Flavone tetraglycosides and benzyl alcohol glycosides from the Mongolian Medicinal plant dracocephalum ruyschiana

Article abstract of DOI:10.1021/np300609u

From an extract of the aerial parts of Dracocephalum ruyschiana, five new flavone tetraglycosides, five new benzyl alcohol glycosides, and 19 known compounds were isolated. The tetraglycosides contain a 7-O-β-D- glucopyranosyl-(1→2)-β-D-glucopyranosyl-( 1→2)-[α-L- rhamnopyranosyl-(1→6)]-β-D-glucopyranosyl moiety. The benzyl alcohol glycosides had acyl groups on their glycosyl or aglycone moieties. The compounds were tested for antioxidant activity using DPPH. Although the new compounds were not active, phenylpropanoylquinic acid derivatives were revealed as radical scavengers in D. ruyschiana.

Full text of DOI:10.1021/np300609u

Article
pubs.acs.org/jnp
Flavone Tetraglycosides and Benzyl Alcohol Glycosides from the
Mongolian Medicinal Plant Dracocephalum ruyschiana
Erdenechimeg Selenge,† Toshihiro Murata,*,† Kyoko Kobayashi,† Javzan Batkhuu,‡
and Fumihiko Yoshizaki†
†Department of Pharmacognosy, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
‡School of Biology and Biotechnology, National University of Mongolia, POB-617, Ulaanbaatar-46A, Mongolia
S
* Supporting Information
ABSTRACT: From an extract of the aerial parts of Dracocephalum ruyschiana, five new flavone tetraglycosides, five new benzyl
alcohol glycosides, and 19 known compounds were isolated. The tetraglycosides contain a 7-O-β-D-glucopyranosyl-(1→2)-β-D-
glucopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranosyl moiety. The benzyl alcohol glycosides had acyl
groups on their glycosyl or aglycone moieties. The compounds were tested for antioxidant activity using DPPH. Although the
new compounds were not active, phenylpropanoylquinic acid derivatives were revealed as radical scavengers in D. ruyschiana.
hypertrophy protective, anticonvulsant, and neuroprotective
Dracocephalum ruyschiana L. (Lamiaceae) is a traditional herbal
medicinal plant in Mongolia. The aerial parts are widely used
for the treatment of gastric ulcers, laryngitis, headache, acute
respiratory infection, diarrhea, and rheumatoid arthritis.1 It has
also been shown to have hepatoprotective effects, antimicrobial
activity against Gram-positive bacteria, and antispasmodic
effects.1 Caffeic acid and α-hydroxydihydrocaffeic acid were
reported as the chemical constituents of D. ruyschiana.2
Herein, we report the isolation of five new flavone
tetraglycosides (1−5) and five new benzyl alcohol glycosides
(7−9, 12, and 13) from D. ruyschiana. Similar tetraglycosides
comprising a rhamnopyranosyl and three glucopyranosyl units
were identified in Peganum harmala,3 P. nigellastrum,4 and
Coptis japonica var. dissecta.5 Similar triglycosides were found in
species such as Calamintha glandulosa, Micromeria spp.,6
Valeriana jatamansi,7 Sclerochiton vogelii,8 and Robinia pseudoa-
cacia.9 Some of the flavone tri- and tetraglycosides have an O-
acetyl group on a glycosidic moiety. Flavone monodesmosides
containing four glycosidic moieties seemed to be rare. On the
other hand, benzyl alcohol, the aglycone moiety of 7−9, is
known as an important aroma substance with a variety of
biological activities.10 Two of the new benzyl alcohol glycosides
(12 and 13) are esters of gastrodin, one of the major bioactive
components from Gastrodia elata Bl., exhibiting cardiac
effects.11
RESULTS AND DISCUSSION
■
From an extract of D. ruyschiana, 29 compounds (1−29) were
isolated. Known compounds were identified from spectroscopic
data as diosmetin 7-O-β-D-glucopyranosyl-(1→2)-β-D-gluco-
pyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyr-
anoside (6),4 benzyl-O-α-L-rhamnopyranosyl-(1→6)-β-D-gluco-
pyranoside (10),12 benzyl-O-β-D-glucopyranoside (11),13
kaempferol 3-O-β-D-glucopyranoside (14),14 quercetin 3-O-β-
D-glucopyranoside (15),14 quercetin 3-O-β-D-glucuronide
(16),15 chlorogenic acid (17),16 3,5-dicaffeoylquinic acid
(18),17 3,4-dicaffeoylquinic acid (19),17 3-p-(E)-coumaroyl-5-
(E)-caffeoylquinic acid (20),16,17 (7S,8R)-dihydrodehydrodico-
niferyl alcohol-9′-O-β-D-glucopyranoside (21),18 (7S,8R)-uro-
lignoside (22),18 citrusin C (23),19 trans-p-coumaric acid
(24),20 methyl trans-p-coumaric acid (25),21 trans-ferulic acid
(26),20 cis-p-coumaric acid (27),20 p-hydroxybenzaldehyde
(28),22 and 4,4′-dihydroxydiphenylmethane (29).23
Compounds 1−5 were isolated as amorphous solids.
Compound 1 was demonstrated to have the molecular formula
Received: September 4, 2012
Published: January 28, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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C40H52O24 on the basis of HRFABMS (m/z 939.2752, [M +
Na]+). In the 13C NMR spectrum, 40 carbons (Table 1) were
observed; 14 aromatic/olefinic carbons (δC 94.9, C-8; 99.8, C-
6; 103.8, C-3; 105.5, C-10; 114.8, C-3′ and -5′; 122.7, C-1′;
128.5, C-2′ and -6′; 157.0, C-9; 161.1, C-5; 162.5, C-4′; 162.7,
C-7; 164.0, C-2) and a carbonyl carbon (δC 182.1) suggested a
flavone skeleton. An O-methyl resonance at δH 3.85 (3H, H-4′-
OMe) was long-range coupled with C-4′ (δC 162.5) in the
HMBC spectrum. These results demonstrated the presence of
1
an acacetin skeleton. Four anomeric protons in the H NMR
spectrum and a methyl (δC 17.8) and 23 oxygenated carbons in
the 13C NMR spectrum suggested the presence of four
glycosidic units. The glycosidic components and their absolute
configurations were determined as one L-rhamnose and three D-
glucose units from the NMR data and HPLC sugar analysis, as
described in the Experimental Section.24 The coupling
constants of the anomeric protons of the three D-glucopyr-
anosyl moieties were 7.5 Hz, indicating anomeric β-
configurations.9 Similarly, the anomeric proton (δH 4.54, br s,
H-Rha-1) of the L-rhamnopyranosyl moiety indicated an α-
anomeric configuration.9 An anomeric proton at δH 5.20 (d, J =
7.5 Hz, H-Glc-I-1) was correlated with a proton at δH 3.48 (dd,
J = 9.5, 7.5 Hz, H-Glc-I-2) and with the corresponding carbon
at δC 83.0 (C-Glc-I-2) according to COSY and HMQC data. In
addition, an anomeric proton at δH 4.62 (1H, d, J = 7.5 Hz, H-
Glc-II-1) was correlated with a proton at δH 3.25 (dd, J = 9.0,
7.5 Hz, H-Glc-II-2) and with the corresponding carbon at δC
83.1 (C-Glc-II-2). The resonances of the 13C NMR spectrum
were similar to those of acacetin-7-β-D-glucopyranosyl-(1→2)-
[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside,7 except
for the presence of the third β-D-glucopyranosyl unit of 1.
The C-Glc-II-2 signal was shifted downfield relative to that of
the acacetin triglycoside, which suggested that the third
glucopyranosyl unit is coupled to the carbon. In the HMBC
spectrum, an anomeric proton at δH 4.51 (d, J = 7.5 Hz, H-Glc-
III-1) was long-range coupled with C-Glc-II-2. The anomeric
proton of the second glucopyranosyl unit (H-Glc-II-1) was
long-range coupled with C-Glc-I-2. A lower shifted C-6 signal
at δC 65.9 (C-Glc-I-6) was long-range coupled with H-Rha-1.
The signal of the acacetin moiety (C-7) was long-range coupled
with H-Glc-I-1. From these results, the structure of 1 was
determined as acacetin-7-O-β-D-glucopyranosyl-(1→2)-β-D-glu-
copyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-gluco-
pyranoside (Figure 1).
Figure 1. Structures of 1−5 and 7−9.
d6, Table 1) were similar to those of 2. The H and 13C NMR
spectra recorded in pyridine-d5 (see Experimental Section)
were almost superimposable onto those of peganetin.3 The
HMBC spectrum showed that an anomeric proton at δH 5.63
(d, J = 7.5 Hz, H-Glc-I-1) was long-range coupled with C-7 (δC
164.0) of an acacetin moiety. Similarly, H-Glc-II-1 (δH 5.27, d, J
= 8.0 Hz) was long-range coupled with C-Glc-I-2 (δC 84.7), H-
Glc-III-1 (δH 5.31, d, J = 7.5 Hz) with C-Glc-II-2 (δC 85.2), and
H-Rha-I-1 (δH 5.41, br s) with C-Glc-I-6 (δC 67.2). In the
differential HOHAHA spectrum (in pyridine-d5), the anomeric
proton of Glc-II correlated with H-Glc-II-6 (δH 4.80, dd, J =
12.0, 3.0 Hz and 4.86, dd, J = 12.0, 1.0 Hz). The H-Glc-II-6
protons were long-range coupled with an O-acetyl carbonyl
carbon at δC 170.9 in the HMBC spectrum. Hence, the
structure of 3 was determined as acacetin-7-O-β-D-glucopyr-
anosyl-(1→2)-6-O-acetyl-β-D-glucopyranosyl-(1→2)-[α-L-
rhamnopyranosyl-(1→6)]-β-D-glucopyranoside. Assignments
of NMR signals were as shown in Table 1 (in DMSO-d6)
and the Experimental Section (in pyridine-d5).
1
The molecular formula of 2 was established as C42H54O25 on
the basis of the HRFABMS (m/z 959.3031 [M + H]+), which
The molecular formula of 4 was suggested as C42H54O26 on
the basis of HRFABMS (m/z 975.2988 [M + H]+), which was
one oxygen atom more than that of 3. Signals of glycosyl and an
O-acetyl moiety in the 1H and 13C NMR spectra (in DMSO-d6,
Table 1) were similar to those of 3. The aromatic protons (δH
7.44, d, J = 2.0 Hz, H-2′; 7.13, d, J = 8.5 Hz, H-5′; 7.57, dd, J =
8.5, 2.0 Hz, H-6′), and an O-methyl singlet at δH 3.88 suggested
that the aglycone moiety of 4 is diosmetin. In the differential
HOHAHA spectra, correlations of glycosidic resonances such
as from H-Glc-II-1 (δH 4.68, d, J = 8.0 Hz) to H-Glc-II-6 (δH
4.00, 1H, m; 4.01, 1H, m) were observed. Consequently,
compound 4 was identified as diosmetin-7-O-β-D-glucopyr-
anosyl-(1→2)-6-O-acetyl-β-D-glucopyranosyl-(1→2)-[α-L-
rhamnopyranosyl-(1→6)]-β-D-glucopyranoside.
1
was C2H2O more than that of 1. In the H and 13C NMR
spectra, signals of an O-acetyl group (δH 2.03, 3H, s; δC 21.1
and 169.5) were observed. Other signals were similar to those
of 1. The differential HOHAHA spectrum showed the
correlations of the resonances of the second glucosyl moiety
[δH 4.73 (d, J = 7.5 Hz, H-Glc-II-1), 3.49 (dd, J = 9.0, 7.5 Hz,
H- Glc-II-2), 5.00 (dd, J = 9.5, 9.0 Hz, H-Glc-II-3), 3.44 (t, J =
9.5 Hz, H-Glc-II-4), 3.30 (overlapped, H-Glc-II-5), 3.30
(overlapped, H-Glc-II-6), and 3.47 (dd, J = 11.5, 2.0 Hz, H-
Glc-II-6)]. The H-Glc-II-3 signal was long-range coupled with
the carbonyl carbon signal of the O-acetyl group (δC 169.5).
Thus, the structure of 2 was identified as acacetin-7-O-β-D-
glucopyranosyl-(1→2)-3-O-acetyl-β-D-glucopyranosyl-(1→2)-
[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside.
1
In the H NMR spectrum of 5 (Table 1), an AA′BB′ spin
Compound 3 had an [M + H]+ ion peak in HRFABMS at m/
z 959.3026, corresponding to the molecular formula C42H54O25,
the same as that of 2. Its 1H and 13C NMR spectra (in DMSO-
system (δH 6.95, 2H, d, J = 9.0 Hz, H-3′ and -5′; 7.94, 2H, d, J
= 9.0 Hz, H2′ and -6′), two m-coupled protons (δH 6.46, 1H, J
= 2.0 Hz, H-6; 6.71, 1H, J = 2.0 Hz, H-8), and a singlet (δ 6.84,
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1
Table 2. H and 13C NMR Spectroscopic Data (in methanol-d4) of Compounds 7−9
7
8
9
HMBC (H to
C)
position
δH (J in Hz)
δC
HMBC (H to C)
δH (J in Hz)
δC
δH (J in Hz)
δC
HMBC (H to C)
aglycone
1
2
3
4
5
6
7
138.9
129.3
139.0
129.4
129.3
128.7
129.3
129.4
71.8
138.8
7.41, d (7.0)
7.29, m
4
7.39, br d (8.0)
7.28, m
4, 6, 7
1, 5
7.42, d (8.5)
7.30, br t (8.5)
7.23, m
129.2 4, 7
129.3
128.7 2, 3
129.3
129.2 4, 7
129.3 1, 4
128.7 2, 3
129.3 1, 4
1
7.23, m
7.25, m
2
7.29, m
7.28, m
1, 3
7.30, br t (8.5)
7.42, d (8.5)
4.65, d (12.0)
4.90, d (12.0)
1
7.41, d (7.0)
4.64, d (11.5)
4.87, d (11.5)
129.3
71.8
4
1
7.39, br d (8.0)
4.63, d (12.0)
4.84, d (12.0)
2, 4, 7
Glc-1
71.9
1, Glc-1
p -coumaroyl
1′
2′
3′
4′
5′
6′
7′
8′
127.2
127.6
134.0
115.9
160.2
115.9
134.0
145.9
116.3
167.6
127.1
7.43, d (8.5)
6.80, d (8.5)
131.3 4′, 6′, 7′
116.8 l′, 4′, 5′
161.3
7.66, d (8.5)
6.71, d (8.5)
4′, 7′
l′, 5′
7.39, d (8.5)
6.81, d (8.5)
131.2 3′, 6′, 7′
116.8
161.1
6.80, d (8.5)
7.43, d (8.5)
7.59, d (16.0)
6.36, d (16.0)
116.8 1′, 3′, 4′
131.3 2′, 4′, 7′
147.1 2′, 6′, 8′, 9′
114.9 1′, 9′
168.6
6.71, d (8.5)
7.66, d (8.5)
6.88, d (13.0)
5.82, d (13.0)
1′, 3′
4′, 7′
9′
6.81, d (8.5)
7.39, d (8.5)
7.63, d (16.0)
6.31, d (16.0)
116.8
131.2
146.8 1′, 2′, 8′, 9′
115.1 l′, 9′
168.9
1′
9′
sugar
Glc-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6
4.36, d (8.0)
103.3
7
4.33, d (8.0)
103.3
75.2
78.1
71.9
77.4
68.0
7
4.39, d (8.0)
103.3 7, Glc-5
a
a
3.26, dd (9.0, 8.0) 75.1
3.24
3.31
75.0
78.0
71.6
a
3.36, t, (9.0)
78.0
71.8
77.4
67.8
3.34, t, (9.0)
3.39
a
a
a
3.29
3.28
3.36
3.43, m
3.42, m
3.44, m
76.6
68.2
Glc-4
Rha-1
3.72, dd (11.5,
6.5)
Rha-1
3.70, dd (12.0,
6.0)
Rha-1
3.73, dd (11.5,
6.0)
4.00, dd (11.5,
2.0)
4.00, dd (12.0,
2.0)
4.03, dd (11.5,
2.0)
Rha-1
4.95, d (1.5)
99.4
Glc-6, Rha-2,
Rha-5
4.91, d (1.5)
99.6
Glc-6
4.87, br s
102.1 Glc-6, Rha-2,
Rha-5
a
Rha-2
Rha-3
Rha-4
Rha-5
Rha-6
5.20, dd (3.5, 1.5) 74.0
3.94, dd (9.5, 3.5) 70.7
9′
5.17, dd (3.5, 1.5) 73.9
3.91, dd (9.5, 3.5) 70.7
9′
3.97
72.3
70.4
75.5
a
3.98
3.48, t (9.5, 9.5)
3.77, dd (9.5, 6.5) 69.9
1.32, d (6.5) 18.1
74.4
3.36, t (9.5)
3.73, dd (9.5, 6.0) 70.0
1.28, d (6.5) 18.1
74.3
5.08, t (9.0)
a
3.97
67.7
18.0
Rha-4
Rha-4, Rha-5
1.19, d (6.5)
Rha-4, Rha-5
a
Unclear signal pattern due to overlapping.
1H, H-3) were observed in the aromatic/olefinic region. These
showed the presence of apigenin as the aglycone moiety. The
molecular formula of 5 was suggested to be C41H52O25 on the
basis of the HRFABMS (m/z 967.2713 [M + Na]+), which was
CH2O less than that of 4. These results suggested that 5 was
apigenin-7-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-
(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside.
Compounds 7−9, 12, and 13 were isolated as colorless
powders. Compound 7 was established to have the molecular
formula, C28H34O12, based on the HRFABMS (m/z 563.2117,
[M + H]+). Five aromatic protons (δH 7.23, 1H, m, H-4; 7.29,
2H, m, H-3 and -5; 7.41, 2H, d, J = 7.0 Hz, H-2 and -6) and two
oxygenated methylene protons in the 1H NMR spectrum
(Table 2) at δH 4.64 (d, J = 11.5 Hz, H-7) and 4.87 (d, J = 11.5
Hz, H-7) suggested the presence of a benzyl group. Spin
systems at δH 6.80 (2H, d, J = 8.5 Hz, H-3′ and -5′), 7.43 (2H,
d, J = 8.5 Hz, H-2′ and -6′), 6.36 (1H, d, J = 16.0 Hz, H-8′),
and 7.59 (1H, d, J = 16.0 Hz, H-7′) and a carbonyl carbon at δC
168.6 (C-9′) indicated a trans-p-coumaroyl moiety. Two
anomeric, a methyl, and nine oxygenated carbons suggested
the presence of two glycosidic moieties. After acid hydrolysis of
7, the sugars were identified as D-glucose and L-rhamnose by
the same method as for 1.24 The coupling constant of H-Glc-1
(δH 4.36, d, J = 8.0) indicated a β-D-glucopyranosyl moiety.9,12
The H-Glc-1 signal was long-range coupled with a benzylic
oxygenated carbon at δC 71.8 (C-7) in the HMBC spectrum.
The anomeric proton at δH 4.95 (d, J = 1.5 Hz, H-Rha-1) and
the methyl doublet (δH 1.32, d, J = 6.5 Hz, H-Rha-6) indicated
the presence of an α-L-rhamnopyranosyl moiety.9,12 The H-
Rha-1 signal correlated with H-Rha-2 (δH 5.20, dd, J = 3.5, 1.5
Hz) in the COSY spectrum. In the HMBC spectrum, the H-
Rha-1 signal was long-range coupled with C-Glc-6 (δC 67.8),
and H-Rha-2 was long-range coupled with the acyl carbonyl
carbon (C-9′). From these results, the structure of 7 was
determined as benzyl-2-O-trans-p-coumaroyl-α-L-rhamnopyra-
nosyl-(1→6)-β-D-glucopyranoside.
The molecular formula of 8 was determined as C28H34O12 on
the basis of the HRFABMS (m/z 563.2129 [M + H]+), which
1
was the same as that of 7. The H and 13C NMR spectra were
similar to those of 7, except for signals of the acyl group. The
coupling constant of H-7′ (δH 6.88, d, J = 13.0 Hz) and H-8′
(δH 5.82, d, J = 13.0 Hz) suggested that the acyl group was a cis-
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p-coumaroyl moiety. Therefore, the structure of 8 was
determined as benzyl-2-O-cis-p-coumaroyl-α-L-rhamnopyrano-
syl-(1→6)-β-D-glucopyranoside.
protons at δH 2.70 (2H, br s, H-2′) and the H-7 signal
correlated with C-1′ in the HMBC spectrum. These data
suggested the presence of the ester of 3-hydroxy-3-methyl-
pentanedioic acid. A glycosidic unit was determined as the β-D-
glucopyranosyl by sugar analysis using HPLC (see Exper-
imental Section)24 and a coupling constant of the anomeric
proton at δH 4.90 (d, J = 7.5 Hz, H-Glc-1). The H-Glc-1 signal
correlated with C-4 (δC 159.1) in the HMBC spectrum. These
results suggested that compound 13 was [(3-hydroxy-3-
methylglutaryl)methyl]phenyl-4-O-β-D-glucopyranoside. The
absolute configuration of C-3′ could not be defined.
Compound 9 also had the molecular formula C28H34O12 on
the basis of the HRFABMS (m/z 563.2143, [M + H]+).
Although its NMR spectra were similar to those of 7, the H-
Rha-4 signal (δH 5.08, t, J = 9.0 Hz) in 9 was shifted downfield,
instead of the H-Rha-2 proton. In the HMBC spectrum, the H-
Rha-4 signal correlated with the carbonyl carbon at δC 168.9
(C-9′). Hence, the structure of 9 was determined as benzyl-4-
O-trans-p-coumaroyl-α-L-rhamnopyranosyl-(1→6)-β-D-gluco-
pyranoside.
As described in the introduction, D. ruyschiana is used to
treat inflammatory diseases such as gastric ulcers, laryngitis,
headaches, and rheumatoid arthritis. Excess oxidative stress is
considered to invoke inflammatory reactions and various
diseases including rheumatism and hepatic disorders.25
Consequently, anti-free radical effects are one of the main
targets of drug discovery. Antioxidant activity using 1,1-
diphenyl-2-picrylhydrazyl (DPPH) was measured for com-
pounds 1−29, as shown in Table 3. Caffeoylquinic acid
The molecular formula of 12 was established as C26H32O12
on the basis of the HRFABMS (m/z 559.1797 [M + Na]+).
Aromatic ring protons (δH 7.96, 2H, br d, J = 7.5 Hz, H-2′ and
-6′; 7.65, 1H, m, H-4′; 7.40, 2H, m, H-3′ and -5′), an AA′BB′
spin system (δH 7.40, d, J = 9.0 Hz, H-2 and -6; 7.00, d, J = 9.0
Hz, H-3 and -5), an oxymethylene singlet (δH 5.27, 2H, H-7),
an oxygenated carbon at δC 157.1 (C-4), and an ester carbonyl
1
carbon at δC 165.6 (C-7′) were observed in the H and 13C
NMR spectra of 12, indicating the presence of a benzyl and a
benzoyl moiety. The H-7, -2′, and -6′ signals were long-range
coupled with C-7′ (Figure 2), demonstrating the presence of a
Table 3. DPPH Radical-Scavenging Activity of Compounds
a
from D. ruyschiana, Rutin, and Trolox
compound
IC50 (μM)
11
223
15
60.6
37.2
45.5
32.4
28.9
57.4
284
16
17
18
19
20
21
23
328
26
62.4
38.7
25.9
rutin
Trolox
Figure 2. Structures of 12 and 13 and key HMBC correlations.
a
Compounds 1−10, 12−14, 22, 24, 25, and 27−29: IC50 >200 μg/
mL.
4-hydroxybenzyl benzoate aglycone moiety. Signals of two
1
glycosidic units were also observed in the H and 13C NMR
derivatives 17−20, flavone glycosides 15 and 16, and phenolic
compounds 11 and 26 showed moderate activity (IC50 28.9−
223 μM). The positive controls were rutin (IC50 38.7 μM) and
Trolox (IC50 25.9 μM). The active compounds had catechol
groups. The catechol group in flavonoids and phenolic
compounds is known to be important for free radical
scavenging activity.26,27 Although potent biological activities
for these components were not identified in the present study,
flavones and benzyl alcohols with a variety of biological
activities are known. The new compounds and the DPPH
radical scavengers are expected to contribute to pharmaco-
logical effects and usage of this Mongolian traditional medicinal
herb.
spectra. HPLC sugar analysis, as described in the Experimental
Section, and coupling patterns of anomeric protons at δH 5.00
(d, J = 7.5 Hz, H-Glc-1) and 5.12 (d, J = 1.5 Hz, H-Rha-1)
confirmed the β-D-glucopyranosyl and α-L-rhamnopyranosyl
moieties.9,24 In the COSY spectrum, the H-Glc-1 signal
correlated with H-Glc-2 (δH 3.47, overlapped). The corre-
sponding carbon (δC 76.3, C-Glc-2) was determined using the
HMQC data. In the HMBC spectrum (Figure 2), the H-Rha-1
signal correlated with C-Glc-2, and H-Glc-1 with C-4 (δC
157.1). From these results, the structure of 12 was formulated
as [(benzoxy)methyl]phenyl-4-O-α-L-rhamnopyranosyl-(1→6)
-β-D-glucopyranoside.
Compound 13 had an [M + H]+ ion peak in the HRFABMS
at m/z 431.1558, corresponding to a molecular formula of
C19H26O11. The oxymethylene protons at δH 5.07 (2H, s, H-7)
and AA′BB′ systems (δH 7.31, 2H, d, J = 9.0 Hz, H-2 and -6;
7.08, 2H, d, J = 9.0 Hz, H-3 and -5) indicated that 13 also
contains a 4-hydroxy benzyl moiety. Two carbonyl carbons [δC
174.8 (C-5′) and 172.5 (C-1′)] and an oxygenated carbon [δC
70.7 (C-3′)] were observed in the 13C NMR spectrum. In the
HMBC spectrum (Figure 2), a methyl singlet at δH 1.35 (3H,
H-6′) was long-range coupled with C-3′ and two methylene
signals [δC 46.3 (C-2′) and 45.8 (C-4′)]. The methylene
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
recorded on a Jasco P-2300 polarimeter. H NMR (400 MHz), 13C
1
1
NMR (100 MHz), COSY, HMQC (optimized for JC−H = 145 Hz),
and HMBC (optimized for nJC−H = 8 Hz) spectra were recorded on a
Jeol JNM-AL400 FT-NMR spectrometer, and chemical shifts are given
as δ values with TMS as an internal standard. HRFABMS data were
obtained on a Jeol JMS700 mass spectrometer, using either an m-
nitrobenzyl alcohol or a glycerol matrix. A porous polymer gel
(Mitsubishi Chemical, Diaion HP-20, 60 × 300 mm) and octadecyl
191
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Journal of Natural Products
Article
silica (ODS) (Cosmosil 140 C18-OPN, Nacalai Tesque, 150 g) were
used for column chromatography. Preparative HPLC was performed
on a Jasco 2089 and detected with UV at 210 nm (columns, Cosmosil
AR-II, Nacalai Tesque, 20 × 250 mm; Cosmosil 5PE-MS, Nacalai
Tesque, 20 × 250 mm; Mightysil RP-18 GP, Kanto Chemical, 10 ×
250 mm).
Plant Material. The aerial parts of D. ruyschiana were collected in
Mongolia (N 48°04.550′; E 106°22.611′) in July 2011. Prof. Ch.
Sanchir from the Institute of Botany, Mongolian Academy of Sciences,
identified the plant species. A voucher specimen (sample number:
90.7.7.11A) was deposited at the herbarium of the Laboratory of
Bioorganic Chemistry and Pharmacognosy, National University of
Mongolia.
d, J = 9.0 Hz, H-2′, 6′), 7.18 (2H, d, J = 9.0 Hz, H-3′, 5′), 3.71 (3H, s,
H-OMe of C-4′), 5.63 (1H, d, J = 7.5 Hz, H-Glc-I-1), 4.10 (1H, dd, J =
9.0, 7.5 Hz, H-Glc-I-2), 4.49 (1H, t, J = 9.0 Hz, H-Glc-I-3), 4.19 (1H,
t, J = 9.0 Hz, H-Glc-I-4), 4.22 (1H, m, H-Glc-I-5), 4.07 (1H, m, H-
Glc-I-6), 4.60 (1H, br d, J = 11.5 Hz, H-Glc-6), 5.27 (1H, d, J = 8.0
Hz, H-Glc-II-1), 4.08 (1H, dd, J = 9.0, 8.0 Hz, H-Glc-II-2), 4.27 (1H, t,
J = 9.0 Hz, H-Glc-II-3), 4.09 (1H, t, J = 9.0 Hz, H-Glc-II-4), 4.05 (1H,
m, H-Glc-II-5), 4.80 (1H, dd, J = 12.0, 3.0 Hz, H-Glc-II-6), 4.86 (1H,
dd, J = 12.0, 1.0 Hz, H-Glc-II-6), 2.00 (3H, s, H-Ac), 5.31 (1H, d, J =
7.5 Hz, H-Glc-III-1), 4.09 (1H, dd, J = 9.0, 7.0 Hz, H-Glc-III-2), 4.18
(1H, dd, J = 9.5, 9.0 Hz, H-Glc-III-3), 4.11 (1H, t, J = 9.5 Hz, H-III-4),
4.03 (1H, m, H-Glc-III-5), 4.32 (1H, dd, J = 12.0, 5.0 Hz, H-Glc-III-6),
4.58 (1H, br d, J = 12.0 Hz, H-Glc-III-6), 5.41 (1H, br s, H-Rha-1),
4.64 (1H, dd, J = 3.5, 1.5 Hz, H-Rha-2), 4.52 (1H, dd, J = 9.0, 3.5 Hz,
H-Rha-3), 4.18 (1H, m, H-Rha-4), 4.25 (1H, m, H-Rha-5), 1.54 (1H,
d, J = 1.5 Hz, H-Rha-6); 13C NMR (pyridine-d5, 100 MHz) δ 164.6
(C-2), 104.6 (C-3), 182.8 (C-4), 162.6 (C-5), 100.9 (C-6), 164.0 (C-
7), 95.5 (C-8), 157.8 (C-9), 106.6 (C-10), 123.6 (C-1′), 128.8 (C-2′,
-6′), 115.0 (C-3′, -5′), 163.1 (C-4′), 55.5 (C-OMe of 4′), 100.0 (C-
Glc-I-1), 84.7 (C-Glc-I-2), 77.3 (C-Glc-I-3), 69.9 (C-Glc-I-4), 77.2
(C-Glc-I-5), 67.2 (C-Glc-I-6), 104.4 (C-Glc-II-1), 85.2 (C-Glc-II-2),
77.5 (C-Glc-II-3), 70.6 (C-Glc-II-4), 74.9 (C-Glc-II-5), 64.2 (C-Glc-II-
6), 179.0 (C-Ac-CO), 20.7 (C-Ac-Me), 106.7 (C-Glc-III-1), 76.4
(C-Glc-III-2), 77.7 (C-Glc-III-3), 71.1 (C-Glc-III-4), 79.3 (C-Glc-III-
5), 62.4 (C-Glc-III-6), 102.3 (C-Rha-1), 72.0 (C-Rha-2), 72.7 (C-Rha-
3), 74.0 (C-Rha-4), 69.7 (C-Rha-5), 18.5 (C-Rha-6); HRFABMS
(positive) m/z 959.3026 [M + H]+ (calcd for C42H55O25, 959.3032).
Diosmetin-7-O-β-D-glucopyranosyl-(1→2)-6-O-acetyl-β-D-gluco-
pyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyrano-
side (4): colorless, amorphous solid; [α]21D −49.2 (c 1.30, DMSO); 1H
NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz),
see Table 1; HRFABMS (positive) m/z 975.2988 [M + H]+ (calcd for
C42H55O26, 975.2981).
Extraction and Isolation. Dried aerial parts of D. ruyschiana (95
g) were extracted with acetone−H2O (8:2) at room temperature for
two weeks (3 L). The extract was concentrated at reduced pressure,
and the solid (60.6 g) was suspended in H2O (1.5 L) and subjected to
extraction with Et2O (3 × 1 L). The aqueous layer extract (25.1 g) was
dissolved in H2O, passed through a porous polymer gel (Mitsubishi
Diaion HP-20, 70 × 180 mm), and eluted with H2O (fraction 1A,
13.29 g), MeOH−H2O (50:50) (fr. 1B, 2.55 g), and MeOH (fr. 1C,
9.52 g). Fr. 1B was chromatographed on a reversed-phase column
using an ODS-packed column (ODS-SM-50C-M, Yamazen, Japan)
and eluted with 10% MeOH−H2O (frs. 2A−2E) and 30% MeOH−
H2O (frs. 2F−2I). Compounds 10 (18.6 mg, from fr. 2H), 11 (1.9 mg,
from fr. 2G), 13 (3.0 mg, from fr. 2D), 29 (1.9 mg, from fr. 2D), and
17 (9.2 mg, from fr. 2B) were purified by HPLC [columns: AR-II,
mobile phases 5, 10, and 15% CH3CN−H2O containing 0.2% TFA;
5PE-MS, mobile phases 10% CH3CN−H2O containing 0.2% TFA;
Mightysil RP-18, mobile phases 5 and 10% CH3CN−H2O containing
0.2% TFA]. Fr. 1C was chromatographed on a reversed-phase column
using an ODS-packed column (ODS-SM-50C-M) and eluted with
30% MeOH−H2O (frs. 3A−3I) and 50% MeOH−H2O (frs. 3J−3M).
Each fraction was subjected to HPLC [columns: AR-II, mobile phases
15, 20, and 25% CH3CN−H2O containing 0.2% TFA; 5PE-MS,
mobile phases 15 and 20% CH3CN−H2O containing 0.2% TFA;
Mightysil RP-18, mobile phases 15, 20, and 25% CH3CN−H2O
containing 0.2% TFA and 30% MeOH−H2O containing 0.2% TFA] to
yield the compounds described below. Compound 9 (1.7 mg) was
purified from fr. 3M (228 mg). Compounds 3 (47.6 mg), 7 (28.3 mg),
8 (6.0 mg), and 9 (12.6 mg) were purified from fr. 3L (522 mg); 3
(267 mg) and 12 (123 mg) were purified from fr. 3K (2.04 g); 1 (31.9
mg) and 2 (13.3 mg) were purified from fr. 3J; 1 (55.7 mg) and 25
(0.5 mg) were purified from fr. 3I (309 mg); 1 (2.9 mg) was purified
from fr. 3H (273.0 mg); 1 (1.5 mg), 4 (14.5 mg), 6 (3.5 mg), 14 (7.0
mg), 20 (2.3 mg), and 26 (0.6 mg) were purified from fr. 3G (224
mg); 5 (4.3 mg), 14 (2.0 mg), 15 (12.8 mg), 18 (2.9 mg), 19 (1.7
mg), 23 (3.5 mg), and 26 (6.7 mg) were purified from fr. 3F (215
mg); 16 (58.2 mg), 18 (48.4 mg), 21 (5.4 mg), and 26 (3.1 mg) were
purified from fr. 3E (466.7 mg); 22 (5.4 mg), 24 (18.6 mg), and 27
(2.1 mg) were purified from fr. 3D (163 mg); 10 (18.6 mg), 26 (12.8
mg), and 28 (6.9 mg) were purified from frs. 3B and 3C (215 mg).
Acacetin-7-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-
Apigenin-7-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-
(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside (5): col-
1
orless, amorphous solid; [α]21 −71.0 (c 0.31, DMSO); H NMR
D
(DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz), see
Table 1; HRFABMS (positive) m/z 967.2713 [M + Na]+ (calcd for
C41H52O25Na, 967.2694).
Benzyl-2-O-trans-p-coumaroyl-α-L-rhamnopyranosyl-(1→6)-β-D-
glucopyranoside (7): colorless powder; [α]21 −32.7 (c 2.81,
D
MeOH); 1H NMR (methanol-d4, 400 MHz) and 13C NMR
(methanol-d4, 100 MHz), see Table 2; HRFABMS (positive) m/z
563.2117 [M + H]+ (calcd for C28H35O12, 563.2129).
Benzyl-2-O-cis-p-coumaroyl-α-L-rhamnopyranosyl-(1→6)-β-D-
glucopyranoside (8): colorless powder; [α]23 −26.7 (c 0.39,
D
MeOH); 1H NMR (methanol-d4, 400 MHz) and 13C NMR
(methanol-d4, 100 MHz), see Table 2; HRFABMS (positive) m/z
563.2129 [M + H]+ (calcd for C28H35O12, 563.2129).
Benzyl-4-O-trans-p-coumaroyl-α-L-rhamnopyranosyl-(1→6)-β-D-
glucopyranoside (9): colorless powder; [α]21 −20.2 (c 9.68,
D
MeOH); 1H NMR (methanol-d4, 400 MHz) and 13C NMR
(methanol-d4, 100 MHz), see Table 2; HRFABMS (positive) m/z
563.2143 [M + H]+ (calcd for C28H35O12, 563.2129).
(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside (1): col-
1
[(Benzoxy)methyl]phenyl-4-O-α-L-rhamnopyranosyl-(1→6)-β-D-
orless, amorphous solid; [α]23 −61.1 (c 0.37, DMSO); H NMR
D
glucopyranoside (12): colorless powder; [α]21 −56.3 (c 1.44,
(DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz), see
Table 1; HRFABMS (positive) m/z 939.2752 [M + Na]+ (calcd for
C40H52O24Na, 939.2745).
D
1
MeOH); H NMR (DMSO-d6, 400 MHz) δ 7.40 (2H, d, J = 9.0
Hz, H-2, -6), 7.00 (2H, d, J = 9.0 Hz, H-3, -5), 5.27 (2H, s, H-7), 7.96
(2H, d, J = 7.5 Hz, H-2′, -6′), 7.51 (2H, br t, J = 7.5 Hz, H-3′, -5′),
7.65 (1H, br t, J = 7.5 Hz, H-4′), 5.00 (1H, d, J = 7.5 Hz, H-Glc-1),
3.47 (overlapped, H-Glc-2), 3.45 (overlapped, H-Glc-3), 3.18
(overlapped, H-Glc-4), 3.34 (overlapped, H-Glc-5), 3.44 (1H, dd, J
= 12.5, 5.0 Hz, H-Glc-6), 3.68 (overlapped, H-Glc-6), 5.12 (1H, d, J =
1.5 Hz, H-Rha-1), 3.67 (overlapped, H-Rha-2), 3.33 (dd, J = 9.5, 2.5
Hz, H-Rha-3), 3.18 (overlapped, H-Rha-4), 3.84 (m, H-Rha-5), 1.17
(3H, d, J = 6.5 Hz, H-Rha-6); 13C NMR (DMSO-d6, 100 MHz) δ
129.3 (C-1), 129.1 (C-2, -6), 115.8 (C-3, -5), 157.1 (C-4), 65.9 (C-7),
129.6 (C-1′), 129.8 (2′, 6′), 128.7 (C-3′, -5′), 133.3 (C-4′), 165.6 (C-
7′), 98.2 (C-Glc-1), 76.3 (C-Glc-2), 77.4 (C-Glc-3), 71.9 (C-Glc-4),
76.8 (C-Glc-5), 60.5 (C-Glc-6), 100.3 (C-Rha-1), 70.4 (C-Rha-2),
Acacetin-7-O-β-D-glucopyranosyl-(1→2)-3-O-acetyl-β-D-gluco-
pyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyrano-
side (2): colorless, amorphous solid; [α]21D −51.5 (c 0.54, DMSO); 1H
NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz),
see Table 1; HRFABMS (positive) m/z 959.3031 [M + H]+ (calcd for
C42H55O25, 959.3032).
Acacetin-7-O-β-D-glucopyranosyl-(1→2)-6-O-acetyl-β-D-gluco-
pyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyrano-
side (3): colorless, amorphous solid; [α]21D −59.8 (c 4.28, DMSO); 1H
NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz),
1
see Table 1; H NMR (pyridine-d5, 400 MHz) δ 6.87 (1H, s, H-3),
7.00 (1H, d, J = 2.0 Hz, H-6), 7.07 (1H, d, J = 2.0 Hz, H-8), 8.02 (2H,
192
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Journal of Natural Products
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70.5 (C-Rha-3), 69.8 (C-Rha-4), 68.2 (C-Rha-5), 18.0 (C-Rha-6);
HRFABMS (positive) m/z 559.1797 [M + H]+ (calcd for
C26H32O12Na, 559.1791).
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[(3-Hydroxy-3-methylglutaryl)methyl]phenyl-4-O-β-D-glucopyra-
noside (13): colorless powder; [α]21 −35.7 (c 0.28, MeOH); 1H
D
NMR (methanol-d4, 400 MHz) δ 7.31 (2H, d, J = 9.0 Hz, H-2, -6),
7.08 (2H, d, J = 9.0 Hz, H-3, -5), 5.07 (2H, s, H-7), 2.70 (2H, br s, H-
2′), 2.63 (2H, s, H-4′), 1.35 (3H, s, H-6′), 4.90 (1H, d, J = 7.5 Hz, H-
Glc-1), 3.35−4.00 (overlapped, H-Glc-2, -3, -4, -5), 3.70 (dd, J = 12.0,
5.0 Hz, H-Glc-6), 3.89 (dd, J = 12.0, 2.0 Hz, H-Glc-6); 13C NMR
(methanol-d4, 100 MHz) δ 131.4 (C-1), 130.9 (C-2, 6), 117.7 (C-3,
5), 159.1 (C-4), 66.9 (C-7), 172.5 (C-1′), 46.3 (C-2′), 70.7 (C-3′),
45.8 (C-4′), 174.8 (C-5′), 27.7 (C-6′), 102.3 (C-Glc-1), 74.9 (C-Glc-
2), 78.2 (C-Glc-3), 71.4 (C-Glc-4), 78.0 (C-Glc-5), 62.5 (C-Glc-6);
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431.1553).
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(each 5.0 mg), and 13 (1.0 mg) were separately hydrolyzed with 7%
HCl (1 mL) at 60 °C for 2 h. The reaction mixture was neutralized
using an Amberlite IRA400 column, and the eluate was concentrated.
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tolyl isothiocyanate (10 μL) in pyridine (0.5 mL), using the procedure
reported by Tanaka et al.24 The mixtures were analyzed by HPLC
(column, Cosmosil 5C18-AR II column, 4.6 × 250 mm; mobile phase,
CH3CN−0.2% TFA in H2O (25:75), 1.0 mL/min; detector, UV at
210 nm) at 20 °C. D-Glucose (tR 22.3 min) was identified as the
glycosidic moieties of 1, 7, 12, and 13 by comparison with authentic
samples of D-glucose (tR 22.3 min) and L-glucose (tR 20.3 min)
derivatives. L-Rhamnose (tR 37.6 min) was identified as the glycosidic
moieties of 1, 7, and 12 by comparison with authentic samples of L-
rhamnose (tR 37.6 min) and D-rhamnose (using D-cysteine methyl
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100 μL of 0.06 mM DPPH in MeOH and mixed well. Absorbance was
measured at 510 nm exactly at 30 min by a microplate reader
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values express the concentration of sample required to scavenge 50%
of the DPPH free radicals. All samples were run in triplicate. Rutin
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ASSOCIATED CONTENT
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S
* Supporting Information
1H, 13C, COSY, HMQC, and HMBC NMR spectra for the new
compounds are available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
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(26) Rice-Evans, C. A.; Miller, N. J.; Paganda, G. Free Radical Biol.
Med. 1996, 20, 933−956.
Corresponding Author
*Tel: +81-22-727-0220. Fax: +81-22-727-0220. E-mail: murata-
t@tohoku-pharm.ac.jp.
(27) Burda, S.; Oleszek, W. J. Agric. Food Chem. 2001, 49, 2774−
2779.
Notes
(28) Mensor, L. L.; Menezes, F. S.; Leitao, G. G.; Reis, A. S.; dos
Santos, T. C.; Coube, C. S.; Leitao, S. G. Phytother. Res. 2001, 15,
127−130.
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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We thank Mr. S. Sato and Mr. T. Matsuki of Tohoku
Pharmaceutical University for assisting with the MS measure-
ments.
REFERENCES
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(1) Ligaa, U. In Medicinal Plants of Mongolia Used in Western and
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