A new triterpenoid with antimicrobial activity from Anemone rivularis

Article abstract of DOI:10.1007/s10600-012-0387-x

A new triterpene ester 1 and six known triterpenoids were isolated from Anemone rivularis by repeated column chromatography. Their structures were identified as olean-9(11),12-dien-3-O-palmitate (1), lupeol (2), betulin (3), betulic acid (4), oleanolic acid (5), ursolic acid (6), and β-amyrin (7) by spectral analysis and comparison with the published data. In addition, compound 1 was evaluated in vitro for its antimicrobial activity. It was found to exhibit moderate activity against the Gram-positive bacteria B. subtilis and S. aureus.

Full text of DOI:10.1007/s10600-012-0387-x

Chemistry of Natural Compounds, Vol. 48, No. 5, November, 2012 [Russian original No. 5, September–October, 2012]
A NEW TRITERPENOID WITH ANTIMICROBIAL ACTIVITY
FROM Anemone rivularis
Chun-Chao Zhao,1* Jian-Hua Shao, and Ju-Di Fan
1
2
UDC 547.918
A new triterpene ester 1 and six known triterpenoids were isolated from Anemone rivularis by repeated
column chromatography. Their structures were identified as olean-9(11),12-dien-3-O-palmitate (1), lupeol
(
2), betulin (3), betulic acid (4), oleanolic acid (5), ursolic acid (6), and ꢀ-amyrin (7) by spectral analysis
and comparison with the published data. In addition, compound 1 was evaluated in vitro for its antimicrobial
activity. It was found to exhibit moderate activity against the Gram-positive bacteria B. subtilis and S. aureus.
Keywords: Anemone rivularis, chemical constituents, triterpenoid.
Anemone rivularis is widespread in China. Its roots have been used as an antitumor agent in Chinese traditional
medicine [1]. Although some triterpene glycosides were isolated from the genus Anemone [2–4], there have been few reports
concerning the secondary metabolites of A. rivularis. In this paper, we describe the isolation of seven triterpenoids from
A. rivularis as well as the identification of their structures by spectroscopic analysis and comparison with the published data.
The antimicrobial activity of compound 1 is also reported.
Compound 1 was isolated as a white powder, mp 249–251ꢁC. It showed a positive reaction with the Liebermann–
Burchard reagent. The molecular formula of 1 was suggested as C H O on the basis of HR-ESI-MS (found m/z 663.6112
4
6 78 2
+
1
[
[
1
M + H] ; calcd for C H O , 663.6080). The H NMR spectrum of 1 was very similar to that of olean-9(11),12-dien-3ꢀ-ol
5] except for the chemical shift at ꢂ 4.54 (1H, t like, J = 8.1 Hz, H-3) and additional signals including 28 protons at ꢂ: 1.25–1.36,
.63, 2.30 and another additional methyl signal at ꢂ 0.86 (3H, t, J = 6.8 Hz, CH ), which suggested that the proton of 3-OH is
46 79 2
3
replaced by a long-chain fatty acid. The HMBC cross-peak of H-3 with C-1ꢃ (ꢂ 173.2) indicated that the long chain fatty acid
was assigned to C-3.
Compound 1 was hydrolyzed with KOH [6]. The hydrolyzed alkaline solution was neutralizated with HCl and extracted
with CHCl , and a white mixture was obtained. The mixture was chromatographed on Sephadex LH-20 with CH Cl –MeOH
3
2
2
(
3:1) to yield olean-9(11),12-dien-3ꢀ-ol (8.1 mg) [5] and a white fatty acid (5 mg). The EI-MS of the white fatty acid showed
a molecular ion at m/z 256, corresponding to palmitic acid. Based on the above evidence, 1 was elucidated as olean-9(11),12-
dien-3-O-palmitate.
O
H
H
3
C(H
2
C)14C1
O
'
1
HMBC (HꢄC)
1
) College of Bioscience and Biotechnology, Yangzhou University, 225009, Yangzhou, P. R. China, e-mail:
chunchaozhao@hotmail.com; 2) School of Pharmacology, GuiYang Medical College, 550004, Guiyang, P. R. China.
Published in Khimiya Prirodnykh Soedinenii, No. 5, September–October, 2012, pp. 717–718. Original article submitted
September 8, 2011.
0
0
009-3130/12/4805-0803 2012 Springer Science+Business Media New York
803

TABLE 1. The Results of Antibacterial Activity of Compound 1
1
Ciprofloxacin
Tetracycline
Kind of Bacteria
Diameter of inhibition
Diameter of inhibition
zone, mm
MIC values, ꢅg·mL–1
MIC values, ꢅg·mL–1
zone, mm
B. subtilis
S. aureus
P. aeruginosa
E. coli
14.1
12.5
7.5
10.0
8.0
50
75
> 100
90
34.0
33.5
30.0
32.1
33.2
3
3
25
7
S. typhimurium
> 100
3
Compounds 2–7, by comparison with the published data, were identified as lupeol (2) [7], betulin (3) [8], betulic acid
4), ꢀ-amyrin (7) [9], oleanolic acid (5), and ursolic acid (6) [10], respectively.
(
The testing of antimicrobial activity of compound 1 was done in vitro using the Kirby-Bauer disc diffusion method
11]. The MIC values against bacterial strains were obtained using the broth macro-dilution method [12]. Both the Gram-positive
Bacillus subtilis, Staphyloccocus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi)
[
(
bacteria were grown in nutrient agar medium and incubated at 37ꢁC for 48 h. Compound 1 was dissolved in dimethyl sulfoxide
DMSO-d ). We found that compound 1 showed moderate activity against B. subtilis and S. aureus, and potent activity against
(
6
E. coli, S. typhimurium, and P. aeruginosa (Table 1).
EXPERIMENTAL
Melting points were determined on a Yanaco MP-S3 melting point apparatus and are uncorrected. EI-MS spectra
were measured with a VG-5050E mass spectrometer. HR-ESI-MS was performed on a QSTAR LCQ mass spectrometer.
NMR spectra were recorded on a Bruker ARX-300 NMR spectrometer using TMS as an internal standard. Silica gel,
2
00–300 mesh, was from Qingdao Ocean Chemical Group Co. Ltd., P. R. China. TLC was performed on HSGF254 precoated
silica gel plates, 10–40 ꢅm, from Yantai Chemical Plant, Yantai, P. R. China. Sephadex LH-20 gel was from Pharmacia.
1
Olean-9(11),12-dien-3-O-palmitate (1). H NMR (300 MHz, CDCl –CD OD, ꢂ, ppm, J/Hz): 0.83 (3H, s), 0.86
3
3
(
2
3H, t, J = 6.8, H-16ꢃ), 0.87 (3H, s), 0.91–0.92 (9H), 0.98 (3H, s), 1.08 (3H, s), 1.19 (3H, s), 1.25–1.26, 1.63 (26H, m, H-3ꢃ–15ꢃ),
.30 (2H, m, H-2ꢃ), 4.54 (1H, t like, J = 8.1, H-3), 5.50 (1H, d, J = 5.7, H-12), 5.56 (1H, d, J = 5.7, H-11). 1 C NMR (75 MHz,
3
CDCl –CD OD, ꢂ, ppm): 37.1 (C-1), 23.5 (C-2), 80.4 (C-3), 37.8 (C-4), 51.2 (C-5), 18.2 (C-6), 32.0 (C-7), 38.1 (C-8), 153.8
3
3
(
C-9), 40.7 (C-10), 115.9 (C-11), 120.6 (C-12), 147.2 (C-13), 42.6 (C-14), 25.7 (C-15), 27.2 (C-16), 32.2 (C-17), 45.6 (C-18),
6.8 (C-19), 31.2 (C-20), 34.6 (C-21), 36.9 (C-22), 28.6 (C-23), 16.7 (C-24), 20.1 (C-25), 21.0 (C-26), 25.4 (C-27), 28.2
C-28), 23.6 (C-29), 33.2 (C-30), 14.1 (C-16ꢃ), 22.8 (C-15ꢃ), 25.4 (C-3ꢃ), 29.0–30.2 (C-4ꢃ–13ꢃ), 31.7 (C-14ꢃ), 34.5 (C-2ꢃ),
73.2 (C-1ꢃ).
The EI-MS (70 eV) data of palmitic acid in compound 1 after alkaline hydrolysis showed m/z (I , %): 256 [M]
4
(
1
+
rel
(
100), 227 (7), 2132 (21), 185 (17), 157 (16), 129 (36).
ACKNOWLEDGMENT
This work was partially supported by the programs from the Analytical Detection Center, Shenyang Pharmaceutical
University, and theAnalytical Detection Center, Shanghai Second Military Medical University, theAnalytical Detective Center,
Yangzhou University. We are also grateful to Associate Professor Qingde Long, GuiYang Medical College, for identification
of the plant.
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