Salvianolic acids T and U: A pair of atropisomeric trimeric caffeic acids derivatives from root of Salvia miltiorrhiza

Article abstract of DOI:10.1016/j.fitote.2014.08.018

Two new trimeric caffeic acids, named salvianolic acids T and U (1 and 2), were isolated from the underground part of Salvia miltiorrhiza. Their structures, consisting of three caffeic acid units, were determined based on extensive 1D- and 2D-spectroscopic analyses and electronic circular dichroism (ECD) calculations.

Full text of DOI:10.1016/j.fitote.2014.08.018

Fitoterapia 98 (2014) 248–253
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Salvianolic acids T and U: A pair of atropisomeric trimeric caffeic
acids derivatives from root of Salvia miltiorrhiza
a
b,
a
a
b
Wei Li a, Shui-Ping Zhoua, , Yuan-Peng Jin , Xue-Feng Huang , Wei Zhou , Min Han , Yun Yu ,
⁎
⁎
Kai-jing Yana, Shu-Ming Li a, Xiao-Hui Ma a, Zhi-Xin Guoa, Yong-Hong Zhu a, He Suna
Tasly R&D Institute, Tasly Pharmaceutical Co., Ltd., Tianjin 300410, China
a
b
Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 20009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 June 2014
Accepted in revised form 14 August 2014
Available online 27 August 2014
Two new trimeric caffeic acids, named salvianolic acids T and U (1 and 2), were isolated from the
underground part of Salvia miltiorrhiza. Their structures, consisting of three caffeic acid units, were
determined based on extensive 1D- and 2D-spectroscopic analyses and electronic circular
dichroism (ECD) calculations.
© 2014 Elsevier B.V. All rights reserved.
Keywords:
Trimeric caffeic acid
Salvia miltiorrhiza
Electronic circular dichroism (ECD)
1. Introduction
paper, we describe the isolation and structural determination of
these two new products.
The dried root of Salvia miltiorrhiza (Danshen in Chinese) is
one of the most popular traditional Chinese medicines, which
was extensively used for prevention and treatment of coronary
heart disease [1], chronic renal failure [2], atherosclerosis [3],
ischemic myocardium [4], ischemic heart disease, myocardial
infarction [5], liver fibrosis and cirrhosis [6], and showed an
efficacy in relieving angina pectoris [7]. In Japan, Danshen
products are acted as a medicine to promote circulation and
improve blood flow. In our present research, two new trimeric
caffeic acids, salvianolic acids T and U (1 and 2) (Fig. 1), were
isolated from the roots of S. miltiorrhiza. Their structures,
consisting of three caffeic acid units, were determined based on
extensive 1D- and 2D-spectroscopic analyses and chemical
reactions, and their absolute configurations were determined
by electronic circular dichroism (ECD) calculation. In the present
2. Experimental
2.1. General
Optical rotations were measured with a Jasco P-1020
polarimeter. CD spectra were obtained on
a Jasco 810
spectrometer. UV spectra were performed on a Shimadzu UV-
2450 spectrophotometer. IR spectra were obtained on a
Shimadzu FTIR-8400S spectrophotometer. NMR spectra were
recorded by Bruker Avance-III NMR instrument (1H: 500 MHz,
13C: 125 MHz) with TMS as internal standard. Chemical shifts
were given in values of ppm and coupling constants in Hertz.
Mass spectra were obtained on an MS Agilent 1100 Series LC/
MSD iron-trap mass spectrometer (ESI-MS) and an Agilent
6520 B Q-TOF spectrometer (HR-ESI-MS), respectively. Semi-
preparative HPLC was carried out on a Waters Delta prep 4000
instrument (flow rate set at 10 ml/min) using an Agilent
ZORBAX XDB-C18 (21.2 × 150 mm, 5 μm). Silica gel (100–200,
200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., China),
⁎
Corresponding authors. Tel.: +86 25 52716876; fax: +86 25 85301528.
E-mail addresses: zhousp@tasly.com (S.-P. Zhou), hxf@cpu.edu.cn
(X.-F. Huang).
http://dx.doi.org/10.1016/j.fitote.2014.08.018
0367-326X/© 2014 Elsevier B.V. All rights reserved.

W. Li et al. / Fitoterapia 98 (2014) 248–253
249
Fig. 1. Structures of compounds 1 and 2.
macroporous resin D101 (Qingdao Haiyang Chemical Co. Ltd.,
China), Sephadex LH-20 (Pharmacia, U.S.A.), and RP-C18 (40–
63 μm, YMC, Japan) were used for column chromatography. The
pre-coated silica gel GF254 plates (Qingdao Marine Chemical
Plant, China) were used for TLC. Spots were visualized by
heating silica gel plates immersed in vanillin-H2SO4 in ethanol.
All solvents used were of analytical grade (Jiangsu Hanbon
Science and Technology Co. Ltd., China).
16.5 min) and 2 (24 mg, tR = 13.5 min) using MeOH:H2O
(90:10, v/v).
2.3.1. Salvianolic acid T (1)
Yellowish amorphous powder; [α]2D5 + 196.6 (c = 0.25,
MeOH); UV (MeOH) λ max nm: 220, 326; IR (KBr) νmax cm−1
:
3150, 2878, 2817, 1693, 1600, 1519, 1445, 1360, 1250, 1115,
968, 865, 812; negative HR-ESI-MS [M–H]− m/z: 537.1027
(Calcd for C27H21O1−2, 537.1038); 1H and 13C NMR data, see
Table 1.
2.2. Plant material
The roots of S. miltiorrhiza (batch number: 20100301)
were acquired in March 2010 from Shaanxi Tasly Plant
Pharmaceutical Co., Ltd. (Shaanxi, China), which is Good
Agricultural Practices (GAP) base for the agriculture and
process of S. miltiorrhiza licensed by the State Food and Drug
Administration, P. R. China (SFDA). A voucher specimen (No:
20100318) has been deposited in the Modern TCM Depart-
ment, Tasly R&D Institute.
2.3.2. Salvianolic acid U (2)
Yellowish amorphous powder; [α]2D5 − 157.6 (c = 0.16,
MeOH); UV (MeOH) λ max nm: 225, 325; IR (KBr) νmax cm−1
:
3306, 2881, 2822, 1687, 1602, 1519, 1445, 1360, 1250, 1113,
969, 865, 812; negative HR-ESI-MS [M–H]− m/z: 537.1034
(Calcd for C27H21O1−2, 537.1038); 1H and 13C NMR data, see
Table 1.
2.4. Hydrolysis reaction
2.3. Extraction and isolation
Compounds 1 and 2 (10 mg) are respectively stirred in
hydrochloric acid (5 ml, 6 M), at water bath (40 °C). The
reaction is stirred until completion (high performance liquid
chromatography, 12 h) then quenched with water, extracted 3
times with an equal volume of ethyl acetate, the combined
organic phases, concentration in rotary evaporator is done at
30 °C.
Chromatographic conditions: use Agilent Eclipse XDB-C18
(4.6 mm × 250 mm, 5 μm) as the stationary phase and 0.2%
formic acid-acetonitrile as mobile phase as specified in the
following table gradient elution; flow rate: 1 ml/min; The wave
length of the detection is 280 nm and the column temperature
is 25 °C; injection volume 20 μl.
Air-dried roots of the plant material (20 kg) were
extracted with 95% EtOH under reflux for 3 h. The EtOH
extract (2 kg) was suspended in water and partitioned with
CHCl3 (210 g), EtOAc (450 g) and n-BuOH (190 g). The EtOAc
extract was chromatographed over a D101 macroporous
resin column (15 × 60 cm), eluted successively with H2O, 30%,
50%, 80%, and 95% EtOH in H2O. The 80% EtOH fraction was
chromatographed on silica gel column eluted with CHCl3:
MeOH (100: 0–60: 40, v/v) to obtain eight fractions (A–H).
Fraction D (20 g) was applied to a silica gel column eluted with
CHCl3:MeOH:HCOOH (95:5:0–60:38:2, v/v/v) to give four
fractions (DA–DD). Fraction DC (2.5 g) was separated over silica
gel column eluted with CHCl3:MeOH:HCOOH (90:10:3–
40:10:0.5, v/v/v) to afford three fractions (DCA–DCC). Fraction
2.5. Preparation of fragment I by hydrolysis degradation
DCB (200 mg) was separated over RP-C18 column eluted with
MeOH:H2O:HCOOH (60:40:0.3–90:10:0.5, v/v/v) to afford
three fractions (DCBA–DCBD). Fraction DCBD (60 mg) was
purified by Sephadex LH-20 with CHCl3:MeOH (50:50, v/v)
To determine the hydrolysis reaction mixture, major peaks
in the chromatogram attribution were scanned by mass
spectrometry. Centralized collection of degraded fragment I
(m/z = 197) eluent, and extracted three times with an equal
and preparative HPLC to afford compounds 1 (11 mg, tR
=

250
W. Li et al. / Fitoterapia 98 (2014) 248–253
Table 1
1D and 2D data of compounds 1 and 2 (data measured in DMSO at 500 MHz (1H) and 125 MHz (13C), J in Hz).
No.
δH
δc
COSY
1
HMBC
1
1
2
1
2
2
2
1
2
3
4
5
6
7
8
–
–
123.7
126.4
142.9
147.7
115.0
118.4
143.7
113.9
166.0
127.1
116.5
143.9
144.8
115.5
120.0
36.0
123.8
126.3
142.9
147.7
115.0
118.4
143.7
114.0
165.9
127.2
116.5
143.9
144.9
115.5
120.1
36.1
H-5, H-8
H-6, H-7, H-7″
H-5
H-5, H-8
H-6, H-7, H-7″
H-5
–
–
–
–
–
–
H-5, H-6
H-5, H-6
6.85 (1H, d, 8.5)
7.31 (1H, d, 8.5)
7.41 (1H, d, 15.5)
6.27 (1H, d, 15.5)
6.85 (1H, d, 8.5)
7.29 (1H, d, 8.5)
7.41 (1H, d, 15.5)
6.27 (1H, d, 15.5)
H-6
H-5
H-8
H-7
H-6
H-5
H-8
H-7
H-7
H-6
H-7
H-7
H-6
H-7
9
–
–
H-7, H-8, H-8′
H-2′, H-5′, H-8′, H-7′
H-6′
H-2′, H-5′
H-2′, H-5′, H-6′
H-6′
H-7, H-8, H-8′
H-2′, H-5′, H-8′, H-7′
H-6′, H-7′
H-2′, H-5′, H-6′
H-2′, H-5′
1′
2′
3′
4′
5′
6′
7′
–
–
6.62 (1H, d, 2.0)
–
6.62 (1H, d, 2.0)
–
H-6′
H-6′
–
–
6.63 (1H, d, 8.0)
6.47 (1H, dd, 8.0, 2.0)
2.85 (1H, dAB, 14.0, 8.0)
6.63 (1H, d, 8.0)
6.45 (1H, dd, 8.0, 2.0)
2.83 (1H, dAB, 14.0, 8.0)
H-6′
H-2′, 5′
H-8′
H-6′
H-2′, 5′
H-8′
H-2′, H-5′
H-2′, H-5′, H-6′, H-8′
H-2′, H-5′, H-7′
H-2′, H-5′, H-6′, H-8′
2.91 (1H, dAB, 14.0, 4.5)
2.91 (1H, dAB, 14.0, 4.5)
8′
9′
1″
2″
3″
4″
5″
6″
4.93 (1H, dd, 8.0, 4.5)
–
4.92 (1H, dd, 8.0, 4.5)
–
72.8
170.6
126.0
117.3
144.8
147.2
115.3
122.9
141.1
123.4
168.4
72.9
170.6
126.0
117.3
144.8
147.2
115.3
122.9
141.1
123.3
168.4
H-7′
H-6″
H-7′
H-6″
H-7′
H-7′, H-8′
H-2″
H-6″, H-7″
H-2″, H-5″
H-2″, H-5″, H-6″
H-7′
H-7′, H-8′
H-5″
H-6″, H-7″
H-2″, H-5″
H-2″, H-5″, H-6″
–
–
6.44 (1H, d, 2.0)
–
6.43 (1H, d, 2.0)
–
–
–
6.55 (1H, d, 8.5)
6.43 (1H, dd, 8.5, 2.0)
7.69 (1H, s)
–
6.55 (1H, d, 9.0)
6.43 (1H, dd, 8.5, 2.0)
7.69 (1H, s)
–
H-6″
H-2″, 5″
H-6″
H-2″, 5″
H-2″, H-7″
H-6″
H-7″
H-2″, H-7″
H-2″, H-6″
7″
8″
9″
–
–
H-7″
H-7″
−OH
−COOH
8.63–9.93 (6-OH)
12.53 (2-COOH)
8.64–9.91 (6-OH)
12.46 (2-COOH)
volume of ethyl acetate and the combined extracts after drying
under N2 at 40 °C, the residue was dissolved in ethanol, high
speed centrifugation for 3 min (12000 r·min−1), leaving the
supernatant for analysis.
rings [δ 6.62 (1H, d, J = 2.0 Hz), 6.47 (1H, dd, J = 8.0, 2.0 Hz),
and 6.63 (1H, d, J = 8.0 Hz); δ 6.44 (1H, d, J = 2.0 Hz), 6.43 (1H,
dd, J = 8.5, 2.0 Hz), and 6.55 (1H, d, J = 8.5 Hz)], one 1,2,3,4-
tetrasubstituted aromatic ring [δ 6.85 (1H, d, J = 8.5 Hz), 7.31
(1H, d, J = 8.5 Hz)], one set of trans olefinic proton [δ 7.41 (1H,
d, J = 15.5 Hz), 6.27 (1H, d, J = 15.5 Hz)], one set of
trisubstituted olefinic proton [δ 7.69 (1H, s)], and one set of
ABX resonance system [δ 2.85 (1H, dAB, J = 14.0, 8.0 Hz), 2.91
(1H, dAB, J = 14.0, 4.5 Hz), and 4.93 (1H, dd, J = 8.0, 4.5 Hz)].
2.6. Analysis of chiral hydrolysis degradation fragments I
Preparation of reference solution: weigh R/S-salvianic acid A
reference, dissolved with ethanol and diluted to the appropriate
concentration. Chromatographic conditions: use Chiralcel OD-H
(4.6 mm × 250 mm, 5 μm) as the stationary phase and hexane–
ethanol–trifluoroacetic acid (88:12:0.5, v/v) as mobile phase;
Flow rate: 0.5 ml·min−1; The column temperature is 25 °C;
accurately inject 10 μL of salvianic acid A R-reference, R/S-
salvianic acid A reference and degraded fragment I solution.
3. Results and discussion
Salvianolic acid T (1) was isolated as yellowish amorphous
powder and showed [α]2D5 + 196.6 (c = 0.25, MeOH), it was
recognized as a phenolic acid from a positive test with ferric
trichloride and bromocresol green. It possessed a molecular
formula of C27H22O12 as evidenced by its negative high
resolution-electrospray ionization (HR-ESI)-MS at m/z
537.1027 [M–H]− (Calcd for C27H21O1−2, 537.1038, errors in
2.0 ppm). The IR absorptions showed the presence of hydroxyl
groups (3150 cm−1) and aromatic rings (1600, 1519, and
1445 cm−1), and UV maxima at 220, 326 nm suggested the
presence of a highly conjugated system. The 1H NMR spectrum
of 1 showed the presence of two 1,2,4-trisubstituted aromatic
Fig. 2. Key 1H–1H COSY and HMBC correlations for compounds 1 and 2.

W. Li et al. / Fitoterapia 98 (2014) 248–253
251
Fig. 3. The hydrolysis reaction of compounds 1 and 2.
Above information suggested that compound 1 was a trimeric
caffeic acid derivative. Further, HMBC experiment showed that
the H-7″ (δH 7.69) was long range coupled to C-9″ (δC 168.4),
thus showing that the carboxyl group was attached to C-8″. It
has been reported that the olefinic proton signal in the 1H NMR
of carboxystilbene model compounds is more deshielded in the
Z-configuration [8]. The 1H–1H COSY spectrum permitted the
assignment of the aliphatic and aromatic protons and displayed
the following connectivities: H-7′ (δH 2.91)/H-8′ (δH 4.93), and
H-5 (δH 6.85)/H-6 (δH 7.31), and H-5′ (δH 6.63)/H-6′ (δH 6.47)/
H-2′ (δH 6.62), and H-5″ (δH 6.55)/H-6″ (δH 6.43)/H-2″ (δH
6.44). The analysis of NMR data showed compound 1 was
the same with salvianolic acid E excepted substituent of
But their experimental CD confused us for they are almost
mirrored while they have the same chiral centers. The dilemma
phenomena suggested that another chiral factor may existed.
The structure was rechecked and found that the chiral axis
maybe a chiral factor. So we turn to the conformation search for
help [10]. The dihedral angle scanning result indicated that two
conformations existed (Table 2, Fig. 4). Conformation A with
dihedral angle C7″–C8″–C2–C1 being 65.9° was the most stable
conformation, conformation C with C7″–C8″–C2–C1being −66.1°
was the other stable conformation which has an energy
difference of 0.53 KJ/mol to conformation A. With the scanning
results, we found that there are rotational energy barrier of
121.39 and 130.24 KJ/mol from two sides. The rotational energy
barriers arose from the steric bulk of substituents in the o-
positions and the intramolecular hydrogen bonding, which were
sufficiently high to prevent the interconversion of the two
conformations at room temperature. Furthermore, intramolecu-
lar hydrogen bonding is another interaction to form the two
conformations. As a result, conformations A and C could coexist
stably at room temperature, which was in agreement with the
hydrolyzed products. With this in hand, theoretical calculations
of ECD spectra for conformations A and C were performed with
the Gaussian 09 program package [11]. Geometry for them was
optimized at the B3LYP/6-31++G (d, p) level of theory. The ECD
calculations of compounds 1 and 2 were performed with DFT
calculations at the BPV86/6-31++G (d, p) level of theory with
UV correction. By comparing with the experimental ECD of 1 and
2 (Fig. 5), the absolute configurations of 1 and 2 were assigned as
suggested by the structures of conformations A and C, respec-
tively. At last, the structure of compound 1 was identified as (P,
R)-3-(3,4-dihydroxyphenyl)-2-[(E)-3-[(E)-1-carboxy-2-(3,4-di-
hydroxyphenyl)ethenyl-3,4-ihydroxyphenyl]prop-2-enoyl]
oxypropanoic acid, named as salvianolic acid T, and com-
pound 2 was identified as (M, R)-3-(3,4-dihydroxyphenyl)-
C8″-COOH [9], thus the structural compound
1 was
determined as shown in Fig. 2 and proton and carbon
signals had been totally assigned on the basis of 2D NMR
analysis (Table 1).
On the basis of above evidences, the structure of 1 was
identified as (2R)-3-(3,4-dihydroxyphenyl)-2-[(E)-3-[(E)-
1-carboxy-2-(3,4-dihydroxyphenyl)ethenyl-3,4-dihydroxy-
phenyl]prop-2-enoyl]oxypropanoic acid.
Salvianolic acid U (2) possessed a molecular formula of
C
27H22O12, as evidenced by its negative high resolution-
electrospray ionization (HR-ESI)-MS at m/z 537.1034 [M–H]−
(Calcd for C27H21O−12, 537.1038, errors in 0.7 ppm). The data of
1H and 13C NMR were the same with compound 1, further
analysis of the 2D NMR spectroscopic data (1H–1H COSY,
HMQC, and HMBC spectra) indicated that both compounds 1
and 2 had the same molecular structure (Table 1, Fig. 2).
Nonetheless, the rotational direction and CD spectra of the two
timers were different.
The hydrolysis reaction of salvianolic acid T and U indicated
that the chiral center (C8′) in degradation fragments of their
hydrolysis products was identical to R-configuration (Fig. 3).
Table 2
Energy of rotation around the C2\C8″ bond (conformations A–D).
No.
Dihedral angle
Energy
KJ/mol
Boltzmann distribution
Conformation A
Conformation B
Conformation C
Conformation D
65.9
−164.1
−66.1
17.9
−1933.7373
−1933.6911
−1933.7371
−1933.6877
0.00
121.39
0.53
0.56
0
0.44
0
130.24

252
W. Li et al. / Fitoterapia 98 (2014) 248–253
Fig. 4. Optimized geometric structures of rotation around the C2\C8″ bond (A–D) and their rotational energy barriers (in KJ/mol).
2-[(E)-3-[(E)-1-carboxy-2-(3,4-dihydrophenyl) ethenyl-3,4-
ihydroxyphenyl]prop-2-enoyl]oxypropanoic acid, named as
salvianolic acid U.
Acknowledgments
This work was supported by the National Key Special
Project of Science and Technology for Innovation Drugs of
China (Grant No. 2013ZX09402202).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.fitote.2014.08.018.
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