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Wan-Jr Syu,† Chien-Chang Shen,‡ Ming-Jaw Don,‡ Jun-Chih Ou,‡ Gene-Hsiang Lee,§ and Chang-Ming Sun*,‡
Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, 112 Taiwan,
National Research Institute of Chinese Medicine, Taipei, 112 Taiwan, and Instrumentation Center,
College of Science, National Taiwan University, Taipei, 107 Taiwan
The Chinese traditional medicine Curcuma zedoaria of pure 2 and its derivatives, compounds 1-3 were pre-
(Berg.) Rosc. (Zingiberaceae) has been clinically used for pared by reaction of vanillin, 4-hydroxybenzaldehyde, and
the treatment of cervical cancer.1-4 The essential oil of C. acetyl-
zedoaria exhibits antimicrobial activity against Staphylo- acetone.13 The relative cytotoxicities of these curcuminoids
coccus aureus, Vibrio comma, and Escherichia coli.5 A against OVCAR-3 were then compared.
water extract of C. zedoaria demonstrated antimutagenic The cytotoxicity of synthetic 2 was slightly higher than
activity against benzo[R]pyrene-induced mutations in the that isolated from the plant. Compounds 1 and 3 were also
Salmonella/microsomal system.6 Polysaccharides and the cytotoxic to OVCAR-3 cells. Representative growth inhibi-
protein-bound polysaccharides of C. zedoaria showed in- tion activities are shown in Figure 1. Of these three
hibition of sarcoma-180 and Echrlich ascites tumor in mice, effective curcuminoids, compound 3 with the CD50 value
respectively.7,8 Furthermore, two sesquiterpene derivatives of 3.1 µg/mL was the most active followed by 2 (3.8 µg/mL)
(curcumol and curdione) of C. zedoaria showed cytotoxicity and 1 (4.4 µg/mL). These three compounds have been
against sarcoma-37, Echrlich ascites tumor, and cervical previously found to have antipromotor activity against
carcinoma-U14 in mice.9 carcinogenesis,14 and 1, 2, and 3 are in the same order of
More than twenty sesquiterpenes,10 curcuminoids (1- increasing activity as our results of the cytotoxicity against
3),11 and ethyl p-methoxycinnamate12 have been reported OVCAR-3. Compound 1 has been extensively studied as
as chemical constituents of C. zedoaria. In our continuing a chemopreventive agent against cancers in various models.
search for antitumor agents, we found that a crude etha- It was found to be cytotoxic to human HCT-1515 and HT-
nolic extract of C. zedoaria showed inhibitory activity 2915,16 colon cancer cells, erb B2 oncogene-transformed NIH
against OVCAR-3 cells (a human ovarian cancer cell line). 3T3 cells,16 mouse sarcoma cells,16 human 293 kidney
We herein report the bioassay-directed fractionation of an cancer cells,16 and human heptacellular carcinoma Hep G2
ethanolic extract of C. zedoaria and the isolation of the cells,16 but not to several primary fibroblast cells.16 Here
bioactive compound, demethoxycurcumin (2). Three novel we further demonstrated that 1, as well as 2 and 3, had a
components, 3,7-dimethylindan-5-carboxylic acid (4), cur- cytotoxicity effect against ovarian cancer OVCAR-3 cells.
colonol (5), and guaidiol (6), were isolated from the non-
bioactive fractions, and their structures and relative ster-
eochemistry were determined by spectroscopic methods and
X-ray crystallographic analysis.
10.1021/np980269k CCC: $15.00 © 1998 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/17/1998
1532 Journal of Natural Products, 1998, Vol. 61, No. 12 Syu et al.
Table 2. 1H and 13C NMR Assignments of Curcolonol (5) by DEPT, HMQC, HMBC, and NOESY Experiments in Acetone-d6a
positions δC DEPT δH (J)b HMBC (H f C) NOESY
1 77.9 CH 3.69 m C-9, C-10, C-15 H-2eq, H-3, H-5, H-9ax
2 28.8 CH2 eq 1.73 m C-1, C-3, C-4, C-10 H-1, H-3
ax 1.63 m C-1, C-3, C-4 H-3, H-14, H-15
3 39.3 CH2 1.58 m C-2, C-5, C-14 H-1, H-5
4 71.5 C
5 62.8 CH 2.61 s C-1, C-4, C-6, C-9, H-1, H-3, H-9ax
C-10, C-14, C-15
6 198.4 C
7 119.8c C
8 167.7 C
9 40.3 CH2 eq 3.03 d (17) C-5, C-8, C-10, C-15 H-9ax, H-15
ax 2.84 d (17) C-1, C-8, C-10, C-15 H-1, H-5, H-9eq
10 45.4 C
11 140.6 CH 7.29 bs C-8 H-13
12 119.6c C
13 9.1 CH3 2.14 d (1.3) C-11 H-11
14 25.0 CH3 1.40 s C-3, C-4, C-5 H-2ax, H-15
15 15.0 CH3 0.97 s C-1, C-5, C-9, C-10 H-2ax, H-9eq, H-14
a 1H NMR (500 MHz), HMBC (J ) 8 Hz) and NOESY spectra were recorded on a Bruker DMX-500 instrument; 13C NMR (50 MHz),
DEPT, and HMQC (J ) 150 Hz) spectra were performed on a Varian Gemini-200 spectrometer. b J ) coupling constant in hertz.
c Assignments may be interchangeable.
the eluent. Cytotoxic activity was observed in subfractions was selected for X-ray analysis. The crystallographic data
51-55 (900 mL each), which was further chromatographed (2.5 were collected on a Siemens Smart CCD diffractometer using
× 50 cm) with 20% (500 mL) and 25% (4 L) acetone/hexane as graphite-monochromated Mo KR radiation. Structure analysis
eluent. Tubes 146-180 (20 mL each) showed cytotoxicity and was made by using the SHELXTL program on PC.22 The
were purified by preparative TLC using 45% acetone/hexane compound crystallized in the space group P212121, a ) 7.8949(2)
as the developing solvent to yield 2 (11 mg). Nonbioactive Å, b ) 11.1943(2) Å, c ) 16.4296(10) Å, orthorhombic, V )
fractions were rechromatographed using gradient solvents of 1452.01(5) Å3, Z ) 4, Dcalc ) 1.090 g/cm3, λ ) 0.71073 Å, µ(Mo
hexane and EtOAc to afford compounds 4 (7 mg), 5 (16 mg), KR) ) 0.70 cm-1, F(000) ) 528, and T ) 295 K. A total of
and 6 (5 mg). 8775 reflections were collected in the range of 2.20o e θ e
Demethoxycurcumin (2) was an orange solid and identi- 27.35°, of which only 1582 unique reflections with I > 2σ(I)
fied by comparison of its 1H and 13C NMR (acetone-d6) spectra were corrected for the analysis. The structure was solved
with literature data.13,19,20 CIMS: m/z 339 [M + 1]+. using direct methods and refined by full-matrix least-squares
3,7-Dimethylindan-5-carboxylic acid (4): colorless needles on F2 values. Non-hydrogen atoms were refined anisotropi-
(hexane); mp 153-155 °C (dec); [R]25D +18° (c 1.0, benzene); cally. Hydrogen atoms were fixed at calculated positions and
TLC Rf 0.67 (50% hexane/EtOAc); IR (film) νmax 3300-2500, refined using a riding mode. The final indices were R )
1674, 1422, 1302, 1284, 1244, 959, 774 cm-1; 1H and 13C NMR, 0.0448, Rw ) 0.0607 with goodness-of-fit ) 1.035. Scattering
see Table 1; EIMS m/z (rel int) 190 [M]+ (64), 175 (100), 145 factors were taken from International Tables for X-ray Crys-
(59), 131 (73), 129 (29), 128 (29), 115 (33), 105 (10), 91 (34), 77 tallography.23
(15); HREIMS m/z 190.0992 (calcd for C12H14O2, 190.0994).
Curcolonol (5): colorless prisms (acetone); mp 183-184 °C; Acknowledgment. We gratefully acknowledge financial
[R]25D 0° (c 2.0, EtOH); TLC Rf 0.35 (15% acetone/CH2Cl2); IR support through the National Science Council (NSC 87-2315-
(film) νmax 3420, 2934, 2872, 1723, 1653, 1562, 1426, 1381, B-010-007-MH to W.J.S. and NSC 87-2113-M-077-001 to
1275, 1126, 1067, 1040, 922, 742 cm-1; 1H and 13C NMR, see C.M.S.) and NRICM for this work.
Table 2; EIMS m/z (rel int) 264 [M]+ (13), 249 (29), 246 (15),
231 (5), 228 (5), 213 (12), 163 (100), 135 (35), 122 (37), 107 References and Notes
(31), 94 (14); HREIMS m/z 264.1354 (calcd for C15H20O4, (1) Jiangsu New Medical College. Zhong Yao Da Ci Dian (Dictionary of
264.1362). Chinese Materia Medica); Shanghai Scientific and Technological
Publishers: Shanghai, 1988; pp 2450-2452.
Guaidiol (6): colorless prisms (benzene/hexane); mp 134- (2) Lu Da Gynecologic and Obstetric Hospital. Zhong Cao Yao Tong Xun
136 °C; [R]25D +30° (c 1.0, EtOH); TLC Rf 0.26 (15% acetone/ (Chin. Tradition. Herb. Drug.) 1972, 2, 69-72.
CH2Cl2); IR (film) νmax 3363, 3069, 2960, 2913, 2873, 1643, (3) Lu Da Gynecologic and Obstetric Hospital. Xin Yi Yao Xue Za Zhi
1456, 1372, 1274, 1125, 1075, 916, 885, 743 cm-1; 1H and 13C (J. Tradition. Chin. Med.) 1977, 3, 109-111.
(4) Chen Guan Hospital Xin Yi Yao Xue Za Zhi (J. Tradition. Chin. Med.)
NMR, see Table 3; EIMS m/z (rel int) 238 [M]+ (1), 223 (8), 1976, 3, 116-117.
220 (20), 205 (69), 202 (30), 187 (28), 177 (34), 162 (100), 159 (5) Rao, B. G.; Nigam, S. S. Indian J. Med. Res. 1970, 58, 627-633.
(45), 149 (45), 147 (36), 135 (22), 133 (17); HREIMS m/z (6) Lee, H.; Lin, J.-Y. Mutat. Res. 1988, 204, 229-234.
238.1923 (calcd for C15H26O2, 238.1933). (7) Moon, C. K.; Park, K. S.; Lee, S. H.; Yoon, Y. P. Arch. Pharm. Res.
1985, 8, 42-44.
Synthesis of Curcuminoids. Compounds 1-3 were pre- (8) Kokan, Takuo Tsumura Juntendo, Inc. JPN Kokai Tokkyo Koho JP
pared by the modified method reported in the literature.13 2,4- 60 67,428 (85 67,428), 1985; Chem. Abstr. 1985, 103, 92834w.
Pentanedione (5.0 g, 0.050 mol) and boric oxide (2.5 g, 0.035 (9) Xu, H.-X.; Zheng, S.-C.; Zuo, S.-X.; Sun, F.-Y. Zhong Cao Yao Tong
Xun (Chin. Tradition. Herb. Drug.) 1979, 10, 433-437.
mol) were stirred in dry EtOAc (50 mL) for 0.5 h at 40 °C. A (10) Tang, W.; Eisenbrand, G. Chinese Drugs of Plant Origin; Springer-
solution of vanillin (7.6 g, 0.050 mol), 4-hydroxybenzaldehyde Verlag: Berlin, 1992; pp 401-415.
(6.1 g, 0.050 mol), and tributyl borate (46 g, 0.20 mol) in dry (11) Kuroyanagi, M.; Natori, S. Yakugaku Zasshi 1970, 90, 1467-1470.
EtOAc (50 mL) was added, and the mixture was stirred at 40 (12) Gupta, S. K.; Banerjee, A. B.; Achari, B. Lloydia 1976, 39, 218-222.
(13) Roughley, P. J.; Whiting, D. A. J. Chem. Soc., Perkin Trans. 1 1973,
°C for 0.5 h. n-Butylamine (1 mL) in dry EtOAc (10 mL) was 2379-2388.
added dropwise during 10 min. The reaction mixture was kept (14) Anto, R. J.; George, J.; Babu, K. V. D.; Rajasekharan, K. N.; Kuttan,
stirring overnight. A hydrochloric acid solution (0.4 N, 75 mL) R. Mut. Res. 1996, 370, 127-131.
was then added, and the mixture was stirred at 60 °C for 1 h. (15) Hanif, R.; Qiao, L.; Shiff, S. J.; Rigas, B. J. Lab. Clin. Med. 1997,
130, 576-584.
The EtOAc layer was washed with water and dried, and the (16) Jiang, M. C.; Yang-Yen, H. F.; Yen, J. J. Y.; Lin, J. K. Nutr. Cancer
solvent was evaporated. The extract was chromatographed 1996, 26, 111-120.
(5 × 100 cm) using gradient solvents of CHCl3 and MeOH to (17) Hansen, H.-J.; Sliwka, H.-R.; Hug, W. Helv. Chim. Acta 1979, 62,
afford compounds 1 (2.1 g), 2 (2.6 g), and 3 (0.7 g) as orange 1120-1128.
(18) Sun, C.-M.; Syu, W.-J.; Huang, Y.-T.; Chen, C.-C.; Ou, J.-C. J. Nat.
solid. Curcumin (1): mp 178-180 °C (lit.19 mp 182-183 °C); Prod. 1997, 60, 382-384.
13C NMR (acetone-d , 50 MHz) δ 184.5, 150.0, 148.8, 141.4,
6 (19) Pedersen, U.; Rasmussen, P. B.; Lawesson, S.-O. Liebigs Ann. Chem.
128.2, 123.8, 122.3, 116.2, 111.6, 101.6, 56.3; CIMS m/z 369 1985, 1557-1569.
(20) Ali, M.; Bagati, A.; Gupta, J. Indian J. Chem. 1995, 34B, 884-888.
[M + 1]+. Demethoxycurcumin (2): mp 172-174 °C (lit.19 (21) Crystallographic data for compound 6 in this paper have been
mp 172-173 °C); 13C NMR (acetone-d6, 50 MHz) δ 184.5, 184.4, deposited with the Cambridge Crystallographic Data Centre. Copies
160.4, 149.9, 148.7, 141.4, 141.0, 130.9, 128.1, 127.7, 123.8, of the data can be obtained, free of charge, on application to the
122.2, 122.0, 116.8, 116.2, 111.5, 101.7, 56.3; CIMS m/z 339 Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K. (Fax,
+44 1223 336033, or e-mail, deposit@ccdc.cam.ac.uk).
[M + 1]+. Bisdemethoxycurcumin (3): mp 221-223 °C (22) Sheldrick, G. M. 1994 SHELXTL/PC, Version 5.03, Siemens Analyti-
(lit.19 mp 223-224 °C); 13C NMR (acetone-d6, 50 MHz) δ 184.5, cal X-ray Instruments Inc., Madison, WI.
160.5, 141.0, 130.9, 127.7, 122.0, 116.8, 101.7; CIMS m/z 309 (23) Ibers, J. A.; Hamilton, W. C., Eds. International Tables for X-ray
[M + 1]+. Crystallography; The Kynoch Press: Birmingham, U.K., 1974; Vol.
IV.
X-ray Crystal Structure Analysis of Guaidiol (6).21 A
colorless crystal of 6 with dimensions 0.50 × 0.20 × 0.20 mm NP980269K