Biotransformation of sinapic acid catalyzed by Momordica charantia Peroxidase

Article abstract of DOI:10.1021/jf0628072

Biotransformation of sinapic acid (1) with H₂O ₂/Momordica charantia peroxidase, which exists in the widely used food M. charantia, at pH 5.0, 43 °C, in the presence of acetone resulted in six compounds, including four new compounds. Compound 2 was the first isolation of a new unique sinapic acid tetrameric derivate, which is formed by peroxidase catalysis in vitro. Besides 2, three other new sinapic acid dimers, 3-5 have also been isolated. Their structures were established on the basis of spectroscopic data. Compound 5 showed a stronger antioxidative activity than the parent sinapic acid (1). Compounds 4 and 5 significantly inhibited the growth of HL-60 cell at the concentration of 10^-5 mol/L.

Full text of DOI:10.1021/jf0628072

J. Agric. Food Chem. 2007, 55, 1003−1008 1003
Biotransformation of Sinapic Acid Catalyzed by Momordica
charantia Peroxidase
HAI-LI LIU, XIANG WAN, XUE-FENG HUANG, AND LING-YI KONG*
Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang,
Nanjing 210009, People’s Republic of China
Biotransformation of sinapic acid (1) with H2O2/Momordica charantia peroxidase, which exists in the
widely used food M. charantia, at pH 5.0, 43 °C, in the presence of acetone resulted in six compounds,
including four new compounds. Compound 2 was the first isolation of a new unique sinapic acid
tetrameric derivate, which is formed by peroxidase catalysis in vitro. Besides 2, three other new sinapic
acid dimers, 3-5 have also been isolated. Their structures were established on the basis of
spectroscopic data. Compound 5 showed a stronger antioxidative activity than the parent sinapic
acid (1). Compounds 4 and 5 significantly inhibited the growth of HL-60 cell at the concentration of
10-5 mol/L.
KEYWORDS: Biotransformation; Momordica charantia peroxidase; sinapic acid; cinnamic acid; tetramer
INTRODUCTION
19). They redsulted from oxidative coupling of sinapate ester
and represented the products of 8-8 radical coupling. Some
peroxidases such as CWPO-C were able to oxidize syringyl
moieties which were hard to be oxidized by others (20-22).
However, only a few dehydrodimers and higher oligomers of
sinapic acid have been obtained. With the aim of studying the
biotransformation of sinapic acid by purified MCP and screening
the bioactive properties of higher oligomers, the biotransfor-
mation was carried out in this research.
The fruit of Momordica charantia is one of the most widely
used vegetables in China. Previously, we reported the physical
and chemical characterization of M. charantia peroxidase
(MCP), a novel plant peroxidase with high acidic amino acid
purified from the fruits of M. charantia (1). We found that
although MCP shared spectroscopic and kinetic features with
other peroxidases, the enzyme had several unique characteristics,
including enzyme pH stability (pH 3.8∼8.0) and thermostability
(20∼45 °C) wider than those of other peroxidases such as
horseradish peroxidase (2). We also found that the transforma-
tion efficiency could be higher at 43 °C compared with the room
temperature. Therefore MCP can be expected to oxidize a wider
range of substrates, especially cinnamic acid derivatives,
considering its potential applications for useful biotransforma-
tion. Recently, we have applied purified MCP to transform
ferulic acid, which is an extremely abundant and widespread
cinnamic acid derivative, into some oxidation products. Among
them, two novel dehydrotrimers have been obtained and a dimer
has shown more powerful anti-inflammation activity than the
parent ferulic acid (1, 3).
Sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid, 1,
Figure 1), which is also a widespread cinnamic acid derivative
(4) with anti-inflammation and antioxidation capabilities (5, 6),
exists in many foods and fruits, such as the fruit of Citrus limon,
Vitis Vinifera, and the root of Allium cepa (7-9). Sinapic acid
is a good substrate for many oxidoreductases such as polyphenol
oxidase from the white-rot fungus Trametes Versicolor (10-
12), cell wall-bound peroxidases (13), and horseradish peroxi-
dase (14). Some dehydrodimers obtained both in vitro and in
vivo by oxidoreductases have already been characterizied (15-
MATERIALS AND METHODS
General Procedures. Positive-ion HR-FABMS was recorded with
a JEOL HX-110 spectrometer using m-nitrobenzyl alcohol as a matrix.
Positive-ion HR-ESIMS was recorded with an Agilent TOF MSD
1946D spectrometer. UV and IR spectra were recorded with Shimadzu
1
UV-2501PC and Nicolet Impact 410 spectrometers, respectively. H
and 13C NMR, HSQC, HMBC, and NOESY spectra were taken with
Bruker ACF-500 (500 and 125 MHz, respectively) and Bruker ACF-
600 spectrometers (600 and 150 MHz, respectively) in acetone-d6,
DMSO-d6, or CDCl3; chemical shifts are reported in ppm as δ relative
to Me4Si (internal standard). Column chromatography was carried out
with Sephadex LH-20 (20-100 µm, Pharmacia) and ODS-C18 (100-
200 µm, Waters). L-Cysteine hydrochloride (high pure grade) was
purchased from AMRESCO. Sinapic acid, DMSO, cis-diamminedichlo-
roplatinum (cis-DDP), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) were purchased from Sigma. The malon-
dialdehyde (MDA) kit was provided by Nanjing Jiancheng Bioengi-
neering Institute. Other reagents and solvents used were of analytical
grade and were purchased from reliable commercial sources.
Plant Material. Fruits of M. charantia were collected at a suburb
of Nanjing, China, and identified by Dr. Mingjian Qin, China
Pharmaceutical University. A voucher specimen (No. 000804) was
deposited in the Department of Natural Medicinal Chemistry, China
Pharmaceutical University.
Purification of MCP. MCP from fruits of M. charantia was purified
to electrophoretic homogeneity by consecutive treatment of ammonium
* To whom correspondence should be addressed. Tel.: +86 25
85391289. Fax: +86 25 85301528. E-mail: lykong@jlonline.com.
10.1021/jf0628072 CCC: $37.00
© 2007 American Chemical Society
Published on Web 01/06/2007

1004 J. Agric. Food Chem., Vol. 55, No. 3, 2007
Liu et al.
fractions (42 mg) was submitted to RP-C18 column chromatography
(Φ1.5 × 30, 40 g) eluted with MeOH/H2O (50:50, v/v) to give 6 (24
mg), and the elution of 18 to 23 fractions (60 mg) was submitted to
RP-C18 column chromatography (Φ1.5 × 30, 40 g) eluted with MeOH/
H2O (45:55, v/v) to give 7 (30 mg). The combined n-butanol layer
was concentrated to afford a strong brown powder (64 mg) and
subjected to RP-C18 column chromatography (Φ3.0 × 50, 80 g) eluted
with MeOH/H2O (40:60, 50:50, and 60:40, v/v) to afford fraction 3
(25 mg), fraction 4 (18 mg), and fraction 5 (15 mg), respectively.
Fraction 3 was subjected to Sephadex LH-20 column chromatography
(Φ1.5 × 100, 70 g, 8 mL each) eluted with MeOH. The elution of 8
to 12 fractions (20 mg) was submitted to RP-C18 column chromatog-
raphy (Φ1.5 × 30, 40 g) eluted with MeOH/H2O (40:60, v/v) to give
2 (16 mg). Fraction 4 was subjected to Sephadex LH-20 column
chromatography (Φ1.5 × 100, 70 g, 8 mL each) eluted with MeOH.
The elution of 16 to 19 fractions (14 mg) was submitted to RP-C18
column chromatography (Φ1.5 × 30, 40 g) eluted with MeOH/H2O
(45:55, v/v) to give 3 (10 mg). Fraction 5 was subjected to Sephadex
LH-20 column chromatography (Φ1.5 × 100, 70 g, 8 mL each) eluted
with MeOH/CHCl3 (1:1, v/v). The elution of 14 to 16 fractions (12
mg) was submitted to RP-C18 column chromatography (Φ1.5 × 30,
40 g) eluted with MeOH/H2O (55:45, v/v) to give 4 (9 mg).
Compound 2: green powder. IR νmaxKBr (cm-1) ) 3445, 3423, 2939,
2840, 1701, 1610, 1514, 1456, 1332, 1253, 1220, 1114. Positive HR-
FABMS Found: m/z 831.2498, C43H43O17. For 1H and 13C NMR data,
see Table 1.
Compound 3: white powder. IR νmaxKBr (cm-1) ) 3445, 2933, 1992,
1622, 1514, 1458, 1423, 1340, 1280, 1222, 1164,1118. Positive HR-
1
FABMS Found: m/z 419.1336, C21H23O9. For H and 13C NMR data,
see Table 2.
Compound 4: red powder. IR νmax (cm-1) ) 3552, 3439, 2941,
KBr
1745,1614, 1512, 1460, 1336, 1251, 1217, 1164, 1114. Negative HR-
FABMS Found: m/z 399.1085, C21H19O8. For H and 13C NMR data,
1
see Table 2.
Compound 5: white powder. IR νmaxKBr (cm-1) ) 3443, 3425, 2939,
1734, 1639, 1610, 1520, 1461, 1431, 1352, 1323, 1218, 11161, 1116.
Positive HR-ESIMS Found: m/z 469.1113 [M + Na]+, C22H22O10Na.
Negative HR-ESIMS Found: m/ z 401.1252 [M - CO2 - H]-,
C21H21O8. For H and 13C NMR data, see Table 2.
1
1
Compound 6: white powder. ESI-MS: m/z ) 445 [M - H]-. H
NMR (acetone-d6, 500 MHz): δ ) 6.70 (s, 2, 2a-CH and 6a-CH), 5.75
(d, 1, J ) 2.6 Hz, 7a-CH), 4.36 (m, 1, 8a-CH), 7.08 (s, 2, 2b-CH and
6b-CH), 7.63 (d, 1, J ) 2.6, 7b-CH), 3.83 (s, 6, 3a-OCH3 and 5a-
OCH3), 3.88 (s, 6, 3b-OCH3 and 5b-OCH3), 7.39 (br s, 1, 4a-OH), 7.92-
(br s, 1, 4b-OH). 13C NMR (acetone-d6, 125 MHz): δ ) 125.7 (C-1a),
104.6 (C-2a), 149.4 (C-3a), 137.8 (C-4a), 149.4 (C-5a), 104.6 (C-6a),
81.9 (C-7a) 54.6 (C-8a), 172.5 (C-9a), 131.7 (C-1b), 109.8 (C-2b), 149.2
(C-3b), 141.4 (C-4b), 149.2 (C-5b), 109.8 (C-6b), 140.1 (C-7b), 125.7
(C-8b), 172.0 (C-9b), 57.0 (OMe-3a and OMe-5a), 57.1 (OMe-3b and
OMe-5b).
Figure 1. Chemical structures of 1−7.
sulfate fractionation, ion exchange chromatography on DEAE-Sepharose
FF, affinity chromatography on Con A Sepharose, and gel filtration
on Sephadex G-150 as described in our previous report (1). The purified
MCP exhibited a specific activity of 7757 E.U. of peroxidase per mg
of protein, which was 46-fold higher than that of the crude extract.
Biotransformation of Sinapic Acid in Aqueous Acetone. A
solution of sinapic acid (500 mg) in acetone (25 mL) and a solution of
MCP (4 × 103 U) in buffer (100 mM NaOAC-HOAC, pH 5.0, 100
mL) were mixed and treated with hydrogen peroxide (3%, 5 mL) at
43 °C and stirred for 4 h, during which time the mixture gradually
turned a brownish red color.
Isolation and Identification. The reaction mixture was evaporated
to dryness at 40 °C under reduced pressure. The extractum was
dissolved with water. Then the aqueous mixture was extracted with
ethyl acetate and n-butanol, successively. The extract was washed with
water, dried with anhydrous Na2SO4, and evaporated to dryness at 40
°C under reduced pressure. The combined ethyl acetate layer was
concentrated to afford a brown powder (388 mg), which was subjected
to RP-C18 column chromatography (Φ3.0 × 50, 80 g) eluted with
MeOH/H2O (30:70 and 40:60, v/v) to afford fraction 1 (233 mg) and
fraction 2 (118 mg). Fraction 1 was subjected to Sephadex LH-20
column chromatography (Φ1.5 × 100, 70 g, 8 mL each) eluted with
MeOH. The elution of 15 to 20 fractions (197 mg) was submitted to
RP-C18 column chromatography (Φ1.5 × 30, 40 g) eluted with MeOH/
H2O (40:60, v/v) to give 5 (166 mg). Fraction 2 was subjected to
Sephadex LH-20 column chromatography (Φ1.5 × 100, 70 g, 8 mL
each) eluted with MeOH/CHCl3. (1:1, v/v). The elution of 13 to 15
1
Compound 7: white powder. ESI-MS: m/z ) 445 [M - H]-. H
NMR (acetone-d6, 500 MHz): δ ) 6.75 (s, 4, 2a-CH, 5a-CH, 2b-CH,
and 5b-CH), 5.77 (s, 2, 7a-CH and 7b-CH), 4.13 (s, 2, 8a-CH and 8b-
CH), 3.85 (s, 12, 3a-OMe, 5a-OMe, 3b-OMe and 5b-OMe). 13C NMR
(acetone-d6, 125 MHz): δ ) 130.3 (C-1a and C-1b), 104.7 (C-2a, C-6a,
C-2b and C-6b), 149.7 (C-3a, C-5a, C-3b and C-5b), 138.0 (C-4a and
C-4b), 83.8 (C-7a and C-7b), 49.5 (C-8a and C-8b), 176.5 (C-9a and
C-9b), 57.2 (OMe-3a, OMe-5a, OMe-3b and OMe-3a).
Measurement of Antioxidative Activity. Male Sprague-Dawley
SD rats (8-9 weeks old) were used. They were purchased from the
Experimental Animal House of China Pharmaceutical University
(Nanjing, China). All rats were maintained with free access to pellet
food and water in plastic cages at 20 ( 2 °C and kept on a 12 h light/
dark cycle. The animal study protocol was in compliance with the
guidelines of China for animal care, which was conformed to the
internationally accepted principles in the care and use of experimental
animals. Rat was sacrificed by bloodletting and was perfused in situ
via the portal vein with phosphate buffered saline (PBS). Liver tissue
was collected and cut into species. After being weighed, the tissue was
homogenized with PBS (1:4) using a high-speed homogenizer for 10
s each time at 0 °C, and the operation was repeated for 4 to 5 times.

Biotransformation of Sinapic Acid
J. Agric. Food Chem., Vol. 55, No. 3, 2007 1005
a
Table 1. NMR Data for Compound 2 in Acetone-d6
position
1a
2a
3a
4a
5a
6a
7a
8a
1c
2c
3c
4c
5c
6c
7c
1H
13C
HMBC
position
1b
2b
3b
4b
5b
6b
7b
8b
1d
2d
3d
4d
5d
6d
7d
1H
13C
HMBC
123.3
104.7
149.4
137.7
149.4
104.7
149.7
122.5
126.7
110.5
148.8
139.6
148.8
110.5
144.9
120.2
169.0
57.3
123.4
104.6
149.5
137.7
149.5
104.6
150.1
122.3
126.7
110.6
148.7
139.4
148.7
110.6
145.4
119.6
169.1
57.3
6.92 (1H, s)
1a, 4a, 6a, 7a
7.03 (1H, s)
1b, 4b, 6b, 7b
6.92 (1H, s)
6.99 (1H, s)
1a, 2a, 4a, 7a
1c, 4c, 6c, 7c
7.03 (1H, s)
6.96 (1H, s)
1b, 2b, 4b, 7b
1d, 4d, 6d, 7d
6.99 (1H, s)
7.84 (1H, s)
1c, 2c, 4c, 7c
8a, 8c, 9c
6.96 (1H, s)
7.77 (1H, s)
1d, 2d, 4d, 7d
8b, 8d, 9d
8c
9c
8d
9d
3a, 5a-OMe
3c, 5c-OMe
3.72 (6H, s)
3.67 (6H, s)
3.12 (3H, s)
3a, 5a
3c, 5c
9d
3b, 5b-OMe
3d, 5d-OMe
3.70 (6H, s)
3.65 (6H, s)
3b, 5b
3d, 5d
57.0
52.3
57.0
−COOMe
a Chemical shifts are given in
δ
values and followed by proton numbers and multiplicities. The spectra of 1H and 13C signals were measured in acetone-d6 (1H, 500 MHz
and 13C, 125 MHz), and the spectra of HSQC and HMBC were measured in acetone-d6 (1H, 600 MHz and 13C, 150 MHz). The 1H and 13C signals were assigned by HSQC
and HMBC spectra.
Table 2. NMR Data for Compounds 3, 4, and 5a
3
4
5
position
13C
1H
13C
1H
13C
1H
1a
2a
3a
4a
5a
6a
7a
8a
9a
1b
2b
3b
4b
5b
6b
7b
8b
9b
128.8
107.4
149.2
142.8
149.2
107.4
198.8
39.4
118.2
103.2
148.2
138.2
148.2
103.2
155.1
99.1
127.5
105.4
149.4
137.5
149.4
105.4
80.7
7.37 (1H, s)
7.13 (1H, s)
6.81 (1H, s)
7.37 (1H, s)
7.13 (1H, s)
6.81 (1H, s)
5.80 (1H, d, 7.5)
4.71 (1H, dd, 7.5, 1.8)
4.30 (2H, s)
−
7.34 (1H, s)
−
53.4
−
−
172.1
126.0
109.6
149.0
140.0
149.0
109.6
139.7
123.6
171.4
57.1
127.3
108.0
149.1
138.0
149.1
108.0
143.6
126.5
171.4
56.6
125.3
108.8
148.2
138.9
148.2
108.8
134.7
121.8
169.3
56.3
6.60 (1H, s)
7.10 (1H, s)
7.03 (1H, s)
6.60 (1H, s)
7.90 (1H, s)
7.10 (1H, s)
7.25 (1H, s)
7.03 (1H, s)
7.56 (1H, d, 1.8)
3a, 5a-OMe
3b, 5b-OMe
4a-OH
3.64 (6H, s)
3.88 (6H, s)
3.85 (6H, s)
3.89 (6H, s)
9.09 (1H, s)
9.28 (1H, s)
3.83 (6H, s)
3.88 (6H, s)
7.31 (1H, br s)
7.96 (1H, br s)
57.0
56.4
57.1
4b-OH
a Spectra of compound 3 were measured in CD3OD (1H, 500 MHz and 13C, 125 MHz), those of compound 4 were measured in DMSO-d6 (1H, 500 MHz and 13C, 125
MHz), and those of compound 5 were measured in acetone-d6 (1H, 500 MHz and 13C, 125 MHz).
The homogenates (25%) were then centrifuged at 10 000 rpm for 20
min. The supernatants were collected and again centrifuged at 100 000
rpm for 60 min. The supernatants were discarded, and the microsomal
pellet was suspended in 10 volume PBS containing 30% glycerol
(glycerol:PBS ) 30:70 (v/v)). Microsomal suspensions were stored in
small portions at -80 °C and thawed before lipid peroxidation assay.
Microsomal protein was determined by the biuret method.
inhibition ratio was defined according to the following equation:
inhibition ratio ) (the absorbance of vehicle -
the absorbance of tested compound)/
the absorbance of vehicle × 100%
Lipid peroxidation was induced in rat liver microsomes by FeSO4-
cysteine. A mixture of the microsomal suspension (10%, 100 µL),
FeSO4 solution (1 mmol/L, 50 µL), L-cysteine hydrochloride (10 mmol/
L, 20 µL), the sample solution (final concentration of 10-4 mol/L, 10
µL) or vehicle (DMSO, 10 µL), and PBS (0.01 M, pH 7.4, 820 µL)
was incubated at 37 °C for 30 min. Each reaction mixture was stopped
at 4 °C. The concentration of MDA, the second derivative of lipid
peroxidation, in the reaction mixture above-mentioned was determined
using the commercial kit according to the guidelines provided. The
Measurement of Inhibition of HL-60. Human leukemia cell line
(HL-60) cells were obtained from Institute of Biochemistry and Cell
Biology, Chinese Academy of Sciences. HL-60 cells were cultured in
a humidified incubator at 37 °C in RPMI-1640 medium containing 10%
heat-inactivated fetal bovine serum (GIBCO BRL) and antibiotics (100
U/mL penicillin and 100 g/mL streptomycin) in an atmosphere
containing 5% CO2. Cells were passaged twice weekly and routinely
examined for mycoplasma contamination.

1006 J. Agric. Food Chem., Vol. 55, No. 3, 2007
Liu et al.
When HL-60 cells began growth exponential, HL-60 cells were
seeded onto 96-well microplates (Costar) at a density of 2 × 104 cells/
well in RPMI-1640 medium (180 µL) and cultured in a humidified
incubator for 24 h at 37 °C in 5% CO2/air. After the supernatant was
removed, 20 µL of tested compound (with final concentration of 10-5
,
10-6, or 10-7 mol/L) was added and cultured for 48 h. Then cell growth
was evaluated with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) (Sigma) assay. A total of 20 µL of 5 mg/mL MTT
in RPMI-1640 medium was added to each well for a further 4 h
incubation. After the supernatant was removed, 150 µL of DMSO
(dimethyl sulfoxide) was added to dissolve the formazan crystals. The
plate was shaken for 5 min and then scanned with an ELISA reader
(Multiskan Spectrum MSS 1500-320, Thermo Electron Corporation)
at 570 nm. The inhibition ratio was calculated according to the following
equation:
Figure 2. Key HMBC and key NOESY for compound 2.
inhibition ratio ) (the absorbance of negative control -
the absorbance of tested compound)/
the absorbance of negative control × 100%
RESULTS AND DISCUSSION
Compound 2 was obtained as a green powder. Its molecular
formula was determined as C43H42O17 by positive HR-FABMS
spectrometric analysis (data given above), indicating the forma-
tion of a sinapic acid tetramer. The IR spectrum exhibited
absorption bands of hydroxyl (3445, 3423 cm-1), ester carbonyl
(1701 cm-1), and phenyl (1610, 1514 cm-1) groups. In the 1H
and 13C NMR spectra (Table 1), four 1,3,4,5-tetra-substituted
aromatic fragments (A, B, C, and D) were observed, and no
trans-coupled disubstituted double bonds were detected, sug-
gesting that the sinapic radical, the reaction intermediate, did
not react with its aromatic ring but was tetramerized at the side
chain. The side chains of two sinapic radicals involved decar-
boxylation and further formed a furan fragment (E) as shown
by only two carbonyl groups (δC 169.1 and 169.0) and two
oxygen-substituted aromatic carbons (δC 149.7 and 150.1)
observed in the furan ring, with 12 carbons linked with oxygen
atoms located in the four aromatic rings. The carboxylic acid
group of one sinapic was present as a methyl ester, as indicated
by one methoxy group linked to the carbonyl group (δH 3.12,
δC 52.3), with eight methoxy groups located in the four aromatic
rings. In addition, two trisubstituted double bonds (δH 7.84, 7.77;
δC 144.9, 120.2 and 145.4, 119.6) were also characterized.
All the signals of protons and carbons of 2 were assigned
Figure 3. Key HMBC and key NOESY for compound 3.
olefinic bonds (7c, 8c and 7d, 8d) was determined to be
Z-configuration on the basis of the obvious NOE correlation
between H-7c and H-7d. Combining the above analysis, the
structure of 2 was elucidated as 3-(4-hydroxy-3,5-dimethoxy)-
2-[2,5-bis(4-hydroxy- 3,5-dimethoxy)-4-[3-(4-hydroxy-3,5-
dimethoxy)-[(E)-2-carboxyethenyl]-2-yl]furan-3-yl]-(E)-2-pro-
penoic acid methyl ester. This is the first successful directed
conversion of the sinapic acid into a tetramer derivate possessing
two full conjugation systems in the molecule, which would be
rather difficult to obtain through organic synthesis.
Compound 3 was obtained as a white powder. Its molecular
formula was determined as C21H22O9 by positive HR-ESIMS
spectrometric analysis, indicating a sinapic acid dehydrodimer.
The IR spectrum exhibited absorption bands of hydroxyl (3445
cm-1), ketone carbonyl (1992 cm-1), and phenyl (1622, 1514
1
cm-1) groups. In the H and 13C NMR spectra (Table 2), two
1,3,4,5-tetra-substituted aromatic fragments (A and B), one
trisubstituted double bond, one -CH2CO- fragment, one
carboxylic acid, and four methoxy groups were observed,
suggesting that the sinapic radical was dimerized at the side
chain.
1
with detailed analysis of the H and 13C NMR data associated
with HSQC and HMBC spectra (Table 1). An HSQC experi-
ment established all direct 1H-13C connectivities. The HMBC
1
Detailed analysis of the H and 13C NMR data associated
1
spectrum showed many informative H-13C long-range cor-
with HSQC and HMBC spectra allowed assignment of all the
H and C signals (Table 2). An HSQC experiment established
all direct 1H-13C connectivities. The HMBC spectrum showed
relations which allowed us to connect partial fragments. The
correlations of H-2a (6a) to C-7a and H-2b (6b) to C-7b
suggested that aromatic fragments A and B were connected to
the 7a and 7b positions of the furan fragment E through 1a and
1b carbon atoms, respectively. For the trisubstituted CHdC
fragment (7c, 8c), the correlations of H-2c (6c) to C-7c and
H-7c to C-2c (6c, 9c, 8a) supported connection of the aromatic
fragment C to the CHd group with δ 7.84 (7c) through the 1c
carbon atom, the furan fragment E was connected to the dC
group (8c) through the 8a carbon atom, and a carboxylic acid
group was also connected to the dC group (8c). For the other
trisubstituted CHdC fragment (7d, 8d), the correlations of H-2d
(6d) to C-7d, H-7d to C-2d (6d, 9d, 8b), and OMe-9d to C-9d
indicated that the aromatic fragment D was connected to the
CHd group with δ 7.77 (7d) through the 1d carbon atom. The
furan fragment E was connected to the dC group (8d) through
the 8b carbon atom, and a methyl ester group was also connected
to the dC group (8d) (Figure 2). The stereochemistry of two
1
many informative H-13C long-range correlations. The cor-
relations of H-2a (6a) to C-7a suggested that aromatic fragment
A was connected to the carbonyl group of the -CH2CO-
fragment through the 1a atom. For the trisubstituted CHdC
fragment (7b, 8b), the correlations of H-2b (6b) to C-7b, H-7b
to C-2b (6b, 9b, 8a), and H-8a to C-7b (9b) supported that the
aromatic fragment B was connected to the CHd group with δ
7.90 (7b) through the 1b atom, the -CH2CO- fragment was
connected to the dC group (8b) through the 8a atom, and the
carboxylic acid group was also connected to the dC group (8b)
(Figure 3). The stereochemistry of olefin carbon (C-7b) was
determined to be E-configuration, because NOE correlation
peaks between H-8a and H-2a (6a, 2b, 6b) were observed in
the NOESY spectrum. According to the above analysis, the
structure of compound 3 was elucidated as 3-(4-hydroxy-3,5-

Biotransformation of Sinapic Acid
J. Agric. Food Chem., Vol. 55, No. 3, 2007 1007
Figure 4. Key HMBC and key NOESY for compound 4.
Figure 5. Key NOESY for compound 5.
Table 3. Effect of Compounds on Lipid Peroxidation of Rat Liver
dimethoxyphenyl)-2-[1-(4-hydroxy-3,5-dimethoxyphenyl)-
acetyl ]-(E)-2-propenoic acid. Compound 3 was a new com-
pound.
Microsomesa
inhibition ratio
Compound 4 was obtained as a red powder. Its molecular
formula was determined as C21H20O8 by negative HR-ESIMS
spectrometric analysis, indicating a sinapic acid dehydrodimer.
The IR spectrum exhibited absorption bands of hydroxyl (3522,
3439 cm-1), lactone carbonyl (1745 cm-1), and phenyl (1614,
1512 cm-1) groups. In the 1H and 13C NMR spectra (Table 2),
two 1,3,4,5-tetra-substituted aromatic fragments (A, B), two
trisubstituted double bonds, one lactone carbonyl group, and
four methoxy groups were observed, suggesting that the sinapic
radical was dimerized at the side chain.
concn
(mol/L)
-
of MDA
formation
compd
4
4
4
4
4
4
5
6
7
1
1
1
1
1
×
×
×
×
×
10
10
10
10
10
65.6
43.1
45.7
46.7
49.8
×
×
×
×
×
2.5
4.0
4.5
3.3
1.8
-
-
-
-
sinapic acid
a Lipid peroxidation was induced in rat liver microsomes by FeSO4
−cysteine.
The concentration of MDA, the second derivative of lipid peroxidation, was
determined. The inhibition rate was calculated. Data are expressed as the mean
By detailed analysis of the 1H and 13C NMR data associated
with HSQC and HMBC spectra, all the H and C signals could
be assigned (Table 2). An HSQC experiment established all
±
SD of three experiments, and each experiment included triplicate sets.
Table 4. Effects of Compounds on the Growth of HL-60 Cellsa
1
direct H-13C connectivities. The HMBC spectrum showed
1
many informative H-13C long-range correlations. The cor-
inhibition ratio
for HL-60 growth
(%)
concn
(mol/L)
-
relations of H-7b to C-9b (8a) and H-8a to C-1a (7b, 8b, 9b)
supported the presence of the 3-methylene-2(3H)-furanone ring
system. The correlations of H-2a (6a) to C-7a and H-8a to C-1a
as well as the correlations of H-2b (6b) to C-7b and H-7b to
C-2b (6b) suggested that the aromatic fragments A and B were
connected to the 7a and 7b positions of the 3-methylene-2(3H)-
furanone moiety through the 1a and 1b atoms, respectively
(Figure 4). The stereochemistry of olefin carbon (C-7b) was
determined to be E-configuration, because NOE correlation
peaks between H-8a and H-2b were observed in the NOESY
spectrum. According to the above analysis, the structure of 4
was elucidated as (E)-5-(4-hydroxy-3,5-dimethoxyphenyl)-3-
[(4-hydroxy-3, 5-dimethoxy-phenyl)-methylene]-2(3H)-fura-
none. Compound 4 was a new compound.
compd
5
6
7
5
6
7
5
6
7
5
6
7
5
6
7
4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
98.4
34.3
0.0
92.3
0.0
0.00
88.3
0.0
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5
6
0.0
98.2
0.0
7
0.0
cis-DDP
93.9
71.3
51.3
Compound 5 was obtained as a white powder. Its molecular
formula was determined as C22H22O10 by positive HR-ESIMS
spectrometric analysis and negative HR-ESIMS spectrometric
analysis, indicating a sinapic acid dehydrodimer. The IR
spectrum exhibited absorption bands of hydroxyl (3443, 3425
cm-1), lactone carbonyl (1734 cm-1), olefin (1639 cm-1), and
a HL-60 cells (2
×
104 in 180
µ
L of RPMI-1640 medium) were pre-cultured in
L of tested
a 96-well microplate for 24 h. After the supernatant was removed, 20
µ
compound (with final concentration of 10 4, 10 5, or 10 mol/L) was added and
cultured for 48 h. Then cell growth was evaluated with MTT assay. The inhibition
ratio was calculated. Each data represented the mean of three experiments, and
each experiment included triplicate sets.
-
-
-6
1
phenyl (1610, 1520 cm-1) groups. In the H and 13C NMR
spectra (Table 2), two 1,3,4,5-tetra-substituted aromatic frag-
ments, one trisubstituted double bond, one saturated CH-CH
fragment, two carbonyl groups, and four methoxy groups were
observed, suggesting that the sinapic radical was dimerized at
the side chain. It further possessed a mono-lactone ring system.
The stereochemistry of olefin carbon (C-7b) was determined
to be Z-configuration, because NOE correlation peaks between
H-8a and H-2b were observed in the NOESY spectrum and the
chemical shift of 7b (δ 7.56) affected by lactone carbonyl group
(9b) in the 1H NMR spectrum was located relatively downfield
when compared with that in the E-configuration (Figure 5).
The relative configuration of H-7a/H-8a was deduced to be a
cis relationship, because the NOE correlation peak between H-7a
and H-8a was detected and the coupling constant of H-7a/H-8a
was J ) 2.6 Hz compared with that (J ) 7.6 Hz) in trans
relationship in the mono-lactone reported in the literature (6).
According to the above analysis, the structure of 5 was
elucidated as cis-(4)E-2-(4-hydroxy-3,5-dimethoxyphenyl)-4-[(4-
hydroxy-3,5-dimethoxyphenyl)-methylene]tetrahydro-5-oxo-3-
furancarboxylic acid. Compound 5 was a new compound.
The known compounds, trans-(4)E-2-(4-hydroxy-3,5-dimethox-
yphenyl)-4-[(4-hydroxyl-3,5-dimethoxyphenyl)-methylene]tet-
rahydro-5-oxo-3-furancarboxylic acid (6) (6), tetra-hydro-3,6-
bis(4-hydroxy-3,5-dimethoxyphenyl)-1H,4H-furo[3,4-c]furan-

1008 J. Agric. Food Chem., Vol. 55, No. 3, 2007
Liu et al.
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1,4-dione (7) (19, 23), were identified by comparing their
physical and spectroscopic data with those reported in the
literature.
The fruit of M. charantia is one of the most widely used
foods in China for thousands of years. As a vegetable, it showed
obvious hypoglycemic activity (24). A novel peroxidase (M.
charantia peroxidase, MCP) was purified from the fruit of M.
charantia. We applied the purified MCP to catalyze the
transformation of sinapic acid, which exists in many foods, and
showed many activities such as anti-inflammation and antioxi-
dation capabilities (5, 6). A new tetramer and three new dimers
of sinapic acid were successfully obtained. In addition, the
known compound 7 which was obtained exists in Gmelina
arborea (19).
In order to investigate the effects of H2O2 or the polymeri-
zation of sinapic acid, a negative contrast experiment was
conducted. The experimental result has shown that H2O2 cannot
catalyze the polymerization of sinapic acid, when MCP did not
exist in the reaction system. During our research, a new tetramer
derivate (2) of cinnamic acid derivatives has been obtained with
peroxidase in vitro. In the pharmacological research, compound
5 has shown stronger antioxidative activity than the parent
sinapic acid (1), see Table 3. Its antioxidant activity appears to
be related to the existence of a fully conjugated system in the
molecule. Compounds 4 and 5 significantly inhibited the growth
of HL-60 cell at the concentration of 10-5 mol/L, see Table 4.
ACKNOWLEDGMENT
We are grateful to Dr. Bin Ma at Shandong University, China,
for the 2D NMR experiment.
(17) Nishiyama, A.; Eto, H.; Terada, Y.; Iguchi, M.; Yamamura, S.
Anodic oxidation of 4-hydroxycinnamie acids and related
phenols. Chem. Pharm. Bull. 1983, 31, 2845- 2852.
(18) Iguchi, M.; Nishiyama, A.; Eto, H.; Terada, Y.; Yamamura, S.
Anodic oxidation of 4-hydroxycinnamic acids. Chem. Lett. 1979,
1397-1400.
(19) Anjaneyulu, A. S. R.; Madhusudhana, Rao, A.; Kameswara, Rao,
V.; Ramachandra, Row, L. Novel hydroxy lignans from the
heartwood of Gmelina arborea. Tetrahedron 1977, 33, 133-
143.
Supporting Information Available: 1H, 13C, HSQC, HMBC,
1
and NOESY spectra for 2 and 4; H, 13C, HSQC, and HMBC
1
1
spectra for 3; H, 13C, and NOESY spectra for 5; and H and
13C spectra for 6 and 7. This material is available free of charge
via the Internet at http://pubs.acs.org.
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Received for review September 30, 2006. Revised manuscript received
November 30, 2006. Accepted December 4, 2006. This investigation was
supported by the Specialized Research Fund for the Doctoral Program
of Higher Education (No. 20040316002), People’s Republic of China.
JF0628072