Chinese Journal of

Natural Chinese Journal of Natural Medicines 2015, 13(7): 05500553 Medicines

doi: 10.3724/SP.J.1009.2015.00550

A new biflavone glucoside from the roots of Stellera chamaejasme

CHEN Wei, LUO Xiao-Hua, WANG Zhuo, ZHANG Ying-Ying, LIU Li-Ping, WANG Hong-Bing*

School of Life Sciences and Technology, Tongji University, Shanghai 200092, China

Available online 20 July 2015

[ABSTRACT] The present study investigated the chemical constituents of the roots of Stellera chamaejasme (Thymelaeaceae). One new biflavone glucoside (1), along with other thirteen known compounds (2–14), was isolated by repeated column chromatographic methods and their structures were elucidated on the basis of spectral analyses. The cytotoxic activities of selected compounds were evaluated against four human cancer cell lines (A549, BEL-7402, HCT-116, and MDA-MB-231) by the SRB assay method. Compound −1 9 showed remarkable cytotoxicity against BEL-7402 with IC50 value being 0.65 μg·mL ; compounds 7, 8, and 12 exhibited significant −1 cytotoxic activity against A549 with IC50 values being 2.38, 1.57, and 2.35 μg·mL , respectively.

[KEY WORDS] Stellera chamaejasme; Thymelaeaceae; Biflavone glucoside; Cytotoxicity [CLC Number] R284 [Document code] A [Article ID] 2095-6975(2015)07-0550-04

Introduction Results and Discussion

The roots of Stellera chamaejasme L. (Thymelaeaceae) Compound 1 was isolated as a yellow amorphous powder. have been used as a traditional Chinese medicine (‘Lang The molecular formula of Compound 1 was deduced to be Du’ in Chinese) for treating scabies, tinea, stubborn skin C36H32O15 according to its HR-ESI-MS at m/z 705.181 1 [M + + 1 13 ulcers, chronic tracheitis, and tuberculosis [1]. Previous H] (Calcd. 705.182 0). The H and C NMR spectra of phytochemical investigations on this plant have led to the compound 1 (Table 1) exhibited unique signals due to a sugar, and two sets of closely related signals, which were isolation of diterpenoids [2-6], biflavonoids [7-12], and characteristics of a biflavanone [7-12]. The sugar moiety was lignans [13]. In the present study, a new biflavone identified as glucose by analysis of the 1H and 13C NMR glucoside named 7-O-β-D-glucopyranosyl-isochamae- spectroscopic data, and the remaining 1H and 13C NMR jasmin (1), along with thirteen known compounds (2–14), signals of compound 1, two sets of closely related signals, was isolated; five of the compounds (5–8, and 11) were very similar to that of isochamaejasmin, a symmetrical were obtained from Thymelaeaceae for the first time, biflavanone, previously isolated from the title plant, and and one compound (12) was isolated from the genus possessing a C-3/C-3'' linkage [10]. The stereochemical Stellera for the first time. The present paper reports assignments at the C-2/C-3 and C-2''/C-3'' positions were the isolation, characterization, and cytotoxic activity of established on the basis of the J-values of the corresponding these compounds. protons (J = 12.0 Hz), and by comparison of the carbon chemical shifts of C-2/C-3 (δ 82.7, 49.0) and C-2''/C-3'' (δ 82.4, 49.0) with those of isochamaejasmin (δ 82.4, 49.0 for C-2, 2'' and C-3/C-3'') and chamaejasmin (δ 84.5, 51.2 for C-2, [Received on] 06-Oct.-2014 2'' and C-3/C-3''), respectively [10]. In the HMBC spectra (Fig. [Research Funding] This work was supported by the National 2), the correlations of H-1'''' of glucose (δ 4.95) with C-7 (δ Natural Science Foundation of China (Nos. 81001369 and 31170327), 167.0) were observed, suggesting glucosylation at C-7. The and the Fundamental Research Funds for the Central Universities (No. β-glucosyl linkage was deduced from the coupling constant of 2000219078) of China. the anomeric proton at δ 4.95 (d, J = 7.3 Hz). On the basis of [*Corresponding author] Tel: 86-21-65983693, 1 1 E-mail: [email protected] above evidences and analyses of HMBC, HMQC, and H- H These authors have no conflict of interest to declare. COSY spectra, compound 1 was determined to be 7-O-

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1 13 1 13 Table 1 H and C NMR ( H NMR 400 MHz; C NMR 100 MHz, in methanol-d4) data for compound 1

No. δC δH No. δC δH 2 82.7 4.83 (d, J = 12.0 Hz) 5'' 165.5 3 49.0 3.30 (d, J = 12.0 Hz) 6'' 97.3 5.89 (d, J = 2.1 Hz) 4 197.7 7'' 168.4 5 164.9 8'' 96.1 5.75 (d, J = 2.1 Hz) 6 98.3 6.18 (d, J = 2.2 Hz) 9'' 164.2 7 167.0 10'' 102.8 8 96.8 6.05 (d, J = 2.2 Hz) 1''' 128.7 9 164.4 2''' 130.8 7.03 (d, J = 8.6 Hz) 10 104.4 3''' 116.6 6.79 (d, J = 8.6 Hz) 1' 128.9 4''' 159.9 2' 130.9 7.03 (d, J = 8.6 Hz) 5''' 116.6 6.79 (d, J = 8.6 Hz) 3' 116.6 6.79 (d, J = 8.6 Hz) 6''' 130.8 7.03 (d, J = 8.6 Hz) 4' 159.8 1'''' 101.1 4.95 (d, J = 7.3 Hz) 5' 116.6 6.79 (d, J = 8.6 Hz) 2'''' 74.6 3.42 (m) 6' 130.9 7.03 (d, J = 8.6 Hz) 3'''' 77.9 3.41 (m) 2'' 82.4 4.83 (d, J = 12.0 Hz) 4'''' 71.1 3.37 (m) 3'' 49.0 3.30 (d, J = 12.0 Hz) 5'''' 78.2 3.42 (m) 3.85 (dd, J = 12.0, 1.8 Hz), 4'' 196.7 6'''' 62.3 3.67 (dd, J = 12.1, 5.3 Hz)

lines using the SRB method (Table 2). Interestingly, compound 9 displayed potent cytotoxicity against BEL-7402 human hepatocellular carcinoma cells, but no cytotoxicity against the other three cancer cell lines. Moreover, compounds 7, 8, and 12 showed significant cytotoxic activity against A549 human lung cancer cells. The other compounds tested, 2−6, were inactive against any of the cell lines used in the present study.

Experimental Fig. 1 Structures of compounds 1 and 2 General Optical rotations were measured on a Perkin Elmer 341 polarimeter (Perkin Elmer, Boston, MA, USA). UV spectra were obtained on a Shimadzu UV-2500 spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were recorded on a Nicolet Magna IR spectrometer (Nicolet, Waltham, MA, USA). NMR spectra were recorded on a Bruker AM-400 spectrometer (Bruker, Billerica, MA, USA) with Fig. 2 Key HMBC correlations of compound 1 tetramethylsilane (TMS) as internal standard. LRESIMS were measured using a Finnigan LCQ-DECA instrument -D-glucopy- ranosylisoch-amaejasmin. (TheromFinnigan, San Jose, CA USA), and HRESIMS data Additionally, thirteen known compounds were identified were obtained on Micromass LCT Premier Spectrometers [13] [13] as isochamaejasmin (2) , chamaejasmin (3) , (Waters, Milford, MA, USA). Column chromatographic [14] [15] chamaechromone (4) , tangeretin (5) , (+)-afzelechin (6) separations were carried out on silica gel H-60 (Qingdao [16] [17] [18] , dextrobursehernin (7) , (–)-haplomyrfolin (8) , Marine Chemical Group Co., Qingdao, China), Sephadex (–)-pinoresinol (9) [19], (+)-3-hydroxy-1, 5-diphenyl-1- LH-20 (Pharmacia Biotech AB, Uppsala, Sweden), and pentanone (10) [20], 4-ethoxy-3, 5-dimethoxy-phenol (11) [21], LiChroprep Rp-18 (1.5 cm × 47 cm, 40–63 μm, Merck). daphnenone (12) [22], 7-hydroxycoumarin (13) [23], and HSGF254 silica gel TLC plates (Yantai Chemical Industrial β-sitosterol (14) [24] by comparisons with literature data. Institute, Yantai, China) were used for analytical TLC. All The isolated compounds 2−9 and 12 were investigated solvents used were of chemical grade (Shanghai Chemical for their cytotoxic activity against four human cancer cell Co., Ltd., Shanghai, China).

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Table 2 In vitro cytotoxicity data of Compounds 2−9, and 12 on four human cancer cell lines

–1 IC50 /(μg·mL ) Compounds A549 BEL-7402 HCT-116 MDA-MB-231 2 > 100 > 100 > 100 > 100 3 > 100 > 100 > 100 > 100 4 > 100 > 100 > 100 61.63 5 > 100 > 100 > 100 54.28 6 > 100 > 100 > 100 > 100 7 2.38 > 100 > 100 > 100 8 1.57 60.91 > 100 > 100 9 > 100 0.65 > 100 > 100 12 2.35 > 100 > 100 > 100 SN-38 1.96×10–4 0.26 3.60×10-3 0.24 SN-38: 7-Ethyl-10-hydroxycamptothecin

Plant materials (18 mg) were obtained through silica gel column chromato- Stellera chamaejasme roots were collected in Haibei, graphy of Fr. 4.3 eluted with petroleum ether-acetone (4 : 3, Qinhai Province of China in October, 2009, and were V/V). Similarly, compound 4 (30 mg) was obtained by silica identified and authenticated by Dr. GUAN ZB from the gel column chromatography of Fr. 4.4 eluted with

Yunnan Branch Institute of Medicinal Plants, Chinese CHCl3-MeOH (10 : 1, V/V), as well as Sephadex LH-20 Academy of Medical Sciences. Voucher specimens were kept column eluted with MeOH. compounds 1 (5 mg) and 6 (10 in the “Herbarium” of the Institute. mg) were afforded through repeated silica gel column using

Extraction and isolation a gradient solvent system CHCl3-MeOH (10 : 1, 5 : 1, 2 : 1, The air-dried and powdered roots of S. chamaejasme V/V) and Sephadex LH-20 eluted with MeOH. Fr. 5 was (5.0 kg) were extracted exhaustively with 95% EtOH at separated through RP-18 column chromatography eluted room temperature. The extract was concentrated in vacuo to with H2O containing increasing amounts of MeOH (1 : 2, 1 : yield an EtOH extract, which was then suspended in 4, 1 : 8, V/V), and Sephadex LH-20 (MeOH), to give distilled water and partitioned successively with petroleum Compound 13 (25 mg). ether, CHCl3, EtOAc, and n-BuOH. The obtained CHCl3 7-O--D-Glucopyranosyl-isochamaejasmin (1). Yellow extract (300 g) was applied to a silica gel column, eluted 20 ° amorphous powder; αD 12 (c = 0.10, MeOH); IR (KBr) with CHCl3 containing increasing amounts of MeOH to υ : 3 320, 1 638, 1 518, 1 372, 1 304 cm–1; UV λ obtain five fractions (Fr. 1–5). Fr. 2 was subjected to max max (MeOH): 294 nm. 1H and 13C NMR data: see Table 1; repeated silica gel column chromatography using a gradient HR-ESI-MS m/z 705.181 1 [M + H]+ (Calcd. for C H O : solvent system of petroleum ether-acetone (15 : 1, 10 : 1, 5 : 36 33 15 705.182 0). 1, 3 : 1, V/V), and a Sephadex LH-20 column, eluted with Cytotoxicity assay CHCl3-MeOH (1:1, V/V), to yield compounds 5 (17 mg), 10 Cytotoxicity of the compounds was evaluated by the (9 mg), and 11 (6 mg). Fr. 3 was applied to a silica gel [25] column eluting with petroleum ether-acetone (10 : 1, 5 : 1, SRB method . Briefly, the cells were seeded in 96-well 2 : 1, 1 : 1, V/V) to afford three subfractions (Fr. 3.1–3.3). Fr. plates, treated with increasing concentrations of test 3.1 was chromatographed by repeated RP-18 column eluting compounds or SN-38 (positive control), and incubated for 72 h. After the drug-containing medium was removed the with MeOH-H2O (3 : 1, 6 : 1, 10 : 1, V/V), and Sephadex cells were treated with 10% precooled TCA (100 µL/well) LH-20 column, eluted with CHCl3-MeOH (1:1, V/V), to obtain Compounds 14 (150 mg) and 9 (260 mg). Fr. 3.2 was and fixed for 1 h at 4 ºC. The plate was washed with −1 separated over repeated silica gel column eluting with distilled water and dried. SRB (4 mg·mL in 1% acetic petroleum ether-EtOAc (5 : 1, 3 : 1, 1 : 1, V/V), and acid, 100 µL) was added to each well and the plate was

Sephadex LH-20 column, eluted with CHCl3-MeOH (1 : 1, incubated for 15 min. The plate was then washed with V/V), to yield compounds 7 (130 mg), 8 (150 mg), and 12 1% acetic acid and dried. SRB in the cells was dissolved −1 (50 mg). Fr. 4 was fractionated by silica gel column eluted in 10 mmol·L Tris-HCl (150 µL) and the absorbance was with CHCl3-MeOH (15 : 1, 10 : 1, 5 : 1, 3 : 1, V/V) to give then measured at 515 nm. The IC50 values were obtained six subfractions (Fr. 4.1–4.6). Compounds 2 (20 mg) and 3 using the logit method.

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Cite this article as: CHEN Wei, LUO Xiao-Hua, WANG Zhuo, ZHANG Ying-Ying, LIU Li-Ping, WANG Hong-Bing. A new biflavone glucoside from the roots of Stellera chamaejasme [J]. Chinese Journal of Natural Medicines 2015, 13(7): 550-553.

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