Isolation, chemotaxonomic significance and cytotoxic effects of quassinoids from Brucea javanica Qing-Mei Ye, Liang-Liang Bai, Shu-Zhi Hu, Hai-Yan Tian, Li-Jun Ruan, Ya-Fang Tan, Li-Ping Hu, Wen-Cai Ye, Dong-Mei Zhang, Ren-Wang Jiang PII: DOI: Reference:

S0367-326X(15)30020-4 doi: 10.1016/j.fitote.2015.06.004 FITOTE 3199

To appear in:

Fitoterapia

Received date: Revised date: Accepted date:

16 April 2015 5 June 2015 6 June 2015

Please cite this article as: Qing-Mei Ye, Liang-Liang Bai, Shu-Zhi Hu, Hai-Yan Tian, LiJun Ruan, Ya-Fang Tan, Li-Ping Hu, Wen-Cai Ye, Dong-Mei Zhang, Ren-Wang Jiang, Isolation, chemotaxonomic significance and cytotoxic effects of quassinoids from Brucea javanica, Fitoterapia (2015), doi: 10.1016/j.fitote.2015.06.004

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Isolation, chemotaxonomic significance and cytotoxic effects of quassinoids from Brucea javanica

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Qing-Mei Yea,b1, Liang-Liang Baia,1, Shu-Zhi Hua,1, Hai-Yan Tiana, Li-Jun Ruana, Ya-Fang Tana, Li-Ping Hua, Wen-Cai Ye a, Dong-Mei Zhang a,c*, Ren-Wang Jiang a,c*

Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New

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Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China. Department of Pharmacy, Hainan General Hospital, Haikou, 570311 , P. R. China.

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Shenzhen Engineering Laboratory of Lingnan Medicinal Resources Development and

*

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Application, Shenzhen Institute for Drug Control, Shenzhen, 518057, P. R. China.

Corresponding authors. Tel.: +86 20 85221016; Fax: +86 20 85221559.

E-mail addresses: [email protected] (R.-W. Jiang), [email protected] (D.-M.

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Zhang).

These co-authors contributed equally to this work.

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ACCEPTED MANUSCRIPT Abstract: A new quassinoid, bruceene A (1) along with seventeen known quassinoids (2-18) was isolated from the fruits of Brucea javanica. The structure of 1 was elucidated by extensive

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spectroscopic methods, and was further confirmed by single-crystal X-ray diffraction analysis. Isolation of similar quassinoids 1-3 as those in genus Ailanthus from genus Brucea,

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indicated the close chemotaxonomic relationship between these two genera, which further supported the phylogenetic study by DNA analysis. Compounds 5, 7, 10 and 12 with a 3-hydroxy-3-en-2-one moiety showed potent inhibitory activities against the MCF-7 and

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MDA-MB-231 cells with IC50 values in the ranges 0.063-0.182 M and 0.081-0.238 M, respectively; while glycosidation at 3-OH significantly decreased the cytotoxicity. It was

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also found that the most potent compound 7 induced apoptosis in MCF-7 cells via the

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intrinsic mitochondrial apoptotic pathway.

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Keywords: Brucea javanica, Quassinoids, Bruceene A, Breast cancer, Chemotaxonomics

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ACCEPTED MANUSCRIPT 1. Introduction. Brucea javanica (L.) Merr. (Simaroubaceae family), an evergreen shrub, widely distributed from southeast Asia to northern Australia. The fruit of this herb (“Ya-Dan-Zi” in

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Chinese) was used as a Traditional Chinese Medicine since Ming Dynasty (1364 - 1644 AD)

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[1], and was currently recorded in the Pharmacopoeia of the People’s Republic of China

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(2010 edition) for removing heat, treatment of malaria and amoebic dysentery [2]. Phytochemical studies revealed that B. javanica is a rich source of quassinoids such as brusatol, bruceines and bruceosides [3,4], and oil-like lipids such as oleic, linoleic, palmitic,

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and stearic acids [5].

The oil-like lipid part of the fruit has been developed into several well-known

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pharmaceutical products, e.g. Brucea javanica oil soft capsule, Oral Brucea javanica oil emulsion, and Brucea javanica oil injection as single or adjuvant treatments for proliferative

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diseases such as cancer [6].

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The quassinoids from B. javanica were found to have potent antiviral activities against tobacco mosaic virus with IC50 values in the range of 3.42-5.66 μM [7]. Both bruceine A and

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D exhibited significant inhibitory activity against the Dactylogyrus intermedius in goldfish with EC50 values of 0.49 mg/L and 0.57 mg/L, respectively, which were more effective than the positive control mebendazole [8]. Bruceine E and D were reported to decrease the blood

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glucose concentration comparable to glibenclamide [9]. Besides the above biological activities reported, the most significant role of this category of compounds is for the prevention or treatment of cancer. Quassinoids were found to show cytotoxic effect against a variety of cancer cells [10,11]. Especially, chrysoeriol could selectively kill the leukemic cells and potentiate the amplification of ROS levels [12]. Brusatol could activate NF-B and promote

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cell

differentiation

[13].

Furthermore,

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could

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chemoresistance and enhance the efficacy of chemotherapy by inhibiting the Nrf2-mediated defense mechanism [14]. Due to the pronounced clinical effect, B. javanica is widely cultivated in Guangxi and Hainan provinces of China; however, the fruits of B. javanica are only used for extraction of oil and the non-oil part containing the quassinoids was discarded. To promote the utilization of the non-oil part, a systematic phytochemical study was 3

ACCEPTED MANUSCRIPT performed. In this paper, we report the isolation and structural elucidation of one new quassinoid bruceene A (1), along with seventeen known quassinoids (2-18) (Fig. 1) and their inhibitory activities against the breast cancer cells (MCF-7 and MDA-MB-231). In addition,

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the chemotaxonomic significance was discussed.

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2. Experimental 2.1. General experimental procedures

Ultraviolet (UV) spectra were determined in CHCl3 on a Jasco V-550 UV/vis

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spectrophotometer. ESI-MS spectra were carried out on a Finnigan LCQ Advantage Max ion trap mass spectrometer. HR-ESI-MS data were obtained on an Agilent 6210 ESI/TOF mass

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spectrometer. Optical rotation was recorded in CHCl3 on Jasco P-1020 polarimeter at room temperature. Infrared (IR) spectra were measured on a Jasco FT/IR-480 plus Fourier

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Transform infrared spectrometer using KBr pellet. Nuclear magnetic resonance (NMR)

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spectra were measured on Bruker AV-300/400 spectrometers. Thin-layer chromatography (TLC) analyses were carried out using pre-coated silica gel GF254 plates (Qingdao Marine

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Chemical Plant, Qingdao, People’s Republic of China).

2.2. Plant Material

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The fruits of B. javanica were bought from the Guangzhou Qingping herb medicine market in October of 2010, and were identified by Prof. Guang-Xiong Zhou (Jinan University). A voucher specimen (No.YDZ20101011) was deposited in the institute of Traditional Chinese Medicine and Natural Products, Jinan University, P. R. China.

2.3. Extraction and Isolation Dried powder fruits of B. javanica (11 kg) was percolated

with 95% EtOH (20 L  3)

at room temperature to afford 800g of crude extract, which was suspended with water and then extracted with petroleum ether (500 mL  3) , chloroform (500 mL  3), and n-BuOH (500 mL  3), successively. The extracts were then evaporated under vacuum to afford corresponding extracts 121 g, 32 g, and 181 g, respectively. The chloroform extract (32 g) was subjected to silica gel (0.5 kg, 200-300 mesh), eluted 4

ACCEPTED MANUSCRIPT with cyclohexane–acetic ether (10:1, 5:1, 3:1, 2:1 and 0:1) to give 5 fractions (Fr. 1 to Fr. 5). Fr. 3 (522 mg) was recrystallized from MeOH-H2O (1:1) to give the crystalline 5 (392.7 mg) and the mother liquor was further separated through preparative HPLC (ODS column) eluted

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with acetonitrile-H2O (41:59) to furnish compound 12 (11.6 mg), 10 (15.1 mg), respectively. Fr. 4 (33 mg) was subjected to preparative HPLC eluted with acetonitrile-H2O (55:45) to

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yield compound 7 (19.3 mg).

The n-BuOH extract (181 g) of B. javanica was separated by D-101 macroporous resin eluted with H2O/EtOH (1:0, 3:1, 1:1 and 0:1) to afford four fractions (Fr. 1’ to Fr. 4’). Fr. 3’

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(26g) was subjected to a silica gel column chromatography to give four subfractions (3’A-3’D). Fraction 3’A (46 mg) was further purified by preparative HPLC eluted with a

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isocratic of 95% MeOH (95:5) to afford compounds 2 (16.0 mg) and 3 (25.4 mg). Fraction 3’B (35 mg) was separated using preparative HPLC eluted with a isocratic of 80% MeOH to

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yield compounds 4 (8.2 mg). Fraction 3’C (600 mg) was separated using preparative HPLC

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eluted with a isocratic of 75% MeOH to yield compounds 4 (23.0 mg), 6 (26.2 mg), 9 (22.0 mg), 13 (25.1 mg), 14 (18.7 mg) and 16 (42.0 mg), Fraction 3’D (136 mg) was purified by

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preparative HPLC eluted with 90% MeOH to give compounds 8 (57.0 mg), 11 (37.2 mg), 15 (50.3 mg), 17 (20.2 mg) and 18 (6.9 mg). Compound 1

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Colorless needle-like crystals (MeOH); [α]20D = 50.2 (c 0.50, MeOH); UV (CH3OH) λmax (logε): 229 nm; IR 3427, 1735, 1604, 1278, 1018 cm-1; HR-ESI-MS m/z: 433.1645 [M+Na]+; 1

H and 13C NMR data were shown in Table 1.

2.4. X-ray Crystallographic Analysis of 1 Upon crystallization from MeOH at room temperature, needle-like crystals of 1 were obtained. Data were collected using a Sapphire CCD with a graphite monochromated CuKα radiation, λ = 1.54184 Å at 173.0(2) K. Crystal data: C20H26O9, orthorhombic, space group P212121; unit cell dimensions were determined to be a = 6.7162(1) Å, b = 13.6796(2) Å, c = 25.9859(5) Å, V = 2387.45(7) Å3, Z = 4, Dx = 1.448 g/cm3, F (000) = 1104, μ (Cu Kα) = 0.955 mm-1. 4961 reflections were collected until θmax = 62.63°, in which independent unique 3380 reflections were observed [F2 > 4σ (F2)]. The structure was solved by direct methods 5

ACCEPTED MANUSCRIPT using the SHELXS-97 program, and refined by the SHELXL-97 program and full-matrix least-squares calculations. In the structure refinements, nonhydrogen atoms were placed on the geometrically ideal positions by the “ride on” method. Hydrogen atoms bonded to

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refinement gave R = 0.0314, RW = 0.0339, and S = 1.035.

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oxygen were located by the structure factors with isotropic temperature factors. The final

2.5. Cell culture

Human breast cancer cell lines MCF-7 (estrogen-positive) and MDA-MB-231

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(estrogen-negative), obtained from American Type Culture Collection (Manassas, VA, USA), were cultured in RPMI-1640 and DMEM medium containing 10% fetal bovine serum and

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1% (v/v) penicillin-streptomycin in a humidified atmosphere with 5% CO2 at 37 °C.

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2.6. Cytotoxicity assay

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The cytotoxic activities of these compounds were assessed by MTT assay as described previously [15]. Cells (5  103/well) were exposed to different concentrations of tested

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compounds for 72 h. After that, MTT (Sigma-Aldrich, St. Louis, MO, USA) solution (5 mg/mL) was added to each well and incubated for 4 h and then the formazan crystals were solubilized with DMSO. Finally, absorbance of each well was determined at 570 nm by a

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microplate reader (Thermo Multiskan MK3, USA). Cells treated with medium containing 0.2% DMSO was considered as 100% viable. The concentration required to inhibit cell growth by 50% (IC50) was calculated from survival curves. Dox (Sigma-Aldrich, St. Louis, MO, USA) was used as the positive control.

2.7. Apoptosis analysis Cell apoptosis analysis was performed using AnnexinV-FITC/PI staining assay kit (Biouniquer Tech, Nanjing, China) according to the manufacturer’s protocol. MCF-7 cells (3  105/well) were treated with compound 7 (0.05 µM and 0.2 µM) or Dox (0.9 µM) for 48 h. After that, cells were stained with AnnexinV-FITC and PI solution and then examined by Guava EasyCyte 6-2L flow cytometry (Mllipore, Merck KGaA, Germany). Data was analyzed quantitatively with Guava incyte software (Mllipore, Merck KGaA, Germany). 6

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2.8. Western blot Cleavage of PARP (Santa Cruz, CA, USA) and activation of Caspase-9 (Cell Signaling

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Technology, Beverly, MA, USA) were evaluated by western blot. MCF-7 cells (1  106/dish) were cultured with compound 7 (0.05 µM and 0.2 µM ) or Dox (0.9 µM) for 48 h, cells were

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harvested and then lysed in RIPA buffer (0.5 M DTT, 0.1 M PMSF and 20  phosphatase inhibitor) for 30 min on ice. After centrifugation at 14 000  g at 4 °C for 15 min, supernatants were collected as total cellular proteins and stored at -80 °C until use. Protein

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concentration was determined using BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Electrophoresis and immunoblotting analysis was carried out as

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described previously [16].

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2.9. Detection of mitochondrial membrane potential

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Changes in mitochondrial membrane potential were measured using a lipophilic cationic fluorescent probe JC-1 (Life Technologies, Eugene, Oregon, USA) as described previously

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[17]. MCF-7 cells (3  105/well) were treated with compound 7 (0.05 µM and 0.2 µM) or Dox (0.9 µM) for 48 h. Cells were collected and incubated with 10 µM JC-1 in darkness at 37 °C for 30 min. Subsequently, JC-1 fluorescence was measured by EPICS-XL flow

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cytometry (Beckman Coulter, USA). Data was analyzed quantitatively with the WINMDI 2.8 software (The Scripps Institute, USA).

3. Results and discussion. Compound 1 was obtained as needle-like crystals. The quasi-molecular ion at m/z 433.1645 [M+Na]+ (calcd. for C20H26O9Na: 433.1649) in the HR-ESIMS suggested the molecular formula C20H26O9. The maximum UV absorbance peaks were observed at 229 nm in the UV spectrum suggested the existence of conjugated double bonds. The IR spectrum displayed characteristic absorptions for hydroxyl (3411 cm-1), -lactone and ester (1711 cm-1) functionalities. The 1H NMR spectrum showed signals ascribable to one tertiary methyl (δH 1.10) and characteristic signals of conjugated double bonds (δH 6.20, 5.49 and 4.98) (Table 1). 7

ACCEPTED MANUSCRIPT Analysis of the 13C NMR and DEPT spectra revealed that 1 possessed a carbonyl group (δC 176.5), nine oxygenated carbons including two quaternary (δC 84.7, 83.6), five tertiary (δC 80.5, 78.0, 77.3, 75.6, 70.7), one secondary (δC 70.9) and one primary carbons (δC 29.3), and

are reminiscent of the C20-type quassinoids [18].

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four conjugated olefinic carbons (δC 145.3, 131.5, 131.1, 112.6). These spectroscopic data

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The full assignments and connectivity in 1 were determined by 1H1H COSY, HSQC and HMBC spectroscopic analysis. The 1H1H COSY spectrum showed three spin systems as shown in Fig. 2. The HMQC spectrum revealed that a hydroxylated proton at H 4.33 (H-1)

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is attached to the carbon at  77.3 (C-1), and the HMBC spectrum showed that H-1 was correlated to C-2, C-3 and C-10 (Fig. 2), suggesting that the hydroxyl group was located at

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C-1. The exo-methylene protons showed HSQC correlation to the carbon resonating at δC 112.6 (C-22, t). In the HMBC spectrum, H2-22 showed correlations to C-3 and C-5, which

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indicated the location of the exocyclic double bond at C-4. Similarly, the HMQC spectrum

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revealed that two hydroxylated protons at H 4.16 (H-21) and 3.91(H-21) are attached to the carbon at  64.7 (C-21), and the HMBC spectrum showed that H2-21 was correlated to

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C-12, C-13 and C-14, suggesting that the hydroxymethyl group was attached at C-13. In addition, the HMBC spectrum revealed that the characteristic oxygenated proton at H 5.08 (H-7) was correlated to C-8, C-14 and C-16, suggesting that a tetrahydropyran unit could be

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formed by ring closure involving an oxygen atom bridged to C-7 and C-16. The relative configuration of 1 was established from the ROESY spectrum. The key ROESY correlations of 1 were shown in Figure 3. The three-dimensional structure is constructed by Chem3D Pro 9.0. The correlations of H3-19  H-6, H-7  H-20, H-6 H-7 and H-12  H-20indicated that H-6H-7, H-12 and H3-19 were all -oriented. The correlations H-9  H-1, H-5, H-11 and H-15 indicated that H-1, H-5, H-9, H-11, H-15 were all-oriented (Fig. 3). Accordingly, relative configuration of compound 1 was identified as show in Fig. 1. The structure and stereochemistry of 1 were further confirmed by X-ray crystallographic analysis (Fig. 4). The small Flack parameter 0.1 (1) together with the known conserved configuration at C-10 indicated that the geometry shown in Fig. 4 represents the absolute configuration. 8

ACCEPTED MANUSCRIPT Normally quassinoids have a methyl group at C-4 and ester groups at both C-15 and C-21. Compound 1 represents a rare example of quassinoid with an exo-methylene group at C-4 but no ester groups at C-15 and C-21. The only similar compound is bruceene (2) [18].

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Comparison of the NMR data of 1 with those of 2 showed that C-21 (δC 18.7 for 2) was significantly shifted to downfield (δC 64.7 for 1), indicating that the methyl group was

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replaced by a hydroxymethyl group. Accordingly, 1 was proposed to be a new quassinoid and has been accorded the trivial name bruceene A. Compound 2 was reported three decades ago; however, only partial NMR data were reported. Thus, the NMR data of 2 were assigned

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by extensive 1D and 2D NMR analysis and compared with those of 1 in Table 1. It was noteworthy that H 6.19 (H-3) showed HMBC correlations with C 41.1 (C-5) and C 77.3

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(C-1), suggesting that the NMR data of these positions in the literature (assigned to C-9 and C-12, respectively) should be updated.

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The structures of other sixteen known compounds were identified by comparing their

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spectroscopic data (UV, MS, 1H and 13C NMR) with those of reported values and found to be bruceine D (3) [19], yadanzioside E (4) [20], brusatol (5) [21], bruceoside B (6) [22],

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bruceantinol (7) [23], yadanzioside K (8) [23], P (9) [24], bruceine A (10) [20], yadanzioside B (11) [20], bruceantarin (12) [25], bruceoside A (13) [26], yadanzioside C (14) [20], G (15) [20], F (16) [27], A (17) [20], and M (18) [23].

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Quassinoids are a group of bitter compounds solely found in the family Simaroubaceae. Quassinoids in genus Brucea often has a methyl carboxylate moiety at C-13; in contrast, similar compounds in Ailanthus genus bear a methyl or exomethylene group at this position [28]. In the current study, in addition to the common quassinoids 4-18, compounds 1-3 with a methyl or hydroxymethylene group at C-13 were also isolated and identified. Isolation of similar substituted quassinoids as genus Ailanthus from genus Brucea, indicated the close chemotaxonomic relationship between these two genera, which further support the phylogenetic study by combined analysis of plastid genes rbcL, atpB and matK of plants in Simaroubaceae family [29]. All isolated compounds 1-18 were tested for their cytotoxic activities against the breast cancer cell lines MCF-7 and MDA-MB-231. As shown in Table 2, compounds 5, 7, 10 and 12, which containing a diosphenol (3-hydroxy-3-en-2-one) moiety, exhibited potent 9

ACCEPTED MANUSCRIPT antineoplastic activity in both MCF-7 and MDA-MB-231 cells with IC50 values in the ranges of 0.063-0.182 μM and 0.081-0.238 μM, respectively. Exhilaratingly, all of these compounds showed more potent than that of the positive control doxorubicin. Regularly, compounds 5

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with a diosphenol (3-hydroxy-3-en-2-one) moiety strikingly inhibited the growth of MCF-7 and MDA-MB-321 cells, whereas, its antitumor activity decreased markedly with

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glycosylating at 3-hydroxy (compound 6). Similarly, the glycosylation at 3-hydroxy (compounds 8 and 11) profoundly impacted the activities of compounds 7 and 10. These data suggested that (3-hydroxy-3-en-2-one) moiety was necessary for maintaining cytotoxic

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activity. In addition, compounds 1-4 and 13-18, which did not contain diosphenol (3-hydroxy-3-en-2-one) moiety, exhibited weak antitumor activity on MCF-7 and

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MDA-MB-231 cells, further highlighting the importance of the moiety to their activities and consistence with the previous report [28]. Of all the tested compounds, compound 7 showed

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the most potent cytotoxic activities against MCF-7 cells with an IC50 value of 0.063 ± 0.016

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μM. It is noteworthy that MCF-7 (estrogen receptor positive) was more sensitive to these active quassinoids than MDA-MB-231 (estrogen receptor negative); however the

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relationship between their anti-breast cancer activities and estrogen receptor remains an open question, and further investigations are warranted. To elucidate the mechanism of action of compound 7 for its cytotoxicity toward MCF-7

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cells, induction of apoptosis was detected by AnnexinV-FITC/PI staining assay and populations of apoptotic cells were quantified. As shown in Fig. 5A and 5B, The percentage of apoptotic MCF-7 cells including early and late apoptotic cells was increased from 33.59% to 67.43% in a does-dependent manner after treatment with compound 7 for 48 h, indicating that compound 7 could induce apoptosis. It was further supported by the fact that compound 7 trigered the cleavage of PARP (Fig. 5C), a hallmarker of apoptosis [30]. Mitochondrial apoptosis pathway, also known as intrinsic apoptotic pathway, is accompanied by the decrease of mitochondrial membrane potential, the translocation of cytochrome c from the mitochondria to the cytosol and the activation of downstream caspases [16]. It was observed that compound 7 treated cells showed a significant decrease in the mitochondrial membrane potential (Fig. 6A), and the activation of Caspase-9 as indicated by the decrease of pro-Caspase-9 and increase of cleaved Caspase-9 (Fig. 6B). Taken together, our data 10

ACCEPTED MANUSCRIPT suggested that compound 7 induced mitochondrial apoptosis in MCF-7 cells.

Conflict of interest

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The authors have declared that no conflict of interest existed.

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Acknowledgements

This work was supported by the Guangdong High Level Talent Scheme (R.W. Jiang), Natural Science Foundation of Guangdong Province (S2013050014183) and Program for

Appendix A. Supplementary data H,

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C, COSY, HSQC, HMBC and NOESY spectra of compound 1 were available

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1

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New Century Excellent Talents in University (D. M. Zhang).

online. Crystal data of compound 1 was deposited with the Cambridge Crystallographic Data

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Centre (CCDC 964467). References

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structural elucidation of bruceoside-A and -B, novel antileukemic quassinoid glycosides, and brucein-D and -E from Brucea javanica. J Org Chem 1979;44:2180-5.

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[23] Sakaki T, Yoshimura S, Tsuyuki T, Takahashi T, Honda T, Nakanishi T. Structures of yadanziosides K, M, N, and O, new quassinoid glycosides from Brucea javanica (L.) Merr. B Chem Soc Jpn 1986;59:3541-6. [24] Okano M, Fukamiya N, Toyota T, Tagahara K, Lee KH. Antitumor agents, 104. Isolation of yadanziosides M and P from Brucea antidysenterica and identification of bruceantinoside B as a mixture of yadanzioside P and bruceantinoside C. J Nat Prod 1989;52:398-401. [25] Darwish FA, Evans FJ, Phillipson JD. Cytotoxic bruceolides from Brucea javanica. J pharm pharmacol 1979;31:10. [26] Ohnishi S, Fukamiya N, Okano M, Tagahara K, Lee KH. Bruceosides D, E, and F, three new cytotoxic quassinoid glucosides from Brucea javanica. J Nat Prod 1995;58:1032-8.

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ACCEPTED MANUSCRIPT [27] Yoshimura S, Sakaki T, Ishibashi M, Tsuyuki T, Takahashi T, Honda T. Constituents of seeds of Brucea javanica. Structures of new bitter principles, yadanziolides A, B, C, yadanziosides F, I, J, and

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L. B Chem Soc Jpn 1985;58:2673-9.

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[28] Wang Y, Wang WJ, Su C, Zhang DM, Xu LP, He RR, et al. Cytotoxic quassinoids from Ailanthus

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altissima. Bioorg. Med Chem Lett 2013;23:654-7.

[29] Clayton JW. Evolutionary history of Simaroubaceae (Sapindales): Systematics, biogeography and

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diversification. PhD thesis of University of Florida 2008;132:137.

[30] Zhang DM, Liu JS, Tang MK, Yiu A, Cao HH, Jiang L, et al. Bufotalin from Venenum Bufonis

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J Pharmacol 2012;692:19-28.

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inhibits growth of multidrug resistant HepG2 cells through G2/M cell cycle arrest and apoptosis. Eur

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Legends for Tables and Figures

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Table 1 1H and 13C NMR spectral data of compounds 1-2.

Fig. 1. Structural formulae of compounds 1-18

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Table 2 Cytotoxic activities of compounds 1-18 in two human breast cancer cell lines.

Fig. 2. Key H1-H1 COSY and HMBC correlations of compound 1

Fig. 4. X-ray structure of compound 1

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Fig. 3. Key NOESY correlations of compound 1

Fig. 5. Apoptosis induction by compound 7 in MCF-7 cells

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Fig. 6. Compound 7 activated mitochondrial apoptotic pathway

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ACCEPTED MANUSCRIPT Table 1 H- and 13C-NMR spectral data of compounds 1-2.

1 position

a

2

a

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1

δC

δH

1

4.33, s

77.3

4.32, s

2

5.49(d, 10.3)

131.5

5.49(d, 9.9)

3

6.20(d, 10.0)

131.1

6.19(dd,1.8,10.1)

131.1

4

—

145.3

—

145.3

5

2.42(d, 12.0)

41.1

2.40(d, 11.8)

41.1

6α

2.19, m

29.3

2.18(dt, 3.2,3.2,15.0)

29.3

6

1.73, m

7

5.08, s

80.5

8

—

9

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δH ( J in Hz )

δC 77.3 131.5

1.72(ddd, 12.8,12.3,15.1) 81.0

51.3

—

50.9

2.10(d, 4.7)

46.6

2.08(d, 4.0)

46.8

10

—

44.7

—

44.7

11

4.36(d, 5.6)

75.6

4.37(d, 5.4)

75.6

12

3.91, m

78.0

3.77, s

81.7

13

—

84.7

—

85.1

14

—

83.6

—

82.2

15

5.17, s

70.7

5.15, s

70.7

16

—

176.5

—

176.6

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1.10, s

11.5

1.10, s

11.6

20α

3.91, m

70.9

3.81(dd, 1.7,7.3)

70.1

20

4.47(d, 7.1)

21 22α 22

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21α

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5.08(t, 2.8,2.8)

4.16(d, 12.0)

4.42(d, 7.2) 64.7

1.41,s

18.7

112.6

4.97, s

112.6

3.91, m 4.98, s 4.90, s

4.89, s

H and 13C NMR data were measured at 300MHz and 75M respectively in CD3OD. Values are 

a1

ppm. Coupling constants in Hz in parentheses.

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ACCEPTED MANUSCRIPT Table 2 Cytotoxic activities of compounds 1-18 in two human breast Cancer Cell Lines.

T

IC50 ( x ± SD) μMa

Compounds

MDA-MB-231

2

0.880±0.045 >50 >50

0.261±0.020 >50 >50

3

18.999±9.612

4

>50

5

0.083±0.038

6

>50

7

0.063±0.016

8

>50

9

5.669±0.172

10

0.182±0.048

11

>50

12

0.144±0.039

0.238±0.021

13

>50

>50

14

>50

>50

>50

>50

>50

>50

>50

>50

>50

>50

17 18

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0.081±0.017 >50

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0.088±0.012 >50 4.429±0.140 0.228±0.020 >50

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24.064±13.487

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1

D

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MCF-7

b

Cytotoxic activities of compounds 1-18 were measured by MTT assay. All data are presented as means ± standard deviation of at least three independent experiments. a

IC50: Concentration of the tested compound inhibits 50% of cell growth.

b

DOX: Doxorubicin was used as positive control.

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Fig. 1. Structural formulae of compounds 1-18.

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Fig. 2. Key 1H-1H COSY and HMBC correlations of compound 1.

Fig. 3. Key NOESY correlations of compound 1.

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Fig. 4. X-ray crystal structure of 1. The dashed lines represent intramolecular hydrogen bonds.

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B

C

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**

60 40

** **

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Apoptotic ratio (%)

80

0

0

0.05

0.2

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A

DOX

D

HU15 concentration (μM)

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Fig. 5. Apoptosis induction by compound 7 in MCF-7 cells. (A) Flow cytometry analysis of Annexin V-FITC and PI stained MCF-7 cells treated with compound 7

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(0.05 µM and 0.2 µM) or Dox (0.9 µM) for 48 h. (B) The quantitative analysis of the ratio of apoptotic MCF-7 cells. **P < 0.01, one-way ANOVA, post hoc comparisons, Tukey’s test. Columns, mean; error bars, S.D. (C) Western blot analysis of cleaved

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PARP in MCF-7 cells treated with compound 7 (0.05 µM and 0.2 µM) or Dox (0.9 µM ) for 48 h. β-actin served as a loading control.

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B

D

Fig. 6. Compound 7 activated mitochondrial apoptotic pathway. (A) Mitochondrial

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membrane potential of MCF-7 cells treated with compound 7 (0.05 µM and 0.2 µM) or Dox (0.9 µM) for 48 h was analyzed by JC-1 staining assay. Distributions of red

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(JC-1 aggregates) versus green (JC-1 monomers) fluorescence were shown. (B) Western blot analysis of pro-Caspase-9 and cleaved Caspase-9 in MCF-7 cells treated with compound 7 (0.05 µM and 0.2 µM) or Dox (0.9 µM) for 48 h. β-actin served as a

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loading control.

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ACCEPTED MANUSCRIPT Graphical abstract

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Isolation, chemotaxonomic significance and cytotoxic effects of quassinoids from Brucea javanica Qing-Mei Ye, Liang-Liang Bai, Shu-Zhi Hu, Hai-Yan Tian, Li-Jun Ruan,

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Ya-Fang Tan, Li-Ping Hu, Wen-Cai Ye, Dong-Mei Zhang, Ren-Wang Jiang

A new quassinoid, bruceene A (1) along with seventeen known quassinoids (2-17)

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was isolated from the fruits of Brucea javanica.

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