Two novel aromatic glucosides, marylaurencinosides D and E, from the fresh flowers of Cymbidium Great Flower 'Marylaurencin'

Article abstract of DOI:10.1007/s11418-013-0814-8

Two novel aromatic glucosides, named marylaurencinosides D (1) and E (2), were isolated from the fresh flowers of Cymbidium Great Flower 'Marylaurencin'. In addition, eight known aromatic compounds (3-10) were isolated. These structures were determined on

Full text of DOI:10.1007/s11418-013-0814-8

J Nat Med
DOI 10.1007/s11418-013-0814-8
NOTE
Two novel aromatic glucosides, marylaurencinosides D and E,
from the fresh flowers of Cymbidium Great Flower
‘Marylaurencin’
•
Kazuko Yoshikawa Mariko Okahuji
•
•
Kanako Iseki Takuya Ito Yoshinori Asakawa
•
•
•
Sachiko Kawano Toshihiro Hashimoto
Received: 23 October 2013 / Accepted: 30 December 2013
Ó The Japanese Society of Pharmacognosy and Springer Japan 2014
Abstract Two novel aromatic glucosides, named mary-
laurencinosides D (1) and E (2), were isolated from the
fresh flowers of Cymbidium Great Flower ‘Marylaurencin’.
In addition, eight known aromatic compounds (3–10) were
isolated. These structures were determined on the basis of
NMR experiments as well as chemical evidence.
cultivated cymbidium, Cymbidium Great Flower ‘Mary-
laurencin’ [2, 3]. In our continuing search for bioactive
substances from the Orchidaceae, we became interested in
the chemical study of the fresh flowers of this plant. Fresh
flowers were completely extracted with MeOH. This
extract was partitioned to give three soluble fractions in
EtOAc, n-BuOH, and H2O. The n-BuOH-soluble portion
was separated by ordinary-phase and reversed-phase silica
gels to furnish two novel aromatic glucosides, marylau-
rencinosides D (1) and E (2), along with eight known
compounds, grammatophylloside B (3) [4], cronupapine
(4) [5], 2-O-(trans-coumaroyl) malic acid (5) [6], conif-
eroside (6) [7], sinapic acid 4-O-glucoside (7) [8], sakakin
(8) [9], arbutin (9) [10], and gastrodin (10) [11] (Fig. 1).
This paper describes the isolation, purification, and struc-
tural elucidation of 1–10 determined primarily by extensive
NMR experiments and chemical degradation.
Keywords Cymbidium Great Flower ‘Marylaurencin’ ꢀ
Orchidaceae ꢀ Marylaurencinoside ꢀ (R)-2-benzylmalic
acid ꢀ (R)-eucomic acid
Introduction
The Orchidaceae family consists of more than 35,000
species, found in 820 genera, of flowering plants, which are
widely distributed in temperate and tropical regions.
However, only a few have been investigated for their
chemical composition and biological activities [1]. We
previously reported four new phenanthrene derivatives,
ephemeranthoquinone B, marylaurencinols A and B, and
marylaurencinoside A, together with six known phenan-
threnes and their antibacterial and cytotoxic activities from
the fresh root, and marylaurencinosides B and C, along
with six known aromatic compounds and their antioxidant
activity, from the fresh floral stems of the well-known
Results and discussion
Marylaurencinoside D (1) was obtained as an amorphous
powder. HR-FAB-MS of 1 gave molecular formula
C25H30O12 with a [M ? Na]? peak at m/z 545.1639,
indicating 11 unsaturations. The IR spectrum of 1 sug-
gested the presence of hydroxyl (3370 cm-1), carbonyl
(1720 cm-1), and aromatic ring (1600 cm-1) groups. The
1H-NMR spectrum of 1 showed 1,3,4-trisubstituted ben-
zene ring protons at d 7.02 (dd, J = 8.3, 2.0 Hz), 7.15 (d,
J = 2.0 Hz), and 7.52 (d, J = 8.3 Hz), one phenyl group
proton at d 7.25 (1H, dd, J = 8.0, 8.0 Hz), 7.30 (2H, dd,
J = 8.0, 8.0 Hz), and 7.51 (2H, d, J = 8.0 Hz), three sets
of isolated methylene protons at d 3.16, 3.48 (each 1H d,
J = 15.8 Hz), 3.35, 3.41 (each 1H d, J = 13.4 Hz), and
5.33, 5.37 (each 1H d, J = 12.1 Hz), and one methoxyl
K. Yoshikawa (&) ꢀ M. Okahuji ꢀ K. Iseki ꢀ T. Ito ꢀ
Y. Asakawa ꢀ T. Hashimoto
Faculty of Pharmaceutical Sciences, Tokushima Bunri
University, Yamashiro-Cho, Tokushima 770-8514, Japan
e-mail: yosikawa@ph.bunri-u.ac.jp
S. Kawano
Kawano-Mericlone Co., Ltd, Wakimachi,
Tokushima 779-3604, Japan
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J Nat Med
Fig. 1 Chemical structures of compounds 1–10, 1a, and 2a
Table 1 1H- and 13C-NMR spectral data for 1 and 2 (600 MHz and
150 MHz, in C5D5N)
No.
1
2
dC
dH (J Hz)
dC
dH (J Hz)
1
2
3
175.2
76.8
175.4
77.1
44.5 3.16 (d, 15.8)
3.48 (d, 15.8)
44.5 3.18 (d, 15.8)
3.48 (d, 15.8)
Fig. 2 COSY, selected HMBC, and ROESY correlations of 1
4
5
173.7
173.9
rings, two carboxyl carbons at d 173.7 and 175.2, a qua-
ternary carbon at d 76.8, an oxymethylene carbon at d 67.2,
two methylene carbons at d 44.5 and 45.9, and a methoxyl
group at d 55.8. The remaining six carbon resonances at d
102.1, 78.9, 78.5, 74.8, 71.2, and 62.3 could be assigned to
a hexose sugar moiety. The COSY correlations of 1
revealed the presence of phenyl, 3,4-disubstituted benzyl,
and b-glucopyranosyl moieties (Fig. 2). Acid hydrolysis of
1 liberated D-glucose, identified by optical rotation using
chiral detection in HPLC analysis (see Experimental sec-
tion) [12]. The HMBC correlations from three sets of iso-
lated methylene to carbons, H2-3 (d 3.16, 3.48)/C-1 (d
175.2),/C-2 (d 76.8), and/C-4 (d 173.7), H2-5 (d 3.35,
3.41)/C-1,/C-2,/C-6 (d 136.9),/C-7 (d 131.2), and/C-11 (d
131.2), H2-70 (d 5.33, 5.37)/C-1,/C-10 (d 130.5),/C-20 (d
113.5), and/C-60 (d 121.9), revealed that the C-1 position of
2-benzylmaloyl moiety was connected through an ester
bond at the C-70 position of 3-methoxy-4-hydroxybenzyl
alcohol unit (Fig. 2). Furthermore, the HMBC correlation
from H-100 (d 5.67) to C-40 (d 147.9) obviously indicated
that the glucopyranosyl moiety combined with the C-40
position. The following NOEs between H-20 (d 7.15)/OMe
(d 3.66) and H-50 (d 7.52)/H-100 confirmed the substituent
positions of the methoxy group and the glucopyranosyl
linkage. On alkaline hydrolysis, 1 afforded 2-benzylmalic
acid (1a), whose optical rotation showed [a]2D5 –17.4 (c 0.2,
MeOH), suggesting an R configuration [13]. Thus, from the
above findings, the structure of 1 was formulated as shown
in Fig. 1.
45.9 3.35 (d, 13.4)
3.41 (d, 13.4)
45.3 3.31 (d, 13.6)
3.37 (d, 13.6)
6
136.9
127.0
7/11
8/10
9
10
20
30
40
50
60
131.2 7.51 (d, 8.0)
128.4 7.30 (dd, 8.0, 8.0)
127.1 7.25 (dd, 8.0, 8.0)
130.5
132.4 7.42 (d, 8.8)
115.9 7.13 (d, 8.8)
157.9
130.0
113.5 7.15 (d, 2.0)
149.8
130.5 7.40 (d, 8.8)
116.8 7.31 (d, 8.8)
158.5
147.9
116.0 7.52 (d, 8.3)
121.9 7.02 (dd, 8.3, 2.0)
67.2 5.33 (d, 12.1)
5.37 (d, 12.1)
116.8 7.31 (d, 8.8)
130.5 7.40 (d, 8.8)
66.9 5.33 (d, 12.1)
5.35 (d, 12.1)
70
Glc-100 102.1 5.67 (d, 7.4)
102.0 5.61 (d, 7.7)
74.9 4.31 (dd, 8.8, 7.7)
78.5 4.37 (dd, 8.8, 8.8)
71.2 4.35 (dd, 8.8, 8.5)
200
300
400
500
74.8 4.34 (dd, 8.8, 7.4)
78.5 4.36 (dd, 8.8, 8.8)
71.2 4.36 (dd, 8.8, 8.8)
78.9 4.11 (ddd, 8.8, 5.1,
2.5)
78.9 4.11 (ddd, 8.5, 5.2,
2.3)
600
62.3 4.40 (dd, 12.0, 5.1)
4.52 (dd, 12.0, 2.5)
55.8 3.66 (s)
62.3 4.40 (dd, 12.1, 5.2)
4.54 (dd, 12.1, 2.3)
OMe
signal at d 3.66 (3H, s), together with one anomeric proton
at d 5.67 (d, J = 7.4 Hz) (Table 1). The 13C-NMR spec-
trum, in combination with HMQC data, showed 25 carbon
resonances, of which 19 were assignable to two benzene
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J Nat Med
internal standard. NMR experiments included COSY,
HMQC, HMBC, and ROESY. Coupling constants (J val-
ues) are given in Hz. HR-FAB-MS was measured on a
JEOL JMS-700 MStation. For HPLC column chromatog-
raphy, Develosil RPAQUEOUS-AR-5 (Nomura Chemical
Co., Ltd., Japan, 20 mm i.d. 9 250 mm) was used. TLC
was performed on pre-coated silica gel 60F254 (Merck).
Spots were detected by examining plates sprayed with
p-anisaldehyde/H2SO4/MeOH reagent followed by heating
on a hot plate.
Fig. 3 COSY and selected HMBC correlations of 2
Marylaurencinoside E (2) was obtained as an amorphous
powder and showed an [M ? Na]? peak at m/z 531.1455 in
HR-FAB-MS, which corresponded to the molecular for-
mula C24H28O12. The IR spectrum of 2 showed absorptions
at 3327, 1720, and 1590 cm-1. The COSY and HMQC
spectra showed the presence of a para-substituted phenyl,
para-substituted benzyl, two sets of isolated methylene,
and b-glucopyranosyl moieties (Fig. 3). On acid hydroly-
sis, 2 liberated D-glucose identified by optical rotation
using chiral detection in HPLC analysis. The gross struc-
ture of 2 was determined by the same strategy as 1. In the
HMBC data, the correlations of H2-3/C-1,/C-2, and/C-4, of
H2-5/C-1,/C-2,/C-6,/C-7, and/C-11, of H2-70/C-1,/C-10,/C-
20, and/C-60 revealed that eucomic acid [14] and
4-hydroxybenzyl alcohol were connected through an ester
bond at C-1 and C-70 in each unit, in which b-glucopyr-
anosyl moiety was also attached at C-40 (Fig. 3). On alka-
line hydrolysis, 2 afforded eucomic acid (2a), [a]2D5 –10.9
(c 0.3, MeOH), suggesting an R configuration [14]. Thus,
from the above findings, the structure of 2 was formulated
as shown in Fig. 1.
Plant material
Fresh flowers of Cymbidium Great Flower ‘Marylaurencin’
(Ministry of Agriculture, Forestry and Fisheries of Japan,
seed registration No. 2841) were cultivated and harvested in
February 2012 at Kawano Mericlone Co., Ltd. (Tokushima
Prefecture, Japan), and identified by one of the authors
(S.K.). A voucher specimen (TB 5430) has been deposited
in the Herbarium of the Department of Pharmacognosy,
Tokushima Bunri University, Tokushima, Japan.
Extraction and isolation
Fresh flowers of Cymbidium Great Flower ‘Marylaurencin’
(10 kg) were completely extracted with MeOH at room
temperature for 2 weeks. The methanolic extract was parti-
tioned between EtOAc, n-BuOH, and H2O. The n-BuOH-
soluble portion (88 g) was subjected to silica gel column
chromatography with CHCl3–MeOH–H2O (25:6:0.1 ?
6:4:1) to afford eight fractions (frs. 1–8). Fraction 2 (1.2 g)
was passed through Sephadex LH-20 with MeOH and puri-
fied by preparative HPLC (ODS, 30–60 % MeOH) to afford
marylaurencinoside D (1, 15.8 mg), and 2-O-(trans-couma-
royl) malic acid (5, 10 mg). Fraction 3 (9.23 g) was passed
through silica gel with CHCl3–MeOH–H2O (65:30:5) and
purified by preparative HPLC (ODS, 20–50 % MeOH) to
afford cronupapine (4, 350 mg), coniferoside (6, 7.6 mg),
sinapic acid 4-O-glucoside (7, 6.6 mg), and sakakin (8,
25.4 mg). Fraction 4 (17.25 g) was passed through Sephadex
LH-20 to afford six subfractions (frs. 4-1–4-6). Fraction 4-3
was purified by preparative HPLC (ODS, 20–50 % MeOH)
to yield grammatophylloside B (3, 14.7 mg), arbutin (9,
35.6 mg), and gastrodin (10, 145.4 mg). Fraction 7 (2.43 g)
was purified by preparative HPLC (ODS, 40 % MeOH) to
afford marylaurencinoside E (2, 70.8 mg).
The isolated compound (8) and its aglycone, orcinol
(8a), were tested for antimicrobial activity against Bacillus
subtilis and Klebsiella pneumonia. However, neither was
active against the two strains.
The present study reported four glucosyloxybenzyl
derivatives of (R)-2-benzylmalic acid (1, 3, and 4) and of
(R)-eucomic acid (2), which are very specific types of
compounds obtained from the orchid family [3–5]. The
main skeletons of 1–4 seem to be formed from one mole-
cule each of phenylpyruvic acid and acetate unit. These
specific compounds provided further confirmation of the
typical profile of secondary metabolites found in this
family, and might be useful for further biological studies.
Experimental
Marylaurencinoside D (1)
General experimental procedures
An amorphous powder; [a]2D5 –45.8 (c 0.7, MeOH); FT-IR
(dry film) kmax 3370 (OH), 1720 (C=O), 1600 (C=C) cm-1
;
NMR spectra were recorded on a Varian UNITY 600
spectrometer. The chemical shifts are given as d (ppm) in
C5D5N solution, using tetramethylsilane (TMS) as the
UV kmax (MeOH) nm (log e): 218 (3.90), 255 (3.21), 261
(3.21). 1H-NMR (600 MHz, C5D5N) and 13C-NMR
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J Nat Med
(150 MHz, C5D5N) data see Table 1; HRMS-FAB m/z
545.1639 [M ? Na]? (calcd for C25H30O12Na: 545.1635).
96-well plates which contained microbial strains incubated
in YP medium. Each plate was further incubated at 37 °C
(B. subtilis) and 27 °C (K. pneumoniae) overnight and
ampicillin (Nacalai Tesque) was used as a reference
reagent for the bacterial strains.
Marylaurencinoside E (2)
An amorphous powder; [a]2D5 –29.0 (c 3.7, MeOH); FT-IR
(dry film) kmax 3327 (OH), 1720 (C=O), 1590 (C=C) cm-1
;
UV kmax (MeOH) nm (log e): 225 (3.91), 256 (3.38), 278
(3.33). 1H-NMR (600 MHz, C5D5N) and 13C-NMR
(150 MHz, C5D5N) data see Table 1; HRMS-FAB m/z
531.1455 [M ? Na]? (calcd for C24H28O12 Na: 531.1478).
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Bacillus subtilis NBRC 3134 and Klebsiella pneumoniae
NBRC 3512 were used for testing antimicrobial activity.
These strains were tested by using microdilution assays.
Bacterial strains were inoculated on YP agar plates [0.5 %
peptone (Becton, Dickinson and Co., USA), 0.3 % yeast
extract (Becton, Dickinson and Co.), 0.1 % MgSO4ꢀ7H2O
and 2 % agar (Nacalai Tesque, Japan)] and incubated at
37 °C (B. subtilis) and 27 °C (K. pneumoniae) for 12 h. A
stock solution of samples was prepared at 10 mg/mL in
DMSO and further diluted to varying concentrations in
123