Chemical conversions of salvianolic acid B by decoction in aqueous solution

Article abstract of DOI:10.1016/j.fitote.2012.06.015

Salvianolic acid B (Sal B) is the most abundant phenolic compound in Salvia miltiorrhiza, which has been widely used for the treatment of cardiovascular diseases in Oriental medicine. To elucidate structure of the converted compounds of Sal B by decoction

Full text of DOI:10.1016/j.fitote.2012.06.015

Fitoterapia 83 (2012) 1196–1204
Contents lists available at SciVerse ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Chemical conversions of salvianolic acid B by decoction in aqueous solution
⁎
Hyoung Jae Lee, Jeong-Yong Cho, Jae-Hak Moon
Department of Food Science & Technology, and Functional Food Research Center, Chonnam National University, 300 Yongbong-dong, Buk-gu,
Gwangju 500‐757, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Salvianolic acid B (Sal B) is the most abundant phenolic compound in Salvia miltiorrhiza, which
has been widely used for the treatment of cardiovascular diseases in Oriental medicine. To
elucidate structure of the converted compounds of Sal B by decoction in aqueous solution, Sal B
(200 mg) was decocted in an aqueous solution (200 mL) at pH 4.9 and the decocted solution
was purified by Sephadex LH-20 column chromatography and preparative HPLC. The 13
converted compounds were isolated and the chemical structures were determined by NMR
and MS. In addition to the 4 compounds previously reported as conversion products of Sal B by
decoction, 9 compounds were first reported with the complete structure of compounds
isolated from decocted Sal B solution and three of the compounds were determined to be novel
compounds. In addition, a conversion mechanism of compounds converted by decoction was
proposed on the basis of kinetics studies, which reasonably supported the conversion
mechanism of Sal B. The 13 compounds seemed to be produced by the hydrolysis of an ester
bond, decarboxylation, retro oxa-Michael reaction, hydration, and radical reaction during
decoction in aqueous solution.
Received 28 March 2012
Accepted in revised form 23 June 2012
Available online 8 July 2012
Keywords:
Salvianolic acid B
Decoction
Salvia miltiorrhiza
Conversion mechanism
Degradation products
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
which has been widely used for the treatment of cardiovascular
diseases such as coronary heart disease, hyperlipidemia, and
Medicinal herbs have been generally taken as an extract
obtained by decoction in aqueous solution. Recently, the
chemical conversion of the components contained in the
medicinal stuffs by decoction in aqueous solution has been
reported [1,2]. Chemical conversion by decoction of the
components contained in medicinal herbs may be very
important in examining the pharmaceutical effects of medicinal
herbs. However, the majority of studies on oriental medicine
have only evaluated the physiological activity of decocted
solution [3–5] and quantified the main ingredients of raw
materials contained in decocted solution [6–8], and very little
has been done to investigate the conversion by decoction of
constituents contained in medicinal herbs on the molecular
level.
cerebrovascular disease in oriental medicine [9,10]. According
to recent studies, the biological activities of Sal B and its
magnesium salt include strong free radical-scavenging activities
[11–14], inhibition of the angiotensin converting enzyme (ACE)
[15], inhibition of glucose-induced cell proliferation [16], and
hepatoprotection [17]. In addition, Sal B has been shown to
elicit endothelium-dependent vasodilation [18], lower blood
pressure in hypertension [19], and inhibit replication of human
immunodeficiency virus-1 [20]. Therefore, the effects of Sal B
have been extensively examined.
Previous studies showed that Sal B was converted by
decoction in aqueous solution [1] and several converted
products were identified by LC-ESI-MS analysis [2]. In these
studies, nine compounds were reported as degradation
products of Sal B. Five of these compounds were identified as
danshensu, protocatechuic aldehyde, salvianolic acid E,
lithospermic acid, and prolithospermic acid. However, four
other compounds were ambiguously identified as lithospermic
acid isomers and caffeic acid dimer. In the present study, we
Salvianolic acid B (Sal B, lithospermic acid B, Fig. 1) is the
most abundant phenolic compound in Salvia miltiorrhiza Bunge,
⁎
Corresponding author. Tel.: +82 62 530 2141; fax: +82 62 530 2149.
E-mail address: nutrmoon@chonnam.ac.kr (J.-H. Moon).
0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2012.06.015

H.J. Lee et al. / Fitoterapia 83 (2012) 1196–1204
1197
3'''
4'''
4'
2'''
9'''
5'
5'''
6'
6'''
9''
1'''
3'
5''
2''
8'''
7'''
4''
2'
1'
7'
9'
8'
6''
7
2
3''
1''
9
8''
3
1
6
7''
8
4
5
Fig. 1. Structure of salvianolic acid B (lithospermic acid B, Sal B).
confirmed that the converted compounds of Sal B by decoction
in aqueous solution are more than twenty compounds using
HPLC analysis. Sal B is a condensed compound consisting of
four phenylpropanoic acids (caffeic acid tetramer) and has four
carboxylic acid groups including an ester and free forms and
seven phenolic hydroxyl groups. Therefore, various reactions
such as decomposition, condensation, and ring opening may
occur during decoction of Sal B in aqueous solution.
In general, nuclear magnetic resonance (NMR) analysis
provides much more precise information on the stereoscopic
structure as well as the binding positions of substitute groups in
compounds when compared to the information obtained by MS
analysis. Therefore, in this study, the compounds isolated from
the decocted Sal B solution were structurally analyzed by NMR
and MS. In addition, the kinetics of compound conversion during
decoction of Sal B were evaluated and a conversion mechanism
of Sal B by decoction in aqueous solution was proposed.
HPLC-PDA system (Shimadzu, Kyoto, Japan) equipped with
LC-6AD pump, SPD-M20A detector, DGU-20A3 degasser,
CBM-20A controller, and Shimadzu LC solution program,
and using an ODS-80Ts column (4.6×150 mm, Tosoh, Kyoto,
Japan) at room temperature. The mobile phase was com-
posed of CH3CN/H2O/acetic acid (10:88:2, v/v/v, A) and
CH3CN/H2O (40:60, v/v, B). Elution was started with 100% A
and increased to 100% B after 70 min. The flow rate of the
mobile phases was 1.0 mL/min and the constituents were
monitored at 280 nm.
2.3. Large scale of Sal B decoction in aqueous solution and
isolation and purification of the converted compounds
Sal B (200 mg) was dissolved in H2O (200 mL) that was
adjusted to pH 4.9 by HCl. The sample was then heated on a
hot plate under refluxing at 100 °C for 24 h, cooled and
evaporated in vacuo at 39 °C. The concentrate was purified
by column chromatography using a Sephadex LH-20 gel
(70–230 mesh, Pharmacia, Sweden) column (2.0×132 cm,
bed volume 480 mL) using MeOH/H2O (8:2, v/v) as an elution
solvent. Each fraction obtained in the processes of column
2. Materials and methods
2.1. Materials
Sal B (purity, >99%) was isolated from S. miltiorrhiza
Bunge according to the method described in our previous
study [21]. Methanol (MeOH) used for column chromatog-
raphy was of extra pure quality and obtained from Duksan
(Gyeonggi, Korea). Acetonitrile (MeCN) and trifluoroacetic
acid (TFA) used for HPLC analysis were obtained from Fisher
Scientific Korea (Seoul, Korea) and Sigma-Aldrich (St. Louis,
MO, USA), respectively. Hydrogen chloride (HCl) was pur-
chased from Samchun Pure Chemical (Pyeougtaek, Korea).
chromatography was spotted on a silica gel thin-layer
chromatograph (TLC; silica gel 60 F254, 0.25 mm thickness;
Merck, Darmstadt, Germany) and developed using a mixture of
n-butanol/acetic acid/water (4:1:2, v/v/v). The spots were
detected by UV light (254 and 365 nm) and spraying with
1,1-diphenyl-2-picrylhydrazyl (DPPH) solution (200 μM in
EtOH). HPLC analysis of each fraction was also carried out
using the same conditions as described in 2.2. The fractions
grouped based on the TLC and HPLC results were finally
purified by HPLC on a preparative scale.
2.2. Small scale decoction of Sal B in aqueous solution and HPLC
analysis of the solution
The preparative HPLC was carried out using the same
instrument that was utilized for analytical HPLC except a
preparative mode equipped with a Shim-pack PREP-ODS(H)
KIT column (20×250 mm, Shimadzu) was used. The flow rate
was 9.9 mL/min and detection was monitored at 280 nm. The
mobile phase consisted of CH3CN/H2O (10:90, v/v, A), which
was adjusted to pH 2.65 by TFA and CH3CN/H2O (40:60, v/v, B).
The gradient programs (Conditions 1 and 2) were as follows:
Condition 1, starting with 100% A, rising to 100% B after 70 min,
and maintaining 100% B for 15 min; Condition 2, starting with
100% A, rising to 55% B after 30 min, maintaining 55% B for
Sal B (3 mg) was dissolved in H2O (3 mL) that was
adjusted to pH 4.9 by HCl. The sample was then heated on a
heating block under refluxing at 100 °C for 24 h. Aliquots
(100 μL) were withdrawn at regular time intervals (0.5, 1, 2,
4, 8, 12, 16, and 24 h) and MeOH (200 μL) was added to each
aliquot after cooling. Twenty μL of the samples were then
injected into high performance liquid chromatography
(HPLC). HPLC analysis was carried out a using Shimadzu

1198
H.J. Lee et al. / Fitoterapia 83 (2012) 1196–1204
10 min, rising to 100% B after 30 min, and maintaining 100% B
for 15 min.
solution, and aliquots were withdrawn at predetermined time
intervals (0, 0.5, 1, 2, 4, 8, 12, 16, and 24 h). The collected
aliquots were then subjected to HPLC analysis (Fig. 2).
Interestingly, approximately twenty new compounds were
observed and the content of the new compounds mostly
increased with an increase in the decoction time (Figs. 2 and 3),
which was determined based on the peak areas (Fig. 3) in the
HPLC chromatograms (Fig. 2) detected at 280 nm. On the other
hand, the content of Sal B linearly decreased with an increase in
decoction time (Figs. 2 and 3). Therefore, it was hypothesized
that the new compounds were produced by chemical conver-
sion during the decoction of Sal B in aqueous solution.
Fig. 3 shows the quantitative changes of the compounds
(Fig. 3A, main products; Fig. 3B, minor products) converted
during decoction of Sal B in aqueous solution. The content of
most of the compounds increased with decoction time. In
particular, peak I increased rapidly during the first 4 h,
remained relatively constant from 4 h to 12 h, and then
gradually decreased. Peaks D and H did not increase after
16 h of decoction. However, the other converted compounds
increased with an increase in decoction time. Therefore,
purification of the compounds converted by decoction of Sal
2.4. MS and NMR analyses
Mass spectra were acquired on a LC-MS hybrid ion-trap
time-of-flight (IT-TOF) mass spectrometer (Shimadzu),
which was equipped with an ESI source in negative ion
mode at a mass resolution of 10,000 full-width at half
maximum. 1H and 13C NMR were recorded on a 500 MHz
unitINOVA 500 spectrometer (Varian, Walnut Creek, CA, USA).
The chemical shift was measured using tetramethylsilane as an
internal standard. The isolated compounds were dissolved in
CD3OD (Acros Organics, Geel, Belgium), respectively.
3. Results and discussion
3.1. Change of HPLC chromatogram with the elapse of decoction
time of Sal B in aqueous solution
Sal B was decocted in an aqueous solution at pH 4.9, which
is the pH of the S. miltiorrhiza solution decocted in aqueous
A
M
25
D
mAU
20
15
10
5
0
25
E
I
G
F
J
B
C
K
24 h
16 h
12 h
8 h
L
H
20
15
10
5
0
25
20
15
10
5
0
25
20
15
10
5
0
25
20
15
10
5
0
25
4 h
20
15
10
5
0
25
2 h
20
15
10
5
0
25
1 h
20
15
10
5
0
25
20
15
10
5
0.5 h
0 h
Sal B
30
0
0
10
20
40
50
60
70
Retention time (min)
Fig. 2. HPLC chromatograms of decocted solutions as a function of decoction time of salvianolic acid B. Column, ODS-80Ts column (4.6×150 mm); detection,
280 nm; mobile phase, CH3CN/H2O/acetic acid (10:88:2, v/v/v)→CH3CN/H2O (40:60, v/v) (70 min gradient elution); flow rate, 1.0 mL/min.

H.J. Lee et al. / Fitoterapia 83 (2012) 1196–1204
1199
A
1.2
1.0
0.8
0.6
0.4
0.2
0.0
100
80
60
40
20
0
0
5
10
15
20
25
Decoction time (h)
B
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
100
80
60
40
20
0
0
5
10
15
20
25
Decoction time (h)
Fig. 3. Change in the peaks area on HPLC chromatograms (280 nm) of the converted compounds as a function of decoction time of salvianolic acid B. A, main
converted compounds; B, minor converted compounds. ●, Sal B; ▽, Peak A (1); ⋄, Peak B (2); △, Peak C (3); □, Peak D (4); , Peak E (5); , Peak F (6); , Peak G
(7); , Peak H (8); ○, Peak I (9); ♦, Peak J (10); , Peak K (11); , Peak L (12); ■, Peak M (13).
B in aqueous solution was carried out using the Sal B solution
decocted for 24 h.
compounds [3 (Peak C, tR 35.33 min, 0.9 mg), 4 (Peak D, tR
41.19 min, 2.1 mg), 5 (Peak E, tR 40.96 min, 1.1 mg), 7 (Peak G,
tR 43.63 min, 1.6 mg), and 8 (Peak H, tR 47.10 min, 0.7 mg)]
and two compounds [10 (Peak J, tR 53.51 min, 1.4 mg) and 13
(Peak M, tR 63.62 min, 0.8 mg)], respectively. In addition, the
eluates of Ve/Vt 2.05–2.42, 2.58–2.94, and 3.15–3.66 were also
further purified by prep-HPLC under Condition 2 to yield the
following: one compound [9 (Peak I, tR 39.04 min, 1.3 mg)],
two compounds [11 (Peak K, tR 54.39, min 2.2 mg) and 12
(Peak L, tR 64.65 min, 0.4 mg)], and a compound [6 (Peak F, tR
38.32 min, 1.0 mg)]. Finally, fourteen compounds were isolated
from the decocted Sal B solution. The chemical structures of
the isolated compounds were determined by NMR and MS
spectroscopy.
3.2. Isolation and purification of the compounds produced by
decoction of Sal B
The Sal B (200 mg) solution decocted in an aqueous solution
(200 ml) for 24 h was purified over a Sephadex LH-20 column
(80% MeOH) and the fractions were subjected to TLC and HPLC
analyses for grouping. On the basis of TLC and HPLC analyses,
the fractions were divided to 23 groups. Of them, Ve/Vt (elution
volume/total bed volume) 0.96–1.18, 1.23–1.29, and 3.67–3.95
yielded a mixture of 1 (Peak A, 23.3 mg), 2 (Peak B, 2.9 mg),
and 10 (Peak J, 31.5 mg), respectively, with single spots (Rf 0.60,
0.87, and 0.85, respectively) on the TLC plate and peaks (tR 4.04,
8.52, and 43.09 min, respectively) in the analytical HPLC
chromatograms. However, based on the TLC and HPLC analysis,
the other fractions contained various different compounds.
Therefore, the eluates of Ve/Vt 1.30–2.04 and 3.96–4.58 were
further purified by prep-HPLC using Condition 1 to yield five
3.3. Structural elucidation of isolated compounds
3.3.1. Compounds 1–3, 6, 9, 10, and 13
NMR and ESI-MS (negative) data of compounds 1, 3, 6, 9,
10, and 13 were compared with those of previous reports

1200
H.J. Lee et al. / Fitoterapia 83 (2012) 1196–1204
and identified as (R)-danshensu (1) [22], 7,8-dihydroxy-2-(3,
4-dihydroxyphenyl)-1,2-dihydro-naphthalene-1,3-dicarboxylic
acid 3-[1-carboxy-2-(3,4-dihydroxyphenyl)] ethyl ester (3)
[23], salvianolic acid F (6) [24], lithospermic acid (9) [25],
salvianolic acid A (10) [26], and salvianolic acid C (13) [27]
(Fig. 4), respectively. Compound 2 was identified as 3,4-
dihydroxybenzaldehyde by comparison of 1H NMR spectrum
of standard (Fig. 4).
3.3.3. Compound 5
The 1H NMR spectrum (Table 2) of 5 showed the presence
of a tri-substituted benzene ring protons at δ 6.74 (1H, br. s,
H-2′), 6.70 (1H, d, J=8.0 Hz, H-5′), and 6.62 (1H, br. d, J=
8.0 Hz, H-6′), a couple of equivalent hydrogen in benzene
ring [δ 7.16 (1H, d, J=8.5 Hz, H-5) and 6.86 (1H, d, J=
8.5 Hz, H-6)]. In addition, the 1H NMR spectrum contained 2
protons in the trans-configuration for double bonds at δ 7.89
(1H, d, J=15.8 Hz, H-7) and 6.28 (1H, d, J=15.8 Hz, H-8).
Furthermore, the 1H NMR spectrum of
5 showed the
3.3.2. Compound 4
presence of 2 pair of geminal methylene protons at δ 3.84
(2H, s, H-10), 3.09 (1H, m, H-7′a), and 2.99 (1H, m, H-7′b)
and one methine proton at δ 5.14 (1H, m, H-8′). These initial
results were supported by the 13C NMR spectrum (Table 1) of
5. In particular, the HMBC correlations (Table 2) from the
proton signal (δ 3.84) at the C-10 position to carbon signals at
C-1 (δ 127.2), C-3 (145.5), and C-11 (175.7) indicated that
the carbon (C-10) of the carboxymethyl group was attached
at the C-2 position of 5. Moreover, the HMBC correlation
(Table 2) from the proton signal (δ 5.14) at the C-8′ position
to carbon signal (δ 168.5) at the C-9 position indicated that
the carbon (C-8′) was attached at the C-9 position of 5. The
ESI-MS (negative) spectrum also displayed signals related to
the molecular ion (m/z 417.0812 [M-H]−). Therefore, 5 was
unambiguously identified as salvianolic acid D (Fig. 4).
The molecular weight of 4 was analyzed to be 538 based
on its ESI-MS (negative) spectrum with m/z 537.1039
[M-H]−. Its molecular weight was smaller (180) than that
of Sal B (718), which indicated that 4 lost a danshensu from
Sal B by hydrolysis of the ester bond. The 1H and 13C NMR
spectra (Tables 1 and 2) of 4 contained 10 sp2 and 5 sp3
carbon proton signals and 23 sp2 and 4 sp3 carbon signals,
respectively, indicating the presence of
a tri-substituted
aromatic benzene ring, trans type olefinic double bond, and a
danshensu. In particular, the HMBC correlation (Table 2) from
the proton signal (δ 5.13, H-8″) to carbon signal (δ 172.3, C-9′)
indicated that the carbon of 8″ position in danshensu was
attached at the C-9′ position of 4. On the basis of the
spectroscopic analysis data, the structure of 4 was conclusively
identified as 4-(2-carboxyethenyl)-2-(3,4-dihydroxyphenyl)-
2,3-dihydro-7-hydroxy-3-benzofurancarboxylic acid 3-[1-
carboxy-2-(3,4-dihydroxyphenyl)ethyl] ester (Fig. 4), and
to the best of our knowledge, this NMR data has never before
been reported.
3.3.4. Compound 7
The molecular formula of
7 was determined to be
C26H24O11 (M.W. 512) by HR-ESI-MS (negative) analysis
OH
5''
OH
HO
O
O
OH
2'
OH
5'
6''
5'
5'
2'
O
O
OH
OH
HOOC
OH
OH
2
5
2
5
10
6'
7'
OH
7
O
COOH
8
HO
HO
O
HO
HO
HO
2''
HO
6'
H
7
OH
1
4
7''
8''
2
HO
HO
6'
O
7''
2''
O
2'
O
8'
7'
OH
8''
OH
OH
6
6
6
O
8'
8
7
5
6
O
6''
5''
O
5a
1
OH
5
O
OH
8
2
6
HO
3a
5
4
11
OH
8
OH
2''
HO
5''
7
5'
1
4
O
3
COOH
8
6''
5'
2'
5'
2'
O
OH
OH
6'
HO
HO
HO
OH
O
8'
6'
6'
7
HO
12
5''
6''
2''
8'
13
7''
2'
7'
OH
7
OH
OH
OH
O
OH
2'
O
O
8''
O
9
COOH
6
5
O
OH
8''
7''
7'
HO
HO
O
12
O
OH
5''
O
8''
7''
8
3
OH
5'
6
OH
2''
4
1
HO
HO
6'
5
6a
7a,b
7
O
OH
9
8
8a,b
11
OH
HO
HO
HO
HO
12
5
5'
5'
OH
5'
4
HO
6'
7'
3
6'
7'
HO
2'
6'
8'
5''
6''
2''
5''
2''
2'
OH
OH
2'
5''
8
OH
OH
11a
OH
8'
5
8'
OH
OH
O
COOH
7'
O
COOH
6''
1
7
O
COOH
7
9
6''
O
10
7
O
11
O
HO
HO
O
8''
7''
O
8''
7''
13
8
O
8''
8
7''
2''
8
O
6
6
HO
HO
6
12a,b
13a
11a
5
5
10a
Fig. 4. Structures of compounds 1–13 isolated from decocted solution of salvianolic acid B. a, first isolated compounds from decocted salvianolic acid B solution;
b, novel compounds.

H.J. Lee et al. / Fitoterapia 83 (2012) 1196–1204
1201
J=8.0 Hz, H-7), 6.45 (1H, d, J=8.0 Hz, H-8)]; 6 sp3 carbon
proton signals, suggesting a methylene proton [δ 2.85 (2H, d,
J=5.0 Hz, H-7″)], 4 methine protons [δ 4.65 (1H, q, J=
6.5 Hz, H-1), 5.08 (1H, d, J=5.3 Hz, H-3, overlapped with
H-8″), 4.11 (1H, d, J=5.3 Hz, H-4), and 5.08 (1H, t, J=5.0,
H-8″, overlapped with H-3)] and a methyl proton [1.45
(3H, d, J=6.5 Hz, H-11)]. In addition, these initial results
were supported by the 13C NMR (Table 1) and 2D NMR
spectroscopic data. The HMBC cross peaks (Table 3) from
H-11 (δ 1.45) to C-1 (δ 70.5) and C-9 (δ 132.2) indicated that
C-11 was coupled at C-1. In addition, the HMBC spectrum
showed cross peaks from H-7″ (δ 2.85) and H-8″ (δ 5.08) to
C-2″ (δ 117.7) and C-2′ (δ 115.9), respectively. These results
indicated that the carbons of H-7″ and H-8″ were respectively
attached at C-1″ (δ 129.0) and C-1′ (δ 132.3) of each benzene
ring. The HMBC cross peak between H-8″ and C-12 (δ 174.3)
indicated that there was an ester bond between C-8″ (δ 74.8)
and C-12. In addition, the cross peaks from H-3 to C-1,
C-10, and C-13 with other significant cross peaks (Table 3)
provided further evidence of the structure of 7. Therefore,
the structure of 7 was confirmed to be 4,7,8-trihydroxy-
1-methyl-2-benzopyran-3-carboxylic acid 3-[3,4-dihydroxy-
(3,4-dihydroxyphenyl)-benzene ethyl] ester, which was a
novel compound (Fig. 4).
Table 1
13C NMR (125 MHz, CD3OD) data of the converted compounds (4, 5, 7, 8,
11, 12).
No.
4
5
7
8
11
No.
12
1
2
3
4
5
6
7
8
125.0
126.3
149.2
145.0
118.5
122.1
143.3
117.9
170.6
133.9
113.4
146.7
127.2
123.8
145.5
145.0
115.1
119.8
148.7
116.7
168.5
32.2
70.5
–
75.3
–
123.9
129.9
148.5
145.1
117.2
123.1
145.3
116.1
168.5
1
2
3
4
4a
5
5a
6
7
8
9
138.1
138.7
118.8
123.0
129.3
34.5
129.3
141.2
150.6
112.1
127.9
125.7
74.8
46.7
81.3
51.5
144.2
144.3
115.5
116.8
132.2
119.7
22.3
174.3
174.3
132.3
115.9
146.2
146.4
116.0
120.6
144.0
144.8
115.3
116.2
132.8
121.6
21.6
9
10
11
12
13
1′
2′
3′
4′
5′
6′
7′
8′
9′
1″
2″
3″
4″
5″
6″
7″
8″
9″
175.7
174.1
9a
146.9
116.6
118.4
88.3
58.0
172.3
129.6
117.6
146.3
145.4
116.4
122.0
38.1
133.7
115.5
146.4
146.6
116.0
120.4
134.8
114.2
146.6
146.7
116.4
119.0
86.3
10
11
11a
12
140.0
117.1
123.3
21.4
13
168.9
75.3
39.5
173.4
129.1
117.4
146.2
145.3
116.6
122.0
37.7
129.0
117.7
146.0
145.2
116.5
122.4
38.0
128.7
117.8
145.8
145.1
116.3
122.6
37.9
129.5
117.7
146.3
145.4
116.4
121.9
38.1
3.3.5. Compound 8
The molecular ion signal (m/z 511.1246 [M-H]−, 0.0 mmu
for C26H23O11) was observed in the HR-ESI-MS (negative)
spectrum of 8. The 1H NMR spectrum (Table 3) of 8 showed
75.8
74.8
74.4
75.1
8
sp2 carbon proton signals,
172.3
173.0
172.4
signals corresponding to
suggesting 3 benzene rings [δ 6.90 (1H, br. s, H-2′), 6.70
(1H, d, J=8.5 Hz, H-5′), 6.74 (1H, br. d, J=8.5 Hz, H-6′), 6.45
(1H, br. s, H-2″), 6.52 (1H, d, J=8.3 Hz, H-5″), 6.08 (1H, br.
d, J=8.3 Hz, H-6″), δ 6.77 (1H, d, J=8.5 Hz, H-7), 6.63 (1H,
d, J=8.5 Hz, H-8)]; 6 sp3 carbon proton signals, suggesting a
methylene proton [δ 2.65–2.80 (2H, m, H-7″)], 4 methine
protons [δ 4.91 (1H, m, H-1), 4.72 (1H, d, J=9.8 Hz, H-3),
3.99 (1H, d, J=9.8, H-4), and 5.03 (1H, m, H-8″)] and a
(m/z 511.1246 [M-H]−, 0.0 mmu for C26H23O11). The 1H NMR
spectrum (Table 3) of 7 contained signals corresponding to
8 sp2 carbon proton signals, suggesting 3 benzene rings [δ
6.75 (1H, br. s, H-2′), 6.66 (1H, d, J=8.0 Hz, H-5′), 6.60 (1H,
br. d, J=8.0 Hz, H-6′), 6.55 (1H, br. s, H-2″), 6.57 (1H, d, J=
8.3 Hz, H-5″), 6.24 (1H, br. d, J=8.3 Hz, H-6″), δ 6.74 (1H, d,
Table 2
1H NMR (500 MHz, CD3OD) and HMBC data of the converted compounds (4, 5, 11).
No.
4
5
11
δ
H (J/Hz)
HMBC (H-C)
C1, C3, C4
C2, C4, C7
δ
H (J/Hz)
HMBC (H-C)
C1, C3
C2, C4,
δH (J/Hz)
6.72 (1H, d, 8.3)
7.06 (1H, d, 8.3)
HMBC (H-C)
C1, C4
C2, C4, C7
5
6
6.84 (1H, d, 8.5)
7.14 (1H, d, 8.5)
6.75 (1H, d, 8.5)
7.14 (1H, d, 8.5)
C7
7
8
10
2′
5′
6′
7.57 (1H, d, 16.0)
6.18 (1H, d, 16.0)
C2, C6, C8, C9
C1
7.89 (1H, d, 15.8)
6.28 (1H, d, 15.8)
3.84 (2H, s)
6.74 (1H, s)
6.70 (1H, d, 8.0)
6.62 (1H, d, 8.0)
C2, C6, C8, C9
C1, C9
C1, C2, C3, C11
C4′, C6′, C7′
C3′
7.62 (1H, d, 16.0)
6.25 (1H, d, 16.0)
C2, C6, C8, C9
C1, C9
6.75 (1H, br. s)
6.74 (1H, d, 8.1)
6.63 (1H, dd, 1.8, 8.1)
C6′, C7′
C1′, C3′
C2′, C4′,
6.87 (1H, d, 2.0)
6.75 (1H, d, 9.2)
6.78 (1H, d, 9.2)
C4′, C6′, C7′
C1′, C3′
C4′
C2′, C4′, C7′
C7′
7′
8′
5.84 (1H, d, 4.5)
4.32 (1H, d, 4.5)
C2, C3, C1′, C2′, C6′, C9′
3.09 (1H, m)
2.99 (1H, m)
5.14 (1H, m)
C1′, C2′, C6′, C9′
C-9, C1′, C7′, C9′
5.71 (1H, m)
C2′, C6′
C1, C2, C3, C1′, C7′, C9′
3.73 (1H, m)
C2, C3,
3.26 (1H, m)
C1′, C7′
2″
5″
6″
7″
6.59 (1H, d, 2.3)
6.59 (1H, d, 8.0)
6.37 (1H, dd, 2.3, 8.0)
3.03 (1H, dd, 3.4, 14.3)
2.90 (1H, dd, 9.1, 14.3)
5.13 (1H, dd, 3.4, 9.1)
C6″, C7″
C1, C″, C3″
C4″, C7″
C1″, C2″,
C6″
6.73 (1H, d, 2.5)
6.67 (1H, d, 8.0)
6.60 (1H, d, 8.0)
3.09 (1H, m)
2.99 (1H, m)
5.17 (1H, dd, 4.0, 8.0)
C4″, C6″, C7″
C1″, C3″
C2″, C4″, C7″
C1″, C2″, C6″, C8″, C9″
8″
C1″, C9′, C9″
C9

1202
H.J. Lee et al. / Fitoterapia 83 (2012) 1196–1204
Table 3
1H NMR (500 MHz, CD3OD) and HMBC data of the converted compounds (7, 8, 12).
No.
7
8
12
δH (J/Hz)
HMBC (H-C)
δ
H (J/Hz)
HMBC (H-C)
δH (J/Hz)
HMBC (H-C)
1
3
4
4.65 (1H, q, 6.5)
5.08 (1H, d, 5.3)
4.11 (1H, d, 5.3)
C3, C10, C8, C9, C11
C1, C4, C10, C12, C13
C3, C5, C10, C12, C13
4.91 (1H, m)
4.72 (1H, d, 9.8)
3.99 (1H, d, 9.8)
C3, C8, C9, C11
C1, C12, C1′, C2′, C6′
C3, C9, C10, C12, C1′
6.87 (1H, d, 8.0)
6.91 (1H, d, 8.0)
5.07 (1H, q, 7.0)
C1, C4a
C2, C5, C11a
C4, C6, C9a, C11a, C12
5
7
8
6.74 (1H, d, 8.0)
6.45 (1H, d, 8.0)
C5, C9
C1, C6, C10
6.77 (1H, d, 8.5)
6.63 (1H, d, 8.5)
C5, C8, C9
C6, C7, C10
6.79 (1H, d, 8.0)
7.07 (1H, d, 8.0)
7.66 (1H, s)
C6, C9a
C5a, C7, C10
C5a, C9, C11, C11a, C13
9
10
11
12
2′
5′
6′
2″
5″
6″
7″
8″
1.45 (3H, d, 6.5)
C1, C9
1.51 (3H, d, 6.5)
C1, C9
5.71 (3H, d, 7.0)
C4a, C5, C5a
6.75 (1H, br. s)
C4′, C6′, C8″
C1′, C3′
C2′, C4′
C4″, C6″, C7″
C1″, C3″
C2″, C4″, C7″
C2″, C6″
6.90 (1H, br. s)
C3, C4′, C6′
C1′, C3′
C3, C2′, C4′
C4″, C6″, C7″
C1″, C3″
C2″, C4″, C7″
C1″, C2″, C6″
C12
6.66 (1H, d, 8.0)
6.60 (1H, br. d, 8.0)
6.55 (1H, br. s)
6.57 (1H, d, 8.3)
6.24 (1H, br. d, 8.3)
2.85 (2H, d, 5.0)
5.08 (1H, t, 5.0)
6.70 (1H, d, 8.5)
6.74 (1H, br. d, 8.5)
6.45 (1H, br. s)
6.52 (1H, d, 8.3)
6.08 (1H, br. d, 8.3)
2.65–2.80 (2H, m)
5.03 (1H, m)
C12, C2′, C6′, C7″
methyl proton [1.51 (3H, d, J=6.5 Hz, H-11)]. These
initial results were also supported by the 13C NMR (Table 1)
and 2D NMR experiments. Furthermore, based on the HMBC
spectrum (Table 3), the cross peaks from H-11 (δ 1.51) to
C-1 (δ 74.4) and C-9 (δ 132.8) and from H-3 (δ 4.72) to
C-1 (δ 75.3) and C-12 (δ 174.1) together with other
significant cross peaks [from H-8″ (5.03) to C-12 (174.1)
and from H-4 (δ 3.99) to C-9 (δ 132.8) and C-1′ (δ 133.7)]
(Table 3) clearly indicated that 8 was 5,6-dihydroxy-3-(3,4-
dihydroxyphenyl)-1-methyl-2-benzopyran-4-carboxylic acid
4-[1-carboxy-2-(3,4-dihydroxyphenyl)] ethyl ester, which
was a novel compound (Fig. 4).
112.1 (C-8), 127.9 (C-9), 125.7 (C-9a), 140.0 (C-10), 117.1
(C-11), 123.3 (C-11a)], 2 sp3 carbon signals [δ 34.5 (C-5) and
21.4 (C-12)], and a carbonyl carbon (δ 168.9, C-13). These
results indicated the presence of two AB type benzene rings, a
methyl cycloheptene, and a lactone ring. In addition, these
initial results were supported by the 1H NMR (Table 3) and 2D
NMR experiments. In particular, the HMBC correlations
(Table 3) from the proton signal (δ 7.66) of H-10 to the carbon
signals of C-5a (δ 129.3), C-9 (127.9), C-11 (117.1), C-11a
(123.3), and C-13 (168.9) indicated that the carboxylic group
(C-13) was attached at the C-11 position of 12. In addition, the
HMBC correlations from the proton signal of H-12 (δ 5.71) to
the carbon signals of C-5a (δ 129.3) and 4a (34.5) indicated that
the methyl carbon (C-12) was attached at the C-5 position and
C-4a and C-5a of two benzene rings were combined via the
methine carbon of C-5. These results were further supported by
the cross peaks from H-5 to C-9a and C-11a. Consequently, 12
was determined to be 1,2,6,7-tetrahydroxy-5 H-dibenzo[a,d]
cycloheptene-5-methyl-11-carboxylic acid, a novel compound
(Fig. 4).
The structure of 13 compounds as the converted products
of Sal B by decoction in aqueous solution was determined.
Compounds 1, 2, 4, and 9 of the converted compounds have
been reported on previous study by LC-MS analysis [2].
However, compounds 3, 5–8, and 10–13 were revealed as the
converted compounds of Sal B for the first time by the
present study. Moreover, compounds 7, 8, and 12 of the
nine converted compounds were determined to be novel
compounds.
3.3.6. Compound 11
In the ESI-MS (negative) spectrum of 11, a pseudomolecular
ion peak was detected at m/z 493.1142 [M-H]−. The 1H NMR
spectrum (Table 2) of 11 was closely related to that of 9, except
for the presence of methylene protons [δ 3.73 (1H, m, H-8′a)
and 3.26 (1H, m, H-8′b)] instead of the methine proton [δ 4.37
(1H, br. s, H-8′)] of 9. These results indicated the absence of
one carboxylic acid group, which was supported by the 13C
NMR (Table 1) and 2D NMR experiments. In particular, the
HMBC (Table 2) correlation from the proton signal (δ 5.17) of
H-8″ to carbon signal (δ 168.5) at the C-9 position indicated
that the carbon at the C-8″ of danshensu was attached
through an ester bond at the C-9 position of 11. In addition,
the other significant cross peaks are shown in Table 3.
Therefore, 11 was conclusively identified as {{(2E)-3-[2-(3,4-
dihydroxyphenyl)-2,3-dihydro-7-hydroxy-4-benzofuranyl]-1-
oxo-2-propenyl}oxy}-3,4-dihydroxy benzenepropanoic acid
(Fig. 4), and to the best of our knowledge, this NMR data has
never before been reported.
3.4. Proposed conversion mechanisms of the compounds
converted from Sal B by decoction in aqueous solution
3.3.7. Compound 12
The mechanism (Fig. 5) by which the compounds were
converted by decoction of Sal B in aqueous solution was
proposed on the basis of kinetics analysis (Fig. 3). Sal B
seemed to be very easily hydrolyzed by decoction in aqueous
solution. In Fig. 3A, compounds 1 (Peak A), 4 (Peak D), and 9
(Peak I) showed a very high initial formation rate. These
results suggest that the two molecules of danshensu, which
The molecular formula of 12 was determined to be
C
17H12O5 (M.W. 296) by HR-ESI-MS (negative) analysis (m/z
295.0611 [M-H]−, −0.1 mmu for C17H11O5). The 13C NMR
spectrum (Table 1) showed total 17 carbon signals: 14 sp2
carbon signals [δ 138.1 (C-1), 138.7 (C-2), 118.8 (C-3), 123.0
(C-4), 129.3 (C-4a), 129.3 (C-5a), 141.2 (C-6), 150.6 (C-7),

H.J. Lee et al. / Fitoterapia 83 (2012) 1196–1204
1203
HO
OH
OH
O
HO
E
HO
HO
?
O
HO
HO
H
OH
B
O
HO
B
O
O
O
OH
OH
OH
2
9'
O
Hydrolysis
D
C
O
9
HO
OH
A
HO
Radical reaction (?)
Radical reaction
B
Sal B
O
OH
O
OH
O
O
OH
OH
O
OH
OH
OH
O
OH
O
D
C
D
O
2
HO
HO
Hydrolysis
Hydrolysis
O
A
A
9
HO
5
?
HO
OH
OH
O
E
HO
HO
HO
O
O
OH
Decarboxylation
Dehydration
B
HO
OH
Decarboxylation
O
O
1
C
O
OH
1
A
4
HO
Opening of ring
OH
HO
HO
HO
OH
COOH
B
HO
HO
B
B
HO
B
O
OH
OH
O
O
OH
OH
OH
O
OH
OH
OH
O
O
O
D
D
A
D
Hydrolysis
C
C
O
O
HO
HO
O
OH
HO
O
Hydration
A
A
A
OH
HO
10
HO
13
11
6
Fig. 5. The proposed conversion mechanisms of the converted compounds from Sal B by decoction in aqueous solution.
were esterified at the C-9 and C-9′ position of Sal B, were first
hydrolyzed (Fig. 5). In addition, compound 9 showed the
highest initial formation rate, which gradually decreased with
decoction time. This indicated that 9 was rapidly converted to
different compounds. On the basis of the structural character-
istic, 5 might be produced by the elimination of benzyl group
containing B ring through retro oxa-Michael reaction in 9 and 2
might be also formed as an inheritor of this reaction (Fig. 5).
As shown in Fig. 5, compound 9 could be converted into 10
and 13 by different routes. Thus, compounds 6 and 11 were
believed to be produced from 10 to 13 by hydrolysis and
hydration, respectively. In addition, a peak corresponding to 13
was detected from the HPLC chromatogram of decocted 10
(data not shown). However, peak of 10 was not detected from
the HPLC chromatogram of decocted 13 (data not shown).
These results suggested that 13 was produced from 10 as well
as 9 and 10 was not produced from 13. The conversion
mechanisms of 9, 10, and 13 are well explained by the pattern
of quantitative changes shown in Fig. 3A. That is, 13 con-
tinuously showed a rapid formation rate with the decoction
time. However, the formation rate of 10 was very low.
Moreover, it was elucidated that 4 would be produced via
successive hydrolysis of the ester bond at the C-9 position of Sal
B and 6 might be formed by hydrolysis at the C-9′ position of 4,
decarboxylation, and opening of the C-ring (Fig. 5). Further-
more, compounds 6 and 11 could be produced by hydrolysis of
10 and hydration at C ring of 13 (Fig. 5), respectively. The
proposed conversion mechanisms of Sal B as shown in Fig. 5
and these mechanisms were reasonably supported by the
changes (Fig. 3) in the content of converted compounds.
In contrast, there was inadequate data to propose formation
mechanisms of 3, 7, 8, and 12 produced by decoction of Sal B. As
shown in Fig. 3B, the content of 3, 7, 8, and 12 was lower than
that of other compounds converted from Sal B. Therefore, the
compounds are most likely produced through minor routes. In
addition, if the structure of compounds produced by decoction
of Sal B is further confirmed in additional studies, the formation
mechanism of the minor compounds may be clarified.
4. Conclusion
The 13 compounds converted by decoction of Sal B in
aqueous solution were structurally determined by NMR and
MS analyses. Compounds 1, 2, 4, and 9 have already been
reported to be converted compounds of Sal B by LC-MS analysis
[2]. However, the LC-MS study did not sufficiently reveal all
converted compounds of Sal B by decoction in aqueous
solution. Therefore, the present study was performed to
identify all converted compounds using NMR analysis. Of the
13 compounds examined, the complete structure of 9 new
converted compounds of Sal B were first reported in this study.
In addition, 7, 8, and 12 were determined to be novel
compounds. The 13 compounds identified in this study seem
to be produced by the hydrolysis of ester bond, decarboxyl-
ation, retro oxa-Michael reaction, hydration, and radical
reaction during the decoction in aqueous solution.
The present study confirmed that the components
contained in the medicinal herbs are chemically converted
during decoction. These results clearly suggest that “decoction”
is a very important factor in determining the pharmaceutical
effect of medicinal herbs. That is, the pharmaceutical effect of a
medicinal herbs may depend on the presence or absence of
decoction, decoction time, decoction temperature, and decoc-
tion method. The present study identified compounds con-
verted by decoction at the single compound level. However,
the decoction of medicinal herbs is generally carried out using a
mixture of several materials. Thus, numerous reactions be-
tween compounds may be occurring during the decoction

1204
H.J. Lee et al. / Fitoterapia 83 (2012) 1196–1204
[8] Chuda Y, Suzuki M, Nagata T, Tsushida T. Contents and cooking loss of
three quinic acid derivatives from garland (Chrysanthemum coronarium L.).
J Agric Food Chem 1998;46:1437–9.
process. Therefore, there is still a lot of work that must be
conducted to fully understand the conversion of compounds by
decoction. Furthermore, chemical changes in compounds by
decoction may also occur during cooking processes. Therefore,
the methods described in the present study may also be used to
investigate changes in food as a function of cooking.
[9] Zhou L, Zuo Z, Chow MSS. Danshen: an overview of its chemistry,
pharmacology, pharmacolinetics, and clinical use.
J Clin Pharmacol
2005;45:1345–59.
[10] Chang PN, Mao JC, Huang SH, Ning L, Wang ZJ, On T, Duan W, Zhu YZ.
Analysis of cardioprotective effects using purified Salvia miltiorrhiza
extract on isolated rat hearts. J Pharmacol Sci 2006;101:245–9.
With regard to medicinal herbs of Oriental medicines, the
specific reasons for decoction have been largely unstated. It is
presumed that at the time of their original development, the
relationship between decoction and its pharmaceutical effect
was determined through trial and error. S. miltiorrhiza is
generally treated by decoction, and this may be carried out in
order to accelerate the pharmaceutical effect. Perhaps this
enhanced pharmaceutical effect is caused by an elevation of
the absorption of the medicine's active compounds and/or
the partial alteration of some of the active compounds
contained in the treatment. A significant amount of further
research would be required to provide any degree of clarity
as to any one of these propositions. Our investigation of the
chemical conversion of compounds contained in medicinal
herbs may provide important information in clarifying the
mechanism of the medicinal herbs active effects. Additionally,
this research may give way to effective methods of identifying
novel medical compounds.
[11] Shigematsu T, Tajima S, Nishikawa T, Murad S, Pinnell SR, Nishioka I.
Inhibition of collagen hydroxylation by lithospermic acid magnesium
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