New neuritogenic steroidal saponin from Ophiopogon japonicus (Thunb.) Ker-Gawl

Article abstract of DOI:10.1271/bbb.110066

A new steroidal saponin was isolated from Ophiopogon japonicus. This saponin possesses a modification by 2-hydroxy-3-methylvalerylation of the hydroxyl group at C-4' of the sugar, linked to C-1 of the aglycone. It exhibited significant neuritogenic activity for PC12 cells. The structure-activity relationship revealed the aglycone, rather than the sugar moieties and acylation, to be important for the neuritogenic activity.

Full text of DOI:10.1271/bbb.110066

Biosci. Biotechnol. Biochem., 75 (6), 1201–1204, 2011
Note
New Neuritogenic Steroidal Saponin
from Ophiopogon japonicus (Thunb.) Ker-Gawl
1
2
2
1
1
2
Yuan QU, Yang ZHANG, Liang PEI, Yan WANG, Lijuan GAO, Qianming HUANG,
3
3;y
1;y
Makoto OJIKA, Youji SAKAGAMI, and Jianhua QI
1
College of Pharmaceutical Sciences, Zhejiang University, Yu Hang Tang Road 388, Hangzhou 310058, China
College of Biology and Science, Sichuan Agricultural University, Yaan, Sichuan Province 625014, China
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
2
3
Received January 25, 2011; Accepted March 15, 2011; Online Publication, June 13, 2011
[doi:10.1271/bbb.110066]
A new steroidal saponin was isolated from Ophiopo-
gon japonicus. This saponin possesses a modification by
21
18
O
A
20
23
27
17
2
1
O
2
-hydroxy-3-methylvalerylation of the hydroxyl group
OR
O 19
R
H
9
H
0
O
O
1
at C-4 of the sugar, linked to C-1 of the aglycone. It
exhibited significant neuritogenic activity for PC12 cells.
The structure-activity relationship revealed the agly-
cone, rather than the sugar moieties and acylation, to be
important for the neuritogenic activity.
HO
HO
O
O
1'
H
H
1'''
OH
HO
HO
3
5
O
1''
HO
OH
R1
R2
Key words: steroidal saponin; Ophiopogon japonicus;
neuritogenic activity; PC12 cells; structure-
activity relationship
O
O
1
''''
1
H
O
OH
OH
H
H
Nerve growth factor (NGF)1) is one of the most
important neurotrophic factors essential for the growth,
survival, and functional maintenance of neurons. It can
be considered as a candidate for treating Alzheimer’s
2
H
H
H
H
H
HO
4
Me
3
2
)
disease, the most common neurodegenerative disorder.
B
NGF also plays an important role in preventing the loss
of cholinergic neurons, stimulating the cholinergic
function, preventing cholinergic degeneration, and
improving memory functions in animal models of
O
O
O
O
OH
O
O
O
O
HO
HO
3
,4)
Alzheimer’s disease.
cross the blood-brain barrier because of its large
However, NGF is unable to
O
OH
HO
O
HO
5
)
molecular size and hydrophilic properties. The use of
NGF for treating neurodegenerative disorders is thus
difficult. Small molecules that mimic the physiological
HO
OH
COSY and HOHAHA
Selected HMBC correlations
6
)
action of NGF are therefore sought. The PC12 cell line
derived from rat pheochromocytoma cells exhibits
characteristic neuronal responses to NGF and has been
used as a bioassay model to screen for small-molecule
candidates.7 Previous studies on screening for these
compounds with the PC12 cell line have isolated six
Fig. 1. Structural Elucidation of Compound 1.
A, Structures of compounds 1–4. B, Gross structure of compound
1, with selected HMBC correlations.
)
8
)
9)
novel cerebrosides, a series of steroidal glycosides,
1
The freeze-dried samples were ground to a powder and
then stirred in MeOH (50 L) for 3 d at room temperature.
A supernatant was separated by filtration and concen-
trated to obtain 1.2 kg of a crude extract. This extract
was chromatographed on ODS (Cosmosil 75 C18-OPN,
Nacalai Tesque) and eluted stepwise with MeOH/H2O
(50:50, 60:40, 70:30, 80:20, 90:10, 100:0) to yield six
fractions. The active fourth fraction (3.8 g) eluted with
MeOH/H2O (8:2) was chromatographed on silica gel
(200–300 mesh, Yantai Chemical Industry Research
Institute) and eluted stepwise with MeOH/CHCl3
(10:90, 15:85, 20:80, 25:75, 30:70, 100:0) to yield six
0,11)
and eleven alkyl benzoates.
Bioassay-guided puri-
fication of an Ophiopogon japonicus (Liliaceae)
Thunb.) Ker-Gawl methanol extract resulted in the
(
isolation of a new neuritogenic steroidal saponin in the
present study (1, Fig. 1A). Interestingly, spicatoside A, a
steroidal saponin, was reported to show neuritogenic
1
2)
activity by J. Hur et al. in 2009. We describe here the
isolation, structure, biological activity, and brief struc-
ture-activity relationship of this new compound (1).
Commercially available dried roots of O. japonicus
(
9.5 kg, dry wt.) were purchased in Sichuan Province.
y
To whom correspondence should be addressed. Jianhua QI, Tel/Fax: +86-571-88208627; E-mail: qijianhua@zju.edu.cn; Youji SAKAGAMI,
Tel: +81-52-7894116; Fax: +81-52-7894118; E-mail: ysaka@agr.nagoya-u.ac.jp

1202
Y. QU et al.
Table 1. 1H-NMR (compound 1) and 13C-NMR Data (compounds 1 and 2) in Pyridine-d5
1
1
2
1
1
2
Carbon
no.
Carbon
no.
1H
a
13C
b
13C
b
1H
a
13C
b
13C
b
0
0
0
1
3.79 dd (12.1, 3.8)
2.66 m
2.35 m
83.1
37.3
83.7
37.4
1
4.68 d (7.8)
99.9
74.4
82.1
73.2
64.2
100.4
74.2
84.4
69.6
67.0
2
2
3
4
4
5
6
7
7
8
9
0
a
2
4.45 dd (9.5, 7.8)
4.14 dd (9.5, 3.5)
5.70 m
b
3
0
3.85 m
2.68 m
68.1
43.6
68.2
43.8
4
5 a
0
a
4.23 dd (12.7, 1.5)
3.68 d (12.7)
0
b
2.56 dd (12.8, 4.2)
—
5 b
139.3
124.6
31.8
139.5
124.6
32.3
0
0
0
0
0
0
0
0
0
0
0
0
5.57 d (5.6)
1.88 m
1.51 m
1
2
3
4
5
6
6.32 br s
4.77 m
101.9
72.4
72.5
74.1
69.5
19.2
101.8
72.5
72.5
74.0
69.4
19.1
a
b
4.55 dd (9.5, 3.6)
4.28 dd (9.5, 9.6)
4.79 m
1.57 m
1.49 m
33.0
50.1
42.6
24.2
33.1
50.3
42.8
24.0
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
—
2.90 m
1.73 d (6.2)
1a
1b
2a
2b
3
0
0
0
0
0
00
00
00
00
00
1.67 m
1.82 m
1
2
3
4
4.89 d (7.5)
106.9
74.7
78.6
70.8
67.1
106.4
74.6
78.2
70.9
66.9
40.2
40.1
3.93 dd (8.4, 7.9)
4.03 t (8.4)
4.11 m
1.37 m
—
40.5
56.9
32.3
40.2
56.8
31.9
4
1.10 m
1.99 m
5 a
4.25 dd (10.8, 5.6)
3.62 t (10.8)
000
5a
5b
6
5 b
1.43 m
4.46 m
0
0
0
0
0
0
0
000
000
000
000
000
000
000
81.0
62.9
16.7
14.9
41.9
14.9
109.2
31.8
81.1
62.9
16.6
14.9
41.9
15.0
109.2
31.8
1
2
3
4
4
5
6
—
174.9
75.5
39.5
24.5
7
1.76 m
0.92 s
4.53 m
2.14 m
1.93 m
1.47 m
0.93 t (7.4)
1.13 d (6.8)
8
9
1.40 s
1.96 m
a
0
b
1
1.19 d (7.0)
—
1.84 m
11.9
15.8
2
3a
3b
4a
4b
5
1.64 m
1.66 m
29.2
29.2
1.53 m
1.53 m
30.5
66.7
30.5
66.7
6a
6b
7
3.54 dd (10.5, 2.5)
3.47 t (10.5)
0.67 d (5.4)
17.2
17.2
a500 MHz, coupling constants (J in Hz) are in parentheses.
b125 MHz.
fractions. The most active sample (190.1 mg), which
was eluted with MeOH/CHCl (25:75), was subjected to
anomeric resonance of protons and carbons (ꢁ /ꢁ :
H C
4.68/99.9, 6.32/101.9, and 4.89/106.9) reveals the
presence of three sugar moieties. An analysis of the
COSY and HOHAHA spectra led to the determination
of the partial structures depicted by the bonds (in bold-
face type) in Fig. 1B. These partial structures were
connected by the following long-range H-C correlations
obtained by an HMBC experiment to give the overall
structure of 1 (Fig. 1B): methyl protons (H-18) to C-12,
C-13, C-14, and C-17; methyl protons (H-19) to C-1,
C-5, C-9, and C-10; methyl protons (H-21) to C-20 and
C-22; methyl protons (H-27) to C-24, C-25, and C-26;
3
HPLC [Develosil ODS-HG-5 (ꢀ 20 ꢀ 250 mm), Nomura
Chemical, 8 mL/min flow rate, MeOH/H2O (8:2)
mobile phase] to give an active fraction and one pure
active compound. The pure compound was identified
as ophiopogonin D (4) (Fig. 1A) by precise agreement
of the specific rotation and NMR data with those
reported.1
3,14)
The active fraction (15.0 mg) was again
purified by HPLC [Develosil ODS-HG-5 (ꢀ 10 ꢀ 250
mm), Nomura Chemical, 3 mL/min flow rate, MeCN/
H O (5:5) mobile phase] to yield pure compound 1
2
0000 0000 0000
methyl protons (H-5 ) to C-4 ; methyl protons (H-6 )
(
5.7 mg, 0.00006% of dry wt., t ¼ 45 min).
R
Compound 11 has the molecular formula C49H78O18
as determined by HR FT-ICR MS measurements. The
IR spectrum of 1 shows an absorption band at 3425
5)
to C-2 , C-3 , and C-4 ; H-4 to C-5, C-6, and C-10;
H-6 to C-8 and C-10; H-20 to C-22; H-23 to C-22; H-26
0000
0000
0000
0 0
to C-22; and H-5 to C-1 . The correlations from
anomeric H-1 to C-1, H-1 to C-2 , and H-1 to C-3
indicate the location of three sugar moieties. The
ꢁ1
1
13
0
00
0
000
0
cm due to hydroxyl groups. The H-NMR, C-NMR,
and DEPT spectra of 1 indicate the presence of 17
oxymethines, 3 oxymethylenes, 1 oxygenated quater-
0
0000
0000
important HMBC correlations H-4 to C-1 and H-2
0
000
nary carbon, 1 trisubstituted double bond (ꢁH 5.57, ꢁC
to C-1 reveal the sugar linked to C-1 of the aglycone
0
3
1
24.6, and 139.3), 2 quaternary sp carbons (ꢁ 42.6 and
C
to be acylated at the hydroxyl group of C-4 which has a
2-hydroxy-3-methylvaleryl group.
4
remaining proton and carbon signals are attributable to 7
0.5), and 1 carboxyl group (ꢁ 174.9) (Table 1). The
C
Further validation was done by the degradation of 1 to
confirm the structure and determine the stereochemistry.
The hydrolysis of 1 under weakly basic conditions
methyls, 7 methines, and 9 methylenes. The signal at ꢁC
1
74.9 supports the presence of one carboxyl group. The

New Neuritogenic Steroidal Saponin
1203
produced compound 2 and potassium 2-hydroxy-3-
1
A
6)
1
13
methylpentanoate. The H-NMR and C-NMR spec-
tra, MS data, and specific rotation of compound 2 agree
1
7)
1
with those reported. The H-NMR data for 2-hydroxy-
-methylpentanoic acid obtained precisely agree with
3
those for (2S,3S) and (2R,3R)-2-hydroxy-3-methylpen-
tanoic acid.1 Although the concentration was low, the
specific rotation of 2-hydroxy-3-methylpentanoic acid
8)
obtained almost agrees with that of the reported
6,18,19)
B
value.1
These results respectively indicate the
0000
S,3S configuration for the hydroxyl group at C-2
2
0000
and the methyl group at C-3 . The same modification
has been reported for the steroidal saponin from Ruscus
auleatus which belongs to the same Liliaceae group
as O. japonicus.1 The absolute stereochemistry of the
9)
2
-hydroxy-3-methylvaleryl group of compound 1 was
therefore determined to be 2S,3S. The degradation of 2
enabled the aglycone to be obtained to identify the
structure-activity relationship and further confirm the
structure. Acid hydrolysis of 2 in MeOH resulted in the
production of an aglycone which was identified as
ruscogenin (3).2
Fig. 2. Biological Activity of Compounds 1–4.
0,21)
A, Neuritogenic activity of compounds 1–4 48 h after the
treatment. The activity is presented as the percentage of PC12 cells
with neurite outgrowth longer than the diameter of the cell body.
c, control (0.5% DMSO); NGF (40 ng/mL), positive control; 1–4 at
0.3 mM. B, Images of the PC12 cells under a phase-contrast
microscope 48 h after the treatment. a, solvent control (0.5%
The neuritogenic activities of these compounds were
evaluated according to the methods described in
previous papers.8,9) A suspension of 20,000 PC12 cells
in 1 mL of a DMEM medium was inoculated in a 24-
well microplate and then pre-cultured under a humidi-
ꢂ
DMSO); b, NGF (40 ng/mL); c, 1 at 0.3 mM; d, 3 at 0.3 mM.
fied atmosphere of 5% CO at 37 C. The medium was
2
replaced after 24 h by 1 mL of the serum-free DMEM
medium containing a test sample and DMSO (0.5%),
and the morphological changes in the cells were
monitored 48 h later under a phase-contrast microscope.
About 100 cells were counted in a randomly chosen
field, this being conducted in triplicate.
tration. The aglycone, rather than the sugar moieties
and other modifications, played a key role in the
neuritogenic activity. The aglycone may be useful for
studying the mechanism for the neurotrophic effect on
PC12 cells.
Compound 1 showed maximum neuritogenic activity
of 46% at 0.3 mM 48 h after the treatment, compared with
the solvent (0.5% DMSO) and the NGF positive control
at optimum concentration (40 ng/mL, Fig. 2A). Com-
pound 2 obtained from the hydrolysis of compound 1
showed 52% activity at 0.3 mM; this suggests that
Acknowledgments
This work was financially supported by NSFC
(30873152 and 81072536), Qianjiang Talent Program
of Zhejiang Province (2010R10052) and Science Foun-
dation of Chinese Universities (2009QNA7023).
0
acylation at C-4 of the sugar linked at C-1 to the
aglycone had little or no effect on the biological activity
of the steroidal saponin. Compound 3 showed similar
neuritogenic activity (54% at 0.3 mM), suggesting that
the sugar moiety at C-1 of the aglycone did not affect the
biological activity of the saponin toward PC12 cells.
These results are supported by the neuritogenic activity
References and Notes
1)
2)
3)
Levi-Montalcini R, Science, 237, 1154–1162 (1987).
Heese K, Low JW, and Inoue N, Neurosignals, 15, 1–12 (2006).
Fischer W, Wictorin K, Bj o¨ rklund A, Williams LR, Varon S,
and Gage FH, Nature, 329, 65–68 (1987).
(
47% at 0.3 mM) of compound 4 (Fig. 2A). The structure-
4
)
Tuszynski MH, Thal L, Pay M, Salmon DP, Sang UH, Bakay R,
Patel P, Blesch A, Vahlsing HL, Ho G, Tong G, Potkin SG,
Fallon J, Hansen L, Mufson EJ, Kordower JH, Gall C, and
Conner J, Nat. Med., 11, 551–555 (2005).
activity relationship emphasizes that the aglycone, rather
than the sugar moieties and acylation, was important for
the neuritogenic activity.
5
)
Granholm AC, Albeck D, B a¨ ckman C, Curtis M, Ebendal T,
Friden P, Henry M, Hoffer B, Kordower J, Rose GM,
S o¨ derstr o¨ m S, and Bartus RT, Rev. Neurosci., 9, 31–55 (1998).
Brinton RD and Yamazaki RS, Pharm. Res., 15, 386–398
Figure 2B shows the morphological changes to the
PC12 cells after treating with 1 and 3 in comparison
with the solvent control (0.5% DMSO, a) and positive
control (40 ng/mL, b). Cells treated with 1 (c) and 3 (d)
at 0.3 mM showed long bipolar and multipolar neurite
outgrowth 48 h after the treatment. While the NGF
treatment resulted in multipolar neurite outgrowth of the
cells, control cells cultured without additional com-
pounds showed few short neurite outgrowths.
The steroidal saponin exhibited significant neurito-
genic activity toward PC12 cells. The aglycone showed
neuritogenic activity higher than that of the natural
compound (the original saponin) at the same concen-
6
)
)
(1998).
Greene LA and Tischler AS, Proc. Natl. Acad. Sci. USA, 73,
7
2424–2428 (1976).
8) Qi JH, Ojika M, and Sakagami Y, Tetrahedron, 56, 5835–5841
2000).
Qi JH, Ojika M, and Sakagami Y, Bioorg. Med. Chem., 10,
961–1966 (2002).
0) Gao LJ, Li JY, and Qi JH, Bioorg. Med. Chem., 18, 2131–2134
2010).
(
9
)
1
1
1
(
1) Gao LJ, Xiang L, Luo Y, Wang GF, Li JY, and Qi JH, Bioorg.
Med. Chem., 18, 6995–7000 (2010).

1204
Y. QU et al.
ꢁ
1
2) Hur J, Lee P, Moon E, Kang I, Kim SH, Oh MS, and Kim SY,
Eur. J. Pharmacol., 620, 9–15 (2009).
3) Tada A, Kobayashi M, and Shoji J, Chem. Pharm. Bull., 21,
(c 0.10, MeOH); IR (KBr): 3425, 1592, 1060 cm 1; ESI-MS
þ
m=z: 863 ½M þ Naꢃ . 2-Hydroxy-3-methylpentanoic acid
1
1
(approx. 200 mg) was also eluted with MeOH/H2O (90:10)
(later than that of compound 2) and obtained as colorless oil,
308–311 (1973).
2
5
25
D
26
D
4) Ophiopogonin D was obtained as amorphous solid, ½ꢂꢃ
½ꢂꢃ þ18.4 (c 0.03, CHCl3) [lit ½ꢂꢃ þ22.0 (c 0.1, CHCl3)];
D
1
D
4
1
ꢁ
105.7 (c 0.45, pyridine) [lit ½ꢂꢃ ꢁ107.9 (c 0.66, pyridine)];
H NMR (CDCl3) ꢁ: 4.19 (1H, d, J ¼ 3:5 Hz, H-2), 1.91 (1H,
1
3
C NMR (125 MHz, in pyridine-d5) ꢁ: 139.5 (C-5), 124.7 (C-
m, H-3), 1.43 and 1.30 (2H, m, H-4), 1.03 (3H, d, J ¼ 7:0 Hz,
Me-6), 0.93 (3H, t, J ¼ 7:3 Hz, Me-5); ESI-MS m=z: 155
000 00 0
6
), 109.2 (C-22), 106.6 (C-1 ), 101.7 (C-1 ), 100.4 (C-1 ), 85.4
0
000
00
þ
(
C-3 ), 83.4 (C-1), 81.0 (C-16), 78.3 (C-3 ), 74.6 (C-4 ), 74.2
00
½M þ Naꢃ . NMR chemical shifts in ꢁ (ppm) were referenced to
0
000
00
0
0
(
C-2 ), 73.8 (C-2 ), 72.6 (C-3 ), 72.5 (C-4 , 2 ), 70.9 (C-5 ),
the solvent peaks of ꢁH 7.26 for CDCl3.
000
00
000
7
6
4
3
2
0.7 (C-4 ), 69.3 (C-5 ), 68.2 (C-3), 67.0 (C-5 ), 66.7 (C-26),
2.9 (C-17), 57.1 (C-14), 50.5 (C-9), 43.8 (C-4), 42.7 (C-10),
1.9 (C-20), 40.4 (C-13), 40.1 (C-12), 38.0 (C-2), 33.0 (C-8),
2.3 (C-7), 32.0 (C-15), 31.7 (C-23), 30.5 (C-25), 29.2 (C-24),
17) Kuroda M, Mimaki Y, Ori K, Sakagami H, and Sashida Y,
J. Nat. Prod., 67, 1690–1696 (2004).
18) Snowden RL, Grenno R, and Vial C, Flavour Fragr. J., 20,
372–380 (2005).
00
0
4.0 (C-11), 19.1 (Me-6 ), 17.2 (Me-27), 17.0 (Me-6 ), 16.8
19) Mimaki Y, Kuroda M, Kameyama A, Yokosuka A, and Sashida
Y, Chem. Pharm. Bull., 46, 298–303 (1998).
20) Acid hydrolysis of 2 to give 3. Compound 2 (1.4 mg) in
(
Me-18), 14.9 (Me-21), 14.8 (Me-19); ESI-MS m=z: 855
þ
½
M þ Hꢃ . NMR chemical shifts in ꢁ (ppm) were referenced
ꢂ
to the solvent peaks of ꢁC 123.4 for pyridine-d5.
anhydrous 2.0 M HCl in MeOH (1 mL) was heated at 80 C for
2
5
1
1
5) Compound 1 was obtained as amorphous solid, ½ꢂꢃ ꢁ49.4
10 h in a sealed tube. After being cooled down, the reaction
solution was evaporated and partitioned between EtOAc and
water. The EtOAc layer was dried up to afford the compound 3
(0.6 mg). Compound 3 was obtained as amorphous solid;
D
ꢁ1
(
c 0.22, MeOH); IR (KBr): 3425, 1727, 1592, 1055 cm ; HR
FT-ICR MS m=z ½M þ Naꢃþ Calcd. for C49H78O18Na:
9
77.5080. Found: 977.5095.
1
6) Hydrolysis of 1 to give 2 and 2-hydroxy-3-methylpentanoate.
Compound 1 (3 mg) in methanol (1 mL) was treated with
potassium carbonate (4 mg) at room temperature for 24 h. The
sample solution was evaporated and then the crude product was
dissolved in H2O (2 mL). The water solution was applied on an
ODS (Cosmosil 75 C18-OPN, Nacalai Tesque), which was
washed with H2O (4 mL) and then eluated with a MeOH/H2O
in stepwise (50:50, 70:30, 90:10, 100:0) to afford the compound
H NMR (500 MHz, in pyridine-d5) ꢁ: 5.60 (1H, d, J ¼
5:5 Hz, H-6), 4.53 (1H, q-like, J ¼ 7:5 Hz, H-16), 3.96 (1H,
m, H-3), 3.81 (1H, br d, J ¼ 11:5 Hz, H-1), 3.56 (1H, dd,
J ¼ 10:5, 3.5 Hz, H-26b), 3.49 (1H, t, J ¼ 10:5 Hz, H-26a), 1.33
(3H, s, Me-19), 1.09 (3H, d, J ¼ 7:0 Hz, Me-21), 0.90 (3H, s,
Me-18), 0.67 (3H, d, J ¼ 6:0 Hz, Me-27); ESI-MS m=z: 431
þ
1
½M þ Hꢃ . H NMR chemical shifts in ꢁ (ppm) were referenced
to the solvent peaks of ꢁH 7.19 for pyridine-d5.
2
(1.8 mg), which was eluted with MeOH/H2O (90:10).
2
21) Mimaki Y, Kuroda M, Kameyama A, Yokosuka A, and Sashida
Y, Phytochemistry, 48, 485–493 (1998).
5
Compound 2 was obtained as amorphous solid, ½ꢂꢃ ꢁ53.8
D