Reassignment of the configuration of salvianolic acid B and establishment of its identity with lithospermic acid B

Article abstract of DOI:10.1021/np060136w

Salvianolic acid B and lithospermic acid B are the major components of Salvia miltiorrhiza, which is one of the most popular herbal traditional medicines in Asian countries. Salvianolic acid B and lithospermic acid B are reported to have identical structures except for the configurational assignments of two stereocenters. Through chemical correlation between a degradation product of salvianolic acid B and synthetic material, the absolute configuration of Salvianolic acid B has been corrected to establish that Salvianolic acid B and lithospermic acid B are in fact the same compound.

Full text of DOI:10.1021/np060136w

J. Nat. Prod. 2006, 69, 1231-1233
1231
Reassignment of the Configuration of Salvianolic Acid B and Establishment of Its Identity with
Lithospermic Acid B
Anja Watzke, Steven J. O’Malley, Robert G. Bergman,* and Jonathan A. Ellman*
Department of Chemistry, UniVersity of California-Berkeley, Berkeley, California 94720
ReceiVed March 25, 2006
Salvianolic acid B and lithospermic acid B are the major components of SalVia miltiorrhiza, which is one of the most
popular herbal traditional medicines in Asian countries. Salvianolic acid B and lithospermic acid B are reported to have
identical structures except for the configurational assignments of two stereocenters. Through chemical correlation between
a degradation product of salvianolic acid B and synthetic material, the absolute configuration of salvianolic acid B has
been corrected to establish that salvianolic acid B and lithospermic acid B are in fact the same compound.
The dried root of SalVia miltiorrhiza (Danshen in Chinese) is
one of the most popular herbal traditional medicines in Asian
countries and has been used extensively for the treatment of
coronary artery diseases, angina pectoris, myocardial infarction,
cerebrovascular diseases, various types of hepatitis, chronic renal
failure, and dysmenorrhea.1 In the United States and European
countries, Danshen products have been widely used for the treatment
of cardiovascular and cerebrovascular diseases, whereas in China
the specific clinical use is to treat angina pectoris. In Japan, Danshen
products are used to promote circulation and improve blood flow.
Early studies of the chemical constituents of Danshen mainly
focused on the lipophilic compounds. More than 30 diterpenes have
been isolated and identified and their pharmacological activities
studied.2 Recent studies have focused more on the hydrophilic
compounds with caffeic acid derivatives occurring as the major
water-soluble components.3 Twenty-five caffeic acid derivatives
have been isolated and identified from the aqueous extracts of
Danshen including caffeic acid monomers and oligomers that are
called salvianolic acids or lithospermic acids.4 Many of these
derivatives have known pharmacological activities, and a wide
variety of biological activities have been evaluated.1,5 The structures
of isolated caffeic acid derivatives have been elucidated and
classified as caffeic acid monomers, to which 3-(3,4-dihydroxy-
phenyl)lactic acid (1) belongs, caffeic acid dimers, of which
rosmarinic acid (2) is the simplest representative, and caffeic acid
oligomers such as lithospermic acid (3), which is characterized by
the presence of a dihydrobenzofuran nucleus (Scheme 1).
Among the polyphenolic compounds, salvianolic acid B is a
major component in S. miltiorrhiza (Scheme 2),6 and extensive
pharmacological studies have been reported for this compound. In
animal studies, salvianolic acid B has shown several beneficial
cardiovascular effects.7 The configurational assignments for salvi-
anolic acid B were based on chemical degradation and circular
dichroic correlation.6 In particular, the R,â-positions of the dihy-
drobenzofuran core were assigned the R/R-configuration (structure
4, Scheme 2).
Scheme 1. Structures of 3-(3,4-Dihydroxyphenyl)lactic Acid
(1), Rosmarinic Acid (2), and Lithospermic Acid (3)
Scheme 2. Reported Structures of Salvianolic Acid B (4) and
Lithospermic Acid B (5)
Lithospermic acid B (5) has also been identified as a major
component of S. miltiorrhiza (Scheme 2). The therapeutic effects
of lithospermic acid B and its salt, magnesium lithospermate B,
have been investigated extensively, including hepatoprotection,8
elicitation of endothelium-dependent vasodilation,9 lowering of
blood pressure in hypertension,10 and inhibition of HIV-1 replica-
tion.11 The absolute configuration of lithospermic acid B was based
on the chemical degradation of permethylated lithospermic acid B
and circular dichroic correlation.12 Using the exciton chirality
method, the dihydrobenzofuran core was assigned the S/S-config-
uration (Scheme 2).
Previous literature suggests that the structures of lithospermic
acid B and salvianolic acid B differ only in the absolute configu-
ration of the stereocenters present in the dihydrobenzofuran core.
Because each is reported to be the major component from the same
plant using similar isolation techniques,13 we postulated that they
are in fact the same compound. This implies that the configurational
assignments for one of the structures are incorrect. For this reason
* Corresponding author. E-mail: jellman@uclink.berkeley.edu.
10.1021/np060136w CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/02/2006

1232 Journal of Natural Products, 2006, Vol. 69, No. 8
Notes
Scheme 3. Methanolysis of Lithospermic Acid Synthetic
Intermediate
In conclusion, through chemical correlation we have corrected
the configurational assignments for salvianolic acid B and thereby
have established that salvianolic acid B and lithospermic acid B
are identical. These results have profound pharmacological implica-
tions because it means that the extensive pharmacological studies
that have separately been reported for salvianolic acid B and
lithospermic acid B must now both be considered when evaluating
the bioactivity of this natural product. Moreover, the configurational
assignments provided here are of critical importance to any future
synthesis of the natural product or its analogues.
Experimental Section
General Experimental Procedures. Materials were obtained from
commercial suppliers and used without further purification. Salvianolic
acid B was purchased from Ivy Fine Chemicals (Cherry Hill, NJ). All
organic reactions were performed under an atmosphere of N2 in flame-
dried or oven-dried glassware. Thin-layer chromatography was per-
formed on Merck 60 F254 250 µm silica gel plates. Visualization of
the developed chromatogram was performed by fluorescence quenching
or KMnO4 stain. Flash chromatography was carried out using Merck
Scheme 4. Methylation and Degradation of Commercial
Salvianolic Acid B
1
60 230-240 mesh silica gel. H and 13C NMR spectra were obtained
in CDCl3. NMR chemical shifts are reported in ppm and referenced to
residual protonated solvent. A Perkin-Elmer 241 polarimeter with a
sodium lamp was used to determine optical rotations, and concentrations
are reported in g/dL. CD spectra were measured with a AVIV 60DS
apparatus.
Compound 4, salvianolic acid B: 1H NMR ((CD3)2CO, 400 MHz)
δ 7.85 (1H, d, J ) 16.0 Hz, CHdCHCO2CH3), 7.63 (1H, d, J ) 16.0
Hz, ArH), 6.90 (1H, d, J ) 8.4 Hz, ArH), 6.84-6.64 (8H, m, ArH ×
8), 6.44 (1H, dd, J ) 2.0, 8.0 Hz, ArH), 6.29 (1H, d, J ) 16.0 Hz,
CHdCHCO2CH3), 5.88 (1H, d, J ) 4.4 Hz, ArOCHAr), 5.23-5.19
(2H, m, ArCH2CHCO2CH3 × 2), 4.47 (1H, d, J ) 4.4 Hz, ArCHCO2R),
3.12-2.90 (4H, m, ArCH2CHCO2CH3 × 2); 13C NMR ((CD3)2CO, 100
MHz) δ 171.8, 171.5, 170.9, 166.9, 148.9, 146.6, 146.3, 145.9, 145.9,
145.1, 145.0, 144.8, 143.1, 133.6, 129.3, 128.9, 126.4, 124.8, 122.1,
122.0, 121.9, 118.6, 118.5, 117.6, 117.5, 117.0, 116.4, 116.4, 116.3,
113.5, 87.8, 75.1, 74.1, 57.3, 37.7, 37.3; CD (c 0.066, EtOH) {Φ} (nm)
+10 204 (340), +8761 (320), +6592 (300), -3009 (283), -13 629
(265), +25 614 (255), +9692 (240), +3647 (223).
we have carried out further investigations on the absolute config-
uration of these compounds.
Compound 6, heptamethyl-(+)-lithospermic acid. Heptamethyl
lithospermic acid was prepared by total synthesis as previously
Recently, we reported the total synthesis of lithospermic acid
(3, Scheme 1),14 which has attracted interest due to its potent
inhibitory activity against HIV-1 integrase.11 The configurational
assignments were established by X-ray analysis using anomalous
dispersion of a bromo-substituted derivative of a late-stage synthetic
intermediate.14 Here we have used the synthetic sequence to make
the configurational assignments for salvianolic acid B by chemical
correlation. Specifically, the synthetic permethylated lithospermic
acid derivative 6, with S/S-configuration at the dihydrobenzofuran
ring, was treated with sodium methoxide to provide 7 (Scheme 3).
The specific rotation was determined to have a large positive value.
According to the literature, the configurational assignments for
salvianolic acid B were established by permethylation of salvianolic
acid B followed by methanolysis to provide a dihydrobenzofuran
derivative, which by circular dichroic correlation was proposed to
be the enantiomer of 7.6 Interestingly, for lithospermic acid B other
researchers assigned the opposite configuration to the stereocenters
present in the dihydrobenzofuran core using the same degradation
method but different circular dichroism techniques.12
We submitted commercially available salvianolic acid B to the
degradation steps previously reported (Scheme 4). After permethyl-
ation and final methanolysis of salvianolic acid B, compound 9
was found to have the same positive specific rotation and relative
magnitude as that for the synthetic compound 7 (Scheme 3).15 In
addition, the CD spectra for compounds 7 and 9 are identical. These
results clearly show that the absolute configurations previously
assigned to the dihydrobenzofuran stereocenters of salvianolic acid
B were incorrect and demonstrate that salvianolic acid B and
lithospermic acid B are the same compound.
1
reported:14 [R]25 +134.50 (c 0.49, CH2Cl2); H NMR (CDCl3, 400
D
MHz) δ 7.71 (1H, d, J ) 15.9 Hz, CHdCHCO2CH3), 7.17 (1H, d, J
) 8.5 Hz, ArH), 6.89-6.85 (3H, m, ArH × 3), 6.81 (1H, d, J ) 8.2
Hz, ArH), 6.77-6.74 (3H, m, ArH × 3), 6.29 (1H, d, J ) 15.9 Hz,
CHdCHCO2CH3), 6.01 (1H, d, J ) 5.6 Hz, ArOCHAr), 5.31 (1H, dd,
J ) 4.8, 8.2 Hz, ArCH2CHCO2CH3), 4.45 (1H, d, J ) 5.6 Hz,
ArCHCO2CH3), 3.91 (3H, s, OCH3), 3.84 (3H, s, OCH3), 3.83 (3H, s,
OCH3), 3.82 (3H, s, OCH3), 3.81 (3H, s, OCH3), 3.71 (6H, s, CO2CH3
× 2), 3.16 (1H, dd, J ) 4.7, 14.3 Hz, one of ArCH2CHCO2CH3), 3.09
(1H, dd, J ) 8.2, 14.3 Hz, one of ArCH2CHCO2CH3); 13C NMR
(CDCl3, 100 MHz) δ 171.4, 170.0, 165.8, 149.0, 149.0, 148.5, 148.2,
147.8, 146.1, 142.2, 132.1, 128.8, 128.1, 124.7, 124.1, 121.2, 120.6,
117.7, 116.2, 112.7, 112.2, 110.9, 110.9, 108.5, 87.2, 72.8, 55.9, 55.6,
55.6, 55.6, 55.5, 52.5, 52.0, 36.8.; FAB(+)-HRMS m/z 636.2216 (calcd
for C34H36O12 [M]+, 636.2207).
Compound 7. Methanolysis of Synthetic Heptamethyllithosper-
mic Acid. To a solution of 25 mg (0.038 mmol) of heptamethyllithos-
permic acid in 3 mL of CHCl3 was added dropwise 1.5 equiv of NaOMe
(0.094 mmol, 157 µL of a 0.6 N solution in MeOH) at 0 °C. The
reaction mixture was stirred for 1 h at 0 °C and one further hour at
room temperature. After addition of 50 µL of HOAc and 50 µL of
H2O the solvent was evaporated under reduced pressure and the crude
product was purified by column chromatography [silica gel, elution
with CH2Cl2-Et2O (95:5)], yielding 7 as an off-white solid (14 mg,
0.033 mmol, 85%): [R] 25D +131.7 (c 0.6, CHCl3); 1H NMR (CDCl3,
400 MHz) δ 7.67 (1H, d, J ) 15.6 Hz, CHdCHCO2CH3), 7.18 (1H,
s, J ) 8.4 Hz, ArH), 6.90-6.86 (3H, m, ArH × 3), 6.80 (1H, d, J )
8.0 Hz, ArH), 6.26 (1H, d, J ) 16.0 Hz, CHdCHCO2CH3), 6.00 (1H,
d, J ) 5.6 Hz, ArOCHAr), 4.45 (1H, d, J ) 6.0 Hz, ArCHCO2CH3),
3.91 (3H, s, OCH3), 3.84 (3H, s, OCH3), 3.83 (3H, s, OCH3), 3.75
(3H, s, CO2CH3), 3.75 (3H, s, CO2CH3); 13C NMR (CDCl3, 100 MHz)

Notes
Journal of Natural Products, 2006, Vol. 69, No. 8 1233
δ 171.8, 167.3, 149.3, 149.2, 148.4, 146.1, 141.1, 132.3, 125.0, 124.5,
120.5, 118.0, 117.3, 112.9, 111.1, 108.7, 87.5, 56.1, 55.9, 52.8, 51.6;
FAB(+)-HRMS m/z 428.1479 (calcd for C23H24O8 [M]+, 428.1471);
CD (c 0.066, CHCl3) {Φ} (nm) +10 547 (340), +13 237 (320),
+10 230 (300), +40 481 (265), +90 233 (256), +15 654 (240), +5344
(227).
DAAD (to A.W.), and by the Director and Office of Energy Research,
Office of Basic Energy Sciences, Chemical Sciences Division, U.S.
Department of Energy, under Contract DE-AC03-76SF00098 (to
R.G.B.).
1
Supporting Information Available: Includes H and 13C NMR
Compound 8. Methylation of Salvianolic Acid B. To a suspension
of 40 mg (0.0557 mmol) of salvianolic acid B and 20 equiv of K2CO3
(1.11 mmol, 154 mg) in 5 mL of dry acetone was added 30 equiv of
Me2SO4 (1.67 mmol, 158 µL). The reaction mixture was heated to reflux
for 5 h. After filtration of the inorganic salts the solvent was evaporated
under reduced pressure and the crude product was purified by column
chromatography [silica gel, benzene-acetone (9:1)], yielding 8 as an
off-white solid (31 mg, 0.037 mmol, 66%): 1H NMR (CDCl3, 400
MHz) δ 7.52 ) (1H, d, J ) 16.0 Hz, CHdCHCO2CH3), 7.12 (1H, d,
J ) 8.4 Hz, ArH), 6.87-6.73 (7H, m, ArH × 7), 6.65 (1H, d, J ) 8.0
Hz, ArH), 6.52 (2H, m, ArH), 6.16 (1H, d, J ) 16.0 Hz, CHdCHCO2-
CH3), 5.99 (1H, d, J ) 5.6 Hz, ArOCHAr), 5.28-5.20 (2H, m,
ArCH2CHCO2CH3 × 2), 4.40 (1H, d, J ) 6.0 Hz, ArCHCO2R), 3.92
(3H, s, OCH3), 3.83 (3H, s, OCH3), 3.82 (3H, s, OCH3), 3.82 (3H, s,
OCH3), 3.81 (3H, s, OCH3), 3.79 (3H, s, OCH3), 3.77 (3H, s, OCH3),
3.67 (3H, s, CO2CH3), 3.66 (3H, s, CO2CH3), 3.11-2.94 (4H, m,
ArCH2CHCO2CH3 × 2); 13C NMR (CDCl3, 100 MHz) δ 170.2, 170.2,
169.3, 165.9, 149.2, 149.2, 148.7, 148.6, 148.5, 148.0, 147.8, 146.2,
142.0, 132.2, 128.4, 127.9, 124.5, 124.3, 121.5, 120.8, 120.8, 118.0,
116.2, 112.8, 112.3, 111.1, 108.6, 87.2, 74.1, 73.1, 56.3, 56.1, 55.9,
55.8, 55.8, 55.7, 55.6, 52.4, 52.2, 37.1, 36.6; FAB(+)-HRMS m/z
844.2950 (calcd for C45H48O16 [M]+, 844.2942).
spectra of compounds 4, 7, 8, and 9 and CD spectra of compounds 4,
7, and 8. This material is available free of charge via the Internet at
http://pubs.acs.org.
References and Notes
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1345-1359. (c) Lu, Y. R.; Foo, L. Y. Phytochemistry 2002, 59, 117-
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(2) (a) Chang, H. M.; Cheng, K. P.; Choang, T. F.; Chow, H. F.; Chui,
K. Y.; Hon, P. M.; Tan, F. W. L.; Yang, Y.; Zhong, Z. Z.; Lee, C.
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Chem. 1990, 55, 3537-3543. (b) Sabri, N. N.; El-Lakany, A. M.;
Alexandria. J. Pharm. Sci. 1990, 4, 94-105.
(3) (a) Li, X.; Yu, C.; Cai, Y.; Liu, G.; Jia, J.; Wang, Y. J. Chromatogr.
B 2005, 820, 41-47. (b) Yokozawa, T.; Liu, Z. W.; Chen, C. P.;
Tanaka, T. Pharm. Pharmacol. Commun. 1999, 5, 365-370.
(4) Li, L. N. J. Chin. Pharm. Sci. 1997, 6, 57-64.
(5) (a) Kong, D. Y. Zhongguo Yiyao Gongye Zazhi 1989, 20, 279-285.
(b) Zheng, G. H.; Kakisawa, H. Zhongguo Yaoxue Zazhi 1989, 24,
6-10. (c) Cai, D. G. Zhongguo Zhongyao Zazhi 1991, 16, 376-
377. (d) Li, L. N. Zhongguo Yaoxue 1997, 6, 57-64.
(6) Ai, C. B.; Li, L. N. J. Nat. Prod. 1988, 51, 145-149.
(7) O, K.; Cheung, F.; Sung, F. L.; Zhu, D. Y.; Siow, Y. L. Mol. Cell.
Biochem. 2000, 207, 35-39.
Compound 9. Degradation of Nonamethylsalvianolic Acid B. To
a solution of 14 mg (0.0166 mmol) of nonamethyl salvianolic acid B
in 3 mL of CHCl3 was added dropwise at 0 °C 3 equiv of NaOMe
(0.0498 mmol, 83 µL of a 0.6 N solution in MeOH). The reaction
mixture was stirred for 1 h at 0 °C and one further hour at room
temperature. After addition of 50 µL of HOAc and 50 µL of H2O the
solvent was evaporated under reduced pressure and the crude product
was purified by column chromatography [silica gel, CH2Cl2-Et2O (95:
(8) Hase, K.; Kasimu, R.; Basnet, P.; Kadota, S.; Namba, T. Planta Med.
1997, 63, 22-26.
(9) Kamata, K.; Iizuka, T.; Nagai, M.; Kasuya, Y. Gen. Pharmacol. 1993,
24, 977-981.
(10) Kamata, K.; Noguchi, M.; Nagai, M. Gen. Pharmacol. 1994, 25,
69-73.
(11) Abd-Elazem, I.; Chen, H. S.; Bates, R. B.; Huang, R. C. C. AntiViral
Res. 2002, 55, 91-1.
5)], yielding 9 as an off-white solid (4 mg, 0.009 mmol, 56%): [R] 25
D
1
+122.3 (c 4.0, CHCl3); H NMR (CDCl3, 400 MHz) δ 7.68 (1H, d, J
(12) Tanaka, T.; Morimoto, S.; Nonaka, G.; Nishioka, I.; Yokozawa, T.;
Chung, H. Y.; Oura, H. Chem. Pharm. Bull. 1989, 37, 340-344.
(13) Salvianolic acid was extracted from S. miltiorrhiza Radix with water
and isolated by MCI-gel and Sephadex LH-20 column chromatog-
raphy (ref 6). Lithospermic acid B was extracted from S. miltiorrhiza
Radix with EtOH and H2O and isolated by SiO2 and Sephadex LH-
20 column chromatography (ref 12).
) 16.0 Hz, CHdCHCO2CH3), 7.18 (1H, d, J ) 8.8 Hz, ArH), 6.90-
6.85 (3H, m, ArH × 3), 6.80 (1H, d, J ) 8.4 Hz, ArH), 6.26 (1H, d,
J ) 16.0 Hz, CHdCHCO2CH3), 6.00 (1H, d, J ) 5.6 Hz, ArOCHAr),
4.46 (1H, d, J ) 5.6 Hz, ArCHCO2CH3), 3.92 (3H, s, OCH3), 3.84
(3H, s, OCH3), 3.84 (3H, s, OCH3), 3.75 (3H, s, CO2CH3), 3.71 (3H,
s, CO2CH3); 13C NMR (CDCl3, 100 MHz) δ 171.8, 167.3, 149.3, 149.2,
148.4, 146.1, 141.1, 132.3, 125.0, 124.5, 120.5, 118.0, 117.3, 112.9,
111.1, 108.7, 87.5, 56.1, 55.9, 52.8, 51.6; FAB(+)-HRMS 428.1479
m/z (calcd for C23H24O8 [M]+, 428.1471); CD (c 0.066, CHCl3) {Φ}
(nm) +7841 (340), +10 051 (320), +8561 (300), +27 046 (265),
+66 119 (256), +14 445 (240), +1246 (227).
(14) O’Malley, S. J.; Tan, K. L.; Watzke, A.; Bergman, R. G.; Ellman, J.
A. J. Am. Chem. Soc. 2005, 127, 13496-13497.
(15) The NMR data for 9 are identical to the NMR data reported by Ai
and Li for the same degradation product in their configurational
assignment of salvianolic acid B.6 The specific rotation of 9 in EtOH
[+88 (c 0.1, EtOH)] also has the same sign as the specific rotation
reported by Ai and Li [+41 (c 0.092, EtOH)]. The CD spectrum of
compound 9 [Supporting Information] also corresponds to the data
reported by Ai and Li.6 This comparison of analytical data confirms
that the same degradation product, i.e., identical structure and
configuration, was obtained in both of our studies.
Acknowledgment. Professor K. Nakanishi is gratefully acknowl-
edged for his analysis of the previously reported configurational
assignments determined through CD measurements for salvianolic acid
B and lithospermic acid B. This work was supported by the NIH,
GM069559 (to J.A.E.) and 5F32GM071207-02 (to S.O.M.), by the
NP060136W