Original Papers
Two New 2,3-Seco-Hopane Triterpene Derivatives from Megacodon stylophorus and Their Antiproliferative and Antimicrobial Activities
Authors
Chao Liu 1, 3, Zhi-Xin Liao 1, 3, Shi-Jun Liu 1, Lan-Ju Ji 2, Hong-Fa Sun 2
Affiliations
1
2 3
Key words " Megacodon stylophorus l " Gentianaceae l " 2,3‑seco‑hopane l triterpenoids " antiproliferative l " antimicrobial l
Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Southeast University, Nanjing, P. R. China Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, P. R. China Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing, P. R. China
Abstract !
Chemical investigation of the ethanol extract of the whole plant of Megacodon stylophorus led to the isolation and identification of two new secohopane triterpenoids, 2,3-seco-22(29)-hopene2-carboxyl-3-aldehyde (1) and 2,3-seco-4(23),22 (29)-hopene-2-carboxyl-3-aldehyde (2), along with 10 known compounds, 3–12. All the isolates were reported from this plant for the first time. The structures of compounds 1 and 2 were determined by detailed analysis of their spectral data including 1D and 2D NMR. In addition, compound 1 was further analyzed by X‑ray crystallography.
Introduction !
received revised accepted
Nov. 28, 2013 May 18, 2014 May 20, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368612 Published online July 4, 2014 Planta Med 2014; 80: 936–941 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Dr. Zhi-Xin Liao Department of Pharmaceutical Engineering School of Chemistry and Chemical Engineering Southeast University No. 2, Southeast University Road Nanjing 211189 P. R. China Phone: + 86 25 52 09 06 20 Fax: + 86 25 52 09 06 18 [email protected]
Many species of the family Gentianaceae are reported to be medicinal plants for the treatment of hepatobiliary diseases [1–3]. Quite a number of chemical components such as xanthones [4– 5], terpenoids [6], aromatic glycosides [7], iridoids and secoiridoid glucosides [8] have been isolated from these medicinal plants. Megacodon (Hemsl.) H. Smith is a genus of Gentianaceae that comprises only two species: Megacodon stylophorus and Megacodon venosus [9–11]. M. venosus was once reported to be endemic to China and had been declared extinct in 1970s but was rediscovered by Prof. Zhen-Yu Li (Institute of Botany, the Chinese Academy of Sciences) in Chongqing municipality in 2007 [12]. M. stylophorus, a perennial herb distributed in Qinghai-Tibet Plateau and Himalaya areas, is generally used by the aboriginal inhabitants of Qinghai and Xizang provinces to clear heat and treat hepatopathy and gallbladder diseases [13]. Up to the present, no chemical investigations of the two plants have been reported in Chemical Abstracts. As part of our work on the chemical compositions of the genus Megacodon and biologically active
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Compounds 1–3 were evaluated for their in vitro anti-proliferative activities on HeLa, MCF-7, and Hep-G2 tumor cell lines. Compound 2 was active against the three cell lines with IC50 values of 3.6, 7.5, and 13.6 µM, respectively, while compound 1 exhibited cytotoxicity on MCF-7 (IC50 14.0 µM) and HeLa (IC50 18.2 µM) cell lines. Antimicrobial activities of compounds 1–2 (minimum inhibitory concentration values in the range of 3.12– 12.50 mg/mL) were also observed. Supporting information available online at http://www.thieme-connect.de/products
novel structures, the whole plant of M. stylophorus was investigated. As a result, two new 2,3-seco-hopane triterpenoids along with 10 known compounds were obtained from its ethanol extract. In this paper, we report the isolation, structure elucidation, as well as anti-tumor and antimicrobial activities of the new compounds.
Results and Discussion !
Repeated silica gel, MCI gel, or Sephadex LH-20 column chromatography (CC) of an ethanol extract of M. stylophorus afforded 12 compounds " Fig. 1). Their structures were identified (1–12) (l by means of IR, HR‑ESI‑MS, NMR spectroscopy (1H‑NMR, 13C‑NMR, HSQC, HMBC, and ROESY) and X‑ray crystallography, as well as comparing spectroscopic data and physicochemical properties with previous literature. The known compounds were identified as thysanolactone (3) [6], halenic acid A (4) [14], 5-formyl-2,3-dihydroisocoumarin (5) [15], stigmasterol (6), ursolic acid (7), ellagic acid (8) [16], 1,5,8-trihydroxy-3-methoxy-xanthone (9) [17], 1,5,7-trihydroxy-3-methoxy-xanthone (10) [18], 1,7-dihydroxy-3,8-di-
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Chemical structures of compounds 1–12.
Fig. 2 Perspective drawing of the X‑ray structure of 1. (Color figure available online only.)
methoxy xanthone (11) [19], and 1,3,7,8-tetrahydroxy-xanthone (12) [20]. Compound 1 was isolated as colorless crystals (CHCl3–MeOH, 10 : 1). mp 203 ~ 204 °C, [α]20 D = + 7.4 (c = 0.0047, MeOH). Its molecular formula was assigned to be C30H48O3 on the basis of the negative mode HR‑ESI‑MS, which gave a pseudomolecular ion peak at m/z 455.3530 [M – H]−. The structure of 1 was deduced as 2,3-seco-22(29)-hopene-2-carboxyl-3-aldehyde based on a " Fig. 2). The single crystal X‑ray diffraction analysis result (l NMR data further secured the structure. Signals for seven meth-
yls and a carboxyl proton were clearly observed in the 1H‑NMR spectra. The 13C‑NMR and DEPT spectra of 1 exhibited the presence of thirty carbons in the molecule. The assigned NMR data " Table 1. Moreover, the HMBC experiment (l " Fig. 3) of 1 are in l proved the cleavage of ring A and indicated that the fracture path was between C-2 and C-3 rather than C-3 and C-4 [21, 22] according to the long-range correlations of H3-23/C-3, H3-23/C‑5, H2-1/ C‑5, and H2-1/C-25. ROESY correlation analysis and X‑ray crystallography indicated that compound 1 and thysanolactone [6], a
Liu C et al. Two New 2,3-Seco-Hopane …
Planta Med 2014; 80: 936–941
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Fig. 1
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Original Papers
Position
1 δH
1α 1β 2 3 4 5 6α 6β 7α 7β 8 9 10 11α 11β 12α 12β 13 14 15α 15β 16α 16β 17 18 19α 19β 20α 20β 21 22 23α 23β 24 25 26 27 28 29α 29β 30
2.28, 1H, d, 15.7 2.56, 1H, d, 15.6 9.77, 1H, s 2.22, 1H, m 1.52, 1H, m 1.56, 1H, m 0.92, 1H, m 1.25, 1H, m 1.87, 1H, m 1.34, 1H, m 1.68, 1H, m 1.16, 1H, m 1.46, 1H,m 1.45, 1H, t, 2.65 1.30, 1H, t 1.52, 1H, t 1.42, 1H, m 1.86, 1H, m 1.01, 1H, m 1.04, 1H, m 1.51, 1H, m 1.18, 1H, m 1.41, 1H, m 2.25, 1H, m 1.09, 3H, s 1.14, 3H, s 0.89, 3H, s 1.01, 3H, s 0.94, 3H, s 0.68, 3H, s 4.67, 1H, s 4.69, 1H, s 1.68, 3H, s
2 δC 43.21 176.69 207.89 50.78 47.49 20.43 32.40 41.67 43.39 42.84 22.38 23.91 49.00 42.67 32.72 27.38 53.98 44.16 40.22 20.83 47.81 148.07 19.35 24.05 19.58 16.48 16.19 15.28 109.48 19.68
δH
δC
1.80, 1H, d, 14.5 2.34, 1H, d, 14.8 9.51, 1H, s 2.94, 1H, dd, 2.79, 12.55 1.34, 1H, m 1.96, 1H, m 1.27, 1H, m 1.58, 1H, m 2.02, 1H, m 1.43, 1H, m 1.58, 1H, m 1.37, 1H, m 1.77, 1H, m 1.45, 1H, t, 2.65 1.24, 1H, t 1.33, 1H, t 1.43, 1H, m 1.86, 1H, m 1.03, 1H, m 1.06, 1H, m 1.52, 1H, m 1.19, 1H, m 1.42, 1H, m 2.25, 1H, m 6.28, 1H, s 6.49, 1H, s 0.82, 3H, s 1.07, 3H, s 0.99, 3H, s 0.70, 3H, s 4.68, 1H, s 4.70, 1H, s 1.67, 3H, s
45.07 173.47 197.33 151.91 42.09 25.13 31.66 41.99 41.70 40.26 23.77 23.02 49.02 42.82 32.74 27.38 53.98 44.22 40.15 20.83 47.83 148.10 140.80
18.46 16.51 16.41 15.21 109.50 19.70
Fig. 3
natural compound also obtained from a Gentianaceae plant, shared the same stereochemistry. Compound 2 was obtained as a white amorphous powder, [α]20 D = + 7.9 (c = 0.006, MeOH). The negative HR‑ESI‑MS of 2 showed a pseudomolecular ion peak at m/z 439.3215 [M – H]−. Combining 1 " Table 1), the molecular formula was deH and 13C‑NMR data (l termined as C29H44O3. Liu C et al. Two New 2,3-Seco-Hopane …
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Table 1 1H and 13C NMR data of 1 and 2 at 500 and 125 MHz in CDCl3 (δ in ppm; J in Hz).
Key HMBC (H→C) correlations of 1 and 2.
Similarly to compound 1, the NMR spectra data of 2 showed the characteristic signals of one isopropenyl group, one carboxyl group, and one aldehyde group. However, only five methyl group signals appeared in the NMR spectra. Moreover, an additional olefinic bond was in 2 according to its NMR spectra [δC 140.80, δC 151.91, δH 6.28 and 6.49]. The DEPT suggested five tertiary methyl groups, eleven methylenes, six methines, and seven qua-
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Sample
1 2 3 Matrinea a
Table 3 Antimicrobial activity of the two new compounds against all tested microorganisms.
Cytotoxicity (IC50, µM) HeLa
MCF-7
Hep-G2
18.2 ± 1.2b 3.6 ± 0.4 68.2 ± 8.7 11.3
14.0 ± 1.8 7.5 ± 0.8 66.0 ± 10.9 12.1
30.7 ± 3.5 13.6 ± 1.6 70.4 ± 6.5 8.3
Tested microorganism
Salmonella typhimurium Escherichia coli Bacillus subtilis Staphylococcus aureus Aspergillus niger Saccharomyces cerevisiae Penicillium Fusarium oxysporum
Positive control; b Data represent the mean ± SD of three independent experiments
ternary carbons in the molecule. Comparison of the NMR data of 2 with those of 1 showed that both compounds were alike at rings B, C, D, and E except for an exocyclic double bond in 2 instead of two methyl groups at C-4. In the HMBC spectra " Fig. 3), the correlations of H -23 with both C-3 and C-5 con(l 2 firmed that the exomethylene was located between C-3 and C-5. Similar NMR data of compounds 1 and 2 suggested that the relative configuration of 2 was in accordance with that of 1. Therefore, compound 2 was assigned as 2,3-seco-4(23),22(29)hopene-2-carboxyl-3-aldehyde. Quite a number of triterpenoids of different structural types have been shown to be anti-tumor agents [23–24], however, the cytotoxic effects of 2,3-seco-hopane triterpenoids have not yet been reported. Compounds 1–3 were evaluated for their in vitro antiproliferative activities on HeLa, MCF-7, and Hep-G2 tumor cell " Table 2). Compound 2 showed strong activlines in this study (l ity against the three cell lines with IC50 values of 3.6, 7.5, and 13.6 µM, respectively, comparable to the positive control (matrine); and compound 1 exhibited relatively weaker cytotoxicity in MCF-7 (IC50 14.0 µM), HeLa (IC50 18.2 µM), and Hep-G2 (30.7 μΜ) cell lines. However, compound 3 was nearly inactive in the test (IC50≥66.0 μΜ). In addition, the two 2,3-seco-hopane triterpenoids were more potent than their intact hopane counterparts [23–25]. These results suggest that cleavage of ring A at C-2 and C-3 and presence of an α,β-unsaturated aldehyde group could be important for the enhanced cytotoxic activity. The antimicrobial activities of the two new compounds were evaluated against four bacterial strains and four fungal strains " Table 3). It was demonstrated that although varying degrees (l of inhibition (minimum inhibitory concentration (MIC) values in the range of 3.12–6.25 mg/mL for fungi and 6.25–12.50 mg/mL for bacteria) were observed for different strains, the two compounds exhibited similar antimicrobial activities comparable to intact hopane triterpenoids [26]. Cleavage of ring A at C-2 and C3 seemed to have no effect on the antimicrobial activity of this structural type. Although 3,4-seco-hopane triterpenoids were occasionally isolated from natural resources, few natural 2,3-seco-hopane triterpenoids had been reported [27–28]. To the best of our knowledge, only two 2,3-seco-hopane triterpenoids were reported from Alstonia scholaris (Apocynaceae) [28]. The discovery of 1 and 2 and their anti-tumor and antimicrobial activities may facilitate the understanding of the chemical and pharmacological aspects of M. stylophorus as well as the biological effects of 2,3-seco-hopane triterpenoids.
a
MIC (mg/mL) Ampicillina
1
2
0.78 1.56 0.78 1.56 0.78 0.78 0.78 0.78
6.25 12.50 6.25 6.25 6.25 3.12 3.12 6.25
6.25 12.50 6.25 6.25 3.12 3.12 3.12 6.25
Positive control
Materials and Methods !
General Optical rotations were measured on a WZZ-2B spectropolarimeter (Shanghai YiCe Apparatus & Equipment Co., Ltd). IR spectra were recorded on a Nicolet IR200 FT‑IR spectrophotometer. NMR spectra were recorded on a Bruker Avance DRX-500 spectrometer at 500 MHz (1H) and 125 MHz (13C). HR‑ESI‑MS analyses were carried out on an Agilent Technologies 6224 TOF LC‑MS apparatus. CC was performed with silica gel (200–300 mesh, Qingdao Marine Chemical, Inc.), MCI gel CHP20P (75–150 µm, Mitsubishi Kasei Corporation), or Sephadex LH-20 (20–100 µm, Pharmacia). TLC was carried out on silica gel GF254 plates (10– 40 mm; Qingdao Marine Chemical, Inc.). Spots were observed by UV light as well as by spraying with 10 % H2SO4-EtOH followed by heating. Tumor cells were incubated in a HF-212UV CO2 incubator (Shanghai Heal Force Co., Ltd.) and observed on an Olympus CKX41 inverted microscope. Optical density (OD) values were read on a BIO‑RAD Model 680 microplate reader. Ampicillin (HPLC 86.2 %) and matrine (HPLC 98%) were all purchased from National Institutes for Food and Drug Control. Purity of the two new compounds (1, 99.2 %; 2, 98.5%) were determined by HPLC (Agilent 1260: DEACA01043) on a column (Agilent Zorbax SB‑C18; 4.6 × 250 mm, 5 µm) isocratically eluted with 80% methanol and 20 % water containing 0.1 % glacial acetic acid at a flow rate of 1 mL/min.
Plant material M. stylophorus material was collected in Cona County (Tibet Autonomous Region, China) in September 2012. The species was identified by Prof. Xue-Feng Lu of Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, China. A voucher specimen (No. 12–09–18) was deposited at our laboratory.
Extraction and isolation The dried plant (8.0 kg) was ground into powder and then extracted with 90 % ethanol (80.0 L) at room temperature (4 × 7 days). Following filtration and vacuum-concentration of the combined solution, it yielded a crude extract (1189.0 g) which was then suspended in water (3000 mL) and extracted successively with petroleum ether (3 × 1000 mL), EtOAc (3 × 1000 mL), and n-BuOH (3 × 1000 mL) to afford three organic fractions. The petroleum ether fraction (165.0 g) was submitted to CC over silica gel (2000.0 g, column: 100 × 10 cm) eluted with a petroleum ether–ethyl acetate (50 : 1, 40 : 1, 20 : 1, 12 : 1, 10 : 1, 9 : 1, 8 : 1, 6 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1, 0 : 1, 8 L each) gradient to yield five
Liu C et al. Two New 2,3-Seco-Hopane …
Planta Med 2014; 80: 936–941
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Table 2 Citotoxicity (IC50) of compounds 1, 2, and 3.
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subfractions (Fr. 1 – Fr. 5). Fr. 2–4 were firstly subjected to MCI gel CC (80% ethanol) to remove pigments. Fr. 2 (petroleum ether– ethyl acetate 9 : 1–4 : 1, ca. 23.2 g) was separated on silica gel CC (300.0 g, column: 50 × 5 cm) eluted with petroleum ether–ethyl acetate (15 : 1, 12 : 1, 10 : 1, 8 : 1, 7 : 1, 5 : 1, 3 : 1, 2 : 1, 1.5 L each) to yield compounds 6 (between 4800 mL and 5800 mL, 638.7 mg) and 7 (between 6400 mL and 6800 mL, 154.4 mg). Fr. 3 (ca. 21.5 g, petroleum ether–ethyl acetate 3 : 1–2 : 1) was submitted to silica gel CC (300.0 g, column: 50 × 5 cm) eluted with petroleum ether–ethyl acetate (10 : 1, 8 : 1, 6;1, 5 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1, 1.5 L each) to yield 1 (545.3 mg) and 3 (13.2 mg), respectively, with solvent ratios of 6 : 1 and 5 : 1. Purification of Fr. 4 (10.4 g, petroleum ether–ethyl acetate 1 : 1–0 : 1) by silica gel CC (150.0 g, column: 50 × 3 cm) eluted with petroleum ether–ethyl acetate (5 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1, 1 : 2, 0 : 1, 600 mL each) afforded compound 5 (22.2 mg, between 2100 mL and 2400 mL). The extract of EtOAc (86.5 g) was submitted to silica gel CC (1000.0 g, column: 100 × 10 cm) eluted gradiently with petroleum ether–ethyl acetate (50 : 1, 20 : 1, 12 : 1, 10 : 1, 8 : 1, 6 : 1, 4 : 1, 2 : 1, 1 : 1, 1 : 2, 0 : 1, 4 L each) to produce fractions I–IV. Fraction II (16.5 g, petroleum ether–ethyl acetate 9 : 1–4 : 1) was first separated by silica gel CC (200.0 g, column: 50 × 5 cm) eluted with petroleum ether–Me2CO (10 : 1, 8 : 1, 6 : 1, 4 : 1, 2 : 1, 1 : 1, 800 mL each, collection started from 1200 mL to 1800 mL) and then purified using Sephadex LH-20 (CHCl3–MeOH, 1 : 1, 3 L, column: 50 × 2 cm) to yield compound 2 (68.4 mg). Fr. III (11.5 g, petroleum ether–ethyl acetate 2 : 1–1 : 1) was fractionated by silica gel CC (150.0 g, column: 50 × 3 cm) eluted with petroleum ether– ethyl acetate (9 : 1, 8 : 1, 6 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1, 1 : 2, 600 mL each). The fraction (3.2 g, petroleum ether–ethyl acetate 3 : 1) was further separated over Sephadex LH-20 CC eluted with ethanol–water (7 : 3, 8 : 2, 9 : 1) to yield 4 (17.0 mg, 8 : 2) and 8 (12.0 mg, 9 : 1). Compound 9 (109.7 mg, between 2500 mL and 2800 mL), 10 (41.1 mg, between 3200 mL and 3600 mL), 11 (11.7 mg, between 4000 mL and 4400 mL), and 12 (26.5 mg, between 5000 mL and 5200 mL) were obtained from Fr. IV (23.2 g, petroleum ether–ethyl acetate 1 : 2–0 : 1) through silica gel CC (300.0 g, column: 50 × 5 cm) eluted with CHCl3–MeOH (10 : 1, 9 : 1, 8 : 1, 7 : 1, 6 : 1, 5 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1; 1.5 L each).
Characterization of compounds 2,3-seco-22(29)-hopene-2-carboxyl-3-aldehyde (1): colorless crystal (CHCl3–MeOH, 10 : 1). [α]20 D = + 7.4 (c = 0.0047, MeOH), IR (KBr): 3648, 2946, 2865, 1718, 1230, 1213 cm−1. 1H and 13C‑NMR (CDCl3) − " Table 1). HR‑ESI‑MS: 455.3530 ([M – H]−, C (see l 30H47O3 , calc. 455.3525). 2,3-seco-4(23),22(29)-hopene-2-carboxyl-3-aldehyde (2): white amorphous powder. [α]20 D = + 7.9 (c = 0.006, MeOH), IR (KBr): 2948, 2864, 1704, 1693, 1443, 1237 cm−1. 1H and 13C‑NMR " Table 1). HR‑ESI‑MS: 439.3215 ([M – H]−, (CDCl3) (see l C29H43O3−, calc. 439.3212).
X‑ray crystallographic analysis of 1 Colorless blocks, C120H192O12 (4 × C30H48O3), Mr = 1826.74, hexagonal, spacegroup P32, a = 16.66(3) Å, b = 16.66(3) Å, c = 30.07 (13) Å, V = 8190(4) Å3, Z = 3, dx = 1.111 mg/m3, F (000) = 3024, µ (Cu Kα) = 0.069 mm−1. Data collection was performed on a Gemini S Ultra using graphite-monochromated radiation (λ = 0.71 073 Å); 13 969 unique reflections were collected to θmax = 25.16°, where 4167 reflections were observed [F2 > 4σ(F2)]. The structures were solved by direct methods (SHELXTL version 5.1) and refined by full-matrix least-squares on F2. The final
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R = 0.0806, Rw = 0.1398, and S = 1.010. Crystallographic data has been deposited at the Cambridge Crystallographic Data Centre as supplementary publication No. CCDC 970 077.
Cytotoxicity activity experiments The cytotoxic effects of compounds 1–3 were estimated in vitro against the cancer cell lines HeLa, MCF-7, and Hep-G2 (ATCC) by MTT assay [29]. Briefly, the cell suspensions (200 mL) at a density of 5 × 104 cells · mL−1 were distributed into 96-well cell culture plates and cultured at 37 °C in incubator with 5 % CO2 for 24 h. The solution of five different concentrations of the test compounds (2 mL in DMSO) was added to each well and further incubated for 48 h under the same conditions. The MTT solution (20 mL) was then added to each well and incubated for 4 h. Finally, the supernatant was discarded, and limited DMSO was added to each well to dissolve the blue-violet crystals completely; the OD values were then read on the microplate reader at 570 nm. All tests and analyses were carried out in triplicate. Dose-response curves were generated, and the IC50 values were defined as the concentration of compound required to inhibit cell proliferation by 50 %. Matrine, an approved agent for the treatment of many tumors, was applied as the positive control.
Antimicrobial activity All the bacteria and fungi used for antimicrobial activity evaluation were obtained from international culture collections (ATCC) or Jiangnan University (Wuxi, Jiangsu Province), the bacterial strains were: Staphylococcus aureus ATCC 25 923, Escherichia coli ATCC 25 922, Bacillus subtilis ATCC 6633, and Salmonella typhimurium ATCC 14 028; fungal strains were: Aspergillus niger ATCC 16 404, Penicillium and Fusarium oxysporum (offered by Jiangnan University), and Saccharomyces cerevisiae ATCC 9763. MIC was determined by broth microdilution method. The bacterial strains were cultivated in Muller–Hinton agar (MH) (Oxoid Ltd.), and the fungi were cultured on potatoes dextrose agar (PDA) medium. A serial doubling dilution of the two new compounds was prepared in a 96-well microtiter plate over the range 0.78 mg/mL – 25.00 mg/mL. Each strain (broth cultured overnight) was prepared, and the final concentration in each well was adjusted to 104 cfu/mL. The cultures were then incubated at 37 °C for 18 h for bacterial strains and 72 h for fungi at 28 °C. The lowest concentration of the sample at which the tested microorganism did not show any visible growth was determined as MIC.
Supporting information The original spectra of NMR, HR‑ESI‑MS, and HPLC data for the new compounds are available as Supporting Information.
Acknowledgments !
We are grateful to Prof. Xue-Feng Lu of Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, China, for the identification of the plant material. We also thank Dr. Lin Cheng (Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing, P. R. China) for the analysis of compound 1 X‑ray crystallographic diffraction. This work was supported by the State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Science and Technology of China (CMEMR2014-B04).
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Conflict of Interest !
There are no conflicts of interest of all authors with respect to this work.
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15 Valentão P, Andrade PB, Silva AMS, Moreira MM, Seabra RM. Isolation and structural elucidation of 5-formyl-2,3-dihydroisocoumarin from Centaurium erythraea aerial parts. Nat Prod Res 2003; 17: 361–364 16 Ferreira AMVD, Carvalho MJM, Sequeira MM, Silva AMS, Carvalho LMH. Chemical constituents of Euphorbia hyberna L. (Euphorbiaceae). Nat Prod Res 2013; 27: 282–285 17 Tozhiboev MM, Botirov E, Usmanova GA. Xanthones and flavonoids from Gentiana algida Pall. Russ J Bioorg Chem 2011; 37: 866–870 18 Mukherjee KS, Chakraborty CK, Chatterjee TP, Bhattacharjee D, Laha S. 1,5,7-Trihydroxy-3-methoxyxanthone from Hoppea fastigiata. Phytochemistry 1991; 30: 1036–1037 19 Prasad D, Sati SP. A new 2,8-dihydroxy-1,6-dimethoxy xanthone from Cyathula tomentosa. Orient J Chem 2011; 27: 765–767 20 Zhang BB, Han XL, Jiang Q, Liao ZX, Wang HS. Cytotoxic cholestane-type and ergostane-type steroids from the aerial parts of Euphorbia altotibetica. Steroids 2013; 78: 38–43 21 Dallavalle S, Jayasinghe L, Kumarihamy BMM, Merlini L, Musso L, Scaglioni L. A New 3,4-seco-lupane derivative from Lasianthus gardneri. J Nat Prod 2004; 67: 911–913 22 Chen IH, Du YC, Lu MC, Lin AS, Hsieh PW, Wu CC, Chen SL. Lupane-type triterpenoids from Microtropis fokienensis and Perrottetia arisanensis and the apoptotic effect of 28-hydroxy-3-oxo-lup-20(29)-en-30-al. J Nat Prod 2008; 71: 1352–1357 23 Wu SB, Su JJ, Sun LH, Wang WX, Zhao Y, Li H, Zhang SP, Dai GH, Wang CG, Hu JF. Triterpenoids and steroids from the fruits of Melia toosendan and their cytotoxic effects on two human cancer cell lines. J Nat Prod 2010; 73: 1898–1906 24 Wu SB, Bao QY, Wang WX, Zhao Y, Xia G, Zhao Z, Zeng HQ, Hu JF. Cytotoxic triterpenoids and steroids from the bark of Melia azedarach. Planta Med 2011; 77: 922–928 25 Isaka M, Palasarn S, Supothina S, Komwijit S, Luangsa-ard JJ. Bioactive compounds from the scale insect pathogenic fungus Conoideocrella tenuis BCC 18627. J Nat Prod 2011; 74: 782–789 26 Reddy VLN, Ravikanth V, Rao TP, Diwan PV, Venkateswarlu Y. A new triterpenoid from the fern Adiantum lunulatum and evaluation of antibacterial activity. Phytochemistry 2001; 56: 173–175 27 Toriumi Y, Kakuda R, Kikuchi M, Yaoita Y, Kikuchi M. New triterpenoids from Gentiana lutea. Chem Pharm Bull 2003; 51: 89–91 28 Wang F, Ren FC, Liu JK. Alstonic acids A and B, unusual 2,3-secofernane triterpenoids from Alstonia scholaris. Phytochemistry 2009; 70: 650– 654 29 Wu SB, Pang F, Wen Y, Zhang HF, Zhao Z, Hu JF. Antiproliferative and apoptotic activities of linear furocoumarins from Notopterygium incisum on cancer cell lines. Planta Med 2010; 76: 82–85
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Original Papers
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Two New 2,3-Seco-Hopane Triterpene Derivatives from Megacodon stylophorus and Their Antiproliferative and Antimicrobial Activities
Authors
Chao Liu 1, 3, Zhi-Xin Liao 1, 3, Shi-Jun Liu 1, Lan-Ju Ji 2, Hong-Fa Sun 2
Affiliations
1
2 3
Key words " Megacodon stylophorus l " Gentianaceae l " 2,3‑seco‑hopane l triterpenoids " antiproliferative l " antimicrobial l
Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Southeast University, Nanjing, P. R. China Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, P. R. China Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing, P. R. China
Abstract !
Chemical investigation of the ethanol extract of the whole plant of Megacodon stylophorus led to the isolation and identification of two new secohopane triterpenoids, 2,3-seco-22(29)-hopene2-carboxyl-3-aldehyde (1) and 2,3-seco-4(23),22 (29)-hopene-2-carboxyl-3-aldehyde (2), along with 10 known compounds, 3–12. All the isolates were reported from this plant for the first time. The structures of compounds 1 and 2 were determined by detailed analysis of their spectral data including 1D and 2D NMR. In addition, compound 1 was further analyzed by X‑ray crystallography.
Introduction !
received revised accepted
Nov. 28, 2013 May 18, 2014 May 20, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368612 Published online July 4, 2014 Planta Med 2014; 80: 936–941 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Dr. Zhi-Xin Liao Department of Pharmaceutical Engineering School of Chemistry and Chemical Engineering Southeast University No. 2, Southeast University Road Nanjing 211189 P. R. China Phone: + 86 25 52 09 06 20 Fax: + 86 25 52 09 06 18 [email protected]
Many species of the family Gentianaceae are reported to be medicinal plants for the treatment of hepatobiliary diseases [1–3]. Quite a number of chemical components such as xanthones [4– 5], terpenoids [6], aromatic glycosides [7], iridoids and secoiridoid glucosides [8] have been isolated from these medicinal plants. Megacodon (Hemsl.) H. Smith is a genus of Gentianaceae that comprises only two species: Megacodon stylophorus and Megacodon venosus [9–11]. M. venosus was once reported to be endemic to China and had been declared extinct in 1970s but was rediscovered by Prof. Zhen-Yu Li (Institute of Botany, the Chinese Academy of Sciences) in Chongqing municipality in 2007 [12]. M. stylophorus, a perennial herb distributed in Qinghai-Tibet Plateau and Himalaya areas, is generally used by the aboriginal inhabitants of Qinghai and Xizang provinces to clear heat and treat hepatopathy and gallbladder diseases [13]. Up to the present, no chemical investigations of the two plants have been reported in Chemical Abstracts. As part of our work on the chemical compositions of the genus Megacodon and biologically active
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Compounds 1–3 were evaluated for their in vitro anti-proliferative activities on HeLa, MCF-7, and Hep-G2 tumor cell lines. Compound 2 was active against the three cell lines with IC50 values of 3.6, 7.5, and 13.6 µM, respectively, while compound 1 exhibited cytotoxicity on MCF-7 (IC50 14.0 µM) and HeLa (IC50 18.2 µM) cell lines. Antimicrobial activities of compounds 1–2 (minimum inhibitory concentration values in the range of 3.12– 12.50 mg/mL) were also observed. Supporting information available online at http://www.thieme-connect.de/products
novel structures, the whole plant of M. stylophorus was investigated. As a result, two new 2,3-seco-hopane triterpenoids along with 10 known compounds were obtained from its ethanol extract. In this paper, we report the isolation, structure elucidation, as well as anti-tumor and antimicrobial activities of the new compounds.
Results and Discussion !
Repeated silica gel, MCI gel, or Sephadex LH-20 column chromatography (CC) of an ethanol extract of M. stylophorus afforded 12 compounds " Fig. 1). Their structures were identified (1–12) (l by means of IR, HR‑ESI‑MS, NMR spectroscopy (1H‑NMR, 13C‑NMR, HSQC, HMBC, and ROESY) and X‑ray crystallography, as well as comparing spectroscopic data and physicochemical properties with previous literature. The known compounds were identified as thysanolactone (3) [6], halenic acid A (4) [14], 5-formyl-2,3-dihydroisocoumarin (5) [15], stigmasterol (6), ursolic acid (7), ellagic acid (8) [16], 1,5,8-trihydroxy-3-methoxy-xanthone (9) [17], 1,5,7-trihydroxy-3-methoxy-xanthone (10) [18], 1,7-dihydroxy-3,8-di-
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Chemical structures of compounds 1–12.
Fig. 2 Perspective drawing of the X‑ray structure of 1. (Color figure available online only.)
methoxy xanthone (11) [19], and 1,3,7,8-tetrahydroxy-xanthone (12) [20]. Compound 1 was isolated as colorless crystals (CHCl3–MeOH, 10 : 1). mp 203 ~ 204 °C, [α]20 D = + 7.4 (c = 0.0047, MeOH). Its molecular formula was assigned to be C30H48O3 on the basis of the negative mode HR‑ESI‑MS, which gave a pseudomolecular ion peak at m/z 455.3530 [M – H]−. The structure of 1 was deduced as 2,3-seco-22(29)-hopene-2-carboxyl-3-aldehyde based on a " Fig. 2). The single crystal X‑ray diffraction analysis result (l NMR data further secured the structure. Signals for seven meth-
yls and a carboxyl proton were clearly observed in the 1H‑NMR spectra. The 13C‑NMR and DEPT spectra of 1 exhibited the presence of thirty carbons in the molecule. The assigned NMR data " Table 1. Moreover, the HMBC experiment (l " Fig. 3) of 1 are in l proved the cleavage of ring A and indicated that the fracture path was between C-2 and C-3 rather than C-3 and C-4 [21, 22] according to the long-range correlations of H3-23/C-3, H3-23/C‑5, H2-1/ C‑5, and H2-1/C-25. ROESY correlation analysis and X‑ray crystallography indicated that compound 1 and thysanolactone [6], a
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Fig. 1
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Position
1 δH
1α 1β 2 3 4 5 6α 6β 7α 7β 8 9 10 11α 11β 12α 12β 13 14 15α 15β 16α 16β 17 18 19α 19β 20α 20β 21 22 23α 23β 24 25 26 27 28 29α 29β 30
2.28, 1H, d, 15.7 2.56, 1H, d, 15.6 9.77, 1H, s 2.22, 1H, m 1.52, 1H, m 1.56, 1H, m 0.92, 1H, m 1.25, 1H, m 1.87, 1H, m 1.34, 1H, m 1.68, 1H, m 1.16, 1H, m 1.46, 1H,m 1.45, 1H, t, 2.65 1.30, 1H, t 1.52, 1H, t 1.42, 1H, m 1.86, 1H, m 1.01, 1H, m 1.04, 1H, m 1.51, 1H, m 1.18, 1H, m 1.41, 1H, m 2.25, 1H, m 1.09, 3H, s 1.14, 3H, s 0.89, 3H, s 1.01, 3H, s 0.94, 3H, s 0.68, 3H, s 4.67, 1H, s 4.69, 1H, s 1.68, 3H, s
2 δC 43.21 176.69 207.89 50.78 47.49 20.43 32.40 41.67 43.39 42.84 22.38 23.91 49.00 42.67 32.72 27.38 53.98 44.16 40.22 20.83 47.81 148.07 19.35 24.05 19.58 16.48 16.19 15.28 109.48 19.68
δH
δC
1.80, 1H, d, 14.5 2.34, 1H, d, 14.8 9.51, 1H, s 2.94, 1H, dd, 2.79, 12.55 1.34, 1H, m 1.96, 1H, m 1.27, 1H, m 1.58, 1H, m 2.02, 1H, m 1.43, 1H, m 1.58, 1H, m 1.37, 1H, m 1.77, 1H, m 1.45, 1H, t, 2.65 1.24, 1H, t 1.33, 1H, t 1.43, 1H, m 1.86, 1H, m 1.03, 1H, m 1.06, 1H, m 1.52, 1H, m 1.19, 1H, m 1.42, 1H, m 2.25, 1H, m 6.28, 1H, s 6.49, 1H, s 0.82, 3H, s 1.07, 3H, s 0.99, 3H, s 0.70, 3H, s 4.68, 1H, s 4.70, 1H, s 1.67, 3H, s
45.07 173.47 197.33 151.91 42.09 25.13 31.66 41.99 41.70 40.26 23.77 23.02 49.02 42.82 32.74 27.38 53.98 44.22 40.15 20.83 47.83 148.10 140.80
18.46 16.51 16.41 15.21 109.50 19.70
Fig. 3
natural compound also obtained from a Gentianaceae plant, shared the same stereochemistry. Compound 2 was obtained as a white amorphous powder, [α]20 D = + 7.9 (c = 0.006, MeOH). The negative HR‑ESI‑MS of 2 showed a pseudomolecular ion peak at m/z 439.3215 [M – H]−. Combining 1 " Table 1), the molecular formula was deH and 13C‑NMR data (l termined as C29H44O3. Liu C et al. Two New 2,3-Seco-Hopane …
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Table 1 1H and 13C NMR data of 1 and 2 at 500 and 125 MHz in CDCl3 (δ in ppm; J in Hz).
Key HMBC (H→C) correlations of 1 and 2.
Similarly to compound 1, the NMR spectra data of 2 showed the characteristic signals of one isopropenyl group, one carboxyl group, and one aldehyde group. However, only five methyl group signals appeared in the NMR spectra. Moreover, an additional olefinic bond was in 2 according to its NMR spectra [δC 140.80, δC 151.91, δH 6.28 and 6.49]. The DEPT suggested five tertiary methyl groups, eleven methylenes, six methines, and seven qua-
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Sample
1 2 3 Matrinea a
Table 3 Antimicrobial activity of the two new compounds against all tested microorganisms.
Cytotoxicity (IC50, µM) HeLa
MCF-7
Hep-G2
18.2 ± 1.2b 3.6 ± 0.4 68.2 ± 8.7 11.3
14.0 ± 1.8 7.5 ± 0.8 66.0 ± 10.9 12.1
30.7 ± 3.5 13.6 ± 1.6 70.4 ± 6.5 8.3
Tested microorganism
Salmonella typhimurium Escherichia coli Bacillus subtilis Staphylococcus aureus Aspergillus niger Saccharomyces cerevisiae Penicillium Fusarium oxysporum
Positive control; b Data represent the mean ± SD of three independent experiments
ternary carbons in the molecule. Comparison of the NMR data of 2 with those of 1 showed that both compounds were alike at rings B, C, D, and E except for an exocyclic double bond in 2 instead of two methyl groups at C-4. In the HMBC spectra " Fig. 3), the correlations of H -23 with both C-3 and C-5 con(l 2 firmed that the exomethylene was located between C-3 and C-5. Similar NMR data of compounds 1 and 2 suggested that the relative configuration of 2 was in accordance with that of 1. Therefore, compound 2 was assigned as 2,3-seco-4(23),22(29)hopene-2-carboxyl-3-aldehyde. Quite a number of triterpenoids of different structural types have been shown to be anti-tumor agents [23–24], however, the cytotoxic effects of 2,3-seco-hopane triterpenoids have not yet been reported. Compounds 1–3 were evaluated for their in vitro antiproliferative activities on HeLa, MCF-7, and Hep-G2 tumor cell " Table 2). Compound 2 showed strong activlines in this study (l ity against the three cell lines with IC50 values of 3.6, 7.5, and 13.6 µM, respectively, comparable to the positive control (matrine); and compound 1 exhibited relatively weaker cytotoxicity in MCF-7 (IC50 14.0 µM), HeLa (IC50 18.2 µM), and Hep-G2 (30.7 μΜ) cell lines. However, compound 3 was nearly inactive in the test (IC50≥66.0 μΜ). In addition, the two 2,3-seco-hopane triterpenoids were more potent than their intact hopane counterparts [23–25]. These results suggest that cleavage of ring A at C-2 and C-3 and presence of an α,β-unsaturated aldehyde group could be important for the enhanced cytotoxic activity. The antimicrobial activities of the two new compounds were evaluated against four bacterial strains and four fungal strains " Table 3). It was demonstrated that although varying degrees (l of inhibition (minimum inhibitory concentration (MIC) values in the range of 3.12–6.25 mg/mL for fungi and 6.25–12.50 mg/mL for bacteria) were observed for different strains, the two compounds exhibited similar antimicrobial activities comparable to intact hopane triterpenoids [26]. Cleavage of ring A at C-2 and C3 seemed to have no effect on the antimicrobial activity of this structural type. Although 3,4-seco-hopane triterpenoids were occasionally isolated from natural resources, few natural 2,3-seco-hopane triterpenoids had been reported [27–28]. To the best of our knowledge, only two 2,3-seco-hopane triterpenoids were reported from Alstonia scholaris (Apocynaceae) [28]. The discovery of 1 and 2 and their anti-tumor and antimicrobial activities may facilitate the understanding of the chemical and pharmacological aspects of M. stylophorus as well as the biological effects of 2,3-seco-hopane triterpenoids.
a
MIC (mg/mL) Ampicillina
1
2
0.78 1.56 0.78 1.56 0.78 0.78 0.78 0.78
6.25 12.50 6.25 6.25 6.25 3.12 3.12 6.25
6.25 12.50 6.25 6.25 3.12 3.12 3.12 6.25
Positive control
Materials and Methods !
General Optical rotations were measured on a WZZ-2B spectropolarimeter (Shanghai YiCe Apparatus & Equipment Co., Ltd). IR spectra were recorded on a Nicolet IR200 FT‑IR spectrophotometer. NMR spectra were recorded on a Bruker Avance DRX-500 spectrometer at 500 MHz (1H) and 125 MHz (13C). HR‑ESI‑MS analyses were carried out on an Agilent Technologies 6224 TOF LC‑MS apparatus. CC was performed with silica gel (200–300 mesh, Qingdao Marine Chemical, Inc.), MCI gel CHP20P (75–150 µm, Mitsubishi Kasei Corporation), or Sephadex LH-20 (20–100 µm, Pharmacia). TLC was carried out on silica gel GF254 plates (10– 40 mm; Qingdao Marine Chemical, Inc.). Spots were observed by UV light as well as by spraying with 10 % H2SO4-EtOH followed by heating. Tumor cells were incubated in a HF-212UV CO2 incubator (Shanghai Heal Force Co., Ltd.) and observed on an Olympus CKX41 inverted microscope. Optical density (OD) values were read on a BIO‑RAD Model 680 microplate reader. Ampicillin (HPLC 86.2 %) and matrine (HPLC 98%) were all purchased from National Institutes for Food and Drug Control. Purity of the two new compounds (1, 99.2 %; 2, 98.5%) were determined by HPLC (Agilent 1260: DEACA01043) on a column (Agilent Zorbax SB‑C18; 4.6 × 250 mm, 5 µm) isocratically eluted with 80% methanol and 20 % water containing 0.1 % glacial acetic acid at a flow rate of 1 mL/min.
Plant material M. stylophorus material was collected in Cona County (Tibet Autonomous Region, China) in September 2012. The species was identified by Prof. Xue-Feng Lu of Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, China. A voucher specimen (No. 12–09–18) was deposited at our laboratory.
Extraction and isolation The dried plant (8.0 kg) was ground into powder and then extracted with 90 % ethanol (80.0 L) at room temperature (4 × 7 days). Following filtration and vacuum-concentration of the combined solution, it yielded a crude extract (1189.0 g) which was then suspended in water (3000 mL) and extracted successively with petroleum ether (3 × 1000 mL), EtOAc (3 × 1000 mL), and n-BuOH (3 × 1000 mL) to afford three organic fractions. The petroleum ether fraction (165.0 g) was submitted to CC over silica gel (2000.0 g, column: 100 × 10 cm) eluted with a petroleum ether–ethyl acetate (50 : 1, 40 : 1, 20 : 1, 12 : 1, 10 : 1, 9 : 1, 8 : 1, 6 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1, 0 : 1, 8 L each) gradient to yield five
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Table 2 Citotoxicity (IC50) of compounds 1, 2, and 3.
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subfractions (Fr. 1 – Fr. 5). Fr. 2–4 were firstly subjected to MCI gel CC (80% ethanol) to remove pigments. Fr. 2 (petroleum ether– ethyl acetate 9 : 1–4 : 1, ca. 23.2 g) was separated on silica gel CC (300.0 g, column: 50 × 5 cm) eluted with petroleum ether–ethyl acetate (15 : 1, 12 : 1, 10 : 1, 8 : 1, 7 : 1, 5 : 1, 3 : 1, 2 : 1, 1.5 L each) to yield compounds 6 (between 4800 mL and 5800 mL, 638.7 mg) and 7 (between 6400 mL and 6800 mL, 154.4 mg). Fr. 3 (ca. 21.5 g, petroleum ether–ethyl acetate 3 : 1–2 : 1) was submitted to silica gel CC (300.0 g, column: 50 × 5 cm) eluted with petroleum ether–ethyl acetate (10 : 1, 8 : 1, 6;1, 5 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1, 1.5 L each) to yield 1 (545.3 mg) and 3 (13.2 mg), respectively, with solvent ratios of 6 : 1 and 5 : 1. Purification of Fr. 4 (10.4 g, petroleum ether–ethyl acetate 1 : 1–0 : 1) by silica gel CC (150.0 g, column: 50 × 3 cm) eluted with petroleum ether–ethyl acetate (5 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1, 1 : 2, 0 : 1, 600 mL each) afforded compound 5 (22.2 mg, between 2100 mL and 2400 mL). The extract of EtOAc (86.5 g) was submitted to silica gel CC (1000.0 g, column: 100 × 10 cm) eluted gradiently with petroleum ether–ethyl acetate (50 : 1, 20 : 1, 12 : 1, 10 : 1, 8 : 1, 6 : 1, 4 : 1, 2 : 1, 1 : 1, 1 : 2, 0 : 1, 4 L each) to produce fractions I–IV. Fraction II (16.5 g, petroleum ether–ethyl acetate 9 : 1–4 : 1) was first separated by silica gel CC (200.0 g, column: 50 × 5 cm) eluted with petroleum ether–Me2CO (10 : 1, 8 : 1, 6 : 1, 4 : 1, 2 : 1, 1 : 1, 800 mL each, collection started from 1200 mL to 1800 mL) and then purified using Sephadex LH-20 (CHCl3–MeOH, 1 : 1, 3 L, column: 50 × 2 cm) to yield compound 2 (68.4 mg). Fr. III (11.5 g, petroleum ether–ethyl acetate 2 : 1–1 : 1) was fractionated by silica gel CC (150.0 g, column: 50 × 3 cm) eluted with petroleum ether– ethyl acetate (9 : 1, 8 : 1, 6 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1, 1 : 2, 600 mL each). The fraction (3.2 g, petroleum ether–ethyl acetate 3 : 1) was further separated over Sephadex LH-20 CC eluted with ethanol–water (7 : 3, 8 : 2, 9 : 1) to yield 4 (17.0 mg, 8 : 2) and 8 (12.0 mg, 9 : 1). Compound 9 (109.7 mg, between 2500 mL and 2800 mL), 10 (41.1 mg, between 3200 mL and 3600 mL), 11 (11.7 mg, between 4000 mL and 4400 mL), and 12 (26.5 mg, between 5000 mL and 5200 mL) were obtained from Fr. IV (23.2 g, petroleum ether–ethyl acetate 1 : 2–0 : 1) through silica gel CC (300.0 g, column: 50 × 5 cm) eluted with CHCl3–MeOH (10 : 1, 9 : 1, 8 : 1, 7 : 1, 6 : 1, 5 : 1, 4 : 1, 3 : 1, 2 : 1, 1 : 1; 1.5 L each).
Characterization of compounds 2,3-seco-22(29)-hopene-2-carboxyl-3-aldehyde (1): colorless crystal (CHCl3–MeOH, 10 : 1). [α]20 D = + 7.4 (c = 0.0047, MeOH), IR (KBr): 3648, 2946, 2865, 1718, 1230, 1213 cm−1. 1H and 13C‑NMR (CDCl3) − " Table 1). HR‑ESI‑MS: 455.3530 ([M – H]−, C (see l 30H47O3 , calc. 455.3525). 2,3-seco-4(23),22(29)-hopene-2-carboxyl-3-aldehyde (2): white amorphous powder. [α]20 D = + 7.9 (c = 0.006, MeOH), IR (KBr): 2948, 2864, 1704, 1693, 1443, 1237 cm−1. 1H and 13C‑NMR " Table 1). HR‑ESI‑MS: 439.3215 ([M – H]−, (CDCl3) (see l C29H43O3−, calc. 439.3212).
X‑ray crystallographic analysis of 1 Colorless blocks, C120H192O12 (4 × C30H48O3), Mr = 1826.74, hexagonal, spacegroup P32, a = 16.66(3) Å, b = 16.66(3) Å, c = 30.07 (13) Å, V = 8190(4) Å3, Z = 3, dx = 1.111 mg/m3, F (000) = 3024, µ (Cu Kα) = 0.069 mm−1. Data collection was performed on a Gemini S Ultra using graphite-monochromated radiation (λ = 0.71 073 Å); 13 969 unique reflections were collected to θmax = 25.16°, where 4167 reflections were observed [F2 > 4σ(F2)]. The structures were solved by direct methods (SHELXTL version 5.1) and refined by full-matrix least-squares on F2. The final
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R = 0.0806, Rw = 0.1398, and S = 1.010. Crystallographic data has been deposited at the Cambridge Crystallographic Data Centre as supplementary publication No. CCDC 970 077.
Cytotoxicity activity experiments The cytotoxic effects of compounds 1–3 were estimated in vitro against the cancer cell lines HeLa, MCF-7, and Hep-G2 (ATCC) by MTT assay [29]. Briefly, the cell suspensions (200 mL) at a density of 5 × 104 cells · mL−1 were distributed into 96-well cell culture plates and cultured at 37 °C in incubator with 5 % CO2 for 24 h. The solution of five different concentrations of the test compounds (2 mL in DMSO) was added to each well and further incubated for 48 h under the same conditions. The MTT solution (20 mL) was then added to each well and incubated for 4 h. Finally, the supernatant was discarded, and limited DMSO was added to each well to dissolve the blue-violet crystals completely; the OD values were then read on the microplate reader at 570 nm. All tests and analyses were carried out in triplicate. Dose-response curves were generated, and the IC50 values were defined as the concentration of compound required to inhibit cell proliferation by 50 %. Matrine, an approved agent for the treatment of many tumors, was applied as the positive control.
Antimicrobial activity All the bacteria and fungi used for antimicrobial activity evaluation were obtained from international culture collections (ATCC) or Jiangnan University (Wuxi, Jiangsu Province), the bacterial strains were: Staphylococcus aureus ATCC 25 923, Escherichia coli ATCC 25 922, Bacillus subtilis ATCC 6633, and Salmonella typhimurium ATCC 14 028; fungal strains were: Aspergillus niger ATCC 16 404, Penicillium and Fusarium oxysporum (offered by Jiangnan University), and Saccharomyces cerevisiae ATCC 9763. MIC was determined by broth microdilution method. The bacterial strains were cultivated in Muller–Hinton agar (MH) (Oxoid Ltd.), and the fungi were cultured on potatoes dextrose agar (PDA) medium. A serial doubling dilution of the two new compounds was prepared in a 96-well microtiter plate over the range 0.78 mg/mL – 25.00 mg/mL. Each strain (broth cultured overnight) was prepared, and the final concentration in each well was adjusted to 104 cfu/mL. The cultures were then incubated at 37 °C for 18 h for bacterial strains and 72 h for fungi at 28 °C. The lowest concentration of the sample at which the tested microorganism did not show any visible growth was determined as MIC.
Supporting information The original spectra of NMR, HR‑ESI‑MS, and HPLC data for the new compounds are available as Supporting Information.
Acknowledgments !
We are grateful to Prof. Xue-Feng Lu of Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, China, for the identification of the plant material. We also thank Dr. Lin Cheng (Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing, P. R. China) for the analysis of compound 1 X‑ray crystallographic diffraction. This work was supported by the State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Science and Technology of China (CMEMR2014-B04).
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Conflict of Interest !
There are no conflicts of interest of all authors with respect to this work.
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