Advanced
Two New Diphenylethylenes from Arundina graminifolia and Their Cytotoxicity
Two New Diphenylethylenes from Arundina graminifolia and Their Cytotoxicity
Bulletin of the Korean Chemical Society. 2013. Nov, 34(11): 3257-3260
Copyright © 2013, Korea Chemical Society
  • Received : June 03, 2013
  • Accepted : August 09, 2013
  • Published : November 20, 2013
Download
PDF
e-PUB
PubReader
PPT
Export by style
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Yin-Ke Li
Collge of Resource and Environment, Yuxi Normal University, Yuxi 653100, P.R. China
Bin Zhou
Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan University of Nationalities, Kunming 650031, P.R. China
Yan-Qing Ye
Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan University of Nationalities, Kunming 650031, P.R. China
Gang Du
Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan University of Nationalities, Kunming 650031, P.R. China
De-Yun Niu
Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan University of Nationalities, Kunming 650031, P.R. China
Chun-Yang Meng
Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan University of Nationalities, Kunming 650031, P.R. China
Xue-Mei Gao
Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan University of Nationalities, Kunming 650031, P.R. China
Qiu-Fen Hu
Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan University of Nationalities, Kunming 650031, P.R. China

Abstract
Two new diphenylethylenes, gramniphenols H and I ( 1 and 2 ), together with six known diphenylethylenes ( 3 - 8 ), were isolated from Arundina graminifolia . The structures of 1 - 8 were elucidated by spectroscopic methods including extensive 1D- and 2D-NMR techniques. Compounds 1 and 2 were evaluated for their cytotoxicity against five human tumor cell lines. Compound 1 showed cytotoxicity against PC3 cells with IC 50 value of 3.5 μM. Compound 2 showed cytotoxicity against NB4 and PC3 cells with IC 50 values of 3.6 and 3.8 μM, respectively.
Keywords
Introduction
Arundina graminifolia (bamboo orchid) is a terrestrial multiperennial orchid. 1 It has been widely used for clearing heat, detoxicating, and dissipating blood stasis by Dai people lived in Xishuangbanna, Yunnan province. 2 Previous phyto-chemical studies of A. graminifolia have shown the presence of stilbenoids, 3 bibenzyls, 4 phenanthrenes, 5 6 and other phenolic compounds. 7 8 In our previous studies, some new phenolic compounds possessing anti-tobacco mosaic virus (anti-TMV) and anti-HIV-1 properties were isolated from A . gramnifolia grown in the Xishuangbanna and Honghe Prefecture. 7 8 Motivated by a search for new bioactive meta-bolites from local plants, our group has investigated the chemical constituents of the whole plant of A. graminifolia growing in the Wuzhishan Prefecture, Hainan province, which led to the isolation and characterization of two new diphenylethylenes ( 1 and 2 ), and six known diphenyl-ethylenes ( 3 - 8 ). This paper deals with the isolation, structural characterization of the new compounds, and their cytotoxi-city against five human tumor cell lines.
Results and Discussion
The whole plant of A. graminifolia was extracted with 70% aqueous acetone. The extract was subjected repeatedly to column chromatography on silica gel, RP-18, and semi-preparative RP-HPLC separation to afford compounds 1-8. Their structures were shown in Figure 1 . The 1 H- and 13 C NMR data of the compounds 1 and 2 were listed in Table 1 . By compared with the literature, the known compounds were identified as pinosylvin ( 3 ), 9 3,5-dihydroxy-stilbene-3- O - β -D-glucoside ( 4 ), 10 rhapontigen ( 5 ), 11 bauhiniastatin D ( 6 ), 12 3-hydroxy-4,3,5-trimethoxy- trans -stilbene ( 7 ). 13 2,3-dihydroxy-3,5-dimethoxystilbene ( 8 ). 14
PPT Slide
Lager Image
The structures of diphenylethylenes from A. graminifolia.
Compound 1 was obtained as a yellow gum. Its HRESIMS in the positive mode revealed a peak at m/z 353.1008 [M+Na] + indicative of the molecular formula of C 18 H 18 O 6 , corresponding to 10 degrees of unsaturation. Its UV spec-trum showed the maximum absorption at 315, 236 and 210 nm, and its IR spectrum also exhibited the presence of hydroxy group (3412 cm −1 ) and aromatic ring (1612, 1586, 1524, 1458 cm −1 ). Its 1 H, 13 C, and DEPT NMR spectra ( Table 1 ) showed signals for 18 carbons and 18 hydrogen atoms, corresponding to the following functional groups: a 1,2,5,6-tetrasubstituted benzene [C-1 to C-6; δ C 145.1, 141.0, 115.5, 106.5, 150.5, and 120.9; δ H 6.89 d ( J = 8.8) and 6.60 d ( J = 8.8)], a 1',2',3',4',6'-pentasubstituted benzene (C-1' to C-6'; δ C 136.4, 154.0, 112.9, 149.4, 104.1, 127.8; δ H 6.52 s), a pair of double bond [CH-7 and CH-8; δ C 126.0 and 130.0; δ C 7.07 d ( J = 11.6) and 6.79 d ( J = 11.6)], a hydroxy-ethyl unit [CH 2 -7' and CH 2 -8'; δ C 35.6, 63.6; 2.59 t (7.2), 3.64 t (7.2)], two methoxy groups ( δ C 55.9, 61.3; δ H 3.80 s, 3.87 s), and two phenolic hydroxy groups ( δ C 9.43 brs, 9.62 brs). Detailed analysis the functional groups suggested that 1 should be an dibenz[ b , f ]oxepin derivatives. 15 The general features of the 1 H and 13 C NMR spectra of 1 resembled to those of bauhiniastatin C 15 except that a vinyl methyl in bauhiniastatin C was replaced by a hydroxyethyl unit in 1 . In HMBC spectrum, the long-range correlations ( Figure 2 ) of H-7' ( δ H 2.59) to C-2' ( δ C 154.0), C-3' ( δ C 112.9) and C-4' ( δ C 149.4), of H-8' ( δ H 3.64) to C-3' ( δ C 112.9), were observed in 1 . This led us to conclude that the hydroxyethyl unit was located on C-3'. The HMBC correlations of two methoxy protons ( δ H 3.80, 3.87) with C-5 ( δ C 150.5) and C-2 ( δ C 154.0) revealed that two methoxy groups should be located at C-5 and C-2. The HMBC correlations between the phenolic hydroxy proton ( δ H 9.43) and C-1 ( δ C 145.1), C-2 ( δ C 141.0), and C-3 ( δ C 115.5), as well as those between the other hydroxy proton ( δ H 9.26) and C-3' ( δ C 112.9), C-4' ( δ C 149.4), and C-5' ( δ C 104.1), led to the assignment of two phenolic hydroxy groups at C-2 and C-4. The above evidence led to oxepin structure 1 for gramniphenol H.
1H and13C NMR data of compounds1and2(δin ppm, data obtained in C5D5N)
PPT Slide
Lager Image
1H and 13C NMR data of compounds 1 and 2 (δ in ppm, data obtained in C5D5N)
PPT Slide
Lager Image
Selected HMBC correlations of 1.
Compounds 2 was also obtained as yellow gum, and should sodiated molecular ion at m/z 353.0997 [M+Na] + in the HRESIMS. This indicated the compounds 1 and 2 have the same molecular formula. The 1 H- and 13 C NMR spectra of 2 were similar to those of 1 . The obvious chemical shift differences resulted from the down-shift of C-4 from δ C 149.4 ppm to δ C 152.2 ppm, and the up-shift of C-2 form δ C 154.0 ppm to δ C 147.8 ppm. These suggested the substituent groups at C-2 and C-4 should be varied. For compound 2 , two methoxy groups located at C-5 and C-4, two phenolic hydroxy group located at C-2 and C-2' were also be con-cluded by the analysis of its HMBC spectrum. Accordingly, the structure of gramniphenol I ( 2 ) was determined as shown. Compounds 1 and 2 are the first naturally occurring dibenz[ b,f ]oxepin derivatives possessing a hydroxyethyl unit.
Since certain of the stilbenoids from Orchidaceae exhibit potential cytotoxicity, 16 - 18 compounds 1-8 were tested for their cytotoxicity against five human tumor cell lines (NB4, A549, SHSY5Y, PC3, and MCF7) using the MTT method as reported previously. 19 Paclitaxel was used as the positive control. The results were shown in Table 2 . Compound 1 showed cytotoxicity against PC3 cells with IC 50 value of 3.5 μM. Compound 2 showed cytotoxicity against NB4 and PC3 cells with IC 50 values of 3.6 and 3.8 μM, respectively. The other compounds also showed modest cytotoxicity with IC 50 below 10 μM for some selected cells line.
The cytotoxicity data for the compounds1-8
PPT Slide
Lager Image
The cytotoxicity data for the compounds 1-8
Experimental Section
General Experimental Procedures. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. A Tenor 27 spectrophotometer was used for scanning IR spectroscopy with KBr pellets. 1D and 2D NMR spectra were recorded on a DRX-500 NMR spectrometer with TMS as internal standard. Unless otherwise specified, chemical shifts ( δ ) are expressed in ppm with reference to the solvent signals. HRESIMS was performed on a VG Autospec-3000 spectrometer. Semipreparative HPLC was performed on a Shimadzu LC-8A preparative liquid chromatograph with Zorbax PrepHT GF (21.2 mm × 25 cm) or Venusil MP C 18 (20 mm × 25 cm) columns. Column chromatography was performed using silica gel (200-300 mesh, Qing-dao Marine Chemical, Inc., Qingdao, People’s Republic of China), Lichroprep RP-18 gel (40-63 δm, Merck, Darmstadt, Germany), and MCI gel (75-150 δm, Mitsubishi Chemical Corporation, Tokyo, Japan). The fractions were monitored by TLC, and spots were visualized by heating silica gel plates sprayed with 5% H 2 SO 4 in EtOH.
Plant Material. The whole plant of A. graminifolia was collected in Wuzhishan Prefecture, Hainan province, People’s Republic of China, in September 2011. The identification of the plant material was verified by Dr. Yuan. N, of Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (YNNU 2011-9-38) has been deposited in our laboratory.
Extraction and Isolation. The air-dried and powdered A. graminifolia (4.8 kg) were extracted four times with 70% aqueous acetone (4 × 6 L) at room temperature and filtered. The filtrate was evaporated under reduced pressure, and the crude extract (315 g) was decolorized by MCI. The 90% methanol part (230 g) was chromatographed on a silica gel column eluting with a CHCl 3 -MeOH gradient system (20:1, 9:1, 8:2, 7:3, 6:4, 5:5), to give six fractions A–F. The further separation of fraction C (8:2, 38.2 g) by silica gel column chromatography, eluted with petroleum ether-acetone (9:1–1:2), yielded mixtures C1–C7. Fraction C4 (6:4, 5.27 g) was subjected to silica gel column chromatography using petro-leum ether-acetone and semi-preparative HPLC (50% MeOH-H 2 O, flow rate 12 mL/min) to give 1 (8.33 mg), 2 (11.5 mg), 6 (13.8 mg), 7 (16.8 mg), and 8 (13.2 mg). Fraction C5 (1:1 3.85 g) was subjected to silica gel column chromatography using petroleum ether-acetone and semi-preparative HPLC (44% MeOH-H 2 O, flow rate 12 mL/min) to give 5 (13.8 mg). The further separation of fraction D (7:3, 20.8 g) by silica gel column chromatography, eluted with chloroform-acetone (8:2–1:2), yielded mixtures D1–C5. Fraction D3 (6:4, 3.18 g) was subjected to semi-preparative HPLC (30% MeOH-H 2 O, flow rate 12 mL/min) to give 4 (54.8 mg).
Cytotoxicity Assay. Colorimetric assays were performed to evaluate each compound’s activity. NB4 (human acute promyelocytic leukemia cells), A549 (Human lung adeno-carcinoma epithelial cells), SHSY5Y (human neuroblastoma cells), PC3 (Human prostate cancer cell), and MCF7 (human breast adenocarcinoma cells) tumor cells were purchased from the American Type Culture Collection (ATCC). All cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere with 5% CO 2 . Cell viability was assessed by conducting colori-metriccolorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO). Briefly, 100 μL of suspended ad-herent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition. In addition, suspended cells were seeded just before drug addition, with an initial density of 1 × 10 5 cells/mL in 100 μL of medium. Each tumor cell line was exposed to each test compound at various concentrations in triplicate for 48 h; paclitaxel (Sigma, purity > 95%) was used as a positive control. After the incubation, MTT (100 μg) was added to each well, and the incubation was continued for 4 h at 37 °C. The cells were lysed with 100 μL of 20% SDS-50% DMF after removal of 100 μL of the medium. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC 50 value of each compound was calculated by Reed and Muench’s method.
Gramniphenol H: Yellow gum; UV (MeOH), λ max (log ε) 315 (3.92), 236 (4.12), 210 (4.32); IR (KBr) cm −1 : 3412, 3028, 2965, 2875, 1612, 1586, 1524, 1458, 1429, 1398, 1185, 1153, 1126, 1078, 855. 1 H- and 13 C-NMR data (CDCl 3 , 500 MHz and 125 MHz, respectively), see Table 1 . ESIMS (positive ion mode), m/z 353 [M+Na] × HRESIMS (positive ion mode), m/z 353.1008 [M+Na] + (calcd. 353.1001 for C 18 H 18 NaO 6 ).
Gramniphenol I: Yellow gum, UV (MeOH), λ max (log ε) 314 (3.88), 235 (4.10), 210 (4.38); IR (KBr) cm −1 : 3410, 3032, 2968, 2872, 1615, 1583, 1520, 1461, 1426, 1395, 1182, 1150, 1124, 1076, 859. 1 H- and 13 C-NMR data (CDCl 3 , 500 MHz and 125 MHz, respectively), see Table 1 . ESIMS (positive ion mode), m/z 353 [M+Na] × HRESIMS (positive ion mode), m/z 353.0997 [M+Na] × (calcd. 353.1001 for C 18 H 18 NaO 6 ).
Acknowledgements
This research was supported by the National Natural Science Foundation of China (No. 21032085), the excellent Scientific and Technological Team of Yunnan High School (2010CI08), the Yunnan University of Nation-alities Green Chemistry and Functional Materials Research for Provincial Innovation Team (2011HC008), and Open Research Fund Program of Key Laboratory of Ethnic Medi-cine Resource Chemistry (Yunnan University of Nationalities) (2010XY08). And the publication cost of this paper was supported by the Korean Chemical Society.
References
Chen X. Q. , Gale S. W. 2009 Floral of China Science Press Beijing
Tang D. Y. , Wang Y. J. , Li R. Y. , Wang Y. Q. 2005 J. Chin. Med. Mat. 28 263 - 264
Liu M. F. , Han Y. , Xing D. M. , Shi Y. , Xu L. Z. , Du L. J. , Ding Y. 2004 J. Asian Nat. Prod. Res. 6 229 - 232    DOI : 10.1080/10286020310001653219
Liu M. F. , Lv H. R. , Ding Y. 2012 J. Chin. Mat. Med. 37 66 - 70
Liu M. F. , Han Y. , Xing D. M. , Wang W. , Xu L. Z. , Du L. J. , Ding Y. 2005 J. Asian Nat. Prod. Res. 7 767 - 770    DOI : 10.1080/102860204100016890181
Liu M. F. , Zhang D. M. , Ding Y. 2005 J. Chin. Mat. Med. 30 353 - 356
Hu Q. F. , Zhou B. , Huang J. M. , Gao X. M. , Shu L. D. , Yang G. Y. , Che C. T. 2013 J. Nat. Prod. 76 292 - 296    DOI : 10.1021/np300727f
Gao X. M. , Yang L. Y. , Shen Y. Q. , Shu L. D. , Li X. M. , Hu Q. F. 2012 Bull. Korean Chem. Soc. 33 2447 - 2449    DOI : 10.5012/bkcs.2012.33.7.2447
Cardona M. L. , Fernandez M. I. , Garcia M. I. , Pedro J. R. 1986 Tetrahedron 42 2725 - 2730    DOI : 10.1016/S0040-4020(01)90559-9
Feng W. S. , Cao X. W. , Kuang H. X. , Zheng X. 2005 Acta Pharm. Sin. 40 1131 - 1134
Silayo A. , Ngadjui B. T. , Abegaz B. M. 1999 Phytochemistry 52 947 - 955    DOI : 10.1016/S0031-9422(99)00267-8
Pettit G. R. , Numata A. , Iwamoto C. , Usami Y. , Yamada T. , Ohishi H. , Cragg G. M. 2006 J. Nat. Prod. 69 323 - 327    DOI : 10.1021/np058075+
Mannila E. , Talvitie A. , Kolehmainen E. 1994 Phytochemistry 33 813 - 816
Garo E. , Hu J. F. , Goering M. , Hough G. , O'Neil-Johnson M. , Eldridge G. 2007 J. Nat. Prod. 70 968 - 973    DOI : 10.1021/np070014j
Pettit G. R. , Numata A. , Iwamoto C. , Usami Y. , Yamada T. , Ohishi H. , Cragg G. M. 2006 J. Nat. Prod. 69 323 - 327    DOI : 10.1021/np058075+
Kovács A. , Vasas A. , Hohmann J. 2008 Phytochemistry 69 1084 - 1110    DOI : 10.1016/j.phytochem.2007.12.005
Xiao K. , Zhang H. J. , Xuan L. J. , Zhang J. , Xu Y. M. , Bai D. L. 2008 Stud. Nat. Prod. Chem. 34 453 - 646    DOI : 10.1016/S1572-5995(08)80032-4
Zaki M. A. , Balachandran P. , Khan S. , Wang M. , Mohammed R. , Hetta M. H. , Pasco D. S. , Muhammad I. 2013 J. Nat. Prod. 76 679 - 684    DOI : 10.1021/np300893n
Mosmann T. 1983 J. Immunol. Methods 65 55 - 63    DOI : 10.1016/0022-1759(83)90303-4