Further acylated flavonol bisdesmosides from Sinocrassula indica

Article abstract of DOI:10.1080/10286020.2013.800973

Further investigation on the whole herbs of Sinocrassula indica (Crassulaceae) led to the isolation of four new acylated flavonol bisdesmosides, sinocrassosides A13 (1), B6 (2), B7 (3), and D4 (4), together with kaempferol 3-O-β-d-(6-O-acetyl)glucopyranos

Full text of DOI:10.1080/10286020.2013.800973

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Further acylated flavonol
bisdesmosides from Sinocrassula indica
Hai-Hui Xie a & Masayuki Yoshikawa b
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, Misasagi, Yamashina-Ku ,
Kyoto , 607-8412 , Japan
Published online: 14 Jun 2013.
To cite this article: Hai-Hui Xie & Masayuki Yoshikawa (2013) Further acylated flavonol
bisdesmosides from Sinocrassula indica , Journal of Asian Natural Products Research, 15:8, 885-890,
DOI: 10.1080/10286020.2013.800973
To link to this article: http://dx.doi.org/10.1080/10286020.2013.800973
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Journal of Asian Natural Products Research, 2013
Vol. 15, No. 8, 885–890, http://dx.doi.org/10.1080/10286020.2013.800973
Further acylated flavonol bisdesmosides from Sinocrassula indica
Hai-Hui Xiea* and Masayuki Yoshikawab
aKey Laboratory of Plant Resources Conservation and Sustainable Utilization, South China
Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China; Kyoto
Pharmaceutical University, Misasagi, Yamashina-Ku, Kyoto 607-8412, Japan
b
(Received 31 January 2013; final version received 27 April 2013)
Further investigation on the whole herbs of Sinocrassula indica (Crassulaceae) led to
the isolation of four new acylated flavonol bisdesmosides, sinocrassosides A13 (1), B6
(2), B7 (3), and D4 (4), together with kaempferol 3-O-b-D-(6-O-acetyl)glucopyranosyl-
7-O-a-L-rhamnopyranoside (5). Their structures were established by spectral and
chemical methods.
Keywords: Sinocrassula indica; flavonol bisdesmoside; acyl group
1. Introduction
an a-rhamnopyranosyl moiety at dH 5.56
(br s, H-1000) and 1.13 (3H, d, J ¼ 6.1 Hz,
H3-6000), and an acetyl group at dH 2.06
(3H, s) and dC 169.4. The HMBC long-
range correlations from H-100 to C-3 at dC
132.9 and H-1000 to C-7 at dC 161.5
revealed the connections of the glucosyl
moiety to C-3 and the rhamnosyl moiety to
C-7. Alkaline hydrolysis of 1 with aqueous
KOH–1,4-dioxane gave kaempferol 3-O-
b-D-glucopyranosyl-7-O-a-L-rhamnopyr-
anoside (1a) [5]. Moreover, acid hydroly-
sis of the alkaline hydrolysate confirmed
D-glucose and L-rhamnose. Furthermore,
the HMBC long-range correlation from H-
200 at dH 4.76 (dd, J ¼ 8.0, 9.5 Hz) to the
carbonyl at dC 169.4 and consistent NMR
data with 2-O-acetylglucopyranosyl moi-
ety in sinocrassoside D3 [3] clarified the
position of the acetyl group. Thus, the
structure of compound 1 was characterized
as kaempferol 3-O-b-D-(2-O-acetyl)glu-
copyranosyl-7-O-a-L-rhamnopyranoside
and named sinocrassoside A13 (Figure 1).
The molecular formula of compound 2
was deduced as C29H32O17 from its MS
and NMR data, an oxygen atom more than
1. The 1H NMR spectrum (Table 1)
Sinocrassula indica (Decaisne) A. Burger
(Crassulaceae) has been used as a folk
medicine for the treatment of hepatitis and
otitis medium in China [1]. Our previous
studies on the whole herbs disclosed 3
megastigmanes and 35 flavonoids [2–6].
Further investigation of the n-BuOH-
soluble fraction from the methanolic
extract yielded four new acylated flavonol
bisdesmosides (1–4) and kaempferol 3-O-
b-D-(6-O-acetyl)glucopyranosyl-7-O-a-L-
rhamnopyranoside (5). This article deals
with the isolation and structure elucidation
of these compounds.
2. Results and discussion
Compound 1 was obtained as a yellow
amorphous powder and assigned the
molecular formula C29H32O16 from
quasi-molecular ion peaks at m/z 637
[M þ H]þ and 635 [M 2 H]2 in the FAB-
MS as well as m/z 659.1592 [M þ Na]þ in
the HR-FAB-MS. The H and 13C NMR
1
spectra (Tables 1 and 2) showed signals for
a b-glucopyranosyl moiety at dH 5.63
(d, J ¼ 8.0 Hz, H-100) and dC 98.3 (C-100),
*Corresponding author. Email: xiehaih@scbg.ac.cn
q 2013 Taylor & Francis

886
H.-H. Xie and M. Yoshikawa
d

Journal of Asian Natural Products Research
887
Table 2. 13C NMR spectral data (d) of
compounds 1–4 in DMSO-d6.
established as quercetin 3-O-b-D-(3-O-
acetyl)glucopyranosyl-7-O-a-L-rhamno-
pyranoside and named sinocrassoside B6.
The 1H and 13C NMR spectra of
compound 3 (Tables 1 and 2) exhibited
similar signals to those of 2 except for an
isobutyryl group at dH 2.60 (dq, J ¼ 7.0,
7.0 Hz, H-20000), 1.14 (6H, d, J ¼ 7.0 Hz,
H3-30000 and H3-40000), and dC 176.0 (C-10000)
[2] instead of an acetyl group in 2.
Alkaline hydrolysis of 3 liberated 2a.
The isobutyryl group was assigned to C-3000
by the HMBC correlation from H-3000 at dH
4.94 (dd, J ¼ 3.4, 9.2 Hz) to C-10000 and
consistent 1H and 13C NMR data with 3-O-
isobutyrylrhamnopyranosyl moiety in
sinocrassoside A4 [5]. Consequently, the
structure of compound 3 was determined
as quercetin 3-O-b-D-glucopyranosyl-7-
O-a-L-(3-O-isobutyryl)rhamnopyranoside
and named sinocrassoside B7.
C
1
2
3
4
2
3
4
5
156.7
132.9
177.3
160.7
99.3
156.6
133.3
177.4
160.8
99.3
156.6
133.5
177.5
160.8
99.3
156.7
133.3
177.8
152.1
99.3
6
7
8
9
161.5
94.4
161.5
94.2
161.1
94.4
150.5
127.3
144.7
105.7
121.2
116.6
145.0
148.5
115.0
121.8
100.6
74.0
76.4
69.8
77.5
60.9
99.3
67.3
73.0
68.8
155.9
105.5
120.4
130.9
115.1
160.2
115.1
130.9
98.3
74.2
73.9
69.9
77.6
60.5
98.2
69.7
70.1
71.5
70.0
17.8
169.4
20.9
155.8
105.5
120.8
116.3
144.8
148.7
115.2
121.5
100.3
71.8
77.5
67.6
77.2
60.5
98.3
69.7
70.2
71.5
155.8
105.7
120.9
116.3
144.8
148.6
115.1
121.6
100.6
74.0
76.4
69.9
77.6
60.9
98.1
67.1
73.2
68.6
10
10
20
30
40
500
600
100
200
300
400
500
6000
1000
2000
3
The molecular formula of compound 4
was deduced as C32H38O18 from its MS
and NMR data. The 1H and 13C NMR
spectra (Tables 1 and 2) exhibited signals
for a gossypetin aglycone [3], a 2-
methylbutyryl group at d 2.43 (ddq,
J ¼ 7.0, 7.0, 7.0 Hz, H-20000), 1.48 and
1.65 (ddq each, J ¼ 7.0, 13.0, 7.3 Hz, H2-
30000), 0.90 (3H, t, J ¼ 7.3 Hz, H3-40000), and
1.12 (3H, d, J ¼ 7.0 Hz, H3-50000) [2], in
addition to a b-glucopyranosyl and an a-
rhamnopyranosyl moiety. Alkaline
hydrolysis of 4 gave sinocrassoside D2
(4a) [3]. The presence of (S)-(þ)-2-
methylbutyric acid in the hydrolysate was
characterized by high-performance liquid
chromatography (HPLC) analysis of its 4-
nitrobenzyl ester using an optical rotation
detector [2,3]. The acyl group was
assigned to C-3000 by the HMBC correlation
from H-3000 at dH 5.12 (dd, J ¼ 3.4, 9.8 Hz)
to C-10000 at dC 175.4 and consistent 1H and
13C NMR data with 3-O-(S)-2-methylbu-
tyrylrhamnopyranosyl moiety in sinocras-
soside D1 [3]. Hence, the structure of
compound 4 was elucidated as gossypetin
3-O-b-D-glucopyranosyl-7-O-a-L-(3-O-
4000
5000
6000
10000
20000
30000
40000
50000
70.0
17.8
169.7
21.0
70.0
17.7
176.0
33.3
18.7
69.9
17.7
175.4
40.2
26.2
18.8
11.2
16.3
exhibited similar proton signals to 1 except
for an ABX system at d 7.65 (d, J ¼ 2.5 Hz,
H-20), 6.88 (d, J ¼ 8.6 Hz, H-50), and 7.61
(dd, J ¼ 8.6, 2.5 Hz, H-60) instead of an
A2B2 system in 1, indicating an aglycone
of quercetin. Alkaline hydrolysis of 2
yielded quercetin 3-O-b-D-glucopyrano-
syl-7-O-a-L-rhamnopyranoside (2a) [5].
The acetyl group was assigned to C-300 by
the HMBC correlation from H-300 at dH
4.85 (dd, J ¼ 9.2, 9.5 Hz) to the carbonyl
at dC 169.7 and consistent 1H and 13C
NMR data with 3-O-acetylglucopyranosyl
moiety in sinocrassoside A3 [5]. There-
fore, the structure of compound 2 was

888
H.-H. Xie and M. Yoshikawa
OH
OH
OH
OH
OH
4'
OH
OH
O
O
O
O
H
O
O
O
H
O
O
7
2
HO
HO
HO
1'
O
O
O
O
1"'
O
H
O
H
5
OH
4
H
OH
HO
R2O OH
RO OH
HO OH
R2O
HO
O
O
O
OH
1"
OR1
OH
HO
H
HO
HO
OR1
OH
OH
R1
Ac
H
R2
R1
Ac
H
R
R2
1
H
H
2
2a
3
H
H
4
(S)-2MB
H
1a
5
4a
H
Ac
H
IB
O
O
O
Ac =
IB =
(S)-2MB =
1""
1""
4""
5""
Figure 1. Structures of compounds 1–5.
(S)-2-methylbutyryl)rhamnopyranoside
and named sinocrassoside D4 (Figure 1).
Compound 5 was determined as
kaempferol 3-O-b-D-(6-O-acetyl)gluco-
pyranosyl-7-O-a-L-rhamnopyranoside by
5C18-MS-II packed columns (5 mm,
250 £ 4.6 mm i.d. for analysis and
250 £ 20 mm i.d. for preparation; Nacalai
Tesque Inc., Kyoto, Japan). For column
chromatography, silica gel BW-200 and
Chromatorex ODS DM1020T (150–300
mesh; Fuji Silysia Chemical, Aichi, Japan)
were used. Thin-layer chromatography
was performed on precoated silica gel 60
F254 or RP-18 F254S plates (0.25 mm
thickness; Merck KGaA, Darmstadt,
Germany) and visualized by spraying 1%
aqueous Ce(SO4)2 in 10% aqueous H2SO4
and followed by heating. p-Nitrobenzyl-
N,N0-diisopropylisourea, Dowex HCR W
resin (Hþ form), Amberlite IRA-400 resin
(OH2 form), and S-(þ)-2-methylbutyric
acid were purchased from Sigma-Aldrich
Japan Co. (Tokyo, Japan). D-(þ)-Glucose
and L-(2)-rhamnose were obtained from
Wako Pure Chemical Industries (Osaka,
Japan).
1
comparison of its H and 13C NMR and
FAB-MS data with the reported values in
the literature [7].
3. Experimental
3.1 General experimental procedures
Optical rotations were measured on a
Perkin-Elmer 341 polarimeter (Waltham,
MA, USA); UV spectra were recorded on a
Perkin-Elmer Lambda 650 UV–vis spec-
trophotometer; IR spectra were recorded
on a WQF-410 FT-IR spectrometer
(Liuzhou Zinc Products Co., Beijing,
China); FAB-MS and HR-FAB-MS were
gained on a JEOL JMS-SX 102A mass
spectrometer; and 1D and 2D NMR
(13C–1H COSY and HMBC) spectra
were measured on a JEOL JNM-LA 500
spectrometer using tetramethylsilane as
internal standard. HPLC was performed on
a Shimadzu LC-6AD liquid chromato-
graph equipped with a Shimadzu RID-10A
refractive index detector and Cosmosil
3.2 Plant material
The whole herbs of Sinocrassula indica
were purchased from Yulin Traditional
Chinese Medicine Market (Yulin, Guangxi,
China)viaTochimotoTenkaidoCo.(Osaka,

Journal of Asian Natural Products Research
889
Japan). The plant was botanically identified
by Prof. D.X. Zhang in South China
Botanical Garden, Chinese Academy of
Sciences (Guangzhou, China). A voucher
specimen (WT041220) has been deposited
at the Herbarium of South China Botanical
Garden.
635 [M 2 H]2; HR-FAB-MS: m/z
659.1592 [M þ Na]þ (calcd for
C29H32O16Na, 659.1583).
3.3.2 Sinocrassoside B6 (2)
20
Yellow amorphous powder; ½aꢀD 258.2
(c 1.50, MeOH); UV (MeOH) lmax (log 1)
260 (4.30), 355 (4.15) nm; IR (KBr) nmax
1
3432, 1725, 1660, 1602, 1075 cm21; H
3.3 Extraction and isolation
The powder of air-dried herbs (9.9 kg) was
extracted three times with methanol by
heating under reflux for 3 h each time.
Evaporation of the solution under reduced
pressure gave a methanolic extract (764 g,
7.7%). The extract (725 g) was dissolved
in water and sequentially partitioned 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 CHCl3–
MeOH (10–50% MeOH, v/v) as eluents to
afford eight fractions. Fraction 2 (30.4 g of
the total 31.6 g) was separated by Chro-
matorex ODS (610 g) column chromatog-
raphy using aqueous MeOH (20–30%,
v/v) to yield fractions 2-1–2-7. Fraction 2-4
(600 mg) was purified by HPLC using 45%
aqueous MeOH (v:v) as a mobile phase
at the flow rate of 9 ml/min to furnish
compounds 2 (tR 24.9 min, 76.3 mg,
0.0035%) and 5 (tR 27.6 min, 38.6 mg,
0.0018%). Fraction 2-5 (602 mg) was
purified by HPLC using 45% aqueous
MeOH as a mobile phase at the flow
rate of 9 ml/min to give compounds
1 (tR 45.3 min, 43.0 mg, 0.0008%),
3 (tR 50.8 min, 35.8 mg, 0.0007%), and 4
(tR 43.2 min, 22.0 mg, 0.0004%).
(500 MHz) and 13C NMR (125 MHz)
spectral data, see Tables 1 and 2; FAB-
MS: m/z 653 [M þ H]þ, 675 [M þ Na]þ,
651 [M 2 H]2; HR-FAB-MS: m/z
675.1541 [M þ Na]þ (calcd for
C29H32O17Na, 675.1532).
3.3.3 Sinocrassoside B7 (3)
20
Yellow amorphous powder; ½aꢀD 265.2
(c 0.80, MeOH); UV (MeOH) lmax (log 1)
258 (4.26), 355 (4.19) nm; IR (KBr) nmax
1
3435, 1726, 1662, 1603, 1075 cm21; H
(500 MHz) and 13C NMR (125 MHz)
spectral data, see Tables 1 and 2; FAB-
MS: m/z 681 [M þ H]þ, 703 [M þ Na]þ,
679 [M 2 H]2; HR-FAB-MS: m/z
703.1854 [M þ Na]þ (calcd for
C31H36O17Na, 703.1845).
3.3.4 Sinocrassoside D4 (4)
20
Yellow amorphous powder; ½aꢀD þ 138.5
(c 0.50, MeOH); UV (MeOH) lmax (log 1)
260 (4.12), 348 (3.97) nm; IR (KBr) nmax
1
3432, 1720, 1655, 1605, 1062 cm21; H
(500 MHz) and 13C NMR (125 MHz)
spectral data, see Tables 1 and 2; FAB-
MS: m/z 711 [M þ H]þ, 733 [M þ Na]þ,
709 [M 2 H]2; HR-FAB-MS: m/z
733.1962 [M þ Na]þ (calcd for
C32H38O18Na, 733.1950).
3.3.1 Sinocrassoside A13 (1)
20
Yellow amorphous powder; ½aꢀD 272.5
(c 1.00, MeOH); UV (MeOH) lmax (log 1)
267 (4.20), 349 (4.11) nm; IR (KBr) nmax
3.4 Alkaline hydrolysis of 1–4
1
3430, 1721, 1655, 1597, 1060 cm21; H
A solution of each compound (1–3: ca.
4 mg, 4: 6.2 mg) in 10% aqueous KOH–
50% aqueous 1,4-dioxane (1:1, v/v, 1 ml)
was stirred at 378C for 1 h, neutralized
(500 MHz) and 13C NMR (125 MHz)
spectral data, see Tables 1 and 2; FAB-
MS: m/z 637 [M þ H]þ, 659 [M þ Na]þ,

890
H.-H. Xie and M. Yoshikawa
with Dowex HCR W2 resin (Hþ form),
and filtrated to give a hydrolysate.
was added to 1 M HCl (0.5 ml) and stirred
at 808C for 3 h. The solution was extracted
with EtOAc (0.5 ml £ 3) and the aqueous
layer was neutralized with Amberlite IRA-
400 resin (OH– form) and then filtrated.
The solution was condensed to ca. 0.15 ml
and passed through a filter (0.45 mm),
and then subjected to HPLC analysis
[column: Kaseisorb LC NH2-60-5 (5 mm,
250 £ 4.6 mm i.d.; Tokyo Kasei Co.,
Tokyo, Japan); detection: Shodex OR-2
optical rotation detector; mobile phase:
85% aqueous CH3CN (v/v); flow rate:
0.8 ml/min] to identify D-glucose and
L-rhamnose by comparison of the retention
time and optical rotation with authentic
samples (L-rhamnose: tR 7.8 min, negative
optical rotation; D-glucose: tR 13.9 min,
positive optical rotation) [3,4].
3.4.1 Characterization of 1a, 2a, and 4a
A part of the hydrolysate (1–3: 0.6 ml, 4:
0.4 ml) was condensed under reduced
pressure to give a residue, which was
dissolved in MeOH (0.1 ml) and passed
through a filter (0.45 mm) and then
subjected to HPLC analysis to characterize
kaempferol 3-O-b-D-glucopyranosyl-7-
O-a-L-rhamnopyranoside (1a) from 1,
quercetin 3-O-b-D-glucopyranosyl-7-O-
a-L-rhamnopyranoside (2a) from 2 and 3,
and sinocrassoside D2 (4a) from 4 by
direct comparison with the samples
previously obtained from this herb [3,5].
3.4.2 Characterization of (S)-(þ)-2-
methylbutanoic acid
A part of the hydrolysate of 4 (0.4 ml) was
condensed under vacuum to yield a
residue, which was dissolved in dichlor-
oethane (1 ml) and added p-nitrobenzyl-
N,N0-diisopropylisourea (5 mg) and stirred
at 808C for 1 h. Evaporation of the reaction
mixture gave a residue, which was
dissolved in MeOH (0.1 ml) and passed
through a filter (0.45 mm), and then
subjected to HPLC analysis [column:
YMC-Pack ODS-AQ (5 mm, 250 £
4.6 mm i.d.; YMC Co., Kyoto, Japan);
mobile phase: 20% CH3CN in 0.1%
aqueous H3PO4 (v/v); detection: Shodex
OR-2 optical rotation detector (Showa
Denko Co., Tokyo, Japan); flow rate:
1.0 ml/min] to identify S-(þ)-2-methylbu-
tyric acid (tR: 15.4 min, positive optical
rotation) by direct comparison of its
retention time and optical rotation with
the commercial standard [2,3].
Acknowledgements
The research 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 Guangzhou Science and
Technology Project (Grant No. 12C14061591).
References
[1] State Administration of Traditional Chi-
nese Medicine, Chinese Materia Medica
(Shanghai Science & Technology Press,
Shanghai, China, 1999), Vol. 9, p. 778.
[2] M. Yoshikawa, T. Wang, T. Morikawa, H.
Xie, and H. Matsuda, Chem. Pharm. Bull.
55, 1308 (2007).
[3] T. Morikawa, H. Xie, T. Wang, H.
Matsuda, and M. Yoshikawa, Chem.
Pharm. Bull. 56, 1438 (2008).
[4] K. Ninomiya, T. Morikawa, H. Xie, H.
Matsuda, and M. Yoshikawa, Heterocycles
75, 1983 (2008).
[5] T. Morikawa, H. Xie, T. Wang, H.
Matsuda, and M. Yoshikawa, Chem.
Biodiver. 6, 411 (2009).
[6] H. Xie and M. Yoshikawa, J. Asian Nat.
Prod. Res. 14, 503 (2012).
[7] H. Xie, T. Morikawa, H. Matsuda, S.
Nakamura, O. Muraoka, and M. Yoshikawa,
Chem. Pharm. Bull. 54, 669 (2006).
3.4.3 Characterization of D-glucose and
L-rhamnose
The rest of the hydrolysate was condensed
under vacuum to afford a residue, which