Furostanol saponins from the rhizomes of Dioscorea japonica and their effects on NGF induction

Article abstract of DOI:10.1016/j.bmcl.2011.02.003

The rhizome of Dioscorea japonica is a food and medicinal source known as 'San Yak' in Korea. Two new furostanol saponins, coreajaponins A (1) and B (2), together with 10 known compounds (3-12) were isolated from the rhizomes of D. japonica. Their structures were determined by spectroscopic methods, including 1D and 2D NMR techniques, HRMS, and chemical methods. Nerve growth factor (NGF), a crucial factor for neuronal survival and differentiation, can potentially improve neurodegenerative diseases and diabetic polyneuropathy. We evaluated the effects of isolates (1-12) on NGF induction in a C6 rat glioma cell line. Coreajaponin B (2) upregulated NGF content without significant cell toxicity, as did 6, 8, 9, and 11.

Full text of DOI:10.1016/j.bmcl.2011.02.003

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2075–2078
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Furostanol saponins from the rhizomes of Dioscorea japonica and their
effects on NGF induction
a
a
b
b
c
c
a,
⇑
Ki Hyun Kim , Min Ah Kim , Eunjung Moon , Sun Yeou Kim , Sang Zin Choi , Mi Won Son , Kang Ro Lee
a
Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, 300 Chonchon-dong, Jangan-ku, Suwon, Gyeonggi-do 440-746, Republic of Korea
Graduate School of East-West Medical Science, Kyung Hee University Global Campus, #1 Seocheon-dong, Giheung-gu, Yongin, Gyeonggi-do 446-701, Republic of Korea
Dong-A Pharm Institute, Kiheung, Yongin, Gyeonggi-do 449-905, Republic of Korea
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
The rhizome of Dioscorea japonica is a food and medicinal source known as ‘San Yak’ in Korea. Two new
furostanol saponins, coreajaponins A (1) and B (2), together with 10 known compounds (3–12) were iso-
lated from the rhizomes of D. japonica. Their structures were determined by spectroscopic methods,
including 1D and 2D NMR techniques, HRMS, and chemical methods. Nerve growth factor (NGF), a crucial
factor for neuronal survival and differentiation, can potentially improve neurodegenerative diseases and
diabetic polyneuropathy. We evaluated the effects of isolates (1–12) on NGF induction in a C6 rat glioma
cell line. Coreajaponin B (2) upregulated NGF content without significant cell toxicity, as did 6, 8, 9,
and 11.
Received 28 October 2010
Revised 29 January 2011
Accepted 2 February 2011
Available online 23 February 2011
Keywords:
Dioscorea japonica
Dioscoreaceae
Furostanol saponins
NGF regulation
Ó 2011 Elsevier Ltd. All rights reserved.
Nerve growth factor (NGF) was first discovered by Levi-Montal-
cini in 1966. NGF has neurotrophic actions that protect choliner-
Dioscorea species,12 but have been rarely described and sparsely
investigated.
1
gic neurons of the basal forebrain against axotomy-induced
neurodegeneration and aged-related atrophy.2 Exogenous NGF
improves impaired function of the cholinergic neuron system, such
In the present work, to identify the active constituents respon-
sible for induction of NGF secretion, we performed a phytochemi-
cal investigation of the rhizomes of D. japonica. Two new furostanol
saponins (1–2), together with 10 known compounds (3–12), were
isolated from its methanolic extract. Their structures were deter-
mined by spectroscopic methods, including 1D and 2D NMR tech-
niques, HRMS, and chemical methods. Herein, we report the
isolation and structural elucidation of the new compounds and
NGF regulation of isolates (1–12).
,3
4
as neuronal degeneration, and has therapeutic potential for neu-
rodegenerative diseases, including Parkinson’s disease, Alzheimer’s
5
6
disease and diabetic polyneuropathy. However, NGF cannot pass
through the blood–brain barrier (BBB) and therefore requires neu-
rosurgical approaches for administration.7 The development of
small molecules that induce NGF secretion or mimic NGF activity
is therefore desirable.
Dried and pulverized rhizomes of D. japonica were extracted
with 50% aqueous EtOH. The extract suspended in water was
In our continuing search for bioactive constituents from Korean
plant sources, we found that the rhizomes of Dioscorea japonica
Thunb. (Dioscoreaceae) induce increases in endogenous NGF levels,
3
successively extracted with n-hexane, CHCl , EtOAc, n-BuOH, and
acetone. Purification of the CHCl -soluble and n-BuOH-soluble frac-
3
8
and thereby have a protective effect against diabetic neuropathy.
tions by multiple chromatographic steps (Supplementary data) led
to the isolation of two new furostanol saponins, coreajaponins A (1)
and B (2), and ten known compounds (3–12) (Fig. 1). The known
The rhizome of D. japonica, naturally distributed in East Asia, China,
Japan, and Korea, is a food and medicinal source known as ‘San Yak’
in Korea. The plant has been used to strengthen stomach function,
improve anorexia, eliminate diarrhea, dilute sputum, and moistur-
ize skin in traditional Chinese medicine.9 Previous phytochemical
investigations of D. japonica revealed active hypoglycemic com-
pounds (dioscorans A–F),10 sesquiterpene, and acetophenone.
The steroidal constituents of this herb, including spirostane, furos-
tane, and cholestane types, are main secondary metabolites of
1
3
compounds were identified as batatasin IV (3), raspberry ketone
1
4
0
15
(4), 2-methoxy-4 -hydroxyacetophenone (5), (3R,5S)-3,5-dihy-
16
droxy-1,7-bis(4-hydroxy-3-methoxyphenyl)heptane (6), b-sitos-
terol (7), blumenol A (8), dihydropinosylvin (9), stilbostemin
N (10), butyl-b-D-fructofuranoside (11), and allantoin (12),
1
7
18
19
11
20
21
22
by comparison of their spectroscopic and physical data with re-
ported values. To the best of our knowledge, four isolates (5–6, 8,
and 11) are here reported for the first time from Dioscorea species.
Coreajaponin A (1) was obtained as a white amorphous powder.
⇑
Corresponding author. Tel.: +82 31 290 7710; fax: +82 31 290 7730.
E-mail address: krlee@skku.ac.kr (K.R. Lee).
The molecular formula was determined to be C50
the molecular ion peak [M+H] at m/z 1035.5387 (calcd for
H
82
O
22 from
+
0
960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.003

2
076
K. H. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2075–2078
HO
O
RO
HO
1
''''
HO
O
OH
H
HO
21
OR
26
23
2
0
25
1
13
8
8
22
27
1
2
17
24
O
3
R = H
1
1
9
OCH3
19
H
16
10 R = CH3
1
4
H
14
OH
HO
HO
2
15
1
0
O
O
3
H
H
O
O
O
1
'''
5
7
HO
O
'
6
1
OCH3
OH
O
1
R = H
1
''
H C
O
2 R = CH3
3
HO
4
HO
5
HO
HO
OH
OH
OH OH
H CO
OCH3
OH
3
OH
O
6
HO
8
H
HO
O
O
H
OH
H
HO
O
O
HN
N
NH
NH
H
2
H
H
OH
HO
OH
7
9
12
1
1
O
OH
Figure 1. Chemical structures of compounds 1–12.
Table 1
1
1
3
a
H
(
5
0
0
M
H
z
)
a
n
d
C
N
M
R
(
1
2
5
M
H
z
)
d
a
t
a
o
f
c
o
m
p
o
u
n
d
s
1
–
2
(
d
i
n
p
p
m
,
J
v
a
l
u
e
s
i
n
p
a
r
e
n
t
h
e
s
e
s
)
Aglycone
1
2
Sugar
1
2
d
H
d
C
d
H
d
C
d
H
d
C
d
H
d
C
0
1
1.78 m, 1.05 m
1.58 m
2.11 m
3.58 m
2.30 t (13.0)
2.45 dd (13.0, 2.5)
37.1
29.3
1.78 m, 1.04 m
1.59 m
2.10 m
3.58 m
2.29 t (13.0)
2.44 dd (13.0, 2.5)
37.4
30.1
Glc-1
2
4.53 d (7.5)
3.41 m
98.9
77.0
4.51 d (7.5)
3.42 m
100.1
77.8
0
2
2
3
4
4
5
6
ax
eq
0
0
77.9
39.4
78.5
39.2
3
4
3.42 m
3.35 m
77.1
79.5
3.44 m
3.34 m
77.3
81.4
ax
eq
0
140.4
121.2
140.8
121.5
5
3.28 m
76.1
60.4
3.27 m
76.3
61.6
0
5.40 br d (4.5)
5.38 br d (4.0)
6 a
3.86 dd (12.5, 1.5)
3.81 dd (12.5, 3.5)
5.23 d (0.5)
3.88 m
3.67 dd (9.5, 3.0)
3.52 m
3.81 dd (9.5, 6.0)
1.25 d (6.0)
4.27 d (7.5)
3.68 dd (8.0, 7.0)
3.51 m
3.87 dd (12.5, 1.5)
3.80 dd (12.5, 3.5)
5.22 d (0.5)
3.87 m
3.66 dd (9.5, 3.0)
3.52 m
3.80 dd (9.5, 6.0)
1.23 d (6.0)
4.26 d (7.5)
3.66 dd (8.0, 7.0)
3.50 m
0
6
b
0
0
7
8
9
1
1
1
1
1
1
1
1
1.99 m, 1.60 m
1.80 m
0.99 m
31.7
31.3
50.2
36.6
20.5
39.4
40.4
56.3
31.3
81.0
63.6
1.97 m, 1.60 m
1.82 m
0.99 m
32.2
31.6
50.3
37.1
21.1
39.7
40.7
56.5
32.0
81.3
64.1
Rha-1
100.5
70.7
71.1
72.9
68.5
16.5
104.0
70.9
73.7
68.3
66.3
102.5
72.5
72.8
74.2
69.4
18.9
105.9
72.6
74.6
69.6
67.8
0
0
0
0
0
0
0
0
0
0
2
3
4
5
6
0
1
2
3
4
5
6
7
1.66 m, 1.63 m
1.78 m, 1.17 m
1.69 m, 1.63 m
1.78 m, 1.18 m
0
00
Ara-1
0
0
0
0
0
00
00
00
00
00
1.15 m
2.03 m, 1.57 m
4.37 m
1.16 m
2.01 m, 1.55 m
4.37 m
2
3
4
5
5
3.80 m
3.80 m
1.73 dd (8.0, 6.5)
1.73 dd (8.0, 6.5)
a
b
3.92 dd (12.0, 1.5)
3.60 dd (12.0, 3.0)
4.25 d (8.0)
3.39 m
3.28 m
3.29 m
3.40 m
3.85 dd (12.5, 1.5)
3.68 dd (12.5, 3.5)
3.92 dd (12.0, 1.5)
3.60 dd (12.0, 3.0)
4.23 d (7.5)
3.38 m
3.27 m
3.30 m
3.40 m
3.84 dd (12.5, 1.5)
3.68 dd (12.5, 3.5)
0
000
1
1
2
2
2
2
8
9
0
1
2
3
0.84 s
1.06 s
2.15 m
1.01 d (7.0)
15.3
18.4
39.7
14.6
112.5
29.9
0.83 s
1.04 s
2.17 m
0.99 d (7.0)
17.1
19.3
41.2
16.2
112.6
30.6
Glc-1
103.1
74.7
76.7
70.3
76.4
61.4
104.9
75.1
78.3
71.7
78.6
62.5
0
0
0
0
0
0
000
000
000
000
000
000
2
3
4
5
6
6
1.78 m
1.75 m
a
b
2
2
2
2
2
4
5
6a
6b
7
1.82 m, 1.75 m
1.79 m
3.72 dd (10.5, 6.0)
3.40 m
27.5
33.5
74.5
1.82 m, 1.75 m
1.79 m
3.72 dd (10.5, 6.0)
3.40 m
0.95 d (7.0)
3.14 s
28.1
34.2
75.1
0.96 d (7.0)
15.8
18.6
47.2
OCH
3
a
1H and 13C NMR data of 1 and 1H NMR data of 2 in CD
OD; 13C NMR data of 2 in C
N; The assignments were based on DEPT, 1H, H-COSY, HMQC and HMBC experiments.
1
3
5
D
5
C
50
H
83
O
22, 1035.5376) in the positive-ion HRFABMS. The glycosidic
for two tertiary methyl groups at d
and two secondary methyl groups at d
d, J = 7.0 Hz), typical steroid methyls, as well as a signal for an
H
1.06 and 0.84 (each 3H, s)
nature of 1 was shown by strong IR absorptions at 3400 and
H
1.01 and 0.96 (each 3H,
ꢀ1
1
1
060 cm . The H NMR spectrum of 1 (Table 1) showed signals

K. H. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2075–2078
2077
HO
O
HO
HO
O
H
C
OH
H
CH3
H
H
3
C
H
H
H
H
H
H
CH3
OH
H
H
OH
HO
H
H
HO
H
CH3
H
H
H
O
O
O
H
H
H
O
HO
O
H
H
OH
O
H
H C
O
H
3
HO
HO
OH
HMBC
H
C
NOESY
H
Figure 2. Key HMBC and NOESY correlations of 1.
olefinic proton at d
for four anomeric protons at d
J = 7.5 Hz), 4.27 (1H, d, J = 7.5 Hz), and 4.25 (1H, d, J = 8.0 Hz) show-
ing HMQC correlations with four anomeric carbon signals at d
H
5.40 (1H, br d, J = 4.5 Hz). Furthermore, signals
sis, the chiral inversion is possible, but was not detected in same
2
8,31
H
5.23 (1H, d, J = 0.5 Hz), 4.53 (1H, d,
reactions of relative furostanol saponins.
tion of 1 was confirmed to be -configuration by analysis of its
NOESY experiment showing a correlation between the methoxy
proton at d 3.14 and the H-16 proton at d 4.37. Thus, the
structure of 1 was established as (25R)-26-[(b- -glucopyrano-
syl)oxy]-22 -hydroxyfurost-5-en-3b-yl O- -rhamnopyranosyl-
(1?2)-O-[ -arabinopyranosyl-(1?4)]-b- -glucopyranoside.
Coreajaponin B (2), obtained as a white amorphous powder, had
the molecular formula C51 22 by the positive-ion HRFABMS
(m/z 1071.5330 [M+Na] ). The glycosidic nature of 1 was shown
The C-22 configura-
a
C
1
00.5, 98.9, 104.0, and 103.1, respectively, were assigned, and the
H
H
signal for the methyl group of a 6-deoxyhexopyranose at d
(
H
1.25
D
3H, d, J = 6.0 Hz) was also observed in the 1H NMR spectrum.
a
a-L
The above NMR data, an acetalic carbon signal at d
C NMR spectrum, and a positive red color reaction in Ehrlich’s
C
112.5 in the
a
-L
D
1
3
test indicated that 1 is a furostanol saponin with four attached gly-
84
H O
cosidic units. The chemical shifts of 1H and C NMR (including
DEPT) and evaluation of 2D NMR, including COSY, HMBC, and
NOESY allowed the identification of two b-glucopyranosyl (Glc),
2
3
13
+
ꢀ
1
by strong IR absorptions at 3397 and 1061 cm . Compound 2
was suggested to be a 22-methoxyfurostanol saponin by Ehrlich’s
2
3
1
13
one
units. The relatively large J values (7.5–8.0 Hz) indicated a b-ori-
entation for the anomeric center of Glc and an -orientation for
that of Ara. The J coupling (d, J = 0.5 Hz) of the anomeric proton
signal at d 5.23 of Rha indicated an -orientation. The monosac-
charides obtained from the acidic hydrolysis of 1 were identified
as -arabinose, -glucose, and -rhamnose by GC analysis of their
chiral derivatives. The sequence and interglycosidic linkages
a
-arabinopyranosyl (Ara), and one
a
-rhamnopyranosyl (Rha)
test, and by H NMR [d
H C
3.14 (3H, s)] and C NMR [d 112.6
2
4
27
1
13
(C-22) and 47.2 (OCH )] spectrum. Analysis of the H and C NMR
3
a
spectrum of 2 and comparison with those of 1 revealed that com-
pound 2 possessed sugar moieties identical to those of 1, but
differed slightly from 1 in the aglycone, with the presence of an
additional methoxy group. Acidic hydrolysis of 2 afforded dios-
H
a
L
D
L
genin, together with
were confirmed by GC analysis.
confirmed by HMBC correlation between a methoxy group at d
L
-arabinose,
D
25,26
-glucose, and
The methoxy group at C-22 was
L
-rhamnose, which
2
5,26
among the four sugar units and the aglycone were revealed by
the HMBC and NOESY experiments (Fig. 2). In the HMBC spectrum,
H
3.14 and C-22 of the aglycone at d
between the methoxy signal at d
C
112.6. An NOESY correlation
3.14 and the H-16 signal at d
H
0000
a correlation peak between H-1 of Glc (terminal glucosyl) at d
.25 and C-26 of the aglycone at d 74.5 implied that one glucose
H
H
4
C
4.37 was consistent with the C-22
a
configuration. Thus, the
was elucidated as (25R)-26-[(b- -glucopyra-
-methoxyfurost-5-en-3b-yl O- -rhamnopyrano-
-arabinopyranosyl-(1?4)]-b- -glucopyranoside.
unit is attached to C-26 of the aglycone, which is a structural fea-
structure of
nosyl)oxy]-22
2
a
D
ture in plant furostanol saponins.27 That one of the glucose units is
a-L
D
attached to C-3 of the aglycone was ascertained by the HMBC cor-
syl-(1?2)-O-[
a
-L
0
relation between H-1 of Glc at d
7
H
4.53 and C-3 of the aglycone at d
C
Compound 2 may be an artifact produced from 1, since 22-hydrox-
0
7.9, and by the NOESY correlation between H-1 of Glc at d
H
4.53
yfurostane derivatives are readily converted into 22-methoxyfuros-
3
1
and H-3 of the aglycone at d
H
3.58. Furthermore, the anomeric pro-
tane ones in solutions containing MeOH.
0
ton of Rha at d
that of Ara at d
H
5.23 was correlated with C-2 of Glc at d
C
77.0, and
79.5,
Then, we evaluated the effects of isolates (1–12) on cell viability
0
H
4.27 was correlated with C-4 of Glc at d
C
by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide
3
2
which supported the proposed sequence of the oligosaccharidic
(MTT) assay and on NGF secretion using ELISA development kit
(R&D System, Minneapolis, MN, USA) from C6 glial cells to measure
chain linked at the C-3 of the aglycone.24 This connection was also
0
0
33
deduced by the NOESY correlations between H-1 of Rha at d
H
5.23
NGF release into the medium. As shown in Table 2, compound 2
0
000
and H-2 of Glc at d
H
3.41, and between H-1 of Ara at d
3.35. The identity of the aglycone of 1 was con-
firmed by the acidic hydrolysis that afforded diosgenin.
H
4.27 and
was the most potent stimulant of NGF release, with 132.9 ± 3.6%
stimulation and normal cell viability (98.0 ± 1.5%) at concentration
of 20 lM. Interestingly, although the structures of 1 and 2 are quite
0
H-4 of Glc at d
H
2
8,29
The
C-25 configuration of 1 was deduced based on the difference
similar except of the presence of a methoxy group, they differed
substantially with respect to their effect on NGF synthesis induc-
tion. The obtained data suggest that the presence of the methoxy
group at C-22 in furostanol saponins is important for the effect
on NGF induction though more saponins need to be tested to con-
firm this hypothesis. Compounds 6, 8, 9 and 11 also increased NGF
secretion to 120.7 ± 4.6%, 122.2 ± 4.5%, 117.0 ± 3.4% and 117.5 ±
3.3% of untreated control, without cell toxicity. No other com-
pounds affected NGF release, and no compound was effective at
1
(
D
ab = d
a
ꢀ d
b
) of the H NMR chemical shifts of the H
protons. The 25R configuration of 1 was assigned by the observed
difference ( ab = 0.32) of the chemical shifts of 1, which was in
agreement with that of 25R furostane-type steroidal saponins
ab <0.48 for 25R;
ab >0.57 for 25S).30 To identify the configura-
tion of C-22, compound 1 was converted to the 22-methoxy form
2
-26 geminal
3
0
D
(D
D
28,31
by treatment with hot MeOH,
which was identical to com-
1
pound 2 by measurement of H NMR data. During the methanoly-

2
078
K. H. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2075–2078
Table 2
Effects of compounds 1–12 on NGF secretion and cell viability in C6 cells
Supplementary data
a
Compounds
NGF secretion
107.2 ± 1.2
Cell viability
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.003.
1
2
3
4
5
6
7
8
9
105.9 ± 4.5
98.0 ± 1.5
95.0 ± 4.7
100.9 ± 2.8
99.8 ± 1.3
102.5 ± 1.7
103.5 ± 2.7
101.0 ± 2.3
104.9 ± 1.5
103.7 ± 4.0
101.6 ± 1.9
100.2 ± 2.7
b
132.9 ± 3.6
106.1 ± 4.3
106.8 ± 6.3
105.6 ± 4.1
References and notes
1
2
.
.
Levi-Montalcini, R. Harvey Lect. 1966, 60, 217.
Whittemore, S. R.; Friedman, P. L.; Larhammar, D.; Persson, H.; Gonzalez-
Carvajal, M.; Holets, V. R. J. Neurosci. Res. 1988, 20, 403.
b
120.7 ± 4.6
106.1 ± 2.8
122.2 ± 4.5
117.0 ± 3.4
b
3. Hartikka, J.; Hefti, F. J. Neurosci. 1988, 8, 2967.
4. Kromer, L. F. Science 1987, 235, 214.
5. Wyman, T.; Rohrer, D.; Kirigiti, P.; Nichols, H.; Pilcher, K.; Nilaver, G.; Machida,
C. Gene Ther. 1999, 6, 1648.
b
1
1
1
0
1
2
104.6 ± 1.1
117.5 ± 3.3
b
6.
Fernyhough, P.; Diemel, L. T.; Brewster, W. J.; Tomlinson, D. R. Neuroscience
994, 62, 337.
102.4 ± 1.4
1
a
C6 cells were treated with 20
l
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4
This study was supported by a grant from the Korea Healthcare
Technology R&D Project, Ministry for Health, Welfare & Family
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S.G. Kim, and J.J. Seo at the Korea Basic Science Institute for their
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