A New Cytotoxic Phenylbutenoid Dimer from the Rhizomes of Zingiber cassumunar

• Abstract
• Materials and Methods
• Acknowledgement
• References

Abstract

A new phenylbutenoid dimer, (±)-trans-3-(4-hydroxy-3-methoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene, was isolated from the rhizomes of Zingiber cassumunar along with the three known compounds, (±)-trans-3-(3,4-dimethoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene, 4-(3,4-dimethoxyphenyl)but-1,3-diene, and 4-(2,4,5-trimethoxyphenyl)but-1,3-diene by bioassay-directed fractionation using the A549 human cancer cell line cytotoxicity assay. Structure of the new compound was elucidated by spectral analysis, including 1D and 2D NMR experiments.

Zingiber cassumunar Roxb. (Zingiberaceae) is the tropical ginger that is widely distributed in Southeast Asia [1]. The ginger rhizoma have been traditionally used for gastrointestinal distress and to prevent motion sickness [2]. Several compounds including phenylbutenoids [3], [4], [5], [6], [7], curcuminoids [8], and sesquiterpenoids [9] have been reported previously as the constituents of Z. cassumunar. This plant was found to have diverse biological activities such as antioxidant [8], anti-inflammatory [9], insecticidal [10], and uterine relaxant activity [11]. However, there have not been many studies related to anticancer activity of Z. cassumunar except for only few previous reports [12], [13]. As a part of our research program to discover potential anticancer agents from higher plants, Z. cassumunar was selected for phytochemical investigation since a CHCl₃ extract exhibited significant cytotoxic activity during preliminary screening (A549, IC₅₀ = 18.5 μg/mL). The bioassay-guided fractionation using the A549 (human lung carcinoma) cell line, led to the isolation of a new phenylbutenoid dimer, (±)-trans-3-(4-hydroxy-3-methoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene (1) along with the three known phenylbutenoids 2 - 4.

Compound 1 gave a molecular ion peak at m/z = 366.1831 in its HR-EI-MS, corresponding to the elemental formula C₂₃H₂₆O₄. The IR spectrum showed an absorption band at 3443 cm^-1 for one or more hydroxy groups. The UV spectrum of 1 showed absorption maxima at 253, 262, and 276 nm, indicating the presence of separate aromatic systems. Its ¹H- and ¹³C-NMR spectra were similar to those of the known phenylbutenoid dimer (2) [7], which was also isolated in the present work, except for the absence of a methoxy signal that was observed in the spectra of 2. The ¹H-NMR spectrum of 1 showed three methoxy signals at δ_H = 3.87 (3H, s), 3.86 (3H, s), and 3.82 (3H, s), which were correlated to the ¹³C-NMR signal at δ_C = 56.1 (OCH₃ × 3) in the HSQC experiment. The three methoxy signals exhibited three-bond connectivities with the ¹³C-NMR signals at δ_C = 149.1, 148.4, and 146.3, respectively, in the HMBC spectrum of 1. Therefore, the three methoxy groups were assigned to C-3′′′, C-4′′′, and C-3′, respectively. The oxygenated ¹³C-NMR signal at δ_C = 144.0 indicated the presence of a hydroxy group at C-4′ of 1, which was the only structural difference with the known compound 2. Further detailed analysis of ¹H-¹H COSY, ¹H-¹³C HSQC, and ¹H-¹³C HMBC NMR data (Fig. [1]) allowed unambiguous assignments for all of the ¹H- and ¹³C-NMR signals of 1. The relative stereochemistry between H-3 and H-4 was proposed as trans each other by the NOESY correlations of H-3/H-1″, H-2″ and H-4/H-2′, H-6′ as shown in Fig. [1]. The optical rotation value of [α]_D ²⁵ 0.00° (acetone, c 0.12) indicated that compound 1 existed as a racemic mixture. Therefore, compound 1 was assigned to the new compound, namely, (±)-trans-3-(4-hydroxy-3-methoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene. Compounds 2 {[α]_D ²⁵: 0.00° (acetone, c 0.17)} - 4 were identified as (±)-trans-3-(3,4-dimethoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene [7], 4-(3,4-dimethoxyphenyl)but-1,3-diene [7], and 4-(2,4,5-trimethoxyphenyl)but-1,3-diene [3], [6], respectively, by physical and spectroscopic methods as well as by comparison of their data with those of published values. The cytotoxicity of compounds 1 - 4 were evaluated against several human cancer cell lines as shown in Table [1]. Compounds 1 and 2 exhibited moderate cytotoxicity against all the human cancer cell lines tested. Whereas, compound 3 showed significant cytotoxicity against the HT-1080 fibrosarcoma cells (IC₅₀ value of 7.9 μM) in a selective manner. This is the first report on their cytotoxicity although compounds 2 - 4 have been reported previously from the same plant. In addition, when we analyzed the cell cycle distribution using flow cytometry compound 2 (13 μM) exhibited an accumulation of G0/G1 phase at the early time (control: 56 G0/G1, 33 S, 11 % G2/M; treated: 64 G0/G1, 26 S, 10 % G2/M at 16 h) and the induction of sub-G0 phase in the late time (control: 67 G0/G1, 20 S, 7 G2/M, 6 % sub-G0; treated: 47 G0/G1, 22 S, 4 G2/M, 27 % sub-G0 at 48 h). This result suggests that one possible mechanism of action by isolated compounds might be related to the cell cycle arrest and induction of apoptosis.

Materials and Methods

Optical rotations were measured with a P-1010 polarimeter (Jasco, Japan) at 25 °C. UV and IR spectra were recorded on a U-3000 spectrophotometer (Hitachi, Japan) and a FTS 135 FT-IR spectrometer (Bio-Rad, CA), respectively. 1D and 2D NMR experiments were performed on a UNITY INOVA 400 MHz FT-NMR instrument (Varian, CA). TMS was used as internal standard. EI-MS was obtained on a JMS 700 Mstation HRMS spectrometer (JEOL, Japan). The rhizomes of Zingiber cassumunar Roxb. (Zingiberaceae) were collected in Surabaya, Indonesia, in June 2001 and were identified by one of the authors, Tri Windono (University of Surabaya, Indonesia). The voucher specimen (No. 20/DT/VI/2001) has been deposited at the University of Surabaya.

The dried rhizomes of Z. cassumunar (500 g) were extracted with MeOH (3 × 2 L) overnight at room temperature. The solvent was evaporated under vacuum to afford a concentrated MeOH extract, which was then suspended in water (200 mL). It was partitioned with n-hexane (4 × 200 mL), CHCl₃ (3 × 200 mL), and n-BuOH (2 × 200 mL), subsequently. The CHCl₃ layers were concentrated to dryness under vacuum to afford a residue (13 g). The active CHCl₃ extract (A549, IC₅₀ = 18.5 μg/mL) was separated by a silica gel flash column chromatography (φ 5.5 cm; 230 - 400 mesh, 500 g) with a gradient of MeOH in CH₂Cl₂ (0, 0.25, 0.5, 1, 2, 5, 10, 100 %, 10 L each) as mobile phase, affording 13 fractions (FI - FXIII). The first fraction, FI (eluted with CH₂Cl₂, 1.7 g) which showed significant cytotoxic activity (A549, IC₅₀ 10.2 μg/mL), was subjected to silica gel flash CC (φ 3 cm; 230 - 400 mesh, 40 g), using a gradient of EtOAc in n-hexane (1, 2, 5, 10, 50, 100 %, 3 L each) to afford 15 fractions (FI-1 - FI-15). The first two fractions eluted with 1 % EtOAc in n-hexane, contained two pure compounds 3 (140 mg; TLC Rf = 0.5, n-hexane-EtOAc = 2 : 1) and 4 (60 mg; TLC Rf = 0.4, n-hexane-EtOAc = 2 : 1), respectively. The tenth fraction (600 mg) eluted with 15 % EtOAc in n-hexane from the CC of FI, was further separated by silica gel CC (φ 2 cm; 230 - 400 mesh, 20 g) with a gradient of acetone in n-hexane (50 : 1, 3 l; 40 : 1, 1.4 L) as a solvent system, providing two pure compounds, 1 (8 mg; TLC Rf = 0.4, n-hexane-acetone = 3 : 2) and 2 (150 mg; TLC Rf = 0.5, n-hexane-acetone = 3 : 2).

(±)-trans-3-(4-Hydroxy-3-methoxyphenyl)-4-[(E)-3,4-dimethoxy-styryl]cyclohex-1-ene (1): a pale yellow gum; [α]_D ²⁵: 0.00° (acetone, c 0.12). IR (liquid film): ν_max = 3443, 1650, 1541, 1507, 1255, 1027 cm^-1. UV (MeOH): λ_max (log ε) = 276 (3.8), 262 (3.8), 253 (3.9) nm. ¹H-NMR (CDCl₃, 400 MHz): δ = 6.83 (1H, br s, H-5′), 6.81 (1H, d, J = 1.4 Hz, H- 2′′′), 6.78 (1H, dd, J = 9.4, 1.4 Hz, H-6′′′), 6.77 (1H, d, J = 9.4 Hz, H-5′′′), 6.69 (1H, dd, J = 8.0, 1.8 Hz, H-6′), 6.67 (1H, d, J = 1.8 Hz, H-2′), 6.10 (1H, d, J = 16.2 Hz, H-2′′), 6.02 (1H, dd, J = 16.2, 7.2 Hz, H-1′′), 5.89 (1H, br dd, J = 10.1, 2.6 Hz, H-1), 5.67 (1H, dd, J = 10.1, 2.4 Hz, H-2), 3.87 (3H, s, OCH₃), 3.86 (3H, s, OCH₃), 3.82 (3H, s, OCH₃), 3.16 (1H, dd, J = 8.2, 2.4 Hz, H-3), 2.33 (1H, br d, J = 8.2 Hz, H-4), 2.21 (2H, m, H-6), 1.92 (1H, br dd, J = 12.8, 2.8 Hz,H-5), 1.66 (1H, m, H-5). ¹³C-NMR (CDCl₃, 100 MHz): δ = 149.1 (C-3′′′), 148.4 (C-4′′′), 146.3 (C-3′), 144.0 (C-4′), 137.1 (C-1′), 132.4 (C-1′′), 131.1 (C-1′′′), 130.5 (C-2), 129.0 (C-2′′), 127.7 (C-1), 121.3 (C-6′), 118.9 (C-6′′′), 114.1 (C-5′), 111.3 (C-5′′′), 111.1 (C-2′), 108.9 (C-2′′′), 56.1 (OCH₃ - 3′ and OCH₃ - 3′′′, interchangeable with OCH₃ - 4′′′), 56.0 (OCH₃ - 4′′′, interchangeable with OCH₃ - 3′ or OCH₃ - 3′′′), 48.3 (C-3), 45.6 (C-4), 28.0 (C-5), 24.7 (C-6). EI-MS: m/z (%) = 366 [M]⁺ (100), 284 (10), 266 (10), 98 (15). HR-EI-MS: m/z = 366.1831 [M]⁺; calcd. for C₂₃H₂₆O₄ : 366.4570.

Cytotoxicity testing: Human cancer cells (A549, lung, ATCC; Col2, colon, UIC; SNU-638, stomach, Korea Cell Bank; HT-1080, fibrosarcoma, ATCC) were maintained in minimal essential medium with Earles’ salt (MEME, GIBCO-BRL, USA) with 10 % fetal bovine serum (FBS, GIBCO-BRL, USA). Cells (5 × 10⁴ cells/mL) were treated with different concentrations of compounds for 3 days. After treatment, the cells were fixed with trichloroacetic acid (TCA, Sigma Chemical Co., USA) and viability was determined with a sulforhodamine B (SRB, Sigma Chemical Co., USA) assay as described previously [14]. All treatments were performed in triplicate. The results were expressed as a percentage, relative to solvent-treated controls, and IC₅₀ values were calculated using non-linear regression analyses (percent survival versus concentration).

Acknowledgement

This investigation was supported by a grant of the Korea Health 21 R&D project, Ministry of Health & Welfare, Republic of Korea (01-PJ2-PG6-01NA01-0002).

References

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• 2 Bruneton J. Pharmacognosy. 2nd Edition New York; Lavoisier Publishing Inc 1999: pp 296-302
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• 14 Lee S K, Cui B, Mehta R R, Kinghorn A D, Pezzuto J M. Cytostatic mechanism and antitumor potential of novel 1H-cyclopenta[b]benzofuran lignans isolated from Aglaia elliptica .  Chemico-Biol Interact. 1998;  115 215-28
  PubMedSearch in Google Scholar

Prof. Eun-Kyoung Seo, Ph. D.

Natural Products Chemistry Laboratory

College of Pharmacy

Ewha Womans University

Seoul 120-750

Korea

Phone: +82-2-3277-3047

Fax: +82-2-3277-3051

Email: Yuny@ ewha.ac.kr

References

• 1 Mabberley D J. The Plant Book. 1st Edition Cambridge; Cambridge University Press 1995: pp 623-4
  Search in Google Scholar
• 2 Bruneton J. Pharmacognosy. 2nd Edition New York; Lavoisier Publishing Inc 1999: pp 296-302
  Search in Google Scholar
• 3 Masuda T, Andoh T, Yonemori S, Takeda Y. Phenylbutenoids from the rhizomes of Alpinia flabellata .  Phytochemistry. 1999;  50 163-6
  CrossrefPubMedSearch in Google Scholar
• 4 Masuda T, Jitoe A. Phenylbutenoid monomers from the rhizomes of Zingiber cassumunar .  Phytochemistry. 1995;  39 459-61
  CrossrefPubMedSearch in Google Scholar
• 5 Jitoe A, Masuda T, Nakatani N. Phenylbutenoid dimmers from the rhizomes of Zingiber cassumunar .  Phytochemistry. 1993;  32 357-63
  CrossrefPubMedSearch in Google Scholar
• 6 Tuntiwachwuttikul P, Pancharoen O, Jaipetch T, Reutrakul V. Phenylbutanoids from Zingiber cassumunar .  Phytochemistry. 1981;  20 1164-5
  CrossrefPubMedSearch in Google Scholar
• 7 Kuroyanagi M, Fukushima S, Yosihira K, Natori S, Dechatiwongse T, Mihashi K, Nishi M, Hara S. Further characterization of the constituents of a Thai medicinal plants, Zingiber cassumunar Roxb.  Chem Pharm Bull. 1980;  28 2948-59
  PubMedSearch in Google Scholar
• 8 Masuda T, Jitoe A. Antioxidative and anti-inflammatory compounds from tropical gingers: isolation, structure determination, and activities of cassumunins A, B, and C, new complex curcuminoids from Zingiber cassumunar .  J Agr Food Chem. 1994;  42 1850-6
  CrossrefPubMedSearch in Google Scholar
• 9 Ozaki Y, Kawahara N, Harada M. Anti-inflammatory effect of Zingiber cassumunar Roxb. and its active principles.  Chem Pharm Bull. 1991;  39 2353-6
  CrossrefPubMedSearch in Google Scholar
• 10 Nugroho B W, Schwarz B, Wray V, Proksch P. Insecticidal constituents from rhizomes of Zingiber cassumunar and Kaempferia rotunda .  Phytochemistry. 1996;  41 129-32
  CrossrefPubMedSearch in Google Scholar
• 11 Kanjanapothi D, Soparat P, Panthong A, Tuntiwachwuttikul P, Reutrakul V. A uterine relaxant compound from Zingiber cassumunar .  Planta Medica. 1987;  53 329-32
  PubMedSearch in Google Scholar
• 12 Vimala S, Norhanom A W, Yadav M. Anti-tumour promoter activity in Malaysian ginger rhizobia used in traditional medicine.  Brit J Cancer. 1999;  80 110-6
  PubMedSearch in Google Scholar
• 13 Park G, Lee E J, Min H Y, Choi H Y, Han A R, Lee S K, Seo E K. Evaluation of cytotoxic potential of Indonesian medicinal plants in cultured human cancer cells.  Nat Prod Sci. 2002;  8 165-9
  PubMedSearch in Google Scholar
• 14 Lee S K, Cui B, Mehta R R, Kinghorn A D, Pezzuto J M. Cytostatic mechanism and antitumor potential of novel 1H-cyclopenta[b]benzofuran lignans isolated from Aglaia elliptica .  Chemico-Biol Interact. 1998;  115 215-28
  PubMedSearch in Google Scholar

Prof. Eun-Kyoung Seo, Ph. D.

Natural Products Chemistry Laboratory

College of Pharmacy

Ewha Womans University

Seoul 120-750

Korea

Phone: +82-2-3277-3047

Fax: +82-2-3277-3051

Email: Yuny@ ewha.ac.kr