Two new anthraquinone dimers from the fruit bodies of Bulgaria inquinans


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Ning Li a, Jing Xu b, Xian Li a, Peng Zhang c,d,⁎ a School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China b College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China c Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China d Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China a r t i c l e i n f o a b s t r a c t Article history: Received 18 July 2012 Accepted in revised form 8 October 2012 Available online 23 October 2012 Two new dimeric anthraquinone derivatives, bulgareone A(1) and bulgareone B(2), were isolated from the fruit bodies of Bulgaria inquinans. Their structures were elucidated on the basis of extensive spectroscopic analysis. The in vitro cytotoxic activity of compounds 1 and 2 was assayed. They displayed inhibitive activities against human cancer cell lines HL60 and K562. © 2012 Elsevier B.V. All rights reserved. Keywords: Dimeric anthraquinone Bulgaria inquinans Cytotoxic activity 1. Introduction Fungus is one of the important issues in the development of new medicine. A lot of fungi, in which most are mushroom, were used as traditional medicines for a thousand years in China [1]. Many fungi are used in the treatment of infection, immunity disease, cancer [2] and dermatological disease [3]. The pigments [4], including anthraquinones [5], chromens [6], xanthones [7] etc. isolated from fungus were proved to be active in antitumor assay. Recently, fungal pigment has received considerable attention as a hot class of secondary metabolites in anticancer investigations. Bulgaria inquinans is a wood-inhabiting ascomycete widely distributed in the north of China. The fruit bodies of the mushroom are delicious food after treated by Na2CO3 solution [8]. It has been reported that the extracts of the fruit bodies have a variety of activities such as antibacterial, anti-tumor [9] and photosensitivity [10,11]. Researchers have found that the polysaccharide in B. inquinans is one of the bioactive substances [3,12]. Report also showed that 95% ethanol extract of B. inquinans was active to tumor-bearing mice [13], which suggested that the low polarity components in B. inquinans maybe an antitumor-active substance. In our previous papers, we reported the isolation of phytosterols, triterpene [14], organic acid [15] and perylenequinone [16] from the 70% ethanol extract of the fruit bodies of B. inquinans. In this paper, we wish to report the isolation and structure elucidation of two new dimeric anthraquinones (see Fig. 1) and their cytotoxic activity against cultured HL60 and K562 cells. 2. Experimental 2.1. General Melting points were measured on a Yamaco-hot-stage and are uncorrected. The optical rotations were measured in MeOH, using a 241MC automatic polarimeter made by Perkin-Elmer Limited Company. The IR spectra were taken on a Bruker IR S-55 spectrometer with KBr disks. The UV spectra in MeOH were taken on a UV-260 spectrometer made by Shimadzu Company. ESI-MS was performed on Finnigan LCQ mass spectrometer. The HR-ESIMS spectra were taken by an Agilent 6520 Q-TOF. 1D and 2D NMR spectra were recorded on a Bruker-ARX-300 and Bruker-AV-600 instrument (300 MHz for 1H and 150 MHz for 13C and 2D) with TMS as an internal Fitoterapia 84 (2013) 85–88 ⁎ Corresponding author. Tel./fax: +86 22 59596163. E-mail address: (P. Zhang). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. Contents lists available at SciVerse ScienceDirect Fitoterapia journal homepage: standard. The HPLC system used a Shimadzu CTP-6A pump equipped with a UV detector and a Shimadzu SPD-6A column (Shimadzu Shim-pack Prep-ODS, i.d. 2.5×21.6 cm). Silica gel (200–300 mesh, QingdaoMarine Chemical Group Co. Ltd., P. R. China) was used for column chromatography. Chemical reagents for isolation were of analytical grade and purchased from Tianjin Yuanli Chemical Co. Ltd., P. R. China. 2.2. Fungal material The fungus was collected at Changbai Mountain, Jilin Province, China, in August 2002, and identified by Jilin Provincial Institute of Tradition Chinese Medicine. A voucher specimen (No. 20020801) has been deposited in the Research Department of Natural Medicine, Shenyang Pharmaceutical University. 2.3. Extraction and isolation The air-dried fruit bodies (7.0 kg) of B. inquinans were extracted with 70% EtOH, the extract was concentrated in vacuo, and then the extract (1800.0 g) was partitioned with light petroleum ether, CHCl3, EtOAc and n-BuOH successively. The CHCl3 fraction (199.0 g) was subjected to column chromatography on a silica gel eluted with petroleum ether/EtOAc by a gradient method. Sub-fraction 1 eluted with petroleum ether-EtOAc (100:8, 30.0 mg), was rechromatographied on a silica gel column eluted with petroleum ether-EtOAc (100:11) to give 2 (8.0 mg). Subfraction 10 [eluted with petroleum ether-EtOAc (100:8), 500.0 mg] was separated by semi-preparative HPLC on an ODS column with 93% MeOH as mobile phase, yield 1 (10.0 mg). 2.4. Bulgareone A(1) Dark-red amorphous powder (CHCl3-MeOH); mp.>300 °C; ½  α 20 D : +1.34° (MeOH, 0.4); UV max (MeOH): 289 and 270 nm; IR bands (KBr, MeOH): 3428, 2927, 1699, and 1628 cm− 1; CD (MeOH; c 3.4×10−6); Δε23: 420 (−3.08), 330 (+2.49), 302 (−5.01), 272 (+26.08), and 252 (−7.31); ESI-MS m/z: 583[M-H]+; HR-ESIMS: m/z 584.0961 (calcd. for C31H20O12: 584.0955); 1H NMR (300 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6) data see Table 1. 2.5. Bulgareone B(2) Dark-red amorphous powder (CHCl3-MeOH); mp.>300 °C; ½  α 20 D : +2.28° (MeOH, 0.4); UV max (MeOH): 300 and 265 nm; CD (MeOH; c 3.4×10−6): Δε23: 420 (−3.56), 300 (−2.09), 272 (+30.28), and 254 (−8.20); ESI-MS m/z: 569 [M-H]+; HRESIMS: m/z 570.0704 (calcd. for C30H18O12: 570.0978) 1H NMR (300 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6) data see Table 1. 2.6. Bioassay for cytotoxic activity The human acute myeloid leukemia cell line HL-60 and human chronic myelogenous leukemia cell line K562 were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China) and maintained in RPMI-1640 medium (Gibco BRL) with 3.7 g/L NaHCO3, supplemented with 10% heat-inactivated FBS. For the cytotoxicity assays with K562 and HL-60, Adriamycin was chosen as a reference drug at a concentration of 4 mg/mL. All the above cell lines seeded in 96-well cell culture plates, and maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 °C for 3–6 d before experimentation. The cytotoxicity of all the tested samples and compounds was assayed using the in vitro MTT reduction assay in 96-well microtiter plates. Cells in a logarithmic growth phase were harvested, diluted to 2–103 cells/mL with fresh medium and gently mixed. The test compound was dissolved in DMSO (at a concentration of 50 mg/mL), and this soln. (0.88 mL) was added to the fresh medium (220 mL/well), and then the dilution (50 mL) and cell suspension (150 mL) were transferred into microtiter plates (concentration 50 mg/mL). If the compound was active at 50 mg/mL, a series of solution were prepared by two-fold dilution, and exposed to the cells as mentioned above, in order to obtain the IC50 values. Plates were incubated at 37 °C under 5% CO2 atmosphere for 72 h. After the incubation period, 10% MTT was added, and the plates were incubated again at 37 °C for 4 h. The pure formazan product was solubilized by 150 mL DMSO for 10 min at r.t. The plate was read at 550 nm in a plate reader. Quadruplex wells were used for each drug concentration, and all of the reported experiments were performed at least four times. IC50 values were calculated with non-linear regression analysis. 3. Results and discussion Compound 1 was isolated as a dark-red amorphous powder. The UV absorption bands (λmax 230, 270, 300, 330sh and 430 nm) together with the positive reaction with Bornträger's reagent suggested a hydroxyl-substituted O O OH OH HO OCH 3 O O OH OH OCH 3 H3CO 1 1' 2 O O OH OH HO OCH3 O O OH OH OCH3 H 3CO H H H H H H HMBC NOESY 1 2 O O OH OH HO OH O O OH OH OCH 3 H 3CO 1' 1 1 O O OH OH HO OH O O OH OH OCH 3 H 3CO H H H H H H Fig. 1. Structures and key pertinent correlations observed with compounds 1 and 2. 86 N. Li et al. / Fitoterapia 84 (2013) 85–88 anthraquinone chromophore. The presence of five hydroxy protons (four peri-hydroxy at δ 12.97, 12.79, 12.26, 12.21and a β-hydroxy proton at δ 11.07, see Table 1) in the 1H-NMR spectrum, four carbonyl resonances (δ 189.0, 188.7, 181.6 and 181.8) in the 13C-NMR spectrum and the quasimolecular ion peak in the HRESI-MS (m/z 583.0879 [M-H]−, calcd. for C31H19O12 583.0882) showed this compound to be a dimeric anthraquinone derivative. The 1H-NMR spectrum of compound 1 showed two pairs of doublet signals at δ 6.97 (1H, d, J=2.4 Hz,), 6.95 (1H, d, J=2.3 Hz), 6.80 (1H, d, J=2.4 Hz) and 6.79 (1H, d, J=2.3 Hz), of which the two aromatic protons at higher shift were located at the α-position of the anthraquinone nucleus and the other two at lower shift at the β position. The proximate chemical shift suggested that there were two similar substructures in the molecular of compound 1. The NOESY correlations (Fig. 2) between the methoxy protons at δ 3.83/3.84 (each 3H, s, 6 or 6′-OCH3) and the protons at 6.97 (1H, d, J=2.4 Hz, H-5)/6.95 (1H, d, J=2.3 Hz, H-5′) as well as 6.80 (1H, d, J=2.4 Hz)/6.79 (1H, d, J=2.3 Hz) (H-7 or H-7′), the α-hydroxy proton at 12.21/12.26 (1H, s, 8-OH or 8′-OH) and the aromatic proton at 6.80/6.79 indicated the two similar fragments as shown in Fig. 2a and b. Obviously, neither of them has a position for dimeric linkage. Therefore, the two fragments should be of different anthraquinone units. The presence of two singlet peaks at δ 7.00 (1H, H-4) and 6.70 (1H, H-2′) is consistent with 2-4′ coupled bianthraquinone structure [17]. In the NOESY spectrum, the cross-peak correlations between the methoxy group at δ 3.70 and the proton at δ 7.00, the cross-peak correlations between the hydroxy proton at δ 12.79 (1′-OH) and the proton at δ 6.70, together with the long-range correlation of H-4 and 1-OH (12.97) with C-2, H-2′ with C-4′ in the HMBC experiment led to the establishment of another fragment (Fig. 2c). Thus, the planar structure of this compound was represented in Fig. 1. The 1H-NMR and 13C-NMR data were assigned by means of HSQC, HMBC and NOESY spectra. The CD spectrum of 1 showed a positive Cotton effect due to the long axis of anthraquinone nuclei at 430 nm, showing that 1 is twisted in an anticlockwise manner, opposite from the cotton curves of the compounds in Refs. [18,19]. This indicates an R-configuration of the biphenyl bond in 1. Therefore, 1 was determined as (R)-1,8,1′,3′,8′-pentahydroxy- 3,6-dimethoxy-[2,4′]bianthracene-9,10,9′,10′-tetraone, and was named bulgareone A. Compound 2 was isolated as a dark-red amorphous powder. The UV (λmax 232, 254, 272, 300, and 364 nm) together with the positive reaction with Bornträger's reagent suggested a hydroxyl-substituted anthraquinone chromophore. The 1H-NMR spectrum of compound 2 showed two pairs of Table 1 The NMR data of compounds 1 and 2. No. Compound 1 Compound 2 No. Compound 1 Compound 2 δC δH δC δH δC δH δC δH 1 164.9 163.9A 1′ 164.3A 164.2 2 124.6 124.4 2′ 107.7 6.70 (1H, s) 107.4 6.67 (s) 3 164.7 164.5 3′ 164.4A 163.9A 4 105.0 7.00 (s) 104.7 6.97 (s) 4′ 123.3 123.8 4a 131.1B 130.2B 4′a 130.3B 130.8B 5 107.1C 6.97 (d, 2.4)a 106.8 6.93 (d, 2.1) 5′ 107.2C 6.95 (d, 2.3)a 108.4 6.85 (d, 2.2) 6 165.8D 165.5 6′ 166.0D 165.2 7 107.0 6.80 (d, 2.4)b 106.7 6.79 (d, 2.1) 7′ 107.0 6.79 (d, 2.3)b 107.7 6.55 (d, 2.2) 8 163.9E 163.6 8′ 163.8E 164.1A 8a 109.5 109.4 8′a 109.5 108.3 9 189.0F 188.5C 9′ 188.7F 188.4C 9a 109.3 109.0 9′a 108.9 108.4 10 181.6 181.6 10′ 181.8 181.7 10a 135.3G 134.9D 10′a 135.1G 135.3D 1-OH 12.97 (s) 13.02 (s) 1′-OH 12.79 (s) 12.29 (s) 8-OH 12.21 (s)c 12.16 (s) 3′-OH 11.07 (br.s) 11.25 (br.s) 3-OCH3 57.0 3.70 (s) 56.7 3.70 (s) 8′-OH 12.26 (s)c 11.25 (s) 6-OCH3 56.3H 3.83 (s)d 56.1 3.82 (s) 6′-OCH3 56.4H 3.84 (s)d 12.81 (s) A, B, C, D, E, F, G, H and a, b, c, d: the signals can be exchanged. OH OCH 3 OH OCH 3 5 10a 8a 7 10'a 8'a 5' 7' a OH HO H OH 3CO 1' 3' 4'a 3 1 4a 9a 9'a b c H H H H H H Fig. 2. Partial structures determined by 2D NMR in compound 1. N. Li et al. / Fitoterapia 84 (2013) 85–88 87 doublet aromatic signals at δ 6.50 to 7.00. The 1H-NMR spectrum of compound 2 also presented two singlet peaks just like the spectrum of compound 1. Its 13C-NMR spectrum was also very similar to that of 1. The difference of the spectra between compounds 1 and 2 lied in the absence of the resonances of a methoxy function in 2. So combined the quasimolecular ion peak in the HRESI-MS (m/z 569.0721, calcd for C30H17O12 569.0725), compound 2 was determined to be a dimeric anthraquinone derivative too. Its structure was elucidated as (R)-1,8,1′,3′,6′,8′-hexahydroxy-3,6-dimethoxy-[2,4′] bianthracene-9,10,9′,10′-tetraone by means of analysis of HSQC, HMBC and NOESY and CD spectra, and was named bulgareone B. 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