Glycosidic constituents of the tubers of Gymnadenia conopsea

Article abstract of DOI:10.1021/np070670j

Ten minor new glycosidic constituents (1-10), together with 10 known compounds, have been isolated from a neuroprotective fraction of an ethanolic extract of the tubers of Gymnadenia conopsea. The structures of 1-10 were determined using spectroscopic and chemical methods. The compounds isolated were evaluated for activity in in vitro assays for acetylcholine esterase and monoamine oxidase inhibitory activities.

Full text of DOI:10.1021/np070670j

J. Nat. Prod. 2008, 71, 799–805
799
Glycosidic Constituents of the Tubers of Gymnadenia conopsea
Jiachen Zi,† Shuai Li,† Mingtao Liu,† Maoluo Gan,† Sheng Lin,† Weixia Song,† Yanling Zhang,† Xiaona Fan,†
Yongchun Yang,† Jianjun Zhang,† Jiangong Shi,*,† and Duolong Di*,‡
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (Key Laboratory of BioactiVe
Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education), Beijing 100050, People’s Republic of China, and
Key Laboratory of Natural Medicine of Gansu ProVince, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou
730000, People’s Republic of China
ReceiVed NoVember 23, 2007
Ten minor new glycosidic constituents (1-10), together with 10 known compounds, have been isolated from a
neuroprotective fraction of an ethanolic extract of the tubers of Gymnadenia conopsea. The structures of 1-10 were
determined using spectroscopic and chemical methods. The compounds isolated were evaluated for activity in in vitro
assays for acetylcholine esterase and monoamine oxidase inhibitory activities.
Gymnadenia conopsea R. Br. is a plant belonging to the
Orchidaceae family and is distributed in the provinces of Tibet,
Xinjiang, Inner Mongolia, Sichuan, Qinghai, and Gansu of mainland
China. The dried tubers of this plant and other species of this genus
and the genus Coeloglossum, known as “Wangla” (Chinese), have
been used as a traditional Tibetan remedy to treat cough, asthma,
and other syndromes, and as a tonic in Chinese folk medicine, with
multiple indications such as invigorating vital energy, promoting
the production of body fluids, having a tranquilizing effect, and
enhancing intelligence.1–3 Antiallergic phenanthrenes and stilbenes,4
as well as nine glycosyloxybenzyl 2-isobutylmalate derivatives,5
were recently reported from a methanol extract of the tubers of G.
conopsea.
As part of a program to assess the chemical and biological
diversity of traditional Chinese medicines, we have reported
chemical and pharmacological investigations of the “Wangla”
source species Coeloglossum Viride (L.) Hartm. var. bracteatum
(Willd.) Richter, collected from Qinghai Province. A fraction (CE)
from an ethanolic extract of C. Viride var. bracteatum, containing
mainly mono- and bis(4-ꢀ-D-glucopyranosyloxybenzyl) 2-isobu-
tyltartrate and 2-isobutylmalate derivatives,6 showed neuroprotective
effects on memory deficits and pathological changes in senescent
mice.7–9 Meanwhile, a main component of the fraction, dactylorhin
B, reduced toxic effects of ꢀ-amyloid fragment (25–35) on neuron
cells and isolated rat brain mitochondria.9 By using the same
protocol for the tubers of C. Viride var. bracteatum,10 a fraction
was obtained in the present investigation from an ethanolic extract
of the tubers of G. conopsea collected in Sichuan Province. HPLC
analysis indicated that the fraction contained constituents similar
to those of the active fraction from the tubers of C. Viride var.
bracteatum. An in vivo preliminary pharmacological test indicated
it was also active in improving the impaired memory in mice caused
by scopolamine and cycloheximide at the same dosage (5.0 mg
kg-1).10 A further chemical investigation of the active fraction has
led to the isolation and structural elucidation of 20 glycosidic
constituents including 10 minor new compounds (1-10). By
comparing with corresponding literature data, the known compounds
were identified as gastrodin,11 loroglossin,12,13 militarine,13 dac-
tylorhins A, B, and E,14 and coelovirins A-D.6 This paper deals
with the structural elucidation of the new compounds (1-10) and
Results and Discussion
their preliminary biological evaluation.
The ethanolic extract of the dried tubers of G. conopsea was
suspended in water and then partitioned with EtOAc. The aqueous
solution was chromatographed successively over macroporous resin,
silica gel, and reversed-phase preparative HPLC to yield 10 minor
new glycosidic constituents, 1-10.
* To whom correspondence should be addressed. Tel: 86-10-83154789.
Fax: 86-10-63017757. E-mail: shijg@imm.ac.cn.
† Chinese Academy of Medical Sciences and Peking Union Medical
College.
‡ Lanzhou Institute of Chemical Physics.
10.1021/np070670j CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/19/2008

800 Journal of Natural Products, 2008, Vol. 71, No. 5
Zi et al.
20
Compound 1 was obtained as a colorless gum, [R]D -36.6 (c
of 4 showed a pair of additional signals assignable to an ethoxyl at
δ 1.14 (3H, t, J ) 7.2 Hz, H-2′′′) and 3.47 (2H, q, J ) 7.2 Hz,
H-1′′′) and δ 15.4 (C-2′′′) and 75.5 (C-1′′′). In addition, C-1 and
C-7 of the benzyl moiety in 4 were shifted -2.3 and +4.1 ppm
from those of 3 (Table 1), respectively. These data indicated that
4 is the 7-ethyl ether of 3. Therefore, the structure of 4 was
determined as (-)-4-[ꢀ-D-glucopyranosyl-(1f3)-ꢀ-D-glucopyra-
nosyloxy]benzyl ethyl ether.
0.08, MeOH), and showed the presence of hydroxy (3325 cm-1
)
and aromatic ring (1611 and 1511 cm-1) functional groups in its
IR spectrum. The positive ESIMS gave a quasimolecular ion peak
at m/z 471 [M + Na]+, and the HRESIMS at m/z 471.1476 [M +
Na]+ indicated the molecular formula of 1 as C19H28O12 (calcd for
1
C19H28O12Na, 471.1478). The H NMR spectrum of 1 (CD3OD)
showed characteristic signals due to a 4-substitued benzyl alcohol
moiety at δ 7.22 (2H, d, J ) 9.0 Hz, H-2 and H-6), 7.01 (2H, d,
J ) 9.0 Hz, H-3 and H-5), and 4.48 (2H, s, H2-7). In addition, two
diagnostic doublets attributed to anomeric protons at δ 4.87 (1H,
d, J ) 7.5 Hz, H-1′) and 4.39 (1H, d, J ) 8.0 Hz, H-1′′), together
with partially overlapped signals assigned to oxymethylene and
oxymethine protons between δ 3.17 and 3.85, indicated the presence
of two ꢀ-glycopyranosyl units in 1. Acid hydrolysis of 1 produced
glucose as the sole sugar identified on the basis of TLC by
comparing with an authentic sugar sample. The glucose isolated
from the hydrolysate gave a positive optical rotation, [R]D20 +41.3
(c 0.35, H2O) and indicated that it is D-glucose.15,16 By comparing
Compound 5 was obtained as a colorless gum, [R]D20 -9.9 (c
0.06, MeOH), and showed IR absorption bands for hydroxy (3330
cm-1), carbonyl (1731 cm-1), and aromatic ring (1612 and 1513
cm-1) functional groups. The positive and negative ESIMS data
of 5 displayed quasimolecular ion peaks at m/z 1089 [M + Na]+
and 1105 [M + K]+, and 1065 [M - H]-, respectively, and the
HRESIMS at m/z 1089.3595 [M + Na]+ indicated the molecular
formula of 5 as C46H66O28 (calcd for C46H66O28Na, 1089.3638).
The 1H NMR spectrum of 5 in CD3OD showed diagnostic signals
attributed to an isobutyl group at δ 0.70 and 0.84 (each 3H, d, J )
6.5 Hz, H3-7 and H3-8), 1.70 (1H, m, H-6), and 2.06 and 1.65 (each
1H, dd, J ) 14.0 and 5.5 Hz, H-5a and H-5b), in addition to two
pairs of partially overlapped A2B2 coupling systems and two pairs
of AB systems attributed to two 4-O-substituted benzyloxy groups
at δ 7.21 (2H, d, J ) 9.0 Hz, H-2′ and H-6′) and 7.03 (2H, d, J )
9.0 Hz, H-3′ and H-5′), and 7.23 (2H, d, J ) 8.5 Hz, H-2′′ and
H-6′′) and 7.03 (2H, d, J ) 8.5 Hz, H-3′′ and H-5′′), and 5.02 (1H,
d, J ) 12.5 Hz, H-7′a) and 4.86 (1H, d, J ) 12.5 Hz, H-7′b), and
5.07 (1H, d, J ) 12.5 Hz, H-7′′a) and 4.83 (1H, d, J ) 12.5 Hz,
H-7′′b). Moreover, the 1H NMR spectrum showed signals assignable
to three anomeric protons with a ꢀ-configuration and an anomeric
proton with an R-configuration at δ 4.52 (1H, d, J ) 7.0 Hz, H-1′′′),
4.83 (1H, d, J ) 7.5 Hz, H-1′′′′), and 4.89 (1H, d, J ) 8.0 Hz,
H-1′′′′′), and 5.15 (1H, d, J ) 3.0 Hz, H-1′′′′′′), together with an
isolated oxymethine singlet at δ 4.43 (1H, s, H-3), as well as
partially overlapped signals attributed to oxymethylenes and
oxymethines of four glucosyl moieties (Tables 2 and 3). These
spectroscopic data indicated that 5 is a bis(4-glycosyloxybenzyl)
2-isobutyltartrate derivative similar to the co-occurring dactylorhin
B.14 This was confirmed by the 13C NMR and DEPT spectra of 5
that showed carbon signals corresponding to the above moieties
(Table 3), including two ester carbonyls at δ 173.8 (C-1) and 172.1
(C-4) and an oxygenated quaternary carbon at δ 86.1 (C-2). Acid
hydrolysis of 5 afforded D-glucose, [R]D20 +37.2 (c 0.14, H2O), as
the only sugar, indicating that all of the sugar units in 5 are
D-glucopyranosyl substituents. After basic hydrolysis of 5 with 3.0%
NaOH, gastrodin, coeloverin E,6 and 2 were obtained from the
hydrolysate by chromatographic techniques, and the spectroscopic
data of the products were identical to those of the natural products
obtained from the plant material. These experimental results
demonstrated that 5 is a derivative of dactylorhin B13,14 with a
4-[R-D-glucopyranosyl-(1f4)-ꢀ-D-glucopyranosyloxy]benzyl moi-
ety replacing a 4-ꢀ-D-glucopyranosyloxybenzyl unit.
1
the H NMR data of 1 with those of the co-occurring gastrodin,
the chemical shift and coupling pattern of H2-7 of 1 suggested that
it was a gastrodin derivative with an additional ꢀ-D-glucopyranosyl
unit substituted at the sugar moiety. The above deduction was
supported by the 13C NMR and DEPT spectra of 1 (Table 1).
Analysis of 1H-1H gCOSY and gHSQC spectra led to the
unambiguous assignment of the protons and corresponding carbon
signals in the NMR spectra (Table 1). In the gHMBC spectrum of
1, long-range correlations from H2-7 to C-1 and C-6, from H-2
and H-6 to C-4, from H-3 and H-5 to C-1, and from H-1′ to C-4,
in combination with chemical shifts of these protons and carbons,
confirmed the presence of the gastrodin moiety in 1. Also, HMBC
correlations from H-1′′ to C-4′ and from H-4′ to C-1′′ indicated
unequivocally that the additional ꢀ-D-glucopyranosyl unit was
located at C-4′ of the gastrodin moiety. Therefore, the structure of
1 was determined as (-)-4-[ꢀ-D-glucopyranosyl-(1f4)-ꢀ-D-glu-
copyranosyloxy]benzyl alcohol.
20
Compound 2 was obtained as a colorless gum, [R]D +17.4
(c 0.14, MeOH), and showed IR, ESIMS, and NMR spectroscopic
features similar to those of 1. However, in the H NMR spectrum
1
of 2, an anomeric proton signal attributed to an R-glycosyl unit at
δ 5.14 (1H, d, J ) 3.5 Hz, H-1′′) replaced that of the outer ꢀ-D-
glucopyranosyl of 1.17 Acid hydrolysis of 2 yielded D-glucose with
[R]D20 +42.1 (c 0.15, H2O) as the only sugar. These data suggested
that 2 is an analogue of 1 with an outer R-glucopyranosyl unit.
Unambiguous assignments of the NMR data of 2 (Table 1) were
accomplished from the 1D TOCSY and 2D NMR spectra of 2.
Meanwhile, the location of the R-glucopyranosyl in 2 was indicated
unambiguously by HMBC correlations from H-1′′ to C-4′ and from
H-4′ to C-2′, C-3′, C-6′, and C-1′′. Therefore, the structure of 2
was determined as (+)-4-[R-D-glucopyranosyl-(1f4)-ꢀ-D-glucopy-
ranosyloxy]benzyl alcohol. Although 2 was synthesized earlier, its
spectroscopic data were unavailable in the literature.18
The structure of 5 was substantiated using several 2D NMR
experiments. An analysis of the gCOSY and gHSQC spectra of 5
led to the unambiguous assignment of the protons and corresponding
carbon signals in the NMR spectra (Tables 2 and 3). The presence
of a 2-isobutyltartrate, two 4-O-substituted benzyloxy units, and
four glycosyl moieties was proved by two- and three-bond
correlations from their protons to carbons in the gHMBC spectrum
of 5. In addition, HMBC correlations from H-7′a and H-7′b to C-1,
and from H-1′′′′ to C-4′, in combination with couplings and
chemical shifts of H-1′′′′ to H2-6′′′′ and C-1′′′′ to C-6′′′′, confirmed
that the 4-ꢀ-D-glucopyranosyloxybenzyl unit is esterified at C-1 of
the 2-isobutyltartrate unit in 5. Other HMBC correlations from
H-7′′a and H-7′′b to C-4, from H-1′′′′′ to C-4′′, and from H-1′′′′′′
to C-4′′′′′, together with couplings and chemical shifts of H-1′′′′′
to H2-6′′′′′, H-1′′′′′′ to H2-6′′′′′′, C-1′′′′′ to C-6′′′′′, and C-1′′′′′′ to
C-6′′′′′′, revealed that the 4-[R-D-glucopyranosyl-(1f4)-ꢀ-D-glu-
20
Compound 3 was obtained as a colorless gum, [R]D -17.3
(c 0.10, DMSO), and its spectroscopic data (Table 1 and Experi-
mental Section) indicated that it is another isomer of 1 with a
different connectivity between the two ꢀ-D-glucopyranosyl units.
Acid hydrolysis of 3 released D-glucose, with [R]D20 +39.7 (c 0.11,
H2O). In the 13C NMR spectrum of 3, a characteristic resonance at
δC 87.7 indicated a (1f3) connection between the two ꢀ-D-
glucopyranosyl moieties in 3.19 The NMR data assignments (Tables
1 and 2) and the structure of 3 were confirmed by 2D NMR
experiments. Therefore, the structure of 3 was elucidated as (-)-
4-[ꢀ-D-glucopyranosyl-(1f3)-ꢀ-D-glucopyranosyloxy]benzyl alcohol.
Compound 4 was obtained as colorless needles, [R]D20 -24.0
(c 0.25, MeOH), and its positive-mode ESIMS gave quasimolecular
ion peaks at m/z 499 [M + Na]+ and 515 [M + K]+. The IR and
NMR spectra were similar to those of 3. However, the NMR spectra

Glycosidic Constituents of Gymnadenia conopsea
Journal of Natural Products, 2008, Vol. 71, No. 5 801

802 Journal of Natural Products, 2008, Vol. 71, No. 5
Zi et al.
Table 2. 1H NMR Spectroscopic Data (δ) of Compounds 5–10a
position
5
6
7
8
9b
10
3a
3b
5a
5b
4.43 s
4.43 s
4.42 s
4.22 s
4.28 s
3.12 d (16.5)
2.75 d (16.5)
1.72 d (5.5)
1.72 d (5.5)
1.83 m
2.06 dd (5.5, 14.0)
1.65 dd (5.5, 14.0)
1.70 m
2.05 dd (5.5, 14.0)
1.67 dd (5.5, 14.0)
1.69 m
1.94 dd (14.4, 4.0)
1.71 dd (14.4, 6.8)
1.81 m
1.72 dd (16.0, 8.0)
1.52 dd (16.0, 6.4)
1.53 m
1.83 dd (14.0, 6.0)
1.64 dd (14.0, 6.0)
1.59 m
6
7
8
2′
3′
5′
6′
7′a
7′b
2′′
3′′
5′′
6′′
7′′a
7′′b
1′′′
2′′′
3′′′
0.70 d (6.5)
0.84 d (6.5)
7.21 d (9.0)
7.03 d (9.0)
7.03 d (9.0)
7.21 d (9.0)
5.02 d (12.5)
4.86 d (12.5)
7.23 d (8.5)
7.03 d (8.5)
7.03 d (8.5)
7.23 d (8.5)
5.07 d (12.5)
4.83 d (12.5)
4.52 d (7.0)
3.14 dd (9.0, 7.0)
3.11 t (9.0)
0.70 d (6.5)
0.84 d (6.5)
7.20 d (8.5)
7.03 d (8.5)
7.03 d (8.5)
7.20 d (8.5)
5.02.d (12.0)
4.87.d (12.0)
7.23 d (8.5)
7.03 d (8.5)
7.03d (8.5)
7.23 d (8.5)
5.08 d (12.0)
4.83 d (12.0)
4.53 d (7.5)
3.13 dd (9.0, 7.5)
3.15 t (9.0)
0.63 d (6.4)
0.79 d (6.4)
7.22 d (8.0)
6.98 d (8.0)
6.98 d (8.0)
7.22 d (8.0)
4.97 d (11.6)
4.87 d (11.6)
7.22 d (8.0)
6.98 d (8.0)
6.98 d (8.0)
7.22 d (8.0)
4.97 d (11.6)
4.81 d (11.6)
4.67 d (8.0)
3.00 dd (8.4, 8.0)
3.18 t (8.4)
3.06 t (8.4)
2.94 m
0.66 d (6.4)
0.80 d (6.4)
7.17 d (8.8)
6.97 d (8.8)
6.97 d (8.8)
7.17 d (8.8)
4.84 d (12.0)
4.77 d (12.0)
7.24 d (8.8)
7.02 d (8.8)
7.02 d (8.8)
7.24 d (8.8)
4.86 d (12.0)
4.98 d (12.0)
0.73 d (6.8)
0.87 d (6.8)
7.30 d (8.8)
7.05 d (8.8)
7.05 d (8.8)
7.30 d (8.8)
5.07 s
0.91 d (6.5)
0.91 d (6.5)
5.07 s
7.27 d (8.0)
7.05 d (8.0)
7.05 d (8.0)
7.27 d (8.0)
5.05 d (12.0)
4.99 d (12.0)
4.78 d (7.5)
3.46 dd (7.5, 9.0)
3.91 t (9.0)
4′′′
5′′′
3.32 t (9.0)
2.76 brd (9.0)
3.62 m
3.57 m
4.83 d (7.5)
3.41 m
3.30 t (9.0)
3.32 t (9.0)
2.77 dt (9.0, 3.5)
3.66 dd (11.5, 3.5)
3.56 dd (11.5, 3.5)
4.83 d (7.5)
3.40 t (7.5)
3.20 dd (9.0, 6.0)
3.80 d (12.0)
3.62 dd (12.0, 6.0)
6′′′a
6′′′b
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′a
6′′′′b
1′′′′′
2′′′′′
3′′′′′
4′′′′′
5′′′′′
6′′′′′a
6′′′′′b
1′′′′′′
2′′′′′′
3′′′′′′
4′′′′′′
5′′′′′′
6′′′′′′a
6′′′′′′b
a
3.47 dd (11.6, 4.0)
3.45 m
4.95 d (7.2)
3.43 dd (8.4, 7.2)
3.50 t (8.4)
3.28 t (8.4)
3.31 m
3.65 brd (11.6)
3.42 brd (11.6)
4.81 d (7.6)
3.21 dd (8.0, 7,6)
3.26 dd (8.4, 8.0)
3.18 t (8.4)
3.07 m
4.81 d (7.6)
3.22 m
3.10 t (8.4)
3.04 m
3.10 m
3.65 m
3.38 dd (12, 4.8)
4.80 d (6.5)
3.22 m
3.26 m
3.18 t (8.0)
3.55 m
4.85 d (7.6)
3.39 dd (8.0, 7.6)
3.39 t (8.0)
3.36 dd (8.0, 7.6)
3.34 ddd (7.6, 5.6, 2.0)
3.83 dd (12.0, 2.0)
3.64 dd (12.0, 5.6)
3.41 m
3.35 t (9.0)
3.40 t (7.5)
3.34 t (7.5)
3.36 dd (9.0, 3.0)
3.84 brd (12.0)
3.66 dd (12.0, 3.0)
4.89 d (8.0)
3.45 dd (9.0, 8.0)
3.70 t (9.0)
3.38 dd (7.5, 3.5)
3.82 brd (10.0)
3.64 dd (10.0, 3.5)
4.93 d (7.5)
3.63 m
3.64 m
3.46 m
3.46 m
3.84 brd (11.5)
3.65 m
4.88 d (6.5)
3.40 m
3.40 m
3.32 t (9.0)
3.40 m
3.84 d (12.0)
3.64 dd (12.0, 6.0)
3.59 t (9.0)
3.54 dd (9.0, 4.0)
3.82 d (12.0)
3.64 dd (12.0, 4.0)
5.15 d (3.0)
3.41 dd (9.5, 3.0)
3.59 dd (9.5, 8.5)
3.24 t (8.5)
3.66 m
3.78 brd (11.0)
3.67 m
3.65 brd (11.6)
3.42 m
3.94 d (10.4)
3.72 m
4.55 d (8.0)
3.24 dd (8.5, 8.0)
3.36 t (8.5)
4.37 d (7.6)
3.02 dd (8.4, 7.6)
3.14 t (8.4)
3.14 t (8.4)
3.11 m
4.18 d (8.0)
2.95 dd (8.4, 8.0)
3.01 t (8.4)
3.11 t (8.4)
3.28 m
3.24 t (8.5)
3.30 dd (8.5, 3.5)
3.84 brd (11.5)
3.59 dd (11.5, 3.5)
3.69 m
3.65 m
3.57 m)
3.44 dd (12.0, 5.6)
1H NMR data (δ) were measured in MeOH-d4 for 5, 6, 9, and 10 and DMSO-d6 + D2O for 7 and 8 at 400 or 500 MHz. Proton coupling
constants (J) in Hz are given in parentheses. The assignments were based on DEPT, 1H-1H COSY, HSQC, and HMBC experiments. b Data for
OMe: δ 3.51 (3H, s).
copyranosyloxy]benzyl residue is esterified at C-4 of the 2-isobu-
tyltartrate unit. Furthermore, a HMBC correlation from H-1′′′ to
C-2 indicated unequivocally that the remaining ꢀ-D-glucopyranosyl
is located at C-2 of the 2-isobutyltartrate unit. Therefore, the
structure of 5 was determined as (-)-(2R,3S)-1-(4-ꢀ-D-glucopyra-
nosyloxybenzyl)-2-O-ꢀ-D-glucopyranosyl-4-{4-[R-D-glucopyrano-
syl-(1f4)-ꢀ-D-glucopyranosyloxy]benzyl}-2-isobutyltartrate.
of the 4-[ꢀ-D-glucopyranosyl-(1f3)-ꢀ-D-glucopyranosyloxy]benzyl
moiety was also determined by an extensive analysis of 2D NMR
spectra of 6, which resulted in the unambiguous assignment of NMR
data of 6 (Tables 2 and 3). Especially, in the HMBC spectrum of
6, correlations from both H-7′′a and H-7′′b to C-4, from H-1′′′′′ to
C-4′′, and from H-1′′′′′′ to C-3′′′′′ indicated that the 4-[ꢀ-D-
glucopyranosyl-(1f3)-ꢀ-D-glucopyranosyloxy]benzyl unit was lo-
cated at C-4 of the 2-isobutyltartrate moiety in 6. Therefore, the
structure of 6 was determined as (-)-(2R,3S)-1-(4-ꢀ-D-glucopyra-
nosyloxybenzyl)-2-O-ꢀ-D-glucopyranosyl-4-{4-[ꢀ-D-glucopyrano-
syl-(1f3)-ꢀ-D-glucopyranosyloxy]benzyl}-2-isobutyltartrate.
20
Compound 6 was obtained as a colorless gum, [R]D -33.7
(c 0.05, MeOH), and its ESIMS, IR, and NMR spectra were similar
to those of 5. HRESIMS indicated that it is an isomer of 5. Acid
20
hydrolysis of 6 yielded D-glucose, [R]D +41.6 (c 0.15, H2O).
20
However, the 1H NMR spectra of 6 indicated the presence of four
anomeric protons with a ꢀ-configuration at δ 4.93 (1H, d, J ) 7.5
Hz, H-1′′′′′), 4.83 (1H, d, J ) 7.5 Hz, H-1′′′′), 4.55 (1H, d, J ) 8.0
Hz, H-1′′′′′′), and 4.53 (1H, d, J ) 7.5 Hz, H-1′′′). A comparison
of the NMR data between 5 and 6, and between 3 and 6 (Tables 2
and 3), together with the observation of a diagnostic carbon signal
at δ 87.6 (C-3′′′′′) in the 13C NMR spectrum of 6, suggested the
presence of a 4-[ꢀ-D-glucopyranosyl-(1f3)-ꢀ-D-glucopyranosy-
loxy]benzyl moiety in 6. This was confirmed by basic hydrolysis
of 6, which afforded gastrodin, coeloverin E, and 3. The location
Compound 7 was obtained as a colorless gum, [R]D -36.1
(c 0.03, MeOH), and its spectroscopic data (Tables 2 and 3 and
Experimental Section) were very similar to those of 6. Acid and
basic hydrolysis of 7 yielded products identical to those of 6. This
indicated that 7 is an isomer of 6 in which the location of the 4-[ꢀ-
D-glucopyranosyl-(1f3)-ꢀ-D-glucopyranosyloxy]benzyl and 4-ꢀ-
D-glucopyranosyloxybenzyl units are transposed. The 2D NMR
spectra of 7 confirmed the deduction and led to the assignment of
NMR data of 7 (Tables 2 and 3). Therefore, the structure of 7 was
determined as (-)-(2R,3S)-1-{4-[ꢀ-D-glucopyranosyl-(1f3)-ꢀ-D-

Glycosidic Constituents of Gymnadenia conopsea
Journal of Natural Products, 2008, Vol. 71, No. 5 803
Table 3. 13C NMR Spectroscopic Data (δ) of Compounds
5-10a
hydrolysis of 8 gave gastrodin, 8a, and (+)-(2R,3S)-2-isobutyltartric
acid.14 The structure of 8a was identified as (-)-4-ꢀ-D-glucopyra-
nosyl-(1f6)-ꢀ-D-glucopyranosyloxybenzyl alcohol from its 1H and
13C NMR and ESIMS data (Table 1 and Experimental Section). In
the HMBC spectrum of 8, correlations of C-1 with both H-7′a and
H-7′b, C-4 with both H-7′′a and H-7′′b, C-4′ with H-1′′′′, C-4′′
with H-1′′′′′, and C-6′′′′′ with H-1′′′′′′, in combination with chemical
shifts of these protons and carbons, confirmed the connection among
the structural moieties in 8. Therefore, the structure of 8 was
determined as (-)-(2R,3S)-1-(4-ꢀ-D-glucopyranosyloxybenzyl)-4-
{4-[ꢀ-D-glucopyranosyl-(1f6)-ꢀ-D-glucopyranosyloxy]benzyl}-2-
isobutyltartrate.
Compound 9 was obtained as colorless needles, with [R]D20 –13.4
(c 0.08, MeOH), and its positive-mode ESIMS gave quasimolecular
ion peaks at m/z 511 [M + Na]+ and 527 [M + K]+. The IR and
NMR spectroscopic features of 9 were very similar to those of the
co-occurring coelovirin A.6 However, the NMR spectra of 9 showed
additional signals due to a methoxy group at δH 3.51 and δC 52.5.
A comparison of the 13C NMR data between coelovirin A and 9
indicated that C-4 of 9 was shifted upfield by 1.4 ppm. These
spectroscopic data revealed 9 to be coelovirin A methyl ester.
Therefore, the structure of 9 was determined as (-)-(2R,3S)-1-(4-
ꢀ-D-glucopyranosyloxybenzyl)-4-methyl-2-isobutyltartrate.
position
5
6
7
8
9b
10
1
2
3
4
5
6
7
8
1′
2′
3′
4′
5′
6′
7′
1′′
173.8
86.1
75.8
172.1
46.8
24.9
173.8
86.1
75.8
172.1
46.8
24.9
170.8
84.1
74.5
170.3
43.6
22.8
23.1
24.4
127.7
129.7
116.1
157.1
116.1
129.7
66.1
128.7
130.0
116.2
157.4
116.2
130.0
66.1
97.3
73.9
77.0
70.1
76.7
60.1
99.6
71.9
87.7
67.6
76.5
60.4
100.3
73.2
77.0
69.7
76.0
60.7
104.0
73.8
76.6
68.0
76.4
61.1
173.4
79.7
76.3
171.3
44.3
23.8
174.8
81.0
77.5
173.1
45.1
25.2
172.3
80.9
49.8
171.6
45.3
25.2
24.9
24.0
24.0
24.7
24.0
24.7
23.7
24.6
24.0
24.6
130.3
131.5
117.9
159.4
117.9
131.5
68.4
130.3
132.1
118.0
159.5
118.0
132.1
68.3
99.5
75.5
78.1
70.1
77.2
61.6
102.3
75.1
77.9
71.3
78.1
62.5
102.1
74.5
77.6
80.9
76.7
62.0
102.9
74.2
74.9
71.5
74.9
130.3
131.5
117.9
159.35
117.9
131.5
68.5
130.4
132.0
118.0
159.42
118.0
132.0
68.3
99.5
75.46
78.1
70.1
77.2
61.6
102.3
74.9
77.8
71.3
77.9
62.4
101.8
74.2
87.6
69.8
77.7
62.5
105.2
75.52
78.1
71.6
78.1
129.3
130.2
116.57
157.6
116.57
130.2
66.43
129.3
130.5
116.64
157.6
116.64
130.5
66.40
130.7
131.6
117.7
159.3
117.7
131.6
68.1
130.9
131.3
117.9
159.3
117.9
131.3
67.6
94.9
74.98
81.2
2′′
3′′
4′′
5′′
6′′
7′′
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′
1′′′′′
2′′′′′
3′′′′′
4′′′′′
5′′′′′
6′′′′′
1′′′′”
2′′′′′′
3′′′′′′
4′′′′′′
5′′′′′′
6′′′′′′
a
Compound 10 was obtained as a colorless gum, [R]D20 -13.2
(c 0.43, MeOH), and its positive-mode ESIMS gave a quasimo-
lecular ion peak at m/z 643 [M + Na]+. The UV, IR, and NMR
spectroscopic data were similar to those of 9 (Tables 2 and 3 and
Experimental Section). However, a comparison of the NMR
spectroscopic data of 9 and 10 indicated that the signals assigned
to the oxymethine (H-3 and C-3) of the 2-isobutyltartric acid moiety
in 9 were replaced by those assignable to an isolated aliphatic
methylene of a 2-isobutylmalate moiety at δ 3.12 (1H, d, J ) 16.5
Hz, H-3a) and 2.75 (1H, d, J ) 16.5 Hz, H-3b) and δ 49.8 (C-3).
This indicated that 10 is a 2-isobutylmalate derivative similar to
the co-occurring dactylorhin E.14 Comparison of NMR data between
10 and dactylorhin E showed that the data of C-1 and C-4 of 10
were shifted 3.3 and 2.3 ppm upfield, respectively, from those of
dactylorhin E. This indicated that the 4-ꢀ-D-glucopyranosyloxy-
benzyl was esterified with the terminal carboxylic group (C-4) of
the 2-isobutylmalic acid unit in 10. This was confirmed by 2D NMR
experiments of 10. The basic hydrolysis of 10 yielded gastrodin
71.4
79.6
62.3
100.6
73.5
76.6
70.4
77.0
61.3
100.6
73.5
76.1
69.9
76.7
68.7
103.6
73.8
76.5
70.0
77.2
61.0
102.2
74.9
78.0
71.4
78.2
62.5
102.3
74.92
78.1
71.4
78.0
62.5
and dactylorhin C, [R]D -20.7 (c 1.01, H2O).14 Consequently,
20
62.8
62.6
the structure of 10 was determined as (-)-(2R)-2-O-ꢀ-D-glucopy-
ranosyl-4-(4-ꢀ-D-glucopyranosyloxybenzyl)-2-isobutylmalate.
13C NMR data (δ) were measured in MeOH-d4 for 5, 6, 9, and 10
and DMSO-d6 + D2O for 7 and 8 at 100, 125, or 150 MHz. The
assignments were based on DEPT, 1H-1H COSY, HSQC, and HMBC
experiments. b Data for OMe: δ 52.5.
Compound 2 may be generated from 5, and compound 3 from
6 and 7 during the isolation procedure. However, 2 and 3 may also
be biosynthetic precursors of 5-7. With this hypothesis, the fresh
tubers of this plant were collected in September 2007, from the
initial collection location, and an ethanolic extract of the fresh
material was analyzed by HPLC/UV/ESIMS. However, none of
the new compounds (1-10) were detectable. In addition, in
simulated isolation conditions by heating the aqueous ethanol (50%)
solutions of compounds 5-10 and the purified known compounds
with the ester bond, with or without silica gel at 50 °C for 36 h,
they were not hydrolyzed on the basis of TLC and HPLC analysis.
In addition, the ethylation of 3 did not occur as a result of heating
the ethanol solution of 3 with and without silica gel at 50 °C for
50 h.
glucopyranosyloxy]benzyl}-2-O-ꢀ-D-glucopyranosyl-4-(4-ꢀ-D-glu-
copyranosyloxybenzyl)-2-isobutyltartrate.
Compound 8, obtained as a colorless gum, [R]D20 -29.9 (c 0.07,
MeOH), showed IR absorption bands similar to those of 6. The
positive- and negative-mode ESIMS data showed quasimolecular
ion peaks at m/z 927 [M + Na]+ and 943 [M + K]+, and 903 [M -
H]-, respectively. HRESIMS at m/z 927.3054 [M + Na]+ indicated
the molecular formula of 8 to be C40H56O23, one glucopyranosyl
unit less than in 6. A comparison of the NMR spectroscopic data
between 6 and 8 (Tables 2 and 3) indicated that the data assigned
to the ꢀ-D-glucopyranosyl at C-2 in 6 were absent in the NMR
spectra of 8, and the C-2 signal of 8 was shifted 6.4 ppm upfield
from that of 6. In addition, the data assigned to C-3′′′′′ and C-6′′′′′
of the di-ꢀ-D-glucopyranosyl unit were shifted from δ 87.6 and
62.5 of 6 to δ 76.1 and 68.7 of 8, respectively. This indicated that
the ꢀ-D-glucopyranosyloxy at C-2 and the ꢀ-D-glucopyranosyl-
(1f3)-ꢀ-D-glucopyranosyloxy at C-4′′ in 6 were replaced by a
hydroxy and a ꢀ-D-glucopyranosyl-(1f6)-ꢀ-D-glucopyranosyloxy
in 8, respectively. Acid hydrolysis of 8, with 2 N HCl at 80 °C for
Pharmacological studies have shown that the active fraction
containing mono- and bis(4-ꢀ-D-glucopyranosyloxybenzyl) 2-isobu-
tyltartrate and 2-isobutylmalate derivatives6 showed neuroprotective
effects on memory deficits and pathological changes in senescent
mice,7,8 while the main component, dactylorhin B, reduced toxic
effects of ꢀ-amyloid fragment (25–35) on neuron cells and isolated
rat brain mitochondria.9 In in vitro assays against acetylcholine
esterase (AChE) and monoamine oxidase (MAO), compounds 1-10
were inactive at 10-5 M.
20
6 h, produced D-glucose, [R]D +40.0 (c 0.13, H2O), while basic

804 Journal of Natural Products, 2008, Vol. 71, No. 5
Zi et al.
Experimental Section
(-)-4-[ꢀ-D-Glucopyranosyl-(1f3)-ꢀ-D-glucopyranosyloxy]ben-
20
zyl alcohol (3): colorless gum, [R]D -17.3 (c 0.10, DMSO); UV
General Experimental Procedures. Optical rotations were mea-
sured on a PE model 343 polarimeter. UV spectra were measured
on a Cary 300 spectrometer. IR spectra were recorded on a Nicolet
5700 FT-IR microscope spectrometer (FT-IR microscope transmis-
sion). 1D- and 2D-NMR spectra were obtained at 400, 500, or 600
MHz for 1H and 100, 125, or 150 MHz for 13C, respectively, on INOVA
400, 500, and 600 MHz spectrometers in MeOH-d4 or DMSO-d6, with
solvent peaks as references. ESIMS data were measured with a Q-Trap
LC/MS/MS (Turbo Ionspray source) spectrometer. HRESIMS data were
measured using a JMS-T100CS AccuToF CS spectrometer. Column
chromatography was performed with silica gel (200–300 mesh, Qingdao
Marine Chemical Inc. Qingdao, People’s Republic of China) and
Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). HPLC
separation was performed on an instrument consisting of a Waters 600
controller, a Waters 600 pump, and a Waters 2487 dual λ absorbance
detector, with an Alltima (250 × 10 mm i.d.) preparative column packed
with C18 (5 µm). TLC was carried out with glass precoated silica gel
GF254 plates. Spots were visualized under UV light or by spraying
with 7% H2SO4 in 95% EtOH followed by heating.
(MeOH) λmax (log ꢀ) 201 (3.91), 220 (4.02), 270 (2.93) nm; IR νmax
3457, 3333, 2863, 1611, 1509, 1468, 1411, 1367, 1234, 1171, 1118,
1082, 1028, 992, 902, 822 cm-1;1H NMR (DMSO-d6, 500 MHz) and
13C NMR (DMSO-d6, 125 MHz) data, see Table 1; positive-mode
ESIMS m/z 471 [M + Na]+; negative-mode ESIMS m/z 447 [M -
H]-; HRESIMS m/z 471.1476 [M + Na]+ (calcd for C19H28O12Na,
471.1478).
(-)-4-[ꢀ-D-Glucopyranosyl-(1f3)-ꢀ-D-glucopyranosyloxy]ben-
20
zyl ethyl ether (4): colorless needles, [R]D -24.0 (c 0.25, MeOH);
UV (MeOH) λmax (log ꢀ) 201 (3.84), 222 (3.87), 273 (2.99) nm; IR
νmax 3364, 2879, 1612, 1513, 1412. 1375, 1237, 1164, 1083, 894, 851,
827 cm-1; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150
MHz) data, see Table 1; positive-mode ESIMS m/z 499 [M + Na]+
and 515 [M + K]+; negative-mode ESIMS m/z 475 [M - H]-.
(-)-(2R,3S)-1-(4-ꢀ-D-Glucopyranosyloxybenzyl)-2-O-ꢀ-D-glucopyra-
nosyl-4-{4-[r-D-glucopyranosyl-(1f4)-ꢀ-D-glucopyranosyloxy]benzyl}-
2-isobutyltartrate (5): colorless gum, [R]D20 -9.9 (c 0.06, MeOH); UV
(MeOH) λmax (log ꢀ) 202 (4.33), 224 (4.36), 270 (3.19) nm; IR νmax
3330, 2925, 1731, 1612, 1513, 1413, 1308, 1226, 1164, 1067, 1032,
928, 897, 830 cm-1;1H NMR (CD3OD, 500 MHz) and 13C NMR
(CD3OD, 125 MHz) data, see Tables 2 and 3; positive-mode ESIMS
m/z 1089 [M + Na]+ and 1105 [M + K]+; negative-mode ESIMS m/z
1065 [M - H]-; HRESIMS m/z 1089.3595 [M + Na]+ (calcd for
C46H66O28Na, 1089.3638).
Plant Material. The tubers of G. conoposa were collected at Ganzi,
Sichuan Province, People’s Republic of China, in September 2005. The
plant identification was verified by Associate Professor Lin Ma (Institute
of Materia Medica). A voucher specimen (No. 05916) is deposited at
the Herbarium of the Department of Medicinal Plants, Institute of
Materia Medica.
(-)-(2R,3S)-1-(4-ꢀ-D-Glucopyranosyloxybenzyl)-2-O-ꢀ-D-glucopyra-
Extraction and Isolation. The air-dried tubers of G. conoposa (16.2
kg) were powdered and extracted with 20.0 L of EtOH at room
temperature for 4 × 42 h. The EtOH extract was evaporated under
reduced pressure at <40 °C to yield a residue (618.2 g). The residue
was suspended in H2O (2000 mL) and then partitioned with EtOAc (5
× 1200 mL). The aqueous phase was applied to a HP-20 macroporous
adsorbent resin (1500 g) column. Successive elution of the column with
H2O, 15% EtOH, 40% EtOH, and 95% EtOH (5000 mL each) yielded
four fractions after removing the solvents. The active fraction (100.2
g) eluted by 40% EtOH was suspended in H2O (500 mL) and then
partitioned with n-BuOH (5 × 400 mL) to give H2O and n-BuOH
fractions. The H2O (40 g) fraction was separated by medium-pressure
liquid chromatography over reversed-phase silica gel eluting with a
gradient of increasing MeOH (0–90%) in H2O to give five fractions
(A-E) on the basis of TLC analysis. Fraction B (0.12 g) was subjected
to column chromatography over Sephadex LH-20, using MeOH-H2O
(50:50) as the eluting solvent, to afford four subfractions, B1-B4.
Subfractions B2 (66 mg) and B3 (38 mg) were separately purified by
reversed-phase preparative HPLC, using the mobile phase MeOH-H2O
(12:88), to afford 1 (15 mg, 0.000093%), 2 (17 mg, 0.00011%), 3 (27
mg, 0.00017%), and 4 (13 mg, 0.000080%). Separation of fraction E
(9.72 g) by normal-phase silica gel column chromatography, eluting
with a gradient of increasing MeOH (0–100%) in CHCl3, afforded seven
subfractions (E1-E7). Subfraction E1 (751 mg) was further separated
by silica gel column chromatography, using CHCl3-MeOH-H2O as
the eluting solvent, and then purified by reversed-phase preparative
HPLC, using MeOH-H2O (29:71) as the mobile phase, to yield 5 (43
mg, 0.00026%), 6 (31 mg, 0.00019%), 7 (25 mg, 0.00015%), and 8
(21 mg, 0.00013%). Subfractions E3 (105 mg) and E7 (52 mg) were
separately purified by preparative reversed-phase HPLC, using
MeOH-H2O (32:68 and 42:58) as the mobile phases, respectively, to
afford 9 (91 mg, 0.00056%) and 10 (25 mg, 0.00015%).
nosyl-4-{4-[ꢀ-D-glucopyranosyl-(1f3)-ꢀ-D-glucopyranosyloxy]benzyl}-
20
2-isobutyltartrate (6): colorless gum, [R]D -33.7 (c 0.05, MeOH);
UV (MeOH) λmax (log ꢀ) 202 (4.34), 224 (4.39), 270 (3.23) nm; IR
νmax 3321, 2917, 1731, 1612, 1512, 1413, 1308, 1223, 1163, 1066,
1016, 927, 896, 828 cm-1; 1H NMR (CD3OD, 500 MHz) and 13C NMR
(CD3OD, 125 MHz) data, see Tables 2 and 3; positive-mode ESIMS
m/z 1089 [M + Na]+ and 1105 [M + K]+; negative-mode ESIMS m/z
1065 [M - H]-; HRESIMS m/z 1089.3610 [M + Na]+ (calcd for
C46H66O28Na, 1089.3638).
(-)-(2R,3S)-1-{4-[ꢀ-D-Glucopyranosyl-(1f3)-ꢀ-D-glucopyranosy-
loxy]benzyl}-2-O-ꢀ-D-glucopyranosyl-4-(4-ꢀ-D-glucopyranosyloxyben-
zyl)-2-isobutyltartrate (7): colorless gum, [R]D20 -36.1 (c 0.03, MeOH);
UV (MeOH) λmax (log ꢀ) 202 (4.29), 224 (4.31), 270 (3.15) nm; IR
νmax 3349, 2920, 1730, 1612, 1512, 1414, 1307, 1224, 1163, 1066,
1015, 927, 897, 830 cm-1; 1H NMR (DMSO-d6 +D2O, 400 MHz) and
13C NMR (DMSO-d6 +D2O, 150 MHz) data, see Tables 2 and 3;
positive-mode ESIMS m/z 1089 [M + Na]+ and 1105 [M + K]+;
negative-mode ESIMS m/z 1065 [M - H]- and 1101 [M + Cl]-;
HRESIMS m/z 1089.3682 [M + Na]+ (calcd for C46H66O28Na,
1089.3638).
(-)-(2R,3S)-1-(4-ꢀ-D-Glucopyranosyloxybenzyl)-4-{4-[ꢀ-D-glu-
copyranosyl-(1f6)-ꢀ-D-glucopyranosyloxy]benzyl}-2-isobutyltar-
20
trate (8): colorless gum; [R]D -29.9 (c 0.07, MeOH); UV (MeOH)
λmax (log ꢀ) 203 (4.27), 222 (4.32), 270 (3.12) nm; IR νmax 3297, 2920,
1732, 1612, 1513, 1415, 1308, 1227, 1164, 1071, 1042, 928, 897, 831
cm-1; 1H NMR (DMSO-d6 + D2O, 400 MHz) and 13C NMR (DMSO-
d6 + D2O, 125 MHz) data, see Tables 2 and 3; positive-mode ESIMS
m/z 927 [M + Na]+ and 943 [M + K]+; negative-mode ESIMS m/z
903 [M - H]-; HRESIMS m/z 927.3054 [M + Na]+ (calcd for
C46H66O28Na, 927.3110).
(-)-(2R,3S)-1-(4-ꢀ-D-Glucopyranosyloxybenzyl)-4-methyl-2-isobu-
20
tyltartrate (9): colorless gum; [R]D -13.4 (c 0.08, MeOH); UV
(-)-4-[ꢀ-D-Glucopyranosyl-(1f4)-ꢀ-D-glucopyranosyloxy]ben-
(MeOH) λmax (log ꢀ) 201 (4.30), 223 (4.35), 270 (3.17) nm; IR νmax
20
zyl alcohol (1): colorless gum, [R]D -36.6 (c 0.08, MeOH); UV
3345, 2951, 1731, 1611, 1513, 1440, 1399, 1300, 1218, 1188, 1074,
1
1046, 1018, 979, 900, 839 cm-1; H NMR (CD3OD, 400 MHz) and
(MeOH) λmax (log ꢀ) 201 (3.87), 222 (3.99), 270 (2.97) nm; IR νmax
1
3325, 2883, 1611, 1511, 1411, 1230, 1164, 1023, 898, 830 cm-1; H
13C NMR (CD3OD, 100 MHz) data, see Tables 2 and 3; positive-mode
ESIMS m/z 511 [M + Na]+ and 527 [M + K]+.
NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data,
see Table 1; positive-mode ESIMS m/z 471 [M + Na]+; negative-mode
ESIMS m/z 447 [M - H]-; HRESIMS m/z 471.1476 [M + Na]+ (calcd
for C19H28O12Na, 471.1478).
(-)-(2R)-2-O-ꢀ-D-Glucopyranosyl-4-(4-ꢀ-D-glucopyranosyloxy-
20
benzyl)-2-isobutylmalate (10): colorless gum, [R]D -13.2 (c 0.43,
MeOH); UV (MeOH) λmax (log ꢀ) 201 (4.34), 222 (4.34) 270 (3.20)
nm; IR νmax 3389, 2931, 2888, 1760, 1727, 1713, 1612, 1512, 1398,
(+)-4-[r-D-Glucopyranosyl-(1f4)-ꢀ-D-glucopyranosyloxy]ben-
20
1
1382, 1291, 1233, 1190, 1072, 1051, 1014, 975, 827 cm-1; H NMR
zyl alcohol (2): colorless gum, [R]D +17.4 (c 0.14, MeOH); UV
(CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data, see Tables
2 and 3; positive-mode ESIMS m/z 643 [M + Na]+; negative-mode
ESIMS m/z 619 [M - H]-.
(MeOH) λmax (log ꢀ) 201 (3.82), 221 (3.96), 270 (2.89) nm; IR νmax
3334, 2924, 2882, 1608, 1589, 1511, 1407, 1230, 1145, 1035, 831,
782 cm-1; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125
MHz) data, see Table 1; positive-mode ESIMS m/z 471 [M + Na]+
and 487 [M + K]+; negative-mode ESIMS m/z 447 [M - H]-.
Acidic Hydrolysis of 1–10. A solution of each compound (5-10
mg) in 2 N HCl (5.0 mL) was individually refluxed at 80 °C for

Glycosidic Constituents of Gymnadenia conopsea
Journal of Natural Products, 2008, Vol. 71, No. 5 805
6 h. The reaction mixture was extracted with EtOAc (3 × 5 mL),
and the aqueous phase was neutralized with 1 N NaOH and dried
using a stream of N2. The residue was subjected to column
chromatography over silica gel with MeCN-H2O (8:1) to yield
glucose (1.1-2.6 mg) from compounds 1-10. The solvent systems
CHCl3-MeOH (2.5:1) and MeCN-H2O (6:1) were used for TLC
identification of glucose. Glucose from compounds 1-10 gave
No. 20402025), the National “973” Program of China (Grant No.
2004CB13518906), and the Program for Changjiang Scholars and
Innovative Research Team in University (PCSIRT, Grant no.
IRT0514).
Supporting Information Available: NMR spectra of compounds
1–10 and 8a. This material is available free of charge via the Internet
at http://pubs.acs.org.
20
positive optical rotation, with [R]D values in a range +37.2 to
+44.8 (c in a range of 0.11 to 0.35, H2O).
References and Notes
Basic Hydrolysis of 5–10. A solution of each compound in 3%
NaOH (5 mL) was stirred at room temperature for 2 h. The reaction
mixture was acidified to pH 2 by 2 N HCl and then evaporated
under reduced pressure to give a dry residue. The residue was
dissolved in 5 mL of H2O and then subjected to column chroma-
tography over ODS (20 g). The column was successively eluted
with H2O, 5% MeCN, 15% MeCN, and 50% MeCN (100 mL, each).
The fraction eluted by 5% MeCN was evaporated under reduced
pressure and then subjected to reversed-phase preparative HPLC
using 11% MeOH as mobile phase. From the hydrolysates of
compounds 5-10 (6.1–10.2 mg), gastrodin (1.3–2.4 mg) was
obtained with a yield range of 58%-73%. Coeloverin E (2.4, 1.9,
and 1.3 mg) was obtained from the hydrolysates of 5 (10.1 mg), 6
(8.1 mg), and 7 (6.2 mg), respectively. 2-Isobutyltartric acid (1.1
and 1.6 mg) was obtained from the hydrolysates of 8 (6.1 mg) and
9 (6.0 mg), respectively. Dactylorhin C (2.3 mg) was obtained from
10 (6.1 mg). In addition, compound 2 (2.8 mg) was produced from
5, while compound 3 (2.2 and 1.4 mg) was produced from 6 and 7.
The 1H NMR, ESIMS, and optical rotation data of gastrodin,
coeloverin E, dactylorhin C, 2-isobutyltartric acid, and compounds
1 and 2, obtained from the hydrolysates, were identical to those of
the natural products isolated from the plant material and reported
in the literature.6,11,13,14 Compound 8a (1.9 mg) was obtained as a
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20
colorless gum from the hydrolysate of 8: [R]D -28.3 (c 0.05,
MeOH); UV (MeOH) λmax (log ꢀ) 201 (3.79), 221 (3.92), 270 (2.86)
nm; IR νmax 3272, 2923, 2902, 2865, 1611, 1511, 1378, 1244, 1170,
1083, 1049, 1035, 887, 833, 650 cm-1; 1H NMR (CD3OD, 400 MHz)
and 13C NMR (CD3OD, 150 MHz) data, see Table 1; positive-mode
ESIMS m/z 471 [M + Na]+.
Enzymatic Inhibitory Assay. The fresh rat brains were quickly
prepared into brain homogenate in 9-fold volume ice-cold PBS and
incubated with tested compounds, separately.
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rate of 77.2%.
The activity of monoamine oxidase-B was determined by
quantifying the oxidative product of benzyl amines,21 with pargyline
as the positive control. At 10-5 M, the tested compounds gave
inhibition rates less than 15.2%, and pargyline had an inhibition
rate of 94.5%.
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Acknowledgment. Financial support was provided by the New
Century Excellent Talent (NCET) Program of Chinese Ministry of
Education, the Natural Sciences Foundation of China (NSFC, Grant
(21) Nag, M.; Nandy, N. Indian J. Exp. Biol. 2001, 39, 802–806.
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