Fitoterapia 99 (2014) 334–340
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Cytotoxic biflavones from Stellera chamaejasme Zhi-xin Wang a, Meng-chun Cheng a, Xiao-zhe Zhang a, Zhi-lai Hong a, Ming-zhe Gao a, Xiao-xi Kan b, Qi Li b, Ya-jie Wang b, Xiao-xin Zhu b,⁎, Hong-bin Xiao a,b,⁎⁎ a b
Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
a r t i c l e
i n f o
Article history: Received 29 July 2014 Accepted in revised form 30 September 2014 Available online 12 October 2014 Keywords: Stellera chamaejasme Chamaejasmenin E Chamaejasmin D ECD Cytotoxic activity
a b s t r a c t Bioassay-guided phytochemical studies on Stellera chamaejasme led to the isolation of two new biflavones, chamaejasmenin E (1) and chamaejasmin D (2), together with ten known compounds. The structures of new compounds were elucidated by extensive spectroscopic analyses and their absolute configurations on 2, 3, 2″ and 3″ were confirmed by TDDFT quantum chemical calculated ECD spectra combined with experimental ECD spectra. All isolated biflavones were evaluated for their cytotoxic activities against Bel-7402 and A549 tumor cell lines, and sikokianin D (3) was found to possess the most potential cytotoxic activities against both the two cell lines with IC50 values of 1.29 ± 0.21 and 0.75 ± 0.25 μM, respectively. Moreover, some structure–function relationships of these bioflavones for cytotoxic activities were explored and summarized. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Stellera chamaejasme (Thymelaeaceae), known as “Duanchangcao”, is a famous toxic plant widely distributed in the northern parts of China and Mongolia. The roots of this plant have been used for treatment of scabies, tinea, stubborn skin ulcers, chronic tracheitis, and tuberculosis by local physicians since ancient time [1]. Recently, more and more attentions have been drawn to S. chamaejasme because of its substantial antitumor activity [2,3], and it is not surprising that particularly in Mongolia it is the most widely used form in traditional medicine. Previous studies on chemical constituents of this plant have reported the presence of various compounds such as flavones, coumarins, lignans, diterpenes, etc. Among them, the antitumor activity was mainly focused on biflavones. For example, neochamaejasmin A could induce cell cycle arrest ⁎ Correspondence to: X. Zhu, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, No.16 Nanxiao Street, Dongzhimen Nei, Dongcheng, Beijing 100700, China. Tel./fax: +86 10 64056154. ⁎⁎ Correspondence to: H. Xiao, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Shahekou, Dalian 116023, China. Tel./fax: +86 411 84379756. E-mail addresses: [email protected] (X. Zhu), [email protected] (H. Xiao).
http://dx.doi.org/10.1016/j.fitote.2014.10.002 0367-326X/© 2014 Elsevier B.V. All rights reserved.
and apoptosis involved of p21 and FasL in human prostate LNCaP cancer cells [4]. Chamaejasmin A showed inhibitory action against human HEP-2 epithelial cells, which was related to tubulin protein [5]. Chamaejasmin could arrest cell cycle, induce apoptosis and inhibit nuclear NF-κB translocation in human breast cancer cell line MDA-MB-231 [6]. Also, both chamaejasmenin B and neochamaejasmin C exerted potent anti-proliferative effects in eight human solid tumor cell lines. In terms of the most sensitive A549 and KHOS cells, the mechanisms were that the two compounds induced prominent expression of the DNA damage marker γ-H2AX as well as apoptosis [7]. As part of our continuous efforts to search plant-originated anticancer agents, 95% ethanol extracts of the roots of S. chamaejasme showing considerable cytotoxic activities against Bel-7402 and A549 cell lines were subjected to bioassay-guided fractionation with Bel-7402 cell line as a screening model. As a result, twelve compounds were isolated and identified. Among them, chamaejasmenin E (1) and chamaejasmin D (2) were new compounds, and sikokianin B (8) was isolated from Stellera genus for the first time. All the isolated biflavones (1–10) were further tested against Bel-7402 and A549 cell lines. The results showed that seven of ten
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biflavones exhibited significant cytotoxic activities against both Bel-7402 and A549 cell lines. Especially, sikokianin D (3) was found to possess the most potent cytotoxic activities against both the two cell lines. These results in some degree expanded our scope of knowledge about the cytotoxic biflavones in S. chamaejasme and these biflavones represented promising new leads for development into antitumor clinical trial candidates. Herein, we depict the isolation, structure elucidation and the confirmation of the absolute configurations on 2, 3, 2″ and 3″ of the two new compounds (1, 2), as well as some structure– function relationships of these bioflavones for cytotoxic activities. 2. Experimental 2.1. General experimental procedures UV spectra were obtained using a SP-1901 UV–vis spectrophotometer; FT-IR spectra were recorded on a Spectrum-GX infrared spectrometer, using KBr pellets, over the range 400–4000 cm−1. Optical rotations were measured with a AUTOPOL-IV polarimeter (Na filter, λ = 589 nm). CD spectra were obtained on a MOS-450 spectropolarimeter. Highresolution mass spectrometry experiments were obtained on an Agilent-6520 QTOF mass spectrometer equipped with electrospray ionization (ESI) interface. NMR experiments were carried out on a Bruker Avance III-400 spectrometer with TMS as an internal standard. Chromatographic analyses were performed on an Agilent-1290 Series UHPLC system equipped with a diode array detector. Column chromatography was performed on silica gel (200–300 mesh, Qingdao Marine, China), ODS (40–75 μm, Fuji Silysia, Japan) and Jiangshen LC-100 semi-preparative HPLC system. Bel-7402 cells were purchased from Yinzijing Biotechnologies (Beijing, China), and A549 cells were purchased from China Center for Type Culture Collection (Wuhan, China). RPMI 1640 medium, MEM medium and fetal bovine serums (FBS) were purchased from Gibco Life Technologies (Grand Island, NY, USA). Vincristine was purchased from Aladdin Industrial Corporation (Shanghai, China). 2.2. Plant material The roots of S. chamaejasme were collected from Sichuan, China in October, 2012 and identified by Prof. Xi-rong He (Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing). A voucher specimen (No. SC20121026) had been deposited in our laboratory, the Group of High Performance Preparative Separation and Analysis of Natural Medicine, Dalian Institute of Chemical Physics, Chinese Academy of Sciences. 2.3. Extraction and isolation The air-dried roots of S. chamaejasme (12 kg) were extracted with 95% ethanol for three times (4 h each time) at 50 °C. After that, the extracts were combined and evaporated under reduced pressure. The concentrated residue (1500 g) was subjected to a 200–300 mesh silica gel column chromatography and partitioned sequentially with a gradient of CHCl3–MeOH (1:0, 50:1, 20:1, 10:1, 5:1, 1:1 and 0:1, v/v) to
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afford seven fractions (Fr.1 to Fr.7). Fr.3, Fr.4 and Fr.5 were found to be the most active against Bel-7402 cell line (their IC50 values were 65.74 ± 5.13, 23.34 ± 0.59 and 51.75 ± 1.01 μg/ml, respectively). Accordingly, further isolation was conducted in order to investigate active ingredients of the three fractions. Fr.3 (140 g) was chromatographed on an ODS column eluted with MeOH–H2O (15:85, 30:70, 45:55, 60:40, 70:30, 80:20, 90:10 and 100:0, v/v) to give eight subfractions (Fr.3-1 to Fr.3-8). Fr.3-5 yielded compound 1 (50.1 mg), 3 (18.6 mg), 4 (48.7 mg), 5 (249.2 mg), 6 (14.5 mg) by semi-preparative HPLC (ACN–H2O 50:50, v/v, containing 0.2% formic acid). Fr.4 (85.0 g) was isolated with semi-preparative HPLC (MeOH–H2O 65:35, v/v) to generate three subfractions (Fr.4-1 to Fr.4-3) and compound 11 (97.6 mg), while compounds 7 (65.3 mg) and 8 (70.2 mg) were derived from Fr.4-2 by semi-preparative HPLC (MeOH–H2O 60:40, v/v, containing 0.2% formic acid). Similarly, Fr.5 (330 g) was rechromatographed on an ODS column eluted with methanol– water (20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 and 90:10, v/v) to obtain eight subfractions (Fr.5-1 to Fr.5-8). Then Fr.5-3 was separated by semi-preparative HPLC (ACN–H2O 40:60, v/v, containing 0.2% formic acid), resulting in the purification of four compounds: 2 (23.5 mg), 9 (25.4 mg), 10 (37.8 mg), and 12 (15.2 mg). 2.3.1. Chamaejasmenin E (1) Light yellow powder; UV (MeOH): λmax nm 297 (lg ε 4.69), 220 (lg ε 5.01); IR (KBr): νmax cm−1 3404, 2933, 2838, 1637, 1541, 1516, 1463, 1427, 1381, 1344, 1290, 1253, 1175, 1112, 1085, 1016; [α]19 D = −185.45° (MeOH, c 0.5); CD (MeOH, c 0.01): 309 (Δε −14.25), 287 (Δε +21.35), 254 (Δε −1.00), 241 (Δε +1.55), 217 (Δε −15.37); HR-ESI-MS: [M–H]− m/z 569.1474 (C32H25O10, calcd. 569.1453); 1H and 13C-NMR spectral data were shown in Table 1. 2.3.2. Chamaejasmin D (2) Light yellow powder; UV (MeOH): λmax nm 300 (lg ε 4.55), 217 (lg ε 5.05); IR (KBr): νmax cm−1 3422, 1638, 1542, 1518, 1466, 1350, 1253, 1161, 1087, 1041, 1014; [α]19 D = −196.26° (MeOH, c 0.5); CD (MeOH, c 0.01): 311 (Δε −7.44), 288 (Δε +22.91), 254 (Δε +2.38), 243 (Δε +2.99), 219 (Δε −10.57); HR-ESI-MS: [M–H]− m/z 541.1127 (C30H21O10, calcd. 541.1140); 1H and 13C-NMR spectral data were shown in Table 1. 2.4. Cytotoxic assay All the isolated biflavones (1–10) were tested against Bel7402 and A549 cell lines using MTT method [8]. The cell culture medium for Bel-7402 was RPMI-1640 and for A549 was MEM, both with 10% fetal bovine serum. Vincristine was used as the positive control. Briefly, 8 × 103 exponentially growing cells seeded in 96-well microculture plates were treated with different concentrations (0.63–100 μg/ml) of tested compounds for 48 h, and the viability of the cells was determined by MTT assay. DMSO concentration was the same in all the treatments and did not exceed 0.1% (v/v). The IC50 values (concentrations inducing 50% inhibition of cell growth) were calculated as the sample concentration that caused 50% cell death. The data represent the mean of three experiments in triplicate and were statistically analyzed with Student's t-test. A difference was
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Table 1 1 H and 13C NMR data of compounds 1 and 2. Position
1 δH (mult., J in Hz)
2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 4′-OCH3 4‴-OCH3 a
5.75 d (11.9) 2.81 d (11.9) – – 5.89 d (2.1) – 5.81 d (2.1) – – – 6.99 d (8.7) 6.90 d (8.7) – 6.90 d (8.7) 6.99 d (8.7) 5.75 d (11.9) 2.81 d (11.9) – – 5.89 d (2.1) – 5.81 d (2.1) – – – 6.99 d (8.7) 6.90 d (8.7) – 6.90 d (8.7) 6.99 d (8.7) 3.81 s 3.81 s
2 a
δC
δH (mult., J in Hz)
δC
82.9 49.1 196.0 163.6 96.4 167.3 95.3 162.5 101.7 128.3 129.3 114.2 160.1 114.2 128.3 82.9 49.1 196.0 163.6 96.4 167.3 95.3 162.5 101.7 128.3 129.3 114.2 160.1 114.2 128.3 55.4 55.4
5.70 d (11.9) 2.79 d (11.9) – – 5.88 d (1.8) – 5.80 d (1.8) – – – 6.87 d (8.6) 6.74 d (8.6) – 6.74 d (8.6) 6.87 d (8.6) 5.70 d (11.9) 2.79 d (11.9) – – 5.88 d (1.8) – 5.80 d (1.8) – – – 6.87 d (8.6) 6.74 d (8.6) – 6.74 d (8.6) 6.87 d (8.6) – –
83.0 48.9 195.9 163.4 96.2 167.4 95.1 162.4 101.4 126.3 129.2 115.3 158.2 115.3 129.2 83.0 48.9 195.9 163.4 96.2 167.4 95.1 162.4 101.4 126.3 129.2 115.3 158.2 115.3 129.2 – –
Recorded in DMSO-d6 at 400 MHz (1H NMR) and 100 MHz (13C NMR).
considered statistically significant when p b 0.05. The cytotoxic assay results were shown in Table 2. 3. Results and discussion Chamaejasmenin E (1), obtained as an amorphous powder, had a molecular formula of C32H26O10 established by the negative ion m/z [M–H]− 569.1474 (calcd. 569.1453) in HRESIMS. In the 1H-NMR spectrum observed were signals of two methoxyl protons δH 3.81 (6H, s), two sets of typical 5, Table 2 Cytotoxic activities of ten biflavones against two human tumor cell lines. Compounds
Cell lines (IC50, μM) a Bel-7402
Chamaejasmenin E (1) Chamaejasmin D (2) Sikokianin D (3) Isochamaejasmenin B (4) Chamaejasmenin B (5) Sikokianin C (6) Sikokianin A (7) Sikokianin B (8) Isoneochamaejasmin A (9) Neochamaejasmin B (10) Vincristine b a b
1.17 31.85 1.29 2.70 1.05 6.47 9.31 8.42 46.77 55.13 4.35
± ± ± ± ± ± ± ± ± ± ±
A549 0.07 0.85 0.21 0.26 0.40 0.15 0.93 0.32 0.70 0.31 0.10
4.71 43.81 0.75 4.55 3.60 3.46 7.91 5.53 56.67 56.63 1.05
± ± ± ± ± ± ± ± ± ± ±
0.35 3.30 0.25 0.12 0.42 0.10 0.56 0.17 1.41 1.99 0.13
IC50 values were represented as mean ± standard deviation (n = 3). Vincristine was used as the positive control.
7-dioxygenated A rings δH 5.89 (2H, d, J = 2.10 Hz), δH 5.81 (2H, d, J = 2.10 Hz), and two sets of typical para-oxygenated B rings δH 6.99 (4H, d, J = 8.70 Hz), δH 6.90 (4H, d, J = 8.70 Hz), and the 13C-NMR spectrum showed the presence of two carbonyls (δC 196.0), two methoxyl groups (δC 55.4), and C-2, C-2″ (δC 82.9) and C-3, C-3″ (δC 49.1) (see Table 1). The above spectral information indicated that the structure of 1 was similar to that of the known C-3/C-3″ biflavanone chamaejasmenin B (5). Furthermore, the HMBC correlations of two methoxyl groups (δH 3.81) with C-4′, C-4‴ (δC 160.1) on the B, B′ rings revealed that these two methoxyl groups were also connected to C-4′ and C-4‴, indicating that 1 and chamaejasmenin B (5) had the same plane structure (see Fig. 2). However, comparison of the 1H-NMR spectral data of 1 with those of chamaejasmenin B (5) revealed that the splitting patterns of 1 were different from those of chamaejasmenin B (5), and the signals in chamaejasmenin B (5) were 2, 2″-H δH 5.32 (2H, s) and 3, 3″-H δH 2.98 (2H, s), while those in 1 were 2, 2″-H δH 5.75 (2H, d, J = 11.90 Hz) and 3, 3″-H δH 2.81 (2H, d, J = 11.90 Hz). The coupling constants (11.90 Hz and 11.90 Hz) of 2, 3-H and 2″, 3″-H indicated that 1 had trans- and trans-geometry at the C-2/C-3 and C-2″/C-3″ positions. For the trans-isomers of dihydroflavone, the thermodynamically more stable conformation was when 2-H was axial and 3-H was equatorial, and therefore the absolute configuration had to be either (2R, 3S) or (2S, 3R) [9]. Moreover, for 1, a negative n → π⁎ “cotton effects” at high wavelength (309 nm, Δε − 14.25) by CD spectrum indicated a 2R configuration [10], so the configurations of C-2/C-3 were deduced to be (2R, 3S). Besides, the two parts of the structure of 1 had the same units, rather than antipode for each other (their 1H- and 13C-NMR signals were overlapped and the [α]19 D was not zero), so the two units had the same absolute configurations of (2R, 3S) and (2″R, 3″S) at C-2/C-3 and C-2″/C-3″. Thus, the structure of chamaejasmenin E (1) was proposed as shown in Fig. 1. Chamaejasmin D (2), obtained as an amorphous powder, had a molecular formula of C30H22O10 established by the negative ion m/z [M–H]− 541.1127 (calcd. 541.1140) in HRESI-MS. Compared to 1, the most obvious difference is the absence of two methoxyl groups in 1H and 13C NMR spectra. Furthermore, the existence of two sets of typical 5, 7-dioxygenated A rings δH 5.88 (2H, d, J = 1.85 Hz), δH 5.80 (2H, d, J = 1.85 Hz), and two sets of typical para-oxygenated B rings δH 6.87 (4H, d, J = 8.60 Hz), δH 6.74 (4H, d, J = 8.60 Hz) in the 1H-NMR spectrum, as well as the presence of two carbonyls (δC 195.9) and C-2, C-2″ (δC 83.0) and C-3, C-3″ (δC 48.9) in the 13C-NMR spectrum (see Table 1) indicated that the structure of 2 was similar to that of the known C-3/C-3″ biflavanone chamaejasmine [11], and they had the same planar structures. The comparison of the 1H-NMR data of 2 with those of chamaejasmine revealed that 2 had trans- and trans-geometry at C-2/C-3 and C-2″/C-3″ positions, which were similar to those of chamaejasmine. The corresponding signals in chamaejasmine were 2, 2″-H δH 5.70 (2H, d, J = 11.90 Hz) and 3, 3″-H δH 2.80 (2H, d, J = 11.90), and those were 2, 2″-H 5.70 (2H, d, J = 11.90 Hz) and 3, 3″-H δH 2.79 (2H, d, J = 11.90 Hz) in 2. Therefore, the absolute configurations at C-2/C-3 had to be either (2R, 3S) or (2S, 3R) [9]. Moreover, for 2, a negative n → π⁎ “cotton effects” at high wavelength (311 nm, Δε −7.44) by CD spectrum indicated a 2R configuration [10], so the configurations of C-2/C-3 were deduced to be (2R, 3S). Besides, the two parts of the structure of 2 had the same units, rather than antipode for
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Fig. 1. Chemical structures of compounds 1–12.
each other (their 1H- and 13C-NMR signals were overlapped and the [α]19 D was not zero), so the two units had the same absolute configuration of (2R, 3S) and (2″R, 3″S) at C-2/C-3 and C-2″/C-3″. Thus, the structure of chamaejasmin D (2) was proposed as shown in Fig. 1. In order to further confirm the absolute configurations of 1 and 2, the calculation of electronic circular dichroism (ECD) by using time-dependent density functional theory (TDDFT), which has greatly enhanced the value of ECD in determining absolute configuration in recent years [12], was applied in combination with experimental CD data. Firstly, the proposed 3D structure models of compounds 1 and 2 were constructed based on the above conclusions. The conformation analyses was carried out using Monte Carlo searching with MMFF94 to generate 7 and 2 conformers for 1 and 2, respectively. Then the resulted conformers were re-optimized using DFT method at the B3LYP/6–31G (d) level in gas phase with the GAUSSIAN 09 program [13], respectively. The free energies and vibrational frequencies were calculated at the same level to confirm their stability, and no imaginary frequencies were found. Then the
optimized geometries with Gibbs free energy b 0.35 kcal/mol were considered for ECD calculation. The TDDFT//B3LYP/6–31 G (d, p) method was applied to calculate the excited energies, oscillator strength and rotational strength in the integral equation formalism polarizable continuum model (IEFPCM). The ECD spectra were simulated by the overlapping Gaussian function [14], in which the excited energies and velocity rotatory strengths of the first 60 electronic excitations for 1 and 2 were adopted (σ = 0.3 eV, UV shift = 12 nm). The final ECD spectra of each compound were obtained by averaging all the simulated ECD spectra of selected conformers according to their Gibbs free energies and Boltzmann distribution. As a result, for 1 and 2, the calculated ECD revealed a good agreement with that of the experimentally recorded ECD, which allowed the determination of the absolute configuration of these two compounds (see Fig. 3). The ten known compounds were identified as sikokianin D (3) (its name in reference is isochamaejasmenin C, in which this new compound was found almost at the same time with us, but according to the naming rules of biflavones from
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Fig. 2. Key 1H–1H COSY and HMBC correlations for compounds 1 and 2.
S. chamaejasme, sikokianin D was the more appropriate name) [15], isochamaejasmenin B (4) [16], chamaejasmenin B (5) [17], sikokianin C (6) [18,19], sikokianin A (7) [16,20], sikokianin B (8) [19,20], isoneochamaejasmin A (9) [21], neochamaejasmin B (10) [22], chamaechromone (11) [23] and mohsenone (12) [24], in comparison with their spectral data with those reported in the literatures.
The cytotoxic activities of ten biflavones isolated from S. chamaejasme were evaluated against Bel-7402 and A549 cell lines using vincristine as the positive control [25]. The results showed that seven biflavones exhibited significant cytotoxic activities against these two human cancer cell lines with IC50 values ranging from 1.05 to 9.31 μM for Bel-7402 and 0.75 to 7.91 μM for A549, respectively, as shown
Fig. 3. Experimental ECD and TDDFT calculated ECD at the B3LYP/6–31 G (d, p) level in methanol for 1 and 2.
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Fig. 4. Comparison of cytotoxic activities of ten biflavones and vincristine against Bel-7402 and A549 cell lines.
in Table 2. Chamaejasmenin E (1), sikokianin D (3), isochamaejasmenin B (4) and chamaejasmenin B (5) showed higher cytotoxic activities than vincristine against Bel-7402 cell line, while only sikokianin D (3) showed higher cytotoxic activities than vincristine against A549 cell line (see Fig. 4). Obviously, sikokianin D (3) possessed the most potential cytotoxic activities against both Bel-7402 and A549 cell lines with IC50 values of 1.29 ± 0.21 and 0.75 ± 0.25 μM, respectively. Based on our results, it seemed that for this type of biflavones, 4′-OCH3 contributed more to cytotoxic activity than 4′-OH and other groups, because compounds without 4′-OCH3 (2, 9 and 10) were the least active. The contributions of 4‴-OH and 4‴-OCH3 were nearly the same, since no obvious difference could be observed between IC50 values of compounds with 4‴-OCH3 (1, 4, 5), and compounds with 4‴-OH (6, 7, 8), except 3. As for the relatively higher cytotoxic activities of 3, it seemed that the absolute configuration of C-3″ might contribute to it, because among the four compounds with both 4′-OCH3 and 4‴-OH (3, 6, 7, 8), only 3 had a 3″R configuration. Certainly, these interesting structure–function relationships of biflavanones need to be further evaluated in the future. Acknowledgment This work was supported by grants from the Chinese National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (No. 2011ZX09307002-01). The assistance of Jian Luo and Prof. Ke-li Han at the State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences for the theoretical studies section was also acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.10.002. References [1] Jiangsu College of New Medicine. Dictionary of Chinese materia medica. Shanghai: Shanghai Science and Technology Press; 1986.
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[21] Feng BM, Pei YH, Zhang HL, Hua HM, Wang QY. Chemical constituents from roots of Stellera chamaejasme. Chin Tradit Herb Drugs 2004;35:12–4. [22] Niwa M, Tatematsu H, Liu GQ, Hirata Y. Isolation and structures of two new C-3/C-3″-biflavanones, neochamaejasmin A and neochamaejasmin B. Chem Lett 1984:539–42. [23] Niwa M, Liu GQ, Tatematsu H, Hirata Y. Chamaechromone, a novel rearranged biflavonoid from Stellera chamaejasme L. Tetrahedron Lett 1984;25:3735–8.
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Cytotoxic biflavones from Stellera chamaejasme Zhi-xin Wang a, Meng-chun Cheng a, Xiao-zhe Zhang a, Zhi-lai Hong a, Ming-zhe Gao a, Xiao-xi Kan b, Qi Li b, Ya-jie Wang b, Xiao-xin Zhu b,⁎, Hong-bin Xiao a,b,⁎⁎ a b
Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
a r t i c l e
i n f o
Article history: Received 29 July 2014 Accepted in revised form 30 September 2014 Available online 12 October 2014 Keywords: Stellera chamaejasme Chamaejasmenin E Chamaejasmin D ECD Cytotoxic activity
a b s t r a c t Bioassay-guided phytochemical studies on Stellera chamaejasme led to the isolation of two new biflavones, chamaejasmenin E (1) and chamaejasmin D (2), together with ten known compounds. The structures of new compounds were elucidated by extensive spectroscopic analyses and their absolute configurations on 2, 3, 2″ and 3″ were confirmed by TDDFT quantum chemical calculated ECD spectra combined with experimental ECD spectra. All isolated biflavones were evaluated for their cytotoxic activities against Bel-7402 and A549 tumor cell lines, and sikokianin D (3) was found to possess the most potential cytotoxic activities against both the two cell lines with IC50 values of 1.29 ± 0.21 and 0.75 ± 0.25 μM, respectively. Moreover, some structure–function relationships of these bioflavones for cytotoxic activities were explored and summarized. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Stellera chamaejasme (Thymelaeaceae), known as “Duanchangcao”, is a famous toxic plant widely distributed in the northern parts of China and Mongolia. The roots of this plant have been used for treatment of scabies, tinea, stubborn skin ulcers, chronic tracheitis, and tuberculosis by local physicians since ancient time [1]. Recently, more and more attentions have been drawn to S. chamaejasme because of its substantial antitumor activity [2,3], and it is not surprising that particularly in Mongolia it is the most widely used form in traditional medicine. Previous studies on chemical constituents of this plant have reported the presence of various compounds such as flavones, coumarins, lignans, diterpenes, etc. Among them, the antitumor activity was mainly focused on biflavones. For example, neochamaejasmin A could induce cell cycle arrest ⁎ Correspondence to: X. Zhu, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, No.16 Nanxiao Street, Dongzhimen Nei, Dongcheng, Beijing 100700, China. Tel./fax: +86 10 64056154. ⁎⁎ Correspondence to: H. Xiao, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Shahekou, Dalian 116023, China. Tel./fax: +86 411 84379756. E-mail addresses: [email protected] (X. Zhu), [email protected] (H. Xiao).
http://dx.doi.org/10.1016/j.fitote.2014.10.002 0367-326X/© 2014 Elsevier B.V. All rights reserved.
and apoptosis involved of p21 and FasL in human prostate LNCaP cancer cells [4]. Chamaejasmin A showed inhibitory action against human HEP-2 epithelial cells, which was related to tubulin protein [5]. Chamaejasmin could arrest cell cycle, induce apoptosis and inhibit nuclear NF-κB translocation in human breast cancer cell line MDA-MB-231 [6]. Also, both chamaejasmenin B and neochamaejasmin C exerted potent anti-proliferative effects in eight human solid tumor cell lines. In terms of the most sensitive A549 and KHOS cells, the mechanisms were that the two compounds induced prominent expression of the DNA damage marker γ-H2AX as well as apoptosis [7]. As part of our continuous efforts to search plant-originated anticancer agents, 95% ethanol extracts of the roots of S. chamaejasme showing considerable cytotoxic activities against Bel-7402 and A549 cell lines were subjected to bioassay-guided fractionation with Bel-7402 cell line as a screening model. As a result, twelve compounds were isolated and identified. Among them, chamaejasmenin E (1) and chamaejasmin D (2) were new compounds, and sikokianin B (8) was isolated from Stellera genus for the first time. All the isolated biflavones (1–10) were further tested against Bel-7402 and A549 cell lines. The results showed that seven of ten
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biflavones exhibited significant cytotoxic activities against both Bel-7402 and A549 cell lines. Especially, sikokianin D (3) was found to possess the most potent cytotoxic activities against both the two cell lines. These results in some degree expanded our scope of knowledge about the cytotoxic biflavones in S. chamaejasme and these biflavones represented promising new leads for development into antitumor clinical trial candidates. Herein, we depict the isolation, structure elucidation and the confirmation of the absolute configurations on 2, 3, 2″ and 3″ of the two new compounds (1, 2), as well as some structure– function relationships of these bioflavones for cytotoxic activities. 2. Experimental 2.1. General experimental procedures UV spectra were obtained using a SP-1901 UV–vis spectrophotometer; FT-IR spectra were recorded on a Spectrum-GX infrared spectrometer, using KBr pellets, over the range 400–4000 cm−1. Optical rotations were measured with a AUTOPOL-IV polarimeter (Na filter, λ = 589 nm). CD spectra were obtained on a MOS-450 spectropolarimeter. Highresolution mass spectrometry experiments were obtained on an Agilent-6520 QTOF mass spectrometer equipped with electrospray ionization (ESI) interface. NMR experiments were carried out on a Bruker Avance III-400 spectrometer with TMS as an internal standard. Chromatographic analyses were performed on an Agilent-1290 Series UHPLC system equipped with a diode array detector. Column chromatography was performed on silica gel (200–300 mesh, Qingdao Marine, China), ODS (40–75 μm, Fuji Silysia, Japan) and Jiangshen LC-100 semi-preparative HPLC system. Bel-7402 cells were purchased from Yinzijing Biotechnologies (Beijing, China), and A549 cells were purchased from China Center for Type Culture Collection (Wuhan, China). RPMI 1640 medium, MEM medium and fetal bovine serums (FBS) were purchased from Gibco Life Technologies (Grand Island, NY, USA). Vincristine was purchased from Aladdin Industrial Corporation (Shanghai, China). 2.2. Plant material The roots of S. chamaejasme were collected from Sichuan, China in October, 2012 and identified by Prof. Xi-rong He (Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing). A voucher specimen (No. SC20121026) had been deposited in our laboratory, the Group of High Performance Preparative Separation and Analysis of Natural Medicine, Dalian Institute of Chemical Physics, Chinese Academy of Sciences. 2.3. Extraction and isolation The air-dried roots of S. chamaejasme (12 kg) were extracted with 95% ethanol for three times (4 h each time) at 50 °C. After that, the extracts were combined and evaporated under reduced pressure. The concentrated residue (1500 g) was subjected to a 200–300 mesh silica gel column chromatography and partitioned sequentially with a gradient of CHCl3–MeOH (1:0, 50:1, 20:1, 10:1, 5:1, 1:1 and 0:1, v/v) to
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afford seven fractions (Fr.1 to Fr.7). Fr.3, Fr.4 and Fr.5 were found to be the most active against Bel-7402 cell line (their IC50 values were 65.74 ± 5.13, 23.34 ± 0.59 and 51.75 ± 1.01 μg/ml, respectively). Accordingly, further isolation was conducted in order to investigate active ingredients of the three fractions. Fr.3 (140 g) was chromatographed on an ODS column eluted with MeOH–H2O (15:85, 30:70, 45:55, 60:40, 70:30, 80:20, 90:10 and 100:0, v/v) to give eight subfractions (Fr.3-1 to Fr.3-8). Fr.3-5 yielded compound 1 (50.1 mg), 3 (18.6 mg), 4 (48.7 mg), 5 (249.2 mg), 6 (14.5 mg) by semi-preparative HPLC (ACN–H2O 50:50, v/v, containing 0.2% formic acid). Fr.4 (85.0 g) was isolated with semi-preparative HPLC (MeOH–H2O 65:35, v/v) to generate three subfractions (Fr.4-1 to Fr.4-3) and compound 11 (97.6 mg), while compounds 7 (65.3 mg) and 8 (70.2 mg) were derived from Fr.4-2 by semi-preparative HPLC (MeOH–H2O 60:40, v/v, containing 0.2% formic acid). Similarly, Fr.5 (330 g) was rechromatographed on an ODS column eluted with methanol– water (20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 and 90:10, v/v) to obtain eight subfractions (Fr.5-1 to Fr.5-8). Then Fr.5-3 was separated by semi-preparative HPLC (ACN–H2O 40:60, v/v, containing 0.2% formic acid), resulting in the purification of four compounds: 2 (23.5 mg), 9 (25.4 mg), 10 (37.8 mg), and 12 (15.2 mg). 2.3.1. Chamaejasmenin E (1) Light yellow powder; UV (MeOH): λmax nm 297 (lg ε 4.69), 220 (lg ε 5.01); IR (KBr): νmax cm−1 3404, 2933, 2838, 1637, 1541, 1516, 1463, 1427, 1381, 1344, 1290, 1253, 1175, 1112, 1085, 1016; [α]19 D = −185.45° (MeOH, c 0.5); CD (MeOH, c 0.01): 309 (Δε −14.25), 287 (Δε +21.35), 254 (Δε −1.00), 241 (Δε +1.55), 217 (Δε −15.37); HR-ESI-MS: [M–H]− m/z 569.1474 (C32H25O10, calcd. 569.1453); 1H and 13C-NMR spectral data were shown in Table 1. 2.3.2. Chamaejasmin D (2) Light yellow powder; UV (MeOH): λmax nm 300 (lg ε 4.55), 217 (lg ε 5.05); IR (KBr): νmax cm−1 3422, 1638, 1542, 1518, 1466, 1350, 1253, 1161, 1087, 1041, 1014; [α]19 D = −196.26° (MeOH, c 0.5); CD (MeOH, c 0.01): 311 (Δε −7.44), 288 (Δε +22.91), 254 (Δε +2.38), 243 (Δε +2.99), 219 (Δε −10.57); HR-ESI-MS: [M–H]− m/z 541.1127 (C30H21O10, calcd. 541.1140); 1H and 13C-NMR spectral data were shown in Table 1. 2.4. Cytotoxic assay All the isolated biflavones (1–10) were tested against Bel7402 and A549 cell lines using MTT method [8]. The cell culture medium for Bel-7402 was RPMI-1640 and for A549 was MEM, both with 10% fetal bovine serum. Vincristine was used as the positive control. Briefly, 8 × 103 exponentially growing cells seeded in 96-well microculture plates were treated with different concentrations (0.63–100 μg/ml) of tested compounds for 48 h, and the viability of the cells was determined by MTT assay. DMSO concentration was the same in all the treatments and did not exceed 0.1% (v/v). The IC50 values (concentrations inducing 50% inhibition of cell growth) were calculated as the sample concentration that caused 50% cell death. The data represent the mean of three experiments in triplicate and were statistically analyzed with Student's t-test. A difference was
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Table 1 1 H and 13C NMR data of compounds 1 and 2. Position
1 δH (mult., J in Hz)
2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 4′-OCH3 4‴-OCH3 a
5.75 d (11.9) 2.81 d (11.9) – – 5.89 d (2.1) – 5.81 d (2.1) – – – 6.99 d (8.7) 6.90 d (8.7) – 6.90 d (8.7) 6.99 d (8.7) 5.75 d (11.9) 2.81 d (11.9) – – 5.89 d (2.1) – 5.81 d (2.1) – – – 6.99 d (8.7) 6.90 d (8.7) – 6.90 d (8.7) 6.99 d (8.7) 3.81 s 3.81 s
2 a
δC
δH (mult., J in Hz)
δC
82.9 49.1 196.0 163.6 96.4 167.3 95.3 162.5 101.7 128.3 129.3 114.2 160.1 114.2 128.3 82.9 49.1 196.0 163.6 96.4 167.3 95.3 162.5 101.7 128.3 129.3 114.2 160.1 114.2 128.3 55.4 55.4
5.70 d (11.9) 2.79 d (11.9) – – 5.88 d (1.8) – 5.80 d (1.8) – – – 6.87 d (8.6) 6.74 d (8.6) – 6.74 d (8.6) 6.87 d (8.6) 5.70 d (11.9) 2.79 d (11.9) – – 5.88 d (1.8) – 5.80 d (1.8) – – – 6.87 d (8.6) 6.74 d (8.6) – 6.74 d (8.6) 6.87 d (8.6) – –
83.0 48.9 195.9 163.4 96.2 167.4 95.1 162.4 101.4 126.3 129.2 115.3 158.2 115.3 129.2 83.0 48.9 195.9 163.4 96.2 167.4 95.1 162.4 101.4 126.3 129.2 115.3 158.2 115.3 129.2 – –
Recorded in DMSO-d6 at 400 MHz (1H NMR) and 100 MHz (13C NMR).
considered statistically significant when p b 0.05. The cytotoxic assay results were shown in Table 2. 3. Results and discussion Chamaejasmenin E (1), obtained as an amorphous powder, had a molecular formula of C32H26O10 established by the negative ion m/z [M–H]− 569.1474 (calcd. 569.1453) in HRESIMS. In the 1H-NMR spectrum observed were signals of two methoxyl protons δH 3.81 (6H, s), two sets of typical 5, Table 2 Cytotoxic activities of ten biflavones against two human tumor cell lines. Compounds
Cell lines (IC50, μM) a Bel-7402
Chamaejasmenin E (1) Chamaejasmin D (2) Sikokianin D (3) Isochamaejasmenin B (4) Chamaejasmenin B (5) Sikokianin C (6) Sikokianin A (7) Sikokianin B (8) Isoneochamaejasmin A (9) Neochamaejasmin B (10) Vincristine b a b
1.17 31.85 1.29 2.70 1.05 6.47 9.31 8.42 46.77 55.13 4.35
± ± ± ± ± ± ± ± ± ± ±
A549 0.07 0.85 0.21 0.26 0.40 0.15 0.93 0.32 0.70 0.31 0.10
4.71 43.81 0.75 4.55 3.60 3.46 7.91 5.53 56.67 56.63 1.05
± ± ± ± ± ± ± ± ± ± ±
0.35 3.30 0.25 0.12 0.42 0.10 0.56 0.17 1.41 1.99 0.13
IC50 values were represented as mean ± standard deviation (n = 3). Vincristine was used as the positive control.
7-dioxygenated A rings δH 5.89 (2H, d, J = 2.10 Hz), δH 5.81 (2H, d, J = 2.10 Hz), and two sets of typical para-oxygenated B rings δH 6.99 (4H, d, J = 8.70 Hz), δH 6.90 (4H, d, J = 8.70 Hz), and the 13C-NMR spectrum showed the presence of two carbonyls (δC 196.0), two methoxyl groups (δC 55.4), and C-2, C-2″ (δC 82.9) and C-3, C-3″ (δC 49.1) (see Table 1). The above spectral information indicated that the structure of 1 was similar to that of the known C-3/C-3″ biflavanone chamaejasmenin B (5). Furthermore, the HMBC correlations of two methoxyl groups (δH 3.81) with C-4′, C-4‴ (δC 160.1) on the B, B′ rings revealed that these two methoxyl groups were also connected to C-4′ and C-4‴, indicating that 1 and chamaejasmenin B (5) had the same plane structure (see Fig. 2). However, comparison of the 1H-NMR spectral data of 1 with those of chamaejasmenin B (5) revealed that the splitting patterns of 1 were different from those of chamaejasmenin B (5), and the signals in chamaejasmenin B (5) were 2, 2″-H δH 5.32 (2H, s) and 3, 3″-H δH 2.98 (2H, s), while those in 1 were 2, 2″-H δH 5.75 (2H, d, J = 11.90 Hz) and 3, 3″-H δH 2.81 (2H, d, J = 11.90 Hz). The coupling constants (11.90 Hz and 11.90 Hz) of 2, 3-H and 2″, 3″-H indicated that 1 had trans- and trans-geometry at the C-2/C-3 and C-2″/C-3″ positions. For the trans-isomers of dihydroflavone, the thermodynamically more stable conformation was when 2-H was axial and 3-H was equatorial, and therefore the absolute configuration had to be either (2R, 3S) or (2S, 3R) [9]. Moreover, for 1, a negative n → π⁎ “cotton effects” at high wavelength (309 nm, Δε − 14.25) by CD spectrum indicated a 2R configuration [10], so the configurations of C-2/C-3 were deduced to be (2R, 3S). Besides, the two parts of the structure of 1 had the same units, rather than antipode for each other (their 1H- and 13C-NMR signals were overlapped and the [α]19 D was not zero), so the two units had the same absolute configurations of (2R, 3S) and (2″R, 3″S) at C-2/C-3 and C-2″/C-3″. Thus, the structure of chamaejasmenin E (1) was proposed as shown in Fig. 1. Chamaejasmin D (2), obtained as an amorphous powder, had a molecular formula of C30H22O10 established by the negative ion m/z [M–H]− 541.1127 (calcd. 541.1140) in HRESI-MS. Compared to 1, the most obvious difference is the absence of two methoxyl groups in 1H and 13C NMR spectra. Furthermore, the existence of two sets of typical 5, 7-dioxygenated A rings δH 5.88 (2H, d, J = 1.85 Hz), δH 5.80 (2H, d, J = 1.85 Hz), and two sets of typical para-oxygenated B rings δH 6.87 (4H, d, J = 8.60 Hz), δH 6.74 (4H, d, J = 8.60 Hz) in the 1H-NMR spectrum, as well as the presence of two carbonyls (δC 195.9) and C-2, C-2″ (δC 83.0) and C-3, C-3″ (δC 48.9) in the 13C-NMR spectrum (see Table 1) indicated that the structure of 2 was similar to that of the known C-3/C-3″ biflavanone chamaejasmine [11], and they had the same planar structures. The comparison of the 1H-NMR data of 2 with those of chamaejasmine revealed that 2 had trans- and trans-geometry at C-2/C-3 and C-2″/C-3″ positions, which were similar to those of chamaejasmine. The corresponding signals in chamaejasmine were 2, 2″-H δH 5.70 (2H, d, J = 11.90 Hz) and 3, 3″-H δH 2.80 (2H, d, J = 11.90), and those were 2, 2″-H 5.70 (2H, d, J = 11.90 Hz) and 3, 3″-H δH 2.79 (2H, d, J = 11.90 Hz) in 2. Therefore, the absolute configurations at C-2/C-3 had to be either (2R, 3S) or (2S, 3R) [9]. Moreover, for 2, a negative n → π⁎ “cotton effects” at high wavelength (311 nm, Δε −7.44) by CD spectrum indicated a 2R configuration [10], so the configurations of C-2/C-3 were deduced to be (2R, 3S). Besides, the two parts of the structure of 2 had the same units, rather than antipode for
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Fig. 1. Chemical structures of compounds 1–12.
each other (their 1H- and 13C-NMR signals were overlapped and the [α]19 D was not zero), so the two units had the same absolute configuration of (2R, 3S) and (2″R, 3″S) at C-2/C-3 and C-2″/C-3″. Thus, the structure of chamaejasmin D (2) was proposed as shown in Fig. 1. In order to further confirm the absolute configurations of 1 and 2, the calculation of electronic circular dichroism (ECD) by using time-dependent density functional theory (TDDFT), which has greatly enhanced the value of ECD in determining absolute configuration in recent years [12], was applied in combination with experimental CD data. Firstly, the proposed 3D structure models of compounds 1 and 2 were constructed based on the above conclusions. The conformation analyses was carried out using Monte Carlo searching with MMFF94 to generate 7 and 2 conformers for 1 and 2, respectively. Then the resulted conformers were re-optimized using DFT method at the B3LYP/6–31G (d) level in gas phase with the GAUSSIAN 09 program [13], respectively. The free energies and vibrational frequencies were calculated at the same level to confirm their stability, and no imaginary frequencies were found. Then the
optimized geometries with Gibbs free energy b 0.35 kcal/mol were considered for ECD calculation. The TDDFT//B3LYP/6–31 G (d, p) method was applied to calculate the excited energies, oscillator strength and rotational strength in the integral equation formalism polarizable continuum model (IEFPCM). The ECD spectra were simulated by the overlapping Gaussian function [14], in which the excited energies and velocity rotatory strengths of the first 60 electronic excitations for 1 and 2 were adopted (σ = 0.3 eV, UV shift = 12 nm). The final ECD spectra of each compound were obtained by averaging all the simulated ECD spectra of selected conformers according to their Gibbs free energies and Boltzmann distribution. As a result, for 1 and 2, the calculated ECD revealed a good agreement with that of the experimentally recorded ECD, which allowed the determination of the absolute configuration of these two compounds (see Fig. 3). The ten known compounds were identified as sikokianin D (3) (its name in reference is isochamaejasmenin C, in which this new compound was found almost at the same time with us, but according to the naming rules of biflavones from
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Fig. 2. Key 1H–1H COSY and HMBC correlations for compounds 1 and 2.
S. chamaejasme, sikokianin D was the more appropriate name) [15], isochamaejasmenin B (4) [16], chamaejasmenin B (5) [17], sikokianin C (6) [18,19], sikokianin A (7) [16,20], sikokianin B (8) [19,20], isoneochamaejasmin A (9) [21], neochamaejasmin B (10) [22], chamaechromone (11) [23] and mohsenone (12) [24], in comparison with their spectral data with those reported in the literatures.
The cytotoxic activities of ten biflavones isolated from S. chamaejasme were evaluated against Bel-7402 and A549 cell lines using vincristine as the positive control [25]. The results showed that seven biflavones exhibited significant cytotoxic activities against these two human cancer cell lines with IC50 values ranging from 1.05 to 9.31 μM for Bel-7402 and 0.75 to 7.91 μM for A549, respectively, as shown
Fig. 3. Experimental ECD and TDDFT calculated ECD at the B3LYP/6–31 G (d, p) level in methanol for 1 and 2.
Z. Wang et al. / Fitoterapia 99 (2014) 334–340
339
Fig. 4. Comparison of cytotoxic activities of ten biflavones and vincristine against Bel-7402 and A549 cell lines.
in Table 2. Chamaejasmenin E (1), sikokianin D (3), isochamaejasmenin B (4) and chamaejasmenin B (5) showed higher cytotoxic activities than vincristine against Bel-7402 cell line, while only sikokianin D (3) showed higher cytotoxic activities than vincristine against A549 cell line (see Fig. 4). Obviously, sikokianin D (3) possessed the most potential cytotoxic activities against both Bel-7402 and A549 cell lines with IC50 values of 1.29 ± 0.21 and 0.75 ± 0.25 μM, respectively. Based on our results, it seemed that for this type of biflavones, 4′-OCH3 contributed more to cytotoxic activity than 4′-OH and other groups, because compounds without 4′-OCH3 (2, 9 and 10) were the least active. The contributions of 4‴-OH and 4‴-OCH3 were nearly the same, since no obvious difference could be observed between IC50 values of compounds with 4‴-OCH3 (1, 4, 5), and compounds with 4‴-OH (6, 7, 8), except 3. As for the relatively higher cytotoxic activities of 3, it seemed that the absolute configuration of C-3″ might contribute to it, because among the four compounds with both 4′-OCH3 and 4‴-OH (3, 6, 7, 8), only 3 had a 3″R configuration. Certainly, these interesting structure–function relationships of biflavanones need to be further evaluated in the future. Acknowledgment This work was supported by grants from the Chinese National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (No. 2011ZX09307002-01). The assistance of Jian Luo and Prof. Ke-li Han at the State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences for the theoretical studies section was also acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.10.002. References [1] Jiangsu College of New Medicine. Dictionary of Chinese materia medica. Shanghai: Shanghai Science and Technology Press; 1986.
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