Grasshopper ketone 3-O-primveroside from Sinocrassula indica

Article abstract of DOI:10.1080/10286020.2012.669376

A new megastigmane glycoside, grasshopper ketone 3-O-primveroside (1), was isolated from the methanolic extract of the whole herbs of Sinocrassula indica (Crassulaceae). Its structure was elucidated on the basis of spectral and chemical evidence.

Full text of DOI:10.1080/10286020.2012.669376

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Grasshopper ketone 3-O-primveroside
from Sinocrassula indica
a
b
Hai-Hui Xie & Masayuki Yoshikawa
a
Key Laboratory of Plant Resources Conservation and Sustainable
Utilization, South China Botanical Garden, Chinese Academy of
Sciences , Guangzhou , 510650 , China
b
Kyoto Pharmaceutical University , Kyoto , 607-8412 , Japan
Published online: 24 Apr 2012.
To cite this article: Hai-Hui Xie & Masayuki Yoshikawa (2012) Grasshopper ketone 3-O-primveroside
from Sinocrassula indica , Journal of Asian Natural Products Research, 14:5, 503-507, DOI:
10.1080/10286020.2012.669376
To link to this article: http://dx.doi.org/10.1080/10286020.2012.669376
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Journal of Asian Natural Products Research
Vol. 14, No. 5, May 2012, 503–507
Grasshopper ketone 3-O-primveroside from Sinocrassula indica
b
a
Hai-Hui Xie * and Masayuki Yoshikawa
a
Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China
Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China;
Kyoto Pharmaceutical University, Kyoto 607-8412, Japan
b
(
Received 19 September 2011; final version received 21 February 2012)
A new megastigmane glycoside, grasshopper ketone 3-O-primveroside (1), was
isolated from the methanolic extract of the whole herbs of Sinocrassula indica
(Crassulaceae). Its structure was elucidated on the basis of spectral and chemical
evidence.
Keywords: Sinocrassula indica; Crassulaceae; grasshopper ketone; grasshopper
ketone 3-O-primveroside
1
.
Introduction
Sinocrassula indica (Decaisne) A. Berger
Crassulaceae) is biennial herbs distributed
subjected to silica gel and ODS column
chromatography, respectively, and finally
subjected to preparative HPLC to yield
compound 1.
(
insouthandsouthwest China, Bhutan, India,
Nepal, Pakistan, and Sikkim [1]. It has been
used as a folk medicine for the treatment of
hepatitis and otitis medium and also as
vegetable and herbal tea [2]. Our previous
phytochemical studies on the whole herbs of
this species disclosed 35 flavonoids and two
known megastigmane glycosides [3–6].
Recently, we have re-examined the spectral
data of a compound previously isolated
but its structure remains uncharacterized
and it was found to be a megastigmane,
glycoside possessing a rare grasshopper
ketone aglycone. Herein, we report the
isolation and structural elucidation of this
new compound.
Compound 1, obtained as white amor-
phous powder, was deduced the molecular
formula C H O from its quasi-molecu-
24 38 12
þ
lar ion peaks at m/z 541 [M þ Na] and 517
2
M 2 H] in the FAB-MS as well as m/z
[
þ
5
The IR spectrum showed absorption bands
41.2261 [M þ Na] in the HR-FAB-MS.
21
for hydroxyl (3450 cm ), allenic group
2
1945 cm ), and conjugated carbonyl
1
(
(
(
2
1
1
1673 cm ). The H NMR spectrum
Table 1) exhibited signals readily recog-
nized in C D N for an olefinic proton at d
5
5
5
.90 (s, H-8), two anomeric protons with b-
0
configuration at d 5.05 (d, J ¼ 7.9 Hz, H-1 )
0
0
and 4.99 (d, J ¼ 6.7 Hz, H-1 ), a carbinol
proton at d 4.98 (m, H-3) and four tertiary
methyls at d 2.20 (CH -10), 1.61 (CH -11),
3
3
2
.
Results and discussion
13
1
.52 (CH -13), and 1.14 (CH -12). The C
3 3
The methanolic extract of S. indica was
dissolved in water and sequentially frac-
tionated with EtOAc and n-BuOH. The
resultant n-BuOH-soluble fraction was
NMR and DEPT spectra (Table 1) exhib-
ited 24 carbon signals, in C D N which
consisted of 4 methyls, 4 methylenes
5
5
including 2 oxygenated methylenes at d
*Corresponding author. Email: xiehaih@scbg.ac.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2012.669376
http://www.tandfonline.com

5
04
H.-H. Xie and M. Yoshikawa
1 13
Table 1. H and C NMR (DEPT) spectral data of compound 1.
5
C D
5
N
3
CD OD
H/C
1
d
H
(mult., J in Hz)
d
C
(DEPT)
d
H
(mult., J in Hz)
C
d (DEPT)
36.3 (C)
46.8 (CH2)
37.0 (C)
46.6 (CH2)
2
2
3
4
4
5
6
7
8
9
1
1
1
1
1
2
3
4
5
6
a
b
2.86 (dd, 12.0, 3.0)
1.65 (dd, 12.0, 12.5)
4.98 (m)
2.48 (dd, 12.2, 3.4)
1.70 (dd, 12.2, 12.5)
2.86 (dd, 11.3, 3.0)
1.45 (dd, 11.3, 13.1)
4.33 (m)
2.08 (dd, 11.3, 3.4)
1.46 (dd, 11.3, 13.1)
71.7 (CH)
47.9 (CH2)
72.7 (CH)
48.1 (CH2)
a
b
71.3 (C)
119.8 (C)
197.8 (C)
100.5 (CH)
209.6 (C)
26.4 (CH
29.3 (CH3)
32.0 (CH
31.1 (CH
102.7 (CH)
75.2 (CH)
78.5 (CH)
71.8 (CH)
77.0 (CH)
72.4 (C)
120.1 (C)
200.9 (C)
101.2 (CH)
211.5 (C)
5.90 (s)
5.83 (s)
0
1
2
3
0
0
0
0
0
0
2.20 (3H, s)
1.61 (3H, s)
1.14 (3H, s)
1.52 (3H, s)
5.05 (d, 7.9)
4.03 (m)
4.21 (m)
4.21 (m)
4.01 (m)
4.77 (dd, 11.2, 2.2)
3
)
2.19 (3H, s)
1.39 (3H, s)
1.16 (3H, s)
1.39 (3H, s)
4.45 (d, 8.0)
3.16 (m)
3.38 (m)
3.38 (m)
3.46 (m)
4.10 (dd, 11.6, 2.5)
3.75 (dd, 11.6, 5.2)
4.33 (d, 6.7)
3.61 (dd, 6.7, 8.5)
3.54 (m)
3.81 (m)
3.87 (dd, 12.5, 3.4)
3.55 (br d, 12.5)
26.6 (CH
3
)
29.5 (CH3)
3
)
)
32.3 (CH
30.9 (CH
3
)
)
3
3
102.7 (CH)
75.1 (CH)
77.9 (CH)
71.5 (CH)
76.8 (CH)
69.3 (CH2)
69.2 (CH2)
4.32 (dd, 11.2, 5.5)
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
4.99 (d, 6.7)
4.47 (dd, 6.7, 8.3)
4.21 (m)
4.33 (m)
4.32 (dd, 11.2, 5.5)
105.1 (CH)
72.3 (CH)
74.2 (CH)
69.0 (CH)
104.9 (CH)
72.4 (CH)
74.1 (CH)
69.3 (CH)
66.2 (CH
2
)
2
66.5 (CH )
3.79 (dd, 11.2, 1.5)
1
1
13
1
Note: Assignments in both solvents were based on the HZ H COSY, CZ H COSY, and HMBC spectra.
0
00
0
0
0
00
0
6
9.3 (C-6 ) and 66.2 (C-5 ), 11 oxygen-
C-6 , H -6 to C-1 , H-1 to C-3, and H-3 to
2
bearing methines ranging from d 105.1 (C-
0
C-1 clarified the inter-linkage of two sugar
0
00
1
) to 69.0 (C-4 ), and 5 quaternary
moieties, i.e. b-D-xylopyranosyl-(1 ! 6)-
b-D-glucopyranosyl (primverosyl) [9,10]
and its attachment to C-3 (Figure 1). In
addition, HMBC correlations from H-8 to
C-6 and C-10, both H -11 and H -12 to C-1,
carbons. The carbon signals at d 119.8 (C,
C-6), 197.8 (C, C-7), 100.5 (CH, C-8), and
2
09.6 (C, C-9) were typical of an allenic
3
3
group present in grasshopper ketone and its
derivatives [7,8]. Acid hydrolysis of 1
afforded D-glucose and D-xylose, which
were identified by HPLC analysis with an
optical rotation detector in direct compari-
12
11
9
1
O
3
5
O
son of the retention time (t ) and optical
R
O
O
13
O
OH
OH
1"
rotation (positive or negative) with those of
1
authentic samples. Analysis of the HZ H
OH
1'
1
HO
H
HO
H
1
3
1
OH
COSY and
revealed the connectivity from C-2 via C-
to C-4 (ZCH ZCHOZCH Z). In the
CZ H COSY spectra
OH
1
3
2
2
00
HMBC spectrum, correlations from H-1 to
Figure 1. Structure of compound 1.

Journal of Asian Natural Products Research
505
C-2, and C-6, and H -13 to C-4, C-5, and C-
3
Japan. D-(þ)-Glucose and D-(þ)-xylose
were obtained from Wako Pure Chemical
Industries, Osaka, Japan.
6
led us to construct the planar structure of
. The relative configuration was deter-
mined by the NOESY spectrum, in which
1
correlations between H-3 and H -11, and
3
3
.2. Plant material
between H -12 and H -13 were observed.
3
3
All measured in CD OD, the d value of C-3
3
The whole herbs of S. indica cultivated in
Guangxi, China, were purchased via Tochi-
motoTenkaidoCo., Osaka, Japan.Theplant
was botanically identified by Prof. Dian-
Xiang Zhang in South China Botanical
Garden, Chinese Academy of Sciences.
A voucher specimen (WT041220) has been
deposited at the Herbarium of South China
Botanical Garden, Chinese Academy of
Sciences, Guangzhou, China.
(
d 72.7) was identical to that of staphylio-
noside D [8] but different from that (d 73.4)
of glochidionionoside D [11], indicating 1
to be 3S,5R absolute configurations. More-
over, enzymatic hydrolysis of 1 with
naringinase yielded grasshopper ketone
(
1a), which was identified by comparison
of its spectral data with the reported values
7]. Thus, compound 1 was determined as
grasshopper ketone 3-O-primveroside.
[
3
.3. Extraction and isolation
The powder of air-dried whole herbs
9.9 kg) was extracted three times with
3
.
Experimental
3
.1. General experimental procedures
(
Optical rotations were measured on a Horiba
SEPA-300 digital polarimeter (Horiba,
Kyoto, Japan); the UV spectrum was
recorded on a Shimadzu UV-1600 spec-
trometer (Shimadzu, Kyoto, Japan); the IR
spectrumwasobtainedonaShimadzuFTIR-
methanol under reflux for 3 h each time.
Evaporation of the solvent under reduced
pressure gave a methanolic extract (764 g,
7.70%). The extract (725 g) was dissolved
in water and sequentially fractionated with
EtOAc and n-BuOH. The resultant
n-BuOH-soluble fraction (95 g of the
total 164 g) was subjected to silica gel
(2.5 kg) column chromatography using a
1800 spectrometer (Shimadzu); FAB-MS
and HR-FAB-MS spectra were obtained on a
JEOL JMS-SX 102A mass spectrometer
(JEOL, Tokyo, Japan); EI-MS was recorded
CHCl ZMeOH gradient (10–50% MeOH,
3
on a JEOL JMS-GCMATE mass spec-
trometer (JEOL); 1D and 2D NMR spectra
were recorded on a JEOL JNM-LA 500
spectrometer (JEOL) using TMS as an
internal standard. Analytic and preparative
HPLC were carried out on a Shimadzu LC-
v/v) as eluents to afford eight fractions (Frs
B1 2 B8). Fr. B5 (12.5 g) was separated
by Chromatorex ODS (350 g) column
chromatography using a MeOH–H O
2
gradient (10–40% MeOH, v/v) as eluents
to yield 10 fractions (Frs B5-1 2 B5-10).
Fr. B5-4 (0.51 g of the total 0.71 g) was
purified by preparative HPLC with an RI
6AD liquid chromatograph equipped with a
Shimadzu RID-10A refractive index (RI)
detector(Shimadzu)andYMC-PackODS-A
columns (S-5 mm, 250 £ 4.6 mm and
detector using MeOHZH O (25:75, v/v)
2
as a mobile phase at the flow rate of
9 ml/min to furnish compound 1 (tR
38.2 min, 42.8 mg, 0.0011%).
2
50 £ 20mm i.d., YMC, Kyoto, Japan).
For column chromatography, silica gel BW-
00 (150–300 mesh, Fuji Silysia Chemical,
2
Aichi, Japan) and Chromatorex ODS
DM1020T (100–200 mesh, Fuji Silysia
Chemical) were used. Naringinase and
3.3.1. Grasshopper ketone 3-O-
primveroside (1)
2
20
D
Amberlite IRA-400 (OH form) were
purchased from Sigma-Aldrich, Tokyo,
White amorphous powder; ½aꢀ 283.5 (c
1.00, MeOH); UV (MeOH) l
(log 1)
max

5
06
H.-H. Xie and M. Yoshikawa
2
1
32 (4.09) nm; IR (KBr) nmax 3450, 1945,
3.3.4. Grasshopper ketone (1a)
20
2
673, 1392, 1250, 1168 cm ; H NMR
1 1
White amorphous powder; ½aꢀ 256.8 (c
D
1
3
(
500 MHz) and
C NMR (125 MHz)
spectral data, see Table 1; FAB-MS m/z
1
.15, MeOH); H NMR (C D N, 500 MHz)
0
5
5
d 2.31 (1H, dd, J ¼ 12.5, 4.0 Hz, H-2a), 1.76
41 [M þ Na]þ and 517 [M 2 H] ; HR-
2
5
(
1H, dd, J ¼ 12.5, 12.5 Hz, H-2b), 4.94 (1H,
þ
FAB-MS m/z 541.2261 [M þ Na] (calcd
m, H-3), 2.79 (1H, dd, J ¼ 12.5, 3.5 Hz, H-
þ
for C H O Na , 541.2255).
24 38 12
4a), 1.83 (1H, dd, J ¼ 11.9, 12.5 Hz, H-4b),
5
.92 (1H, s, H-8), 2.26 (3H, s, H -10), 1.63
3
(3H, s, H -11), 1.19 (3H, s, H -12), 1.59 (3H,
3 3
3
.3.2. Acid hydrolysis of 1
A solution of 1 (1.5 mg) in 1 M HCl
2.0 ml) was stirred at 808C for 3 h. The
s, H -13), 6.23 (br s, 3-OH), 6.75 (br s, 5-
3
13
OH); C NMR (C D N, 125 MHz) d 36.4
5
5
(C, C-1), 50.5 (CH , C-2), 63.4 (CH, C-3),
2
(
50.9 (CH , C-4), 71.5 (C, C-5), 120.0 (C, C-
2
cooled reaction mixture was neutralized
2
with Amberlite IRA-400 (OH form), and
6), 197.9 (C, C-7), 100.5 (CH, C-8), 209.8 (C,
C-9), 26.4 (CH , C-10), 29.4 (CH , C-11),
the filtrate was partitioned with EtOAc.
The resultant aqueous layer was passed
through a Sep-Pak Cartridge (Waters, MA,
USA) and then subjected to HPLC analysis
on a Shimadzu LC-6AD liquid chromato-
graph equipped with a Shodex OR-2
optical detector (Showa Denko, Tokyo,
3
3
32.1 (CH , C-12), 31.2 (CH , C-13) (assign-
ments were based on the DEPT, HZ H
3 3
1
1
COSY, HMQC, and HMBC spectra); EI-MS
þ
m/z (rel. int., %) 224 [M] (2), 209 (55), 191
(13), 181 (3), 163 (53), 149 (31), 145 (12),
123 (100), 109 (42), 105 (25), 77 (43).
Japan) and a Kaseisorb LC NH -60-5
2
column (250 £ 4.6 mm, i.d., Tokyo Kasei,
Acknowledgments
Tokyo, Japan) using CH CN–H O (85:15,
3
2
This work was financially supported by the 21st
Centre of Excellence Program, Academic
Frontier Project from the Ministry of Edu-
cation, Culture, Sports, Science and Technol-
ogy of Japan and the Knowledge Innovation
Program of the Chinese Academy of Sciences
v/v) as a mobile phase at the flow rate of
.8 ml/min. Identification of the D-glucose
0
and D-xylose from 1 was carried out by
comparison of their retention times and
optical rotations with those of authentic
(
Grant No. KSCX2-EW-J-28).
samples (D-glucose: t 13.9 min, positive
R
optical rotation; D-xylose: tR 17.4 min,
positive optical rotation) [4,5].
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3
.3.3. Enzymatic hydrolysis of 1
[
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stirred at 378C for 24 h [12]. The reaction
mixture was centrifuged at 4000 rpm for
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[
[
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0 min after 8 ml of EtOH was added. The
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MeOH–H O (45:55, v/v) as a mobile
2
phase at the flow rate of 9 ml/min to furnish
1
a (t 21.4 min, 2.7 mg, 73% yield).
R

Journal of Asian Natural Products Research
507
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