Aromatic constituents of Cymbidium Great Flower Marie Laurencin and their antioxidative activity

Article abstract of DOI:10.1007/s11418-012-0653-z

Two novel aromatic glucosides, named marylaurecinosides B (1) and C (2), were isolated from Cymbidium Great Flower Marie Laurencin, together with six known aromatic compounds (3-8). These structures were determined on the basis of NMR experiments as well as chemical evidence. All of the isolated compounds (1-8) were tested for antioxidative activity using a superoxide dismutase-like assay.

Full text of DOI:10.1007/s11418-012-0653-z

J Nat Med (2013) 67:217–221
DOI 10.1007/s11418-012-0653-z
NOTE
Aromatic constituents of Cymbidium Great Flower Marie
Laurencin and their antioxidative activity
•
Kazuko Yoshikawa Misa Otsu Takuya Ito
•
•
•
Yoshinori Asakawa Sachiko Kawano
•
Toshihiro Hashimoto
Received: 22 December 2011 / Accepted: 26 February 2012 / Published online: 17 March 2012
Ó The Japanese Society of Pharmacognosy and Springer 2012
Abstract Two novel aromatic glucosides, named mary-
laurecinosides B (1) and C (2), were isolated from Cym-
bidium Great Flower Marie Laurencin, together with six
known aromatic compounds (3–8). These structures were
determined on the basis of NMR experiments as well as
chemical evidence. All of the isolated compounds (1–8)
were tested for antioxidative activity using a superoxide
dismutase-like assay.
from the Orchidaceae, we previously reported four new
phenanthrene derivatives, ephemeranthoquinone B, mary-
laurencinols A and B, and marylaurencinoside A, together
with six known phenanthrenes and their antibacterial and
cytotoxic activities, isolated from the fresh roots of the
well-known cultivated cymbidium, Cymbidium Great
Flower Marie Laurencin [2]. In this paper we made a
chemical study of the floral stem of C. Great Flower Marie
Laurencin. Two new phenolic glucosides, marylaurecino-
sides B (1) and C (2) were isolated along with six known
aromatic compounds (3–8). We describe here the isolation,
purification, and structural elucidation of 1 and 2 deter-
mined primarily by extensive NMR experiments, and the
antioxidant activity of all the isolated compounds using a
superoxide dismutase (SOD)-like assay1.
Keywords Cymbidium Great Flower Marie Laurencin ꢀ
Orchidaceae ꢀ Marylaurecinoside ꢀ
2-Benzyl-2-hydroxylsuccinic acid ꢀ Antioxidant activity ꢀ
Superoxide dismutase
Introduction
The Orchidaceae family consists of more than 35,000
species of flowering plants in approximately 750 genera,
which are widely distributed in temperate and tropical
regions, except for the Antarctic. Since ancient times, the
Orchidaceae have been used not only for ornamental pur-
poses but also as medicinal plants, to treat paralysis,
cholera, diarrhea, and sores [1]; therefore, the Orchidaceae
can be said to be a prodigious source of potential new
drugs. In the course of our study of bioactive substances
Results and discussion
Fresh floral stems were completely extracted with MeOH,
and this extract was partitioned to give three soluble frac-
tions in EtOAc, n-BuOH, and H2O. The EtOAc-soluble
portion was separated by ordinary-phase and reverse-phase
silica gels to furnish two novel glucosides, marylaurecin-
osides B (1) and C (2), along with six known compounds,
(2R)-2-benzyl-2-hydroxysuccinic acid (3) [3], 4-hydroxy-
benzoic alcohol (4) [4], protocatechualdehyde (5) [5],
4-hydroxybenzoic acid (6) [6], vanillic acid (7) [7], and
sakakin (8) [8] (Fig. 1).
K. Yoshikawa (&) ꢀ M. Otsu ꢀ T. Ito ꢀ Y. Asakawa ꢀ
T. Hashimoto
Marylaurecinoside B (1) was obtained as an amorphous
powder. Negative-ion high-resolution (HR)-FAB-MS of 1
gave the molecular formula C26H29O12 with a [M - H]-
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
1
This work was presented at the 124th Annual Meeting of the
Pharmaceutical Society of Japan, Kyoto, March, 2009
123

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J Nat Med (2013) 67:217–221
peak at m/z 533.2006, indicating twelve unsaturations. The
IR spectrum of 1 suggested the presence of hydroxyl (3375,
1070 cm-1), carbonyl (1730 cm-1), and aromatic ring
confirmed by optical rotation using chiral detection in
HPLC analysis (see ‘‘Experimental’’ section) [9]. The
HMBC correlations from H2-7 (d 5.07, 5.04) to C-3 (d
131.5), C-4 (d 131.0), C-5 (d 131.5), C-8 (d 175.5), from
H2-10 (d 2.59, 2.96) to C-9 (d 77.1), and C-11 (d 174.0),
from H2-12 (d 2.92, 2.98) to C-8 (d 175.5), C-9 (d 77.1),
C-13 (d 136.6), C-14, and C-18 (d 131.5) revealed that
the 4-hydroxybenzyloxy and 2-benzyl-2-hydroxysuccinyl
moieties were connected between the C-7 and C-8 posi-
tions in each unit. Furthermore, the HMBC correlation
between C-1 (dC 159.0) and H-10 (dH 4.91) indicated that
the b-glucopyranosyl moiety combined with C-1 (Fig. 2).
The remaining location of the acetyl moiety was deter-
mined at H2-6 of glucose by their acylation shifts at d 4.23
(dd, J = 12.0, 6.6 Hz) and d 4.40 (dd, J = 12.0, 2.2 Hz),
and HMBC correlations, as shown in Fig. 2. On alkaline
hydrolysis, 1 afforded 2-benzyl-2-hydroxysuccinic acid
1
groups (1613 cm-1). The H NMR spectrum of 1 showed
AB-type aromatic protons at d 7.05 (2H, d, J = 8.8 Hz)
and d 7.27 (2H, d, J = 8.8 Hz), five overlapped aromatic
protons at d 7.06 (2H, m) and d 7.17 (3H, m), three sets of
isolated methylene protons at d 2.59, 2.96 (each 1H d,
J = 16.2 Hz), d 2.92, 2.98 (each 1H d, J = 13.5 Hz), and
d 5.04, 5.07 (each 1H d, J = 11.9 Hz), and one anomeric
proton at d 4.91 (d, J = 7.7 Hz), together with one acetyl
methyl signal at d 2.02 (3H, s). The 13C NMR spectrum, in
combination with HMQC data, showed 26 carbon reso-
nances, of which 20 were assignable to two aromatic rings,
with a hexose and acetyl group. The other six carbon res-
onances could be assigned to two carboxyl carbons at
d 174.0 and 175.5, a quaternary carbon at d 77.1, an oxy-
methylene carbon at d 67.9, and two methylene carbons at
d 44.1 and 46.3. The COSY correlation of 1 revealed the
presence of phenyl, benzyloxy, and b-glucopyranosyl
moieties (Fig. 2). Acid hydrolysis of 1 liberated D-glucose,
25
(9), and optical roration showed ½aꢁD -20.5° (c = 0.3,
MeOH), suggesting an R configuration [3]. Thus, from the
above findings, the structure of 1 was formulated as shown.
Marylaurenoside C (2) was obtained as an amorphous
powder and showed a [M ? Na]? peak at m/z 415.1389 in
HR-FAB-MS, which corresponded to the molecular for-
mula C20H24O8. The IR spectrum of 2 showed absorptions
at 3375, 1613 and 1070 cm-1. The COSY and HMQC
spectra showed the presence of olefinic methyl, 4-mono-
substituted benzyl, 2,4,6-tri-substituted phenyl, and
b-glucopyranosyl groups (Fig. 3). On acid hydrolysis, 2
liberated D-glucose, identified by optical rotation using
chiral detection in HPLC analysis [9]. The gross structure
of 2 was determined by the same strategy as 1. In the
HMBC data, the connectivity from H2-7 (d 3.82, 4.01) to
C-3 (d 157.9), C-4 (d 112.2), C-5 (d 140.1), C-8 (d 133.7),
C-9 (d 130.2), from H-9 (d 6.93) and H-10 (d 6.61) to C-11
(d 156.0), from H3-14 (d 2.10) to C-4, C-5, and C-6
(d 121.6), and from H-10 (d 4.82) to C-3 revealed that
4-(4-hydroxybenzyl)-5-methylbenzene-1,3-diol, in which
b-glucopyranosyl was attached at C-3 (Fig. 3). The fol-
lowing NOEs between H3-14/H-6 (d 6.31), /H2-7, and H-2
(d 6.55) /H-10 confirmed the substituent positions in the
tetra-substituted aromatic ring. Thus, from the above
findings, the structure of 2 was formulated as shown.
To our knowledge, compound 3, which was identified as
(2R)-2-benzyl-2-hydroxysuccinic acid by optical rotation
AcO
O
HO
HO
O
OH
HO
HO
HO
O
O
OH
O
OH
O
OH
O
2
1
HO
OH
R1
4-Hydroxybenzyl alcohol (4)
Protocatechualdehyde (5)
4-Hydroxybenzoic acid (6)
Vanillic acid (7)
R2=H
R2=OH
R2=H
R1=CH2OH
R1=CHO
R1=COOH
R1=COOH
R2
R2=OMe
OH
HO
COOH
COOH
HO
HO
O
O
(2R)-2-benzyl-2-hydroxysuccinic acid (3)
HO
HO
OH
Sakakin (8)
Fig. 1 Chemical structures of compounds 1–8
O
6'
15
O
25
14
13
16
4'
½aꢁD -14.4° (c = 0.6, MeOH), is here isolated for the first
5'
O
HO
2
time as a natural product.
O
1'
17
1
12
2'
HO
3
3'
The antioxidant activities of 1–8 were studied with a
SOD assay kit. Vitamin C was used as a positive control
(IC50 66.2 lM). Compounds 4 and 5 exhibited marked
SOD-like activity (IC50 24.2 and 11.9 lM, respectively).
In conclusion, we identified eight compounds, including
two new phenolic glucosides, from C. Great Flower Marie
O
18
OH
4
11
9
10
O
6
OH
8
5
7
OH
O
Fig. 2 COSY (thick lines) and HMBC (curved arrows) correlations
for 1
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J Nat Med (2013) 67:217–221
219
Japan, seed registration No. 2841) were cultivated and
harvested in February 2008 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 Pharma-
cognosy, Tokushima Bunri University, Tokushima, Japan.
14
7
9
5
10
6
4
3
8
1
13
11
OH
HO
12
2
Extraction and isolation
O
6'
Fresh floral stems of C. Great Flower Marie Laurencin
(1.4 kg) were completely extracted with MeOH at room
temperature for 3 weeks. The methanolic extract was par-
titioned between EtOAc and H2O. The EtOAc-soluble
portion (21 g) was subjected to silica gel column chroma-
tography with hexane–EtOAc–MeOH (10:1:0 ? 0:1:10) to
afford seven fractions (frs. 1–7). Fraction 2 (4.2 g) was
passed through silica gel with hexane–EtOAc (9:1) and
purified by preparative HPLC (ODS, 95 % MeOH) to afford
4-hydroxybenzyl alcohol (4, 32.6 mg). Fraction 3 (0.62 g)
was passed through silica gel with hexane–EtOAc (1:1) and
purified by preparative HPLC (ODS, 50 % MeOH) to yield
protocatechualdehyde (5, 5.2 mg), 4-hydroxybenzoic acid
(6, 8.1 mg), and vanillic acid (7, 2.7 mg). Fraction 6 (1.9 g)
was purified by preparative HPLC (ODS, 40–50 % MeOH)
to afford marylaurecinosides B (1, 15.8 mg), C (2, 7.7 mg),
(2R)-2-benzyl-2-hydroxysuccinic acid (3, 33.6 mg), and
sakakin (8, 6.4 mg).
HO
O
5'
4'
HO
HO
1'
2'
OH
3'
Fig. 3 COSY (thick lines) and HMBC (curved arrows) correlations
for 2
Laurencin. The major antioxidative compounds were iden-
tified as 4-hydroxybenzoic alcohol (4), and protocatechu-
aldehyde (5). These results suggested that the antioxidative
activities of 1–8 were partly attributed to the catechols, or
the ratio of the phenols in the molecular compounds. The
goal of this study is to identify functional ingredients for the
development and utilization of health-care drugs.The study
of the active ingredient is therefore in progress.
Experimental
Marylaurecinoside B (1)
25
General
An amorphous powder; ½aꢁD -40.3° (c = 1.6, MeOH);
FT-IR (dry film) cm-1: 3375 (OH), 1730 (C=O), 1613
(C=C), 1070 (OH). UV kmax (MeOH) nm (log e): 215
(4.13), 269 (2.83); 1H NMR and 13C NMR data see
Table 1; HR-FAB-MS m/z 533.2006 (calcd for C26H29O12:
533.2001)
Optical rotation was performed on a JASCO P-1030 digital
polarimeter. The UV spectra were recorded using a Shi-
madzu UV-6000 spectrophotometer. IR spectra were
measured on a Shimadzu FT/IR-8400S instrument. NMR
spectra were recorded on a Varian UNITY 600 spectrom-
eter. The chemical shifts are given as d (ppm) in CD3OD
solution, using tetramethylsilane (TMS) as the internal
standard. NMR experiments included COSY, HMQC,
HMBC, and ROESY. Coupling constants (J values) are
given in Hz. HR-FAB-MS was measured on a JEOL JMS-
700 MStation. For HPLC column chromatography, COS-
MOSIL 5C18-AR-II (Nacalai Tesque, Inc., Kyoto, 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.
Marylaurecinoside C (2)
25
An amorphous powder; ½aꢁD -34.7° (c = 0.8, MeOH);
FT-IR (dry film) cm-1: 3375 (OH), 1613 (C=C), 1070
(OH).UV kmax (MeOH) nm (log e): 272 (3.37), 290 (3.12);
1H NMR and 13C NMR data see Table 1; HR-FAB-MS m/z
415.1389 (calcd for C20H24O8Na: 415.1369).
Acid hydrolysis of compounds 1 and 2
Each sample (1 mg) in 5 % H2SO4-dioxane (1:1) was heated
at 100 °C for 2 h. The reaction mixture was diluted with H2O
and then neutralized with Amberlite IRA-35 and evaporated
in vacuo to dryness. The identification and the D or L con-
figuration of glucose was determined by using RI detection
Plant material
Fresh floral stems of Cymbidium Great Flower Marie Lau-
rencin (Ministry of Agriculture, Forestry and Fisheries of
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J Nat Med (2013) 67:217–221
Table 1 1H and 13C NMR
spectral data for 1 and 2 in
CD3OD
Position
Marylaurenoside B (1)
Position
Marylaurenoside C (2)
dC
dH (J Hz)
dC
dH (J Hz)
1
159.0
117.7
131.3
131.0
67.9
1
2
3
4
5
6
7
157.1
101.8
157.9
112.2
140.1
121.6
31.2
2/6
3/5
4
7.05 (d, 8.8)
7.27 (d, 8.8)
6.55 (d, 2.3)
7
5.04 (d, 11.9)
5.07 (d, 11.9)
6.31 (d, 2.2)
3.82 (d, 15.4)
4.01 (d, 15.4)
8
175.5
77.1
44.1
9
10
2.59 (d, 16.2)
2.96 (d, 16.2)
8
133.7
130.2
115.8
156.0
115.8
130.2
20.1
9
6.93 (d, 8.8)
6.61 (d, 8.8)
11
12
174.0
46.3
10
11
12
13
14
2.92 (d, 13.5)
2.98 (d, 13.5)
6.61 (d, 8.8)
6.93 (d, 8.8)
2.10 (s)
13
14/18
15/17
16
10
20
30
40
50
60
136.6
131.5
129.1
127.9
102.1
74.8
7.06 (m)
7.17 (m)
7.17 (m)
4.91 (d, 7.7)
3.46 (m)
10
20
30
40
50
60
103.0
75.0
78.1
71.3
78.2
62.6
4.82 (d, 7.7)
3.41 (m)
77.8
3.47 (m)
3.41 (m)
71.6
3.39 (m)
3.39 (m)
75.3
3.65 (m)
3.40 (m)
64.7
4.23 (dd, 12.0, 6.6)
4.40 (dd, 12.0, 2.2)
2.02 (s)
3.71 (dd, 11.8, 4.7)
3.87 (dd, 11.8, 1.5)
Ac
172.8
20.8
(Shimadzu RID-10A) and chiral detection (Shodex OR-1) by
HPLC (Shodex RSpak NH2P-50 4D, CH3CN–H2O–H3PO4,
95:5:1, 1 mL/min, 47 °C), by comparison with an authentic
sugar (10 mmol D-glc). The sugar portion gave the following
peak of D-(?)-glucose at 20.6 min.
Kumamoto, Japan). A test sample was dissolved in DMSO
to give a final DMSO concentration of 0.8 % (v/v).
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25
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