Isolation and identification of degradation products of salvianolic acid A by NMR and LC-MS

Article abstract of DOI:10.1016/j.fitote.2010.10.008

Salvianolic acid A was subjected to thermal degradation condition in distilled water at 90 °C. Four degradation products, including two novel compounds (1, 2) and two known ones (5, 6), were isolated by reverse-phase semi-preparative liquid chromatography and identified on the basis of NMR and MS data. Other two degradation products (3, 4) were determined by LC-MS analysis. 2010 Published by Elsevier B.V.

Full text of DOI:10.1016/j.fitote.2010.10.008

Fitoterapia 82 (2011) 260–266
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Isolation and identification of degradation products of salvianolic acid A by
NMR and LC-MS
⁎
Jinzhong Xu, Shanshan Zeng, Xueying Chen, Haibin Qu
Department of Chinese Medicine Science and Engineering, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 June 2010
Accepted in revised form 11 October 2010
Available online 26 October 2010
Salvianolic acid A was subjected to thermal degradation condition in distilled water at 90 °C.
Four degradation products, including two novel compounds (1, 2) and two known ones (5, 6),
were isolated by reverse-phase semi-preparative liquid chromatography and identified on the
basis of NMR and MS data. Other two degradation products (3, 4) were determined by LC-MS
analysis.
Keywords:
© 2010 Published by Elsevier B.V.
Salvianolic acid A
Degradation products
NMR
LC-MS
Identification
1. Introduction
solution was found. Undoubtedly, determination of degrada-
tion products of SalA is very important for efficacy and safety
Salvianolic acid A (SalA, Fig. 1) is one of major components
of Salvia miltiorrhiza (Danshen in Chinese) and its traditional
Chinese medicinal preparations [1,2]. This caffeoyl derivative
showed various bioactivities, such as protective effect against
impairment of memory induced by cerebral ischemia-
reperfusion [3], scavenger of oxygen radicals released by
activated neutrophils [4], protective action on hepatocyte
injured by peroxidation, inhibition of lipid peroxidation in
mitochondrial membrane of hepatic cells [5] and apoptosis of
human neuroblastoma SH-SY5Y cells [6], and protective
effect on acute liver injury induced by CCl4 in rats [7]. As a
result, there is a great interest in SalA as a new drug in the
pharmaceutical field. During our research about Danshen
injection, a popular preparation of Danshen, SalA was found
instable either in distilled water even at room temperature
[8]. However, to our knowledge, no publication reporting the
instability and its reaction products of SalA in aqueous
of potential drugs derived from SalA. The present paper
described the isolation and identification of degradation
products formed in aqueous solution.
2. Experimental
2.1. Chemicals and reagents
Acetonitrile was chromatographic grade and purchased
from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China).
Deionized water was generated by a Mill-Q academic water
purification system (Milford, MA, USA). Salvianolic acid A was
isolated from the aqueous extract of Danshen by authors. It
was identified by the MS and NMR technology and its purity
(≥95%) was determined by HPLC analysis.
2.2. High-performance liquid chromatography (HPLC)
The analytical HPLC system HP 1100 series (Agilent
Technologies, Waldbronn, Germany) equipped with the
Chemstation Software (Agilent Technologies) and comprised
⁎
Corresponding author. Tel./fax: +86 571 88208428.
E-mail address: quhb@zju.edu.cn (H. Qu).
0367-326X/$ – see front matter © 2010 Published by Elsevier B.V.
doi:10.1016/j.fitote.2010.10.008

J. Xu et al. / Fitoterapia 82 (2011) 260–266
261
Fig. 1. Chemical structures of SalA and its oxidation products.
a quaternary pump, an online vacuum degasser, an auto-
sampler, a thermostated column compartment, and a UV
detector. The analysis was carried out on a Tigerkin C18
column (200 mm×4.6 mm i.d., 5.0 μm particle size) from
DALIAN SIPORE CO., LTD (Dalian, China) and the elution
programme was as described in other report of our research
group [8]. The detective wavelength was 280 nm.
spectrometer (Thermo Finnigan, San Jose, USA) which
equipped with an ESI interface at 350 °C. Detection of ions
was performed with negative ion mode.
3. Results and discussion
3.1. Enrichment and isolation of degradation products
The preparative HPLC system HP 1200 series (Agilent
Technologies, Waldbronn, Germany) equipped with the
Chemstation Software (Agilent Technologies) and comprised
a quaternary pump and A DAD detector. Reaction products
were semi-prepared by a Zorbax C18 column (9.4 mm×25 cm)
and mobile phases of 0.5% aqueous formic acid (A) and
acetonitrile (B) with the elution program as follows:
0~28 min, A-B (82: 18, v/v); 28~60 min, A-B (82: 18~65: 35,
v/v) with a linear gradient. The flow rate is 4 mL/min.
Purified SalA (about 200 mg) was dissolved in distilled
water (10 mL) and then heated at 90 °C in water bath. The
post-react solvent was analyzed by the analytical HPLC to
ensure that SalA was entirely transformed. At last four
degradation products were isolated and purified from the
post-react solvent by semi-preparative HPLC.
3.2. Structural elucidation of degradation products
2.3. NMR spectroscopy
Compound 1 was obtained as yellow powder. The molecular
formula of compound 1, C26H22O11, was established from
HR-ESI-MS (negative) at m/z 509.1053 [M-H]− (calcd. for
The 1D-NMR (1 H NMR, 13 C NMR and DEPT) and 2D-NMR
(1 H-1 H COSY, HMQC and HMBC) were recorded on Bruker
500 MHz nuclear magnetic resonance spectrometer using
CD3OD as solvent.
C
26H21O11, 509.1078) and 13C-NMR spectral data. Its molecular
is more one oxygen atom than SalA, which indicated that
compound 1 was oxidative product of SalA. The proton NMR
spectrum of 1 showed the presence of eight aromatic proton
signals and two olefinic proton signals, which were distin-
guished by their coupling constants, and five aliphatic proton
signals (Table 1). Comparing with NMR data of SalA, two
olefinic protons were absent while two aliphatic ones were
2.4. LC-MS
HPLC method was followed as described in Section 2.2.
Mass spectra were recorded on an LCQ deca XPplus mass

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J. Xu et al. / Fitoterapia 82 (2011) 260–266
Table 1
Table 2
NMR spectral data for 1 and 2 (CD3OD).
13C-NMR spectral data for 1 and 2 (CD3OD, 125 MHz).
Unit
A
Position
1
2
Position
1
2
Position
1
2
5
6
7
8
2
5
6
7α
7β
8
2
5
7.15, d, J=8.0 Hz
6.82, d, J=8.0 Hz
7.53, d, J=16.0 Hz
6.20, d, J=16.0 Hz
6.70, bs
6.73, d, J=8.0 Hz
6.49, dd, J=8.0, 1.5 Hz
3.05, dd, J=14.5, 4.0 Hz
2.94, dd, J=14.5, 4.0 Hz
5.09, dd, J=4.0 Hz
6.45, bs
7.16, d, J=8.0 Hz
6.81, d, J=8.0 Hz
7.50, d, J=16.0 Hz
6.20, d, J=16.0 Hz
6.73, bs
6.71, d, J=8.0 Hz
6.58, dd, J=8.0, 1.5 Hz
3.05, dd, J=14.5, 4.0 Hz
2.96, dd, J=14.5, 4.0 Hz
5.10, dd, J=4.0 Hz
6.45, bs
A1
A2
A3
A4
A5
A6
A7
A 8
A 9
B1
B2
B3
B4
122.8
130.7
144.0
146.0
121.5
116.1
142.8
114.8
166.8
127.9
116.0
144.7
144.2
122.8
130.8
143.8
145.9
121.2
116.1
142.7
114.8
166.9
127.9
116.1
144.7
144.2
B5
B6
B7
B8
B9
C1
C2
C3
C4
C5
C6
C7
C8
115.4
120.7
36.5
115.4
120.5
36.4
73.3
73.4
B
C
172.1
131.6
113.9
145.4
143.8
115.0
118.8
55.6
172.2
131.7
113.9
145.4
144.0
114.9
118.7
55.6
6.87, d, J=8.0 Hz
6.50, dd, J=8.0, 1.5 Hz
4.38, bs
6.71, d, J=8.0 Hz
6.48, dd, J=8.0, 1.5 Hz
4.35, bs
6
7
108.3
108.3
8
5.65, bs
5.66, bs
(m/z 509, negative ion, Fig. 3) and NMR data as those of
compound 1. The planar structure of 2 was evaluated as
shown in Fig. 1 and its proton and carbon signals were
assigned on the basis of 2D-NMR analysis and comparison
with those of 1 (Tables 1 and 2). As to the stereochemistry of
1 and 2, the configuration of dihydrobenzofuran ring was
determined as S/S or R/R according to the fact of JC7 ~ C8 ≈0 Hz.
Interestingly, other two degradation compounds (3 and 4)
were found in the thermal degradation sample of SalA
(Fig. 4), which showed the same MS spectra as those of
compounds 1 and 2 (Fig. 3). It is conjecturable that
compounds 3 and 4 were epimers of 1 or 2 with the S/R or
R/S configuration in dihydrobenzofuran ring. Compounds 5
and 6 were obtained as yellow powder. The ESI mass spectra
(negative ions) of compounds 5 and 6 gave the same quasi-
molecular ion peaks at m/z 491 amu ([M-H]−) in ESIMS,
which indicated that the two compounds have the same
molecular weight of 492, less 2 Da than SalA. Comparison
with the reported MS and NMR data [9–11], compounds 5 and
6 were identified as isosalvianolic acid C and salvianolic acid C
(iso-SalC and SlaC, Fig. 1).
added, which indicated compound 1 may be degraded from
SalA by oxidation of double bond. Through the analysis of 2D
NMR data (1H-1H COSY, HMQC, and HMBC, Fig. 2), the
structural subunit of A and B in compound 1 (Fig. 1), whose
primaryatomswere carboxylic carbon signals of δC 166.8 and δC
172.1, respectively, were firstly determined. These two sub-
units were identical with SalA. Among unassigned NMR proton
signals, a broad singlet signal (δ 6.45), a doublet signal (δ 6.87,
J=8.0 Hz) and a doublet-doublet signal (δ 6.50, J=8.0, 1.5 Hz)
composed an ABX coupling system identical to SalA. The last
two broad singlet signals (δ 5.65 and δ 4.38) were different
points from the spectrum of SlaA and their directly connected
carbon signals were assigned as δC 55.6 and δC 108.3,
respectively, from HMQC. A hydroxyl proton signal (δ 7.47)
was observed to correlate to proton signal δ 5.52 (δ 5.65 in
CD3OD) in 1H-1H COSY when NMR solvent is DMSO-d6, which
indicated a hemiacetal group. So far, the structural subunit C of
1 was elucidated. Finally the planar structure of compound 1
was determined as shown in Fig. 1 and proton and carbon
signals had been totally assigned on the basis of 2D-NMR
analysis (Tables 1 and 2).
Compound 2 was obtained as yellow powder. This
compound had been baseline separated from compound 1
by HPLC. However this compound showed very similar MS
3.3. Formation mechanism of degradation products
According to their chemical structures, these degradation
compounds 1–6 were oxidation products of SalA, in which
1–4 were the products of mono-oxygenation and 5–6 were
the result of dehydrogenation. The proposed oxidation
pathway is shown in Fig. 5, in which pinacol rearrangement
and hemiacetal formation reaction will produce epimers. The
latter is a stereo-selective step by Cram Rule and the major
products are R/R and S/S configuration at the dihydrobenzo-
furan ring, which consisted with the result of LC-MS analysis
(Fig. 4).
Acknowledgements
This work was supported by the key project from Great
Science & Technology Special Grant of Zhejiang Province (No.
200813004-1) and Program of Education Department of
Zhejiang Province (Y200804910).
Fig. 2. Key 1 H-1 H COSY and HMBC correlations for 1 and 2.

J. Xu et al. / Fitoterapia 82 (2011) 260–266
263

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J. Xu et al. / Fitoterapia 82 (2011) 260–266
Fig. 3. MS spectra of compounds 1–4.

J. Xu et al. / Fitoterapia 82 (2011) 260–266
265
Fig. 4. LC-MS analysis of the thermal degradation sample of SalA (A: TIC, negative ion; B: m/z 509 ion extraction; C: m/z 491 ion extraction).
Fig. 5. Proposed forming pathway of degradation products.

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