,d
75.3,d
71.1,d
67.8,d
17.5,q
4.60,d,1.7
2
70.6,d
73.1,d
71.8,d
67.5,d
17.5,q
5.05,dd, 6.8, 1.7
3.78, overlapped
4.84, overlapped
3.66,dd, 10.0, 6.4
0.96,d, 6.4
5.01,dd, 6.8, 1.7
3.78,dd, 6.8, 2.7
4.84,dd, 10.0, 2.7
3.66,dd, 10.0, 6.4
0.96,d, 6.4
3
4
5
6
2-Acetyl
C=O
–CH3
4-Acetyl
C=O
–CH3
a,b,c,d
2-Acetyl
C=O
171.5,sb
20.6,qc
171.5,sd
20.7,q
1.97,s
–CH3
1.97,s
2.05
4-Acetyl
C=O
171.8,sb
20.7,qc
171.7,sd
20.7,q
2.05
–CH3
Assignments bearing the same superscript may be reversed
observed in an HMBC experiment between the carbonyl
group of the first acetyl functionality (dC 171.4) and one of
the two remained hydrogens of the galactosyl residue at dH
4.82, but there were two possible locations of the first
acetyl group, at C-2 or C-3 of galactose. However, the
1H-NMR signal at dH 4.82 was a doublet of doublets with
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J Nat Med (2012) 66:388–393
391
coupling constants of 10.3 and 3.4 Hz, which obviously
corresponded to an axially and equatorially coupled
hydrogen atom [9]. In galactopyranose, which is the C-4
epimer of glucopyranose, all hydrogens except H-4 are
axial. Since H-4 in galactose is equatorial, it has one Hax–Heq
coupling with H-3. On the other hand, H-2 has two Hax–Hax
couplings. Thus, in 2 only H-3 of the galactoside residue
could show an Hax–Heq (with H-4) and an Hax–Hax (with
H-2) coupling, and the doublet of doublets at dH 4.82 could
be assigned to this hydrogen, corresponding to a 13C-NMR
signal at dC 77.4 (C-3 of galactose) in HMQC experiment.
Hence 2 could unequivocally be identified as quercetin 3-O-
[(2,4-diacetyl-a-L-rhamnopyranosyl)-(1?6)]-3,4-diacetyl-b-
D-galactopyranoside, a new compound. In addition, two
known acetylated quercetin glycosides, quercetin 3-O-[(2,3,4-
triacetyl-a-L-rhamnopyranosyl)-(1?6)-b-D-galactopyranoside
(3) (27.0 mg) and quercetin 3-O-[(2,3,4-triacetyl-a-L-
rhamnopyranosyl)-(1?6)-3-acetyl-b-D-galactopyranoside
(4) (1.5 mg), previously isolated from the same plant [10],
were also isolated and the data were compared with those
previously published. (Fig. 1).
plant enzymes [12]. There are also a number of reports of
the use of lipases to acylate flavonoid glycosides [13, 14].
Most of these studies used Candida antarctica lipase B
(Novozym 435Ò) to acylate common flavonoid glycosides,
e.g. naringin, with fatty acids, in organic solvents at low
water activity. The acylation reactions appear to be selec-
tive for primary sugar residue hydroxyl groups, where
present, and for the secondary 4-hydroxyl of rhamnose
residues [15]. In conclusion, the structure of two new
acetylated flavonoid glycosides from C. spicatum could
1
unequivocally be established, and their H- and 13C-NMR
spectra completely assigned, by 1D- and 2D-NMR tech-
niques. To further elucidate the beneficial activities of the
acetylated flavonoid glycosides, we are currently investi-
gating biological activities such as anti-inflammatory or
anti-melanogenesis of these acetylated flavonoid glyco-
sides, comparing them with the corresponding non-acyl-
ated compounds.
Experimental
Acylated flavonoid glycosides are less common than
flavonoid glycosides in the plant kingdom; acetic acid is
only one of the acylating acids of the sugar hydroxyl
groups that have been described. Acylation changes the
solubility properties of the original flavonoid glycoside,
converting it into a lipophilic substance [11], from which
acylation of anthocyanins in aqueous media using phenolic
acids as acyl donors can be achieved in vitro using natural
Spectroscopic methods
Optical rotations were determined with a Horiba SEPA-
3000 high-sensitivity polarimeter. UV spectra were mea-
sured on a Shimadzu UV–Vis spectrometer. IR spectra
were recorded on a Shimadzu IR-460 IR spectrophotome-
ter. NMR spectra were recorded on a JOEL EC-600
spectrometer operating at 600.17 MHz for 1H and at
150.92 MHz for 13C at 20.1°C in CD3OD (99.5% D) in
5-mm sample tubes. The solvent peaks at dH 3.30 in the
1H- NMR spectra, and at dC 49.00 in 13C-NMR spectra,
respectively, were used as internal references downfield of
tetramethylsilane (TMS) at 0 ppm. Spectral widths were
9008 Hz (26K acquisition points) and 37878 Hz (26K
acquisition points) for 1H- and 13C-NMR, respectively.
Chemical shifts are presented in ppm downfield of TMS.
For CHn groups, n was determined in DEPT-135 yielding a
180° phase difference between –CH2– signals on the one
hand and –CH– and –CH3 signals on the other). Proton–
proton chemical shift correlations were obtained in a
COSY experiment. Proton–carbon chemical shift correla-
tions were obtained in inversely detected HMQC and
HMBC experiments. Fast atom bombardment (FAB) mass
spectra were recorded on a MStation instrument in the
positive ion mode, using glycerol as the liquid matrix.
5'
OH
4'
6'
1'
8
3'
HO
9
O
OH
7
2
2'
10
3
6
4
O
5
OH
O
1''
O
6''
H ax
OR1
H ax
O
5''
2''
R3O
4''
H ax
1'
H ax
3''
H eq
H eq
5'''
6'''
O
R2O
H ax
H ax
H3C
R6O
H eq
OR4
2'''
3'''
OR5
4'''
H ax
R1
R2
R3
R4
R5
H
R6
1
2
3
4
COCH3
H
COCH3 COCH3
COCH3
COCH3
H
H
H
COCH3 COCH3 COCH3
H
Plant material
H
H
H
COCH3 COCH3 COCH3
COCH3 COCH3 COCH3
C. spicatum (L.) Fritsch (Gentianaceae) aerial parts were
collected in May 2009 from New Valley, 200 km south-
west of Assiut City, Egypt. The plant was identified and
COCH3
Fig. 1 Structures of compounds 1–4 from C. spicatum
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J Nat Med (2012) 66:388–393
authenticated by Prof. Dr. A. Fayed, Professor of Plant
Taxonomy, Faculty of Science, Assiut University. A vou-
cher specimen was deposited at the Department of Phar-
macognosy, Faculty of Pharmacy Assiut University,
Assiut, Egypt and at the Department of Pharmacognosy
and Chemistry of Natural Products, School of Pharma-
ceutical Sciences, Kanazawa University, Kanazawa, Japan.
3 h. The reaction mixture was partitioned against EtOAc
(3 9 10 ml). The aglycone was obtained from the EtOAc
layer and identified as quercetin by co-chromatography
on silica gel with a reference sample (Sigma-Aldrich,
St. Louis, MO, USA). The aqueous layer was evaporated
and developed crystal needles with EtOAc–H2O–MeOH–
HOAc (13:3:3:4). Identification of galactose and rhamnose
present in the sugar fraction was carried out by comparison
with authentic samples, galactose (Rf 0.42), glucose (Rf
0.48), and rhamnose (Rf 0.64) (Sigma-Aldrich), in TLC
over silica gel (CHCl3–MeOH–H2O 8:5:1) using 5%
H2SO4 in MeOH as spraying reagent followed by heating
the plates at 120°C for 15–20 min.
Extraction and isolation
Air-dried C. spicatum aerial parts (2 kg) was extracted three
times with MeOH (5 L) at room temperature. The solvents
were combined and filtered through filter paper (Advantec
MFS Incorporated). The solvent was removed under reduced
pressure at 40°C to yield the methanol extract (550 g), which
was partitioned between distilled water and ethylacetate (1 L
of each) to give the aqueous fraction (300 g) and the ethyl
acetate fraction (80 g). The aqueous fraction was further
partitionedbyn-butanoltogive then-butanolfraction(100 g)
and the remaining aqueous fraction (140 g). The ethyl acetate
fraction was in turn partitioned between 90% methanol and
n-hexane to give the 90% methanol fraction (50 g) and
n-hexane fraction (20 g). The n-butanol fraction (100 g) was
separated on a Diaion HP-20 column using water (2 L), and
ethanol (25, 50, 75, and 100%) (2 L of each). The aqueous
fraction was further subjected to Sephadex G-200 (Pharmacia
Fine Chemicals, Sweden) using stepwise gradient 25, 50 and
75% methanol solvent to give three fractions. Individual
fractions were obtained after removal and evaporation of the
respective solvents. A 50% methanol-eluted fraction (33.4 g)
was further separated by chromatography on an ODS column
(80 9 200 mm) (Cosmosil 140 C18 PREP, Nacalai Tesque,
Tokyo, Japan) using six concentrations of CH3CN–H2O (10,
25, 40, 50, 60, 70, and 90% v/v; elution volume 1.5 L of each)
to give six corresponding fractions. The fraction eluted with
40% CH3CN (3.8 g) was further separated by column chro-
matography on silica gel and stepwise gradient eluted with
CHCl3:MeOH(ratiosof9:1, 6:1, 4:1, 3:1, and1:1, v/v, elution
volume 200 ml each) to give five corresponding fractions. A
150-mg portion of the fraction eluted with 6:1 CHCl3:MeOH
was further separated by preparative HPLC, ODS column
C30 UG-5, 20 mm 9 250 mm, particle size 5 lm, flow rate
3 ml/min (Develosil, Nacalai Tesque) equipped with a UV
detector (210 nm). The mobile phase was 25% CH3CN in
H2O. This resulted in elution of compound 1. These pre-
parativeHPLCconditionswerealsousedtoseparatethesame
fraction giving compound 2. Spectroscopic data for com-
pounds 1 and 2 are available as supplementary materials.
Measurements optical rotation of D-galactopyranose
and L-rhamnopyranose tetrabenzoate derivatives
Benzoyl chloride (0.5 ml) was added to each ice-
cooled solution of either D-galactopyranose (18.0 mg) or
L-rhamnopyranose or (15.0 mg) in dry pyridine (1.0 ml),
and each mixture was stirred at room temperature for 15 h.
MeOH (1.0 ml) was added dropwise to the reaction mix-
ture, stirred for 30 min, and then diluted with EtOAc and
aqueous Na2CO3, and the layers were separated. Each
organic layer was washed with brine, and the combined
aqueous layers for each were extracted with EtOAc. Each
combined organic extract was dried over MgSO4 and
concentrated, and the corresponding residual dark brown
oils were individually purified by silica gel CC (eluting
with hexane/EtOAc 5:1) to give either D-galactopyranose
tetrabenzoate (40 mg) [a]3D1 ?53.5 (c = 1.2, CHCl3) or
L-rhamnopyranose tetrabenzoate (22 mg) [a]2D9.6 ?75.0
(c = 1.6, CHCl3) as colorless oils, respectively [7, 8].
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