Aromatic acid derivatives from the lateral roots of Aconitum carmichaelii

Article abstract of DOI:10.1080/10286020.2014.939585

Seven new aromatic acid derivatives (1-7), together with five known analogs, were isolated from the lateral roots of Aconitum carmichaelii. Structures of the new compounds were determined by spectroscopic and chemical methods as 4-methyl (-)-(R)-hydroxyeu

Full text of DOI:10.1080/10286020.2014.939585

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Aromatic acid derivatives from the
lateral roots of Aconitum carmichaelii
Zhi-Bo Jianga, Bing-Ya Jianga, Cheng-Gen Zhua, Qing-Lan Guoa,
Ying Penga, Xiao-Liang Wanga, Sheng Lina & Jian-Gong Shia
a State Key Laboratory of Bioactive Substance and Function of
Natural Medicines, Institute of Materia Medica, Chinese Academy
of Medical Sciences and Peking Union Medical College, Beijing
100050, China
Published online: 27 Aug 2014.
To cite this article: Zhi-Bo Jiang, Bing-Ya Jiang, Cheng-Gen Zhu, Qing-Lan Guo, Ying Peng,
Xiao-Liang Wang, Sheng Lin & Jian-Gong Shi (2014) Aromatic acid derivatives from the lateral
roots of Aconitum carmichaelii, Journal of Asian Natural Products Research, 16:9, 891-900, DOI:
10.1080/10286020.2014.939585
To link to this article: http://dx.doi.org/10.1080/10286020.2014.939585
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Journal of Asian Natural Products Research, 2014
Vol. 16, No. 9, 891–900, http://dx.doi.org/10.1080/10286020.2014.939585
Aromatic acid derivatives from the lateral roots of Aconitum
carmichaelii
Zhi-Bo Jiang, Bing-Ya Jiang, Cheng-Gen Zhu, Qing-Lan Guo, Ying Peng,
Xiao-Liang Wang, Sheng Lin and Jian-Gong Shi*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical
College, Beijing 100050, China
(Received 11 March 2014; final version received 25 June 2014)
Seven new aromatic acid derivatives (1–7), together with five known analogs, were
isolated from the lateral roots of Aconitum carmichaelii. Structures of the new
compounds were determined by spectroscopic and chemical methods as 4-methyl (2)-
(R)-hydroxyeucomate (1), 4-butyl (2)-(R)-hydroxyeucomate (2), 4-butyl-1-methyl
(þ)-(R)-2-O-(40-hydroxy-30-methoxybenzoyl)malate (3), 1-butyl-4-methyl (þ)-(R)-2-
O-(40-hydroxy-30-methoxybenzoyl)malate (4), dimethyl (þ)-(R)-2-O-(40-hydroxy-30-
methoxybenzoyl)malate (5), dimethyl (þ)-(R)-2-O-(40-hydroxybenzoyl)malate (6),
and methyl (^)-3-(40-hydroxy-30-methoxyphenyl)-3-sulfopropionate (7), respectively.
Compounds 1 and 2 are 2-benzylmalates (eucomate derivatives), 3–6 belong to 2-O-
benzoylmalates, and 7 is a rare phenylpropionate containing a sulfonic acid group. The
absolute configurations of eucomate derivatives were confirmed by X-ray crystal-
lographic analysis of 4-methyl eucomate (11).
Keywords: Aconitum carmichaelii; Ranunculaceae; (R)-2-benzylmalates; (R)-2-O-
benzoylmalates; methyl 3-(40-hydroxy-30-methoxyphenyl)-3-sulfopropionate
1. Introduction
medicines and their biological effects [4],
a water extract of the raw lateral roots of A.
carmichaelii was investigated. In previous
work, 4 new C20- and 22 new C19-
diterpenoid alkaloids, together with two
known analogs, were characterized in
several fractions obtained from the extract
[5,6]. Herein, the remaining fractions of
the same extract were investigated, leading
to the isolation of seven new aromatic acid
derivatives (1–7), along with five pre-
viously known analogs (Figure 1). Com-
pounds 1 and 2 are (R)-2-benzylmalates,
3–6 may be classified as (R)-2-O-
benzoylmalate derivatives, and 7 is a rare
phenylpropionate containing a sulfonic
acid group. In this paper, we describe the
isolation and structure elucidation of the
new compounds.
The lateral roots of Aconitum carmichaelii
Debx. (Ranunculaceae), known as ‘fu zi’,
are one of the most important herbal drugs
in traditional Chinese medicine [1]. They
are used in raw or prepared forms to treat
various diseases such as cadianeuria,
neuralgia, and rheumatalgia in China,
Japan, and Korea [2]. Previous studies
have shown that toxic aconitine C19-
diterpenoid alkaloids are the main active
constituents of these drugs, and more than
40 aconitine alkaloids have been isolated
from the raw and prepared forms of ‘fu zi’
[3]. However, the nonalkaloid constituents
have rarely been considered. As part of a
program to systematically study the
chemical diversity of traditional Chinese
*Corresponding author. Email: shijg@imm.ac.cn
q 2014 Taylor & Francis

892
Z.-B. Jiang et al.
1
1
O
7'
COOR1
COOR1
H
R
1'
1'
R
2
3'
2
7' O
HO
3'
COOR2
COOR2
HO
5'
HO
4
4
5'
1 R = OH, R1 = H, R2 = CH3
2 R = OH, R1 = H, R2 = n-But
8 R = OH, R1 = R2 = H
9 R = R1 = R2 = H
3 R = OCH3, R1 = CH3, R2 = n-But
4 R = OCH3, R1 = n-But, R2 = CH3
5 R = OCH3, R1 = R2 = CH3
6 R = H, R1 = R2 = CH3
10 R = H, R1 = R2 = CH3
11 R = R1 = H, R2 = CH3
1" 2" 3" 4"
n-But = CH2CH2CH2CH3
SO3H
1' COOCH3
H3CO
3
1
3'
HO
5'
7
Figure 1. The structures of compounds 1–11.
2. Results and discussion
monoester of hydroxyeucomic acid (8) [7].
The suggestion was supported by compar-
ing the NMR data of 1 with those of the co-
occurring (2)-(R)-eucomic acid analogs
(9–11) and was further confirmed by 2D
NMR data analysis. The HMQC and
1H–1H COSY spectra of 1 provided
unambiguous assignments of proton and
carbon signals in the NMR spectra. In the
HMBC spectrum, correlations from H2-3
to C-1, C-2, and C-4, combined with the
chemical shifts of these proton and carbon
resonances, revealed the presence of a 2-
substituted malic acid moiety in 1. HMBC
correlations of H-20/C-40, C-60, and C-70;
H-50/C-30 and C-40; H-60/C-20, C-40, and C-
70; and H2-70/C-10, C-20, and C-60, together
with their chemical shifts, verified the
occurrence of a 30,40-dihydroxybenzyl
moiety. In addition, HMBC correlations
from H2-3 to C-70 and from H2-70 to C-1,
C-2, and C-3 confirmed that the 30,40-
dihydroxybenzyl moiety was located at C-
2 of the malic acid moiety. The HMBC
correlation from OCH3 to C-4 demon-
strated that the malic acid was esterified at
the 4-carboxyl moiety. Accordingly, the
planar structure of compound 1 was
determined as 4-methyl hydroxyeucomate.
Compound 1 was obtained as a colorless
20
gum with ½aꢀD 242.5 (c 0.18, MeOH).
The IR spectrum of 1 showed absorption
bands for hydroxyl (3390 cm21), carbonyl
(1725 cm21), and aromatic (1608 and
1526 cm21) functional groups. The mol-
ecular formula C12H14O7 was indicated by
HR-ESI-MS at m/z 269.0658 [M 2 H]2,
combined with the NMR spectral data. The
1H NMR spectrum of 1 in acetone-d6
showed signals attributed to a meta–para-
trisubstituted phenyl group at dH 6.79 (d,
J ¼ 1.5 Hz, H-20), 6.69 (d, J ¼ 8.5 Hz, H-
50), and 6.58 (dd, J ¼ 8.5, 1.5 Hz, H-60);
two methylenes at dH 2.95 (d, J ¼ 15.5 Hz,
H-3a), 2.62 (d, J ¼ 15.5 Hz, H-3b),
2.90 (d, J ¼ 14.0 Hz, H-70a), and 2.82
(d, J ¼ 14.0 Hz, H-70b); and a methoxy
group at dH 3.58 (s). The 13C NMR and
DEPT spectra of 1 showed carbon signals
(Table 1) corresponding to the above units
including two oxygen-bearing aromatic
carbons [dC 145.2 (C-30) and 144.8 (C-40)],
in addition to two carbonyl carbons [dC
175.8 (C-1) and 171.4 (C-4)] and an
oxygen-bearing sp3-hybridized quaternary
carbon [dC 76.1 (C-2)]. These spectro-
scopic data suggest that 1 is a methyl

Journal of Asian Natural Products Research
893
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d

894
Z.-B. Jiang et al.
Figure 2. ORTEP diagram of compound 11.
The similarities of the negative optical
rotations for 1 with those of hydroxyeu-
comic acid (8) [7], (2)-(R)-eucomic acid
(9) [8], dimethyl eucomate (10) [7,8], and
yunnanensin C [9] suggest that 1 possesses
the same absolute configuration as these
known natural products. Because only the
absolute configuration of 9 among the
known analogs had been determined by
synthetic correlation with piscidic acid [8],
an available single crystal of the co-
occurring 4-methyl eucomate (11)
was replaced by a butoxy group in 2. This
demonstrated that2 was the butyl monoester
of hydroxyeucomic acid, which was con-
firmed by 2D NMR data analysis and
hydrolysis. In particular, the 1H–1H COSY
correlations of H2-100/H2-200/H2-300/H3-400
and the HMBC correlation of H2-100/C-4
verified that the butoxy group was also
incorporated in the 4-carboxylic acid in 2.
Hydrolysis of 2 with 0.2 N NaOH produced
hydroxyeucomic acid (8), which exhibited
1
20
H NMR, HR-ESI-MS, and ½aꢀD data
20
{½aꢀD 2 18.9 (c 0.23, MeOH)}, which
consistent with those in the literature [7].
Therefore, the structure of compound 2 was
determined as 4-butyl (2)-(R)-hydroxyeu-
comate.
had been previously reported as methyl
eucomate without optical rotation data
[10], was analyzed by X-ray crystal-
lography using Cu Ka radiation. An
ORTEP drawing, with the atom numbering
indicated, is shown in Figure 2. The
absolute configuration was determined on
the basis of the Flack parameter of 20.1
(3), which supported the configuration
assignment for 1. Therefore, the structure
of compound 1 was determined as 4-
methyl (2)-(R)-hydroxyeucomate.
Compound 3, a colorless gum with
20
½aꢀD þ 12.8 (c 0.27, MeOH), has the
molecular formula C17H22O8 as indicated
by HR-ESI-MS at m/z 355.1390 [M þ H]þ
and the NMR spectral data. The NMR
spectral data (Table 1) showed the presence
of a disubstituted benzoyl, a butoxy, and two
methoxy groups, as well as a malic acid
moiety in 3. This was confirmed by 2D
NMR data analysis. The proton and carbon
signals in the NMR spectra were unambigu-
Compound 2 was obtained as a colorless
20
gum with½aꢀD 2 21.8(c0.23, MeOH).The
1
molecular formula C15H20O7 was indicated
from HR-ESI-MS at m/z 313.1280
[M þ Na]þ, combined with the NMR
spectral data. The UV, IR, and NMR
spectroscopic features of 2 (see Section 3
and Table 1) were similar to those of 1.
Comparison of the NMR spectral data of 2
and 1 indicated that the methoxy group in 1
ously assigned by the HMQC and H–1H
COSY experiments. The vicinal coupling
COSY correlation of H-2/H2-3 and two- and
three-bond HMBC correlations from H-2 to
C-1,C-3,andC-4andfromH2-3toC-1,C-2,
and C-4, in combination with the chemical
shiftsoftheseprotonandcarbonresonances,
confirmed the malic acid moiety in 3. The

Journal of Asian Natural Products Research
895
COSY correlation of H-50/H-60 and HMBC
correlations of H-20/C-10, C-30, C-40, C-60,
and C-70; H-50/C-10, C-30, and C-40; and H-
60/C-20, C-40, and C-70, together with their
chemical shifts, verified the 30,40-disubsti-
tuted benzoyl unit. The butoxy group was
confirmed by the COSY correlations of H2-
100/H2-200/H2-300/H3-400 and HMBC corre-
lations of H2-100/C-200 and C-300; H2-200/C-100,
C-300, and C-400; H2-300/C-100, C-200, and C-400;
and H3-400/C-200 and C-300. In addition, the
HMBC correlations from H-2 to C-70 and
from H2-100 to C-4 revealed that the benzoyl
and butyl units wereincorporatedasestersat
the2-OHand4-carboxylgroupsofthemalic
acid moiety in 3, respectively. The HMBC
correlations of 30-OCH3/C-30 and 1-OCH3/
C-1 demonstrated that the two methoxy
groups were located at C-30 and C-1 in 3.
The absolute configuration of compound 3
was determined by comparing its specific
rotationwiththe reported datafor the known
compounds, (S)-benzoyl malic acid, and
(R)-2-O-galloyl malic acid dimethyl ester
[11]. The specific rotation of (S)-benzoyl
correlations of 4-OCH3/C-4 and H2-100 to
C-1 in the HMBC spectrum of 4.
Compound 4 displayed a specific rotation
similar to that of 3, indicating that 4 had
the same R configuration. Therefore, the
structure of compound 4 was determined
as 1-butyl-4-methyl (þ)-(R)-2-O-(40-
hydroxy-30-methoxybenzoyl)malate.
Compound 5, a colorless gum with
20
½aꢀD þ 31.4 (c 0.70, MeOH), has the
molecular formula C14H16O8 as indicated
by HR-ESI-MS at m/z 313.0916 [M þ H]þ
and the NMR spectral data. Comparison of
the NMR spectral data of 5 with those of 4
(Table 1) indicated replacement of the
butoxy group in 4 by a methoxy group in 5,
which was confirmed by 2D NMR data
analysis of 5. In particular, the HMBC
correlations of 1-OCH3/C-1 and 4-OCH3/
C-4 verified that both the 1- and 4-
carboxyl groups of the malic acid moiety
in 5 were methyl ester. The R configur-
ation was indicated by its specific rotation
similar to those of 3 and 4. Therefore, the
structure of compound 5 was determined
as dimethyl (þ)-(R)-2-O-(40-hydroxy-30-
methoxybenzoyl)malate.
21
malic acid has a negative value ½aꢀD 2 7 (c
1.0, CHCl3), whereas that of (R)-galloyl
malic acid dimethyl ester is positive
Compound 6 was obtained a colorless
20
20
½aꢀD þ 21 (c 0.1, MeOH) [11]. Therefore,
gum with ½aꢀD þ 5.5 (c 0.11, MeOH). Its
compound 3 was assigned the R configur-
ation based on its positive specific rotation,
and its structure was designated as 4-butyl-
1-methyl (þ)-(R)-2-O-(40-hydroxy-30-
methoxybenzoyl)malate.
molecular formula C13H14O7 was indi-
cated by HR-ESI-MS at m/z 281.0661
[M 2 H]2 and the NMR spectral data.
Comparison of the NMR spectral data
between 6 and 5 (Table 1) showed
replacement of the 40-hydroxy-30-methox-
ybenzoyl unit in 5 by a 40-hydroxybenzoyl
unit in 6. This was further confirmed by 2D
NMR data analysis of 6. Particularly, the
HMBC correlations of C-70 with H-2, H-20,
and H-60; 1-OCH3/C-1; and 4-OCH3/C-4,
together with their shifts, confirmed that
the 40-hydroxybenzoyl unit and the two
methoxy groups were incorporated as
esters at the 2-OH and the 1- and 4-
carboxyl groups of the malic acid moiety
in 6, respectively. The positive optical
rotation of 6, similar to those of 3–5,
indicated the R configuration. Therefore,
Compound 4, a colorless gum with
20
½aꢀD þ 17.0 (c 0.41, MeOH), is an isomer
of 3 as indicated from the spectroscopic
data. Comparison of the NMR data for 4
with those of 3 indicated that the proton
and carbon resonances of the ester
methoxy group in 4 were deshielded by
DdH þ 0.06 ppm and DdC þ 0.5 ppm,
respectively, whereas the oxymethylene
resonances of the butoxy group in 4 were
shielded by DdH 20.05 ppm and DdC
20.6 ppm, respectively. This suggested
that the 4-butyl-1-methyl ester in 3 was
replaced by a 1-butyl-4-methyl ester in 4.
This was confirmed by the HMBC

896
Z.-B. Jiang et al.
compound 6 was determined as dimethyl
(þ)-(R)-2-O-(40-hydroxybenzoyl)malate.
Compound 7 was obtained a white
amorphous powder with ½aꢀD 0 (c 0.26,
eucomate (10) [8], 4-methyl eucomate
(11) [10], ferulic acid [13], methyl ferulate
[14], butyl ferulate [15], methyl 4-O-b-D-
glucopyranosylferulate [16], and linocin-
namarin [17].
20
MeOH). Its molecular formula C11H14O7S
was indicated by HR-ESI-MS at m/z
313.0353 [M þ Na]þ and the NMR
spectral data. NMR data analysis
(Table 1) revealed the presence of a
methine, a methylene, a carbonyl, and two
methoxy groups, in addition to a 40-
hydroxy-30-methoxyphenyl moiety. This,
combined with the molecular composition,
suggested that 7 was an unusual methyl
(40-hydroxy-30-methoxyphenyl)propionate
derivative containing a sulfo group, which
was further confirmed by 2D NMR data.
The signals in the NMR spectra were
assigned by the HMQC and 1H–1H COSY
experiments of 7. The COSY correlations
of H2-2/H-3 and the HMBC correlations
from H2-2 to C-1 and C-3 and from
1-OCH3 to C-1 verified the presence of a
substituted methyl propionate moiety. The
COSY correlations of H-50/H-60 and the
HMBC correlations of H-20/C-10, C-30, C-
40, and C-60; H-50/C-10, C-30, C-40, and
C-60; H-60/C-20, C-40, and C-50; and from
30-OCH3/C-30 verified the presence of the
40-hydroxy-30-methoxyphenyl moiety.
In addition, the HMBC correlations from
H2-2 to C-10 and from H-20 and H-60 to C-3
demonstrated that both the 40-hydroxy-30-
methoxyphenyl moiety and the sulfo group
were located at C-3 of the methyl
propionate moiety in 7. This was sup-
ported by comparing the NMR data of
7 with those of synthetic analogs reported
in the literature [12]. Compound 7 is
optically inactive, indicating that it was
obtained as a racemate. Therefore, the
structure of compound 7 was determined
as methyl (^)-3-(40-hydroxy-30-methoxy-
phenyl)-3-sulfopropionate.
The methyl and butyl esters, including
1–7, were considered natural products
because methylation or butylation of the
acids 8 and 9 was unlikely to occur during
the isolation procedure. Furthermore,
esterification did not proceed under
simulated conditions heating MeOH or n-
BuOH solutions of the acids or H2O
solutions of the esters, either with or
without silica gel, MCI gel, Sephadex LH-
20, or reversed C18 silica gel at 458C for
48 h. Although the HPLC–ESI-MS tech-
nique was used to detect the occurrence of
these compounds in the crude extract, none
were observed, possibly due to their low
contents and the limit of the ESI-MS
detection technique.
All the compounds isolated in this
study were assayed for their neuroprotec-
tive effects against SK-N-SH neuroblas-
toma cells [18]. As compared with the
blank control, at a concentration of 10 mM,
(2)-(R)-eucomate (10) and butyl ferulate
showed protective activity against oxygen-
glucose deprivation-induced neurotoxi-
city, with cell survival rates improved by
39.96% ^ 4.63% and 23.39% ^ 3.48%,
respectively. Methyl ferulate and linocin-
namarin exhibited protective activity
against L-glutamic acid-induced neuro-
toxicity, with cell survival rates improved
by 14.03% ^ 4.50% and 11.17%
^ 3.48%, respectively. The isolates were
also assessed for their activities against
DL-galactosamine-induced WB-F344 cell
damage [19], and nitric oxide production
in mouse peritoneal macrophages [20], as
well as for their cytotoxicity against
several human cancer cell lines [21], but
were found to be inactive at 10 mM.
The known compounds were identified
by comparison of their spectroscopic data
with those reported in the literature as (2)-
(R)-hydroxyeucomic acid (8) [7], (2)-(R)-
eucomic acid (9) [8], dimethyl (2)-(R)-

Journal of Asian Natural Products Research
897
3. Experimental
Dr Yan Ren (Chengdu University of
Traditional Chinese Medicine, Sichuan
610075, China). A voucher specimen (no.
ID-S-2383) was deposited at the herbar-
ium of the Department of Chemistry of
Natural Products, Institute of Materia
Medica, Chinese Academy of Medical
Sciences and Peking Union Medical
College, Beijing 100050, China.
3.1 General experimental procedures
Optical rotations were measured on P-
2000 polarimeter (JASCO, Tokyo, Japan).
UV spectra were measured on a JASCOP-
650 spectrometer (JASCO). IR spectra
were recorded on a Nicolet 5700 FT-IR
microscope instrument (FT-IR microscope
transmission) (Thermo Electron Corpor-
ation, Madison, WI, USA). NMR spectra
1
were obtained at 500 or 600 MHz for H,
3.3 Extraction and isolation
and 125 or 150 MHz for 13C on Inova 500
or SYS 600 spectrometers (Varian Associ-
ates Inc., Palo Alto, CA, USA) in acetone-
d6, MeOH-d4, or CDCl3 with solvent peaks
used as references. ESI-MS and HR-ESI-
MS data were measured using an Accu-
ToFCS JMS-T100CS spectrometer (Agi-
lent Technologies, Ltd, Santa Clara, CA,
USA). Column chromatography was per-
formed with silica gel (200–300 mesh,
Qingdao Marine Chemical Inc., Qingdao,
China) and Sephadex LH-20 (Pharmacia
Biotech AB, Uppsala, Sweden). HPLC
separation was performed on an instru-
ment consisting of a Waters 600 controller,
a Waters 600 pump, and a Waters 2487
dual l absorbance detector (Waters
Corporation, Milford, MA, USA), with a
YMC-Pack Ph (250 mm £ 10 mm i.d.)
column packed with phenyl-silica gels (5
mm) (YMC Co., Ltd, Kyoto, Japan). TLC
was carried out with glass precoated silica
gel GF254 plates (Qingdao Marine Chemi-
cal Inc.). Spots were visualized under UV
light or by spraying with 7% H2SO4 in
95% EtOH followed by heating. Unless
otherwise noted, all chemicals were
obtained from commercially available
sources and were used without further
purification.
The air-dried lateral roots of A. carmichae-
lii (50 kg) were powdered and extracted
with H2O (3 £ 150 L £ 6 h) at 408C. The
H2O extract was concentrated to 120 L
under reduced pressure, subjected to
chromatography over a macroporous adsor-
bent resin (HPD-110, 19 kg) column
(20 cm £ 200 cm), and eluted successively
with H2O (50 L), 30% EtOH (120L), 50%
EtOH (120L), and 95% EtOH (100L) to
afford the corresponding fractions A–D.
After removal of the solvent, fraction C
(3.5kg) was chromatographed over MCI
gel CHP 20P with successive elution using
H2O (10 L), 30% EtOH (30 L), 50% EtOH
(20 L), and 95% EtOH (10 L) to give
fractions C1–C4. Fraction C2 (600g) was
again fractionated via MCI gel CHP 20P
with successive elution using H2O (10 L),
30% EtOH (30 L), 50% EtOH (20L), and
95% EtOH (10 L) to give subfractions C2-
1–C2-4. Subfraction C2-1 (200 g) was
dissolved in H2O (500mL), basified with
concentrated ammonium hydroxide
(25 mL) to pH 10, and then extracted with
EtOAc (500mL £ 4). The aqueous layer
was acidified with 6.0 N HCl (66 mL) to pH
4, and partitioned with n-butanol (500 mL
£ 3). The n-butanol phase was concentrated
under reduced pressure to give a residue, C-
2-B (12 g), which was chromatographed
over silica gel (100 g) eluting with a
gradient of CHCl3–MeOH (95:5–85:15)
to give fractions C-2-B-1–C-2-B-6. Frac-
tion C-2-B-3 (3.9g) was separated by
reversed-phase medium pressure liquid
chromatography (RP-MPLC, 30% MeCN
3.2 Plant material
The lateral root of A. carmichaelii Debx
was collected in June 2009 from the
culture field in Jiangyou, Sichuan Pro-
vince, China. Plant identity was verified by

898
Z.-B. Jiang et al.
in H2O, containing 0.1% TFA) to give C-2-
B-3-1–C-2-B-3-6, of which C-2-B-3-3
(500mg) was further separated by RP
HPLC (40% MeCN in H2O, containing
0.1% TFA, 1.5 mL/min) to yield 1
(10.5 mg, tR ¼ 18.3min) and 2 (4.1mg,
tR ¼ 22.2 min). Fraction C-4-B-3-4
(300mg) was chromatographed over silica
gel (petroleum ether–EtOAc, 50:1–10:1),
followed by semi-preparative RP HPLC
purification (70% MeOH in H2O, 1.5 mL/
min) to give 3 (7.0mg, tR ¼ 41.3min),
4 (5.2mg, tR ¼ 43.7min), methyl ferulate
(65.0 mg, tR ¼ 17.1min), and butyl ferulate
(73.5 mg, tR ¼ 20.0min). Fraction C-2-B-
3-5 (200mg) was successively chromato-
(4.44), 232 (sh, 4.10), 282 (3.93) nm; IR
max: 3390, 2968, 1725, 1676, 1608, 1527,
n
1443, 1362, 1286, 1202, 1149, 1121, 1046,
1000, 963, 877,796 cm21; for 1H NMR
(acetone-d6, 500 MHz) spectral data, see
Table 1; for 13C NMR (acetone-d6,
125 MHz) spectral data, see Table 1;
ESI-MS: m/z 269 [M 2 H]2; HR-ESI-
MS: m/z 269.0658 [M 2 H]2 (calcd for
C12H13O7, 269.0667).
3.3.2 4-Butyl (2)-(R)-
hydroxyeucomate (2)
20
Colorless gum; ½aꢀD 2 21.8 (c 0.23,
MeOH); UV (MeOH) lmax (log 1): 204
(4.22), 227 (3.60), 282 (3.25) nm; IR nmax:
graphed
over
Sephadex
LH-20
3464, 3325, 3224, 2961, 1747, 1716, 1617,
1598, 1515, 1440, 1376, 1277, 1218, 1178,
(petroleum ether–CHCl3–MeOH, 5:5:1)
and silica gel (petroleum ether–EtOAc,
15:1), followed by RP-HPLC separation
(75% MeOH in H2O, 1.5 mL/min) to give
5 (24.2 mg, tR ¼ 26.5min), 6 (15.6 mg,
tR ¼ 32.0min), 10 (8.5mg, tR ¼ 36.3min),
and 11 (63.0 mg, tR ¼ 17.0min). Fraction
C-2-B-6 (5.3g) was chromatographed over
Sephadex LH-20 (CHCl3–MeOH, 1:1) to
afford C-2-B-6-1–C-2-B-6-3. Fraction C-
2-B-6-1 (600mg) was further chromato-
graphed over Sephadex LH-20 eluting with
petroleum ether–CHCl3–MeOH (5:5:1),
and then purified by RP-HPLC (15%
MeCN in H2O, containing 0.1% TFA,
1.5 mL/min) to yield methyl 4-O-b-D-
1
1114, 1046, 990, 839, 780 cm21; for H
NMR (MeOH-d4, 500 MHz) spectral data,
see Table 1; for 13C NMR (MeOH-d4,
125 MHz) spectral data, see Table 1; ESI-
MS: m/z 335 [M þ Na]þ; HR-ESI-MS: m/z
313.1280 [M þ H]þ (calcd for C15H21O7,
313.1282), 335.1104 [M þ Na]þ (calcd for
C15H20O7Na, 335.1101).
3.3.3 4-Butyl-1-methyl (þ)-(R)-2-O-(40-
hydroxy-30-methoxybenzoyl)malate (3)
20
Colorless gum; ½aꢀD þ 12.8 (c 0.27,
MeOH); UV (MeOH) lmax (log 1): 204
(3.62), 220 (3.55), 265 (3.29), 295
(3.07)nm; IR nmax: 3366, 2919, 2851,
1686, 1598, 1516, 1457, 1390, 1288, 1204,
1141, 1056, 1030, 838, 801,723 cm21; for
1H NMR (acetone-d6, 500 MHz) spectral
data, see Table 1; for 13C NMR (acetone-d6,
125 MHz) spectral data, see Table 1; ESI-
MS: m/z 377 [M þ Na]þ; HR-ESI-MS: m/z
355.1390 [M þ H]þ (calcd for C17H23O8,
355.1387), 377.1210 [M þ Na]þ (calcd for
C17H22O8Na, 377.1207).
glucopyranosylferulate
tR ¼ 23.2 min) and
(40.0 mg,
linocinnamarin
(60.2 mg, tR ¼ 37.1min). Fraction C-2-B-
6-3 (330mg) was isolated by preparative
TLC (mobile phase: CHCl3 –MeOH,
4.5:1), followed by RP-HPLC separation
(10% MeCN in H2O, 1.5 mL/min) to yield
7 (3.7 mg, tR ¼ 21.0 min), 8 (6.7 mg,
tR ¼ 30.0min), 9 (8.2mg, tR ¼ 35.5min),
and ferulic acid (20.3 mg, tR ¼ 24.8min).
3.3.1 4-Methyl (2)-(R)-
hydroxyeucomate (1)
20
3.3.4 1-Butyl-4-methyl (þ)-(R)-2-O-(40-
hydroxy-30-methoxybenzoyl)malate (4)
20
Colorless gum; ½aꢀD 2 42.5 (c 0.18,
Colorless gum; ½aꢀD þ 17.0 (c 0.41,
MeOH); UV (MeOH) lmax (log 1): 205
MeOH); UV (MeOH) lmax (log 1): 204

Journal of Asian Natural Products Research
899
(4.10), 218 (4.07), 265 (3.87), 295 (sh, (4.23), 231 (3.67), 281 (3.20) nm; IR nmax
:
3.70) nm; IR nmax: 3350, 2957, 2919, 2850,
1734, 1681, 1635, 1464, 1207, 1181, 1138,
837, 801, 722 cm21; for 1H NMR (acetone-
d6, 500 MHz) spectral data, see Table 1; for
13C NMR (acetone-d6, 125 MHz) spectral
data, see Table 1; ESI-MS: m/z 377
[M þ Na]þ; HR-ESI-MS: m/z 355.1391
[M þ H]þ (calcd for C17H23O8, 355.1387),
3222, 2920, 2851, 1709, 1679, 1605, 1526,
1438, 1373, 1250, 1203, 119, 1045, 802,
1
724, 646 cm21; for H NMR (MeOH-d4,
600 MHz) spectral data, see Table 1; for
13C NMR (MeOH-d4, 150 MHz) spectral
data, see Table 1; ESI-MS: m/z 289
[M 2 H]2;HR-ESI-MS: m/z 308.0799
[M þ NH4]þ (calcd for C11H18O7NS,
308.0798), 313.0353 [M þ Na]þ (calcd
for C11H14O7SNa, 313.0352).
377.1210
[M þ Na]þ
(calcd
for
C17H22O8Na, 377.1207).
3.3.5 Dimethyl (þ)-(R)-2-O-(40-
3.3.8 X-ray crystallography of 11
hydroxy-30-methoxybenzoyl)malate (5)
C12H14O6, M ¼ 254.23, orthorhombic,
20
˚
Colorless gum; ½aꢀD þ 31.4 (c 0.70,
MeOH); UV (MeOH) lmax (log 1): 197
(4.51), 265 (3.95) nm; IR nmax: 3420, 3009,
2956, 2919, 2850, 1740, 1720, 1597,1515,
1434, 1375, 1284, 1211, 1110, 1055, 1029,
878, 782, 762 cm21; for 1H NMR (CDCl3,
500 MHz) spectral data, see Table 1; for
13C NMR (CDCl3, 125 MHz) spectral data,
see Table 1; ESI-MS: m/z 335.1
[M þ Na]þ; HR-ESI-MS: m/z 313.0916
[M þ H]þ (calcd for C14H17O8, 313.0918),
335.0738 [M þ Na]þ (calcd for
C14H16O8Na, 335.0737).
P212121, a ¼ 5.930 (3) A, b ¼ 6.793
˚
A,
˚
A,
˚
(2)
c ¼ 29.104
(5)
o
3
a ¼ b ¼ g ¼ 90 , V ¼ 1172.4 (7) A ,
Z ¼ 4, Dcalcd ¼ 1.44mg mm23, 3660 col-
lected reflections, 2102 reflections indepen-
dent, R1 ¼ 0.0427, wR2 ¼ 0.1045. The
data were collected on an Agilent Xcalibur
Eos Gemini diffractometer with Cu Ka
radiation (m ¼ 0.994) by using the v scan
technique with 2u from 12.168 to 138.648.
The crystal structures were solved by direct
methods by using SHELXS-97, and all
nonhydrogen atoms were refined anisotro-
pically using the least-squares method. All
hydrogen atoms were positioned by geo-
metrical calculations and difference Fourier
overlapping calculation. The absolute con-
figuration was determined onthe basis ofthe
Flack parameter 20.1(3). Crystallographic
data for the structure of 11 have been
deposited with the Cambridge Crystallo-
graphic Data Centre as supplementary
publication (CCDC 988618). Copies of
these data can be obtained free of charge
via www.ccdc.cam.ac.uk/conts/retrieving.
html (or from the CCDC, 12 Union Road,
Cambridge CB21EZ, UK; fax: (þ44) 1223-
336-033; or deposit@ccdc.cam.ac.uk).
3.3.6 Dimethyl (þ)-(R)-2-O-(40-
hydroxy-30-methoxybenzoyl)malate (6)
20
Colorless gum; ½aꢀD þ 5.5 (c 0.11,
MeOH); UV (MeOH) lmax (log 1): 202
(3.62), 217 (3.21), 259 (3.10) nm; IR nmax
:
3393, 3213, 2919, 2850, 1730, 1679, 1647,
1608, 1516, 1468, 1443, 1420, 1204, 1140,
1050, 802, 724 cm21; for 1H NMR
(acetone-d6, 500 MHz) spectral data, see
Table 1; for 13C NMR (acetone-d6,
125 MHz) spectral data, see Table 1;
ESI-MS: m/z 305 [M þ Na]þ; HR-ESI-
MS: m/z 281.0661 [M 2 H]2 (calcd for
C13H13O7, 281.0667).
3.3.7 Methyl (^)-3-(40-hydroxy-30-
methoxyphenyl)-3-sulfopropionate (7)
20
3.3.9 Hydrolysis of 2
A solution of 2 (7.3 mg) was hydrolyzed
with 0.2 N NaOH (5.0 mL) at 408C for
0.5 h. The reaction mixture was acidified
White amorphous powder; ½aꢀD 0 (c 0.26,
MeOH); UV (MeOH) lmax (log 1): 204

900
Z.-B. Jiang et al.
[6] F.P. Wang, D.L. Chen, H.Y. Deng, Q.H.
Chen, X.Y. Liu, and X.X. Jian, Tetra-
hedron 70, 2592 (2014).
[7] A. Mun’im, O. Negishi, and T. Ozawa,
Biosci. Biotechnol. Biochem. 67, 410 (2003).
[8] P. Sahakitpichan, C. Mahidol, W. Dis-
adee, N. Chimnoi, S. Ruchirawat, and
T. Kanchanapoom, Tetrahedron 69, 1031
(2013).
[9] S.Z. Shang, H. Chen, C.Q. Liang, Z.H.
Gao, X. Du, R.R. Wang, Y.M. Shi, Y.T.
Zheng, W.L. Xiao, and H.D. Sun, Arch.
Pharm. Res. 36, 1223 (2013).
[10] J. Jiang, W. Ye, Z. Chen, F. Lou, and
Z. Min, J. Chin. Pharm. Sci. 11, 1 (2002).
with 0.5 N HCl to pH 2, and evaporated to
dryness under reduced pressure. The
residue was chromatographed on a Sepha-
dex LH-20 column with elution by
MeOH–H2O (1:1), followed by RP-
HPLC purification using the mobile
phase MeOH–H2O (7:93) containing
20
0.1% TFA to yield 8: ½aꢀD 2 18.3
1
(c 0.30, MeOH); HR-ESI-MS and H and
13C NMR spectral data were consistent
with those of hydroxyeucomic acid in the
literature [7].
´
´
[11] H. Ma, T. Yuan, A. Gonzalez-Sarrıas, L.
Li, M.E. Edmonds, and N.P. Seeram, Nat.
Prod. Commun. 7, 45 (2012).
Acknowledgments
Financial support from the National Natural
Sciences Foundation of China (NNSFC; grant
Nos 30825044 and 20932007), the Program for
Changjiang Scholars and Innovative Research
Team in University (PCSIRT, grant No.
IRT1007), and the National Science and
Technology Project of China (No.
2012ZX09301002-002) is acknowledged.
[12] B.F. Lutnaes, B.O. Myrvold, R.A. Lauten,
and M.M. Endeshaw, Magn. Reson.
Chem. 46, 299 (2008).
[13] P. Zhang and X.W. Yang, China J. Chin.
Mater. Med. 33, 2914 (2008).
[14] K. Nakano, K. Murakami, Y. Takaishi,
and T. Tomimatsu, Chem. Pharm. Bull.
34, 5005 (1986).
[15] H.M. Ma, G. Chen, W. Li, X.M. Fan,
Z.F. Li, and Y.H. Pei, J Shenyang Pharm.
Univ. 28, 695 (2011).
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