Fitoterapia 96 (2014) 76–80

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New cytotoxic diarylheptanoids from the rhizomes of Alpinia officinarum Hance Dan Liu a, Yan-Wen Liu a, Fu-Qin Guan b, Jing-Yu Liang c,⁎ a b c

Hubei Key Laboratory of Resource Science and Chemistry in Chinese Medicine, Hubei university of Chinese Medicine, Wuhan 430061, China Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China

a r t i c l e

i n f o

Article history: Received 27 September 2013 Accepted in revised form 11 April 2014 Available online 18 April 2014 Keywords: Alpinia officinarum Hance Diarylheptanoids Cytotoxicity

a b s t r a c t Two new dimeric diarylheptanoids, named Alpinin C (1) and D (2), a new natural product of diarylheptanoid (3) along with three known diarylheptanoids (4–6) were isolated from the rhizomes of Alpinia officinarum Hance. Their structures were elucidated based on extensive spectroscopic analyses (1D and 2D NMR, HRTOFMS, IR). The isolated compounds were evaluated for their cytotoxicity against human tumor cell lines HepG2, MCF-7, T98G and B16-F10. Compound 1 showed selective cytotoxicity against cell lines of MCF-7 and T98G, while compound 6 showed significant cytotoxicity to the all tested tumor cell lines with IC50 in the range from 8.46 to 22.68 μmol/L. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Alpiniae officinarum Rhizoma, named galangal, are the dried rhizomes of Alpinia officinarum Hance (Zingiberaceae). It has been used as a traditional Chinese medicine for relieving stomach ache, treating colds, invigorating the circulatory system and reducing swelling [1]. Pharmacological research found that the diarylheptanoids, which as the characteristic constituents of A. officinarum, possess cytotoxic [2,3], antiinflammatory [4,5], antioxidant [6], antiplatelet [7], antiviral [8] and antiemetic activities [9]. In the earlier studies on the rhizomes of A. officinarum, we have reported a number of diarylheptanoids [10,11]. A further effort to search for more bioactive chemical constituents of A. officinarum, two new dimeric diarylheptanoids, named Alpinin C (1) and D (2), a new natural product of diarylheptanoid (3) along with three known diarylheptanoids (4–6) were obtained. In the present study, we report the isolation, structural elucidation and cytotoxicity of ⁎ Corresponding author at: Department of Natural Medicinal Chemistry, China Pharmaceutical University, #24 Tongjiaxiang, Nanjing 210009, P. R. China. Tel.: + 86 13605154996. E-mail address: [email protected] (J.-Y. Liang).

http://dx.doi.org/10.1016/j.fitote.2014.04.008 0367-326X/© 2014 Elsevier B.V. All rights reserved.

compounds 1–6 isolated from the rhizomes of A. officinarum Hance. 2. Experimental 2.1. General IR spectra were recorded on a Bruker Tensor 27 spectrometer with KBr-disks (Bruker, Karlsruhe, Germany). NMR spectra were recorded on Bruker ACF-500 NMR instrument (1H: 500 MHz, 13C:125 MHz) with TMS as internal standard. Mass spectra were obtained on a Micro Q-TOF-MS (HR-TOF-MS). All solvents used were of analytical grade (Tianjin Chemical Plant). Column chromatography was performed on silica gel (100– 200 mesh, 200–300 mesh, Qingdao Marine Chemical Co., Ltd.) and Sephadex LH-20 (40–75 μm, Pharmacia). Thin-layer chromatography was performed on silica gel GF254 (Qingdao Marine Chemical Co., Ltd.). 2.2. Plant material The rhizomes of A. officinarum Hance were collected in Bobai Prefecture, Guangxi Zhuang Autonomous Region in December

D. Liu et al. / Fitoterapia 96 (2014) 76–80

2008 and identified by Xue-Hua Song, the curator of China Pharmaceutical University, where a voucher specimen is deposited. 2.3. Extraction and isolation Dried powdered rhizomes of A. officinarum (16 kg) were extracted three times with 85% ethanol for 2 h each time. The combined extracts were concentrated under reduced pressure to obtain a brown residue (1214 g), which was suspended in water, then partitioned with petroleum ether, EtOAc and n-BuOH, respectively. The EtOAc extract (741 g) was subjected to CC (silica gel, gradient of petroleum ether– EtOAc 100:1–1:1, v/v) to afford 6 fractions (Fr.1–Fr.6). Fr.2 (41 g) was firstly subjected to silica gel CC (gradient of petroleum ether–acetone 100:1–1:1, v/v) to obtain five fractions (Fr.2a–Fr.2e). Fr.2b was further chromatographed over Sephadex LH-20 (dichloromethane–methanol 1:1, v/v) to yield compounds 3 (4.1 mg) and 6 (3.7 mg). Fr.2c was purified by preparative TLC (developing solvent: cyclohexane–acetone 5:1, v/v) to yield 1 (4.2 mg) and 2 (3.7 mg). Fr.3 (21 g) was firstly subjected to silica gel CC (gradient of petroleum ether–acetone 50:1–1:1, v/v) to obtain five fractions (Fr.3a–Fr. 3e). Fr.3c was further purified by Sephadex LH-20 CC (dichloromethane–methanol 1:1, v/v) to yield 4 (8.4 mg) and 5 (6.9 mg). All the pure compounds were structurally elucidated through 1D, 2D NMR, MS, UV, IR spectra and literature data.

77

617.2899 [M-H]− (calcd. 617.2903 for C40H41O6 −); 1H and 13C NMR (CDCl3) data, see Table 2. 2.3.3. 7-(4-Hydroxyphenyl)-1-phenyl-3-heptanone (3) Colorless oil; ESI-MS: 281 [M-H]−; 1H NMR (CDCl3, 500 MHz) δ: 7.16–7.28 (5H, m, H-2′-6′), 7.01 (2H, d J = 8.4 Hz, H-2″/6″), 6.73 (2H, d J = 8.5 Hz, H-3″/5″), 2.88 (2H, t J = 7.5, 15.3Hz, H-1), 2.71(2H, t J = 7.9, 15.3 Hz, H-2), 2.52 (2H, t J = 7.5, 14.5 Hz, H-4), 2.38 (2H, t J = 6.8, 14.1 Hz, H-5), 1.54–1.60 (4H, m, H-6/7); 13C NMR (CDCl3) data, see Table 2. 2.4. Cytotoxicity assays by MTT method

2.3.2. Alpinin D (2) Pale yellow oil; UV (MeOH) λmax (log ε): 226 (3.23) nm; IR (KBr)νmax: 3443, 1632, 1585, 742 and 701 cm−1; HR-TOF-MS:

Cytotoxicity was determined against HepG2 (human hepatoma), MCF-7 (human breast adenocarcinoma), T98G (human Caucasian glioblastoma), IMR-90, a noncancerous lung (obtained from the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences) and B16-F10 (murine melanoma cells) (kindly provided by Nanjing university) cell lines by the MTT colorimetric method [12]. Briefly, the cell line IMR-90 was grown in modified Eagle's medium (MEM) and the other Cell lines were cultured in DMEM. All medium supplemented with 5% fetal bovine serum (FBS) and 5% calf serum (NBCS), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified incubator under 5% CO2 at 37 °C. The test compounds 1–6 were dissolved in DMSO and stored as stock solution (100 mmol) at 4 °C. The cells were seeded in 96-well plates at a density of 5 × 104 cells per well in 100 μL medium. After being cultivated for 24 h, cells were treated with tested compounds 1–6 at concentration of 0, 2.5, 5, 10, 20, 40 and 80 μM for 72 h. Then 10 μL of MTT (5 mg.mL−1 in phosphate-buffered saline) was added to each well and incubated for 4 h, after this, 100 μL of the solubilisation solution was added into each well, and the plate was allowed to stand overnight in the incubator. Absorbance was recorded on a microplate reader (Tecan, USA) at a wavelength of 570 nm for HepG2, MCF-7, T98G while at 490 nm for B16-F10 cell lines.

Table 1 1 H and 13C NMR spectral data for compounds 1 (500 MHz for 1H and 125 MHz for 13C NMR, in CDCl3, δ in ppm, J in Hz).

Table 2 1 H and 13C NMR spectral data for compounds 2 and 3 (500 MHz for 1H and 125 MHz for 13C NMR, in CDCl3, δ in ppm, J in Hz).

2.3.1. Alpinin C (1) Pale yellow oil; UV (MeOH) λmax (log ε): 208 (3.43) nm; IR (KBr)νmax: 3418, 2922, 1668, 1623, 1510, 1085, 750 and 700 cm−1; HR-TOF-MS: 617.2888 [M-H]− (calcd. 617.2903 1 13 C NMR (CDCl3) data, see Table 1. for C40H41O− 6 ); H and

Unit I

Unit II

2

3

No.

δH

δC

δH

δC

No.

δH

δC

δH

δC

1 2 3 4 5 6 7 1' 2',6' 3',5' 4' 1″ 2″ 3″ 4″ 5″ 6″ 3″-OCH3

2.92 m 2.82 m

30.1 41.8 199.2 130.7 146.1 34.1 34.2 141.2 128.3 128.5 126.1 137.1 112.9 150.4 144.4 119.5 120.6 56.1

2.92 m 2.82 m

30.1 41.8 199.3 130.7 146.0 34.1 34.2 141.2 128.3 128.5 126.1 131.8 107.2 147.9 135.4 144.4 111.5 56.3 5.77 brs

1 2 3 4 5 6 7 1' 2′, 6′ 3′, 5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″ -OCH3 -OH

2.91 (dt 7.2) 2.85 (dt 7.1)

30.1 41.9 199.4 130.7 146.3 34.3 34.2 141.1 128.3 128.5 126.1 132.5 110.5 147.2 140.6 124.3 122.9 56.1

2.88 (t 7.5, 15.3) 2.71 (t 7.9, 15.3)

29.7 44.2 210.2 42.9 34.8 29.8 31.1 141.1 128.3 128.5 126.1 134.4 115.0 115.1 153.6 115.1 115.0

6.10 6.81 2.51 2.73

(d 16.9) (t 14.6,7.2) (q 7.1) (t 14.9,7.4)

7.27 m 7.18 m 7.18 m 6.76 (d 1.8)

6.83 (d 8.1) 6.66 (dd 8.2,1.6) 3.84 s

6.06 6.79 2.42 2.62

(d 16.9) (t 14.6, 7.2) (q 7.1) (t 15.3,7.4)

7.27 m 7.18 m 7.18 m 6.46 (d 1.4)

6.35 (d 1.6) 3.88 s 4″-OH

6.12 6.85 2.52 2.73

(d 15.9) (d 15.9) (t 7.0) (t 7.3)

7.26 m 7.18 m 7.18 6.69 (d 1.9)

6.71 (d 1.9) 3.90 s 5.94 brs

2.52 (t 7.5, 14.5) 1.54–1.60 m 1.54–1.60 m 2.38 ( t 6.8, 14.1) 7.16–7.28 m 7.16–7.28 m 7.16–7.28 m 7.01 (d 8.4) 6.74 (d 8.5) 6.74 (d 8.5) 7.01 (d 8.4) 4.71 brs

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Fig. 1. Structures of compounds 1–6.

5-FU was used as positive control, and DMSO was used as a negative control in all experiments (final concentration of 0.2%). The 50% inhibitory concentration (IC50) values were calculated as characterized previously [13]. 3. Results and discussion The ethyl acetate extract from the rhizomes of A. officinarum was separated by repeated column chromatography and purified by preparative TLC to yield six diarylheptanoids (1–6) (Fig. 1). Compound 1 was obtained as a pale yellow oil, having the molecular formula C40H42O6 by HR-TOF-MS (m/z 617.2888 [M-H]−, calcd. 617.2903). The IR spectrum showed a strong

and broad hydroxyl absorption (3418 cm−1) and an α,βunsaturated keto group (1668 cm−1). The 1H and 13C NMR (Table 1) as well as DEPT data of 1 indicated the presence of two mono-substituted aromatic rings δH: 7.16–7.29 (m, 10H), one 1,3,4-tri-substituted benzene ring δH: 6.83 (d J = 8.1 Hz, 1H), 6.76 (d J = 1.8 Hz, 1H), 6.66 (dd J = 8.2, 1.6 Hz, 1H), one 1,3,4,5-tetra-substituted benzene group δH 6.46 (d J = 1.4 Hz, 1H), 6.35 (d J = 1.6 Hz, 1H), eight methylenes and two α,β-unsaturated keto groups [δC: 199.2, 199.3, δH: 6.10 (d J = 16.9 Hz), 6.81 (t J = 14.6, 7.2 Hz), 6.06 (d J = 16.9 Hz), 6.79 (t J = 14.6, 7.2 Hz)], suggesting 1 to be a diarylheptanoid dimer. The analyses of the DEPT, COSY, HSQC and HMBC spectra (Fig. 2) suggested the diarylheptanoid units to be 7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-4E-hepten-3-

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

D. Liu et al. / Fitoterapia 96 (2014) 76–80

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Fig. 3. The Key HMBC and H-H COSY correlations of 2.

one (unit I) [14] and 7-(4″,5″-dihydroxy-3″-methoxyphenyl)1-phenyl-4E-heptene-3-one (unit II) [15]. In the 13C NMR spectrum, there was an obvious downfield shift of C-II-5″ (δC 144.4) of 1 as compared to the corresponding signal of unit II (δC 124.3) [14], indicated an ether linkage between C-I-4″ and C-II-5″, which was confirmed by the appearance of an IR spectrum peak at 1085 cm−1 that is characteristic of a diphenylether-type diarylheptanoid [16]. Therefore, the structure of 1 was elucidated as shown in Fig. 1, named alpinia C. Compound 2 was obtained as a pale yellow oil, having the molecular formula C40H42O6 by HR-TOF-MS (m/z 617.2899 [M-H]−, calcd. 617.2903). The IR spectrum showed a strong hydroxyl absorption (3418 cm−1). The formula of 2 indicated 20° of unsaturation. The 13C-NMR, however, showed the presence of only 20 C-atoms and the 1H-NMR spectrum exhibited 21 proton signals. This clearly indicated that 2 is a symmetrical dimer. The 1H and 13C NMR (Table 2) as well as the DEPT data of 2 indicated that each half of 2 contained one mono-substituted aromatic rings δH: 7.16–7.28 (m, 5H); one 1,3,4,5-tetra-substituted benzene group δH 6.69 (d J = 1.9 Hz, 1H), 6.71 (d J = 1.9 Hz, 1H) and α,β-unsaturated keto groups δC: 199.4, δH: 6.12 (d J = 16.9 Hz) and 6.815 (t J = 15.9, 7.0 Hz); and one oxygenated Me and five CH2 groups. In the HMBC spectral (Fig. 3), the correlations between H-1 with carbonyl C-atom at δC: 199.4 and C-1′, H-6 with C-4/5 and H-7 with C-1″were observed. Besides, in the H-HCOSY spectral (Fig. 3), the connectivities from H-1 to H-2 and from H-4 to H-7 were displayed. Those NMR data of 2 was similar to 7-(4″hydroxy-3″-methoxyphenyl)-1-phenyl-4E-hepten-3-one [14], but that was approximately double the size of 7-(4″-hydroxy3″-methoxyphenyl)-1-phenyl-4E-hepten-3-one, suggesting that 2 was a dimer of 7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl4E-hepten-3-one through a C-C bond at C-5″[14]. This structure was supported by the presence of HMBC correlations of the benzene-ring protons H-2″with C-4″ (δC: 140.6) and C-6″ (δC:122.9), H-6″with C-2″ (δC: 140.6), C-4″ (δC: 140.6) and C-5″ (δC:110.5). Therefore, the structure of 2 was elucidated as shown in Fig. 1, named alpinia D. Table 3 Cytotoxicity of compounds 1–6 against HepG2, MCF-7, T98G and B16-F10 cancer cell lines. Cell lines (IC50 μg/mL) Compounds

HepG2

MCF-7

T98G

B16-F10

1 2 3 4 5 6 5-FU

90.05 N80 N50 N50 N50 22.68 20.31

38.54 20.747 N50 N50 N50 12.37 17.45

35.43 N80 N80 N80 N80 8.46 30.74

66.45 N80 N50 N80 41.650 4.44 22.82

The known compounds, 7-(4-hydroxyphenyl)-1-phenyl3-heptanone (3) [17], which is a new natural product, 5Sethoxyl-7-(4-hydroxy-3-methoxyphenyl)-1-phenyl-3-hepta none (4) [11], 5R-hydroxy-7-(4-hydroxy-3-methoxyphenyl)1-phenyl-3-heptanone (5) [3] and (4E,6E)-5-hydroxy-1-(4hydroxy-3-methoxyphenyl)-7-phenylhepta-4,6-dien-3-one (6) [18], were determined by comparison of their spectroscopic data with those reported. The isolated compounds 1–6 were evaluated for in vitro cytotoxicity against the HepG2, MCF-7, T98G and B16-F10 tumor cell lines and IMR-90, a noncancerous lung. The results showed that the compounds 1–6 had no effect on cell proliferation for IMR-90 with the dose of 100 μg/mL. The compound 1 has selective cytotoxicity against MCF-7 and T98G, and the compound 6 showed significant cytotoxicity to the all tested tumor cell lines with IC50 values of 8.46, 12.37, 22.68 and 4.44 μmol/L, respectively, as shown in the Table 3. Acknowledgements This work was supported by the Science and Technology Research projects of Education Bureau of Hubei Province, China (no. Q20142005).

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New cytotoxic diarylheptanoids from the rhizomes of Alpinia officinarum Hance.

Two new dimeric diarylheptanoids, named Alpinin C (1) and D (2), a new natural product of diarylheptanoid (3) along with three known diarylheptanoids ...
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