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Phytochemical reinvestigation of labdane-type diterpenes and their cytotoxicity from the rhizomes of Hedychium coronarium
Phytochemistry Letters 2 (2009) 184–187
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
Phytochemical reinvestigation of labdane-type diterpenes and their cytotoxicity from the rhizomes of Hedychium coronarium Nitirat Chimnoi a, Chonticha Sarasuk b, Nisachon Khunnawutmanotham a, Pakamart Intachote a, Suchada Seangsai a, Busakorn Saimanee a, Somchai Pisutjaroenpong a, Chulabhorn Mahidol a, Supanna Techasakul a,b,* a b
Chulabhorn Research Institute, Vipavadee Rangsit Road, Bangkok 10210, Thailand Chemistry Department, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 April 2009 Received in revised form 18 June 2009 Accepted 18 June 2009 Available online 27 June 2009
A comprehensive reinvestigation of chemical constituents from the rhizomes of Hedychium coronarium resulted in the isolation of one new labdane-type diterpene, together with eight known compounds. Their structures were established by spectroscopic methods. Some of the isolated compounds showed significant cytotoxicity with IC50 values lower than 4 mg ml1. ß 2009 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Hedychium coronarium Zingiberaceae Labdane diterpenes Cytotoxicity
1. Introduction The Zingiberaceae plant Hedychium coronarium is widely used in folk medicine, particularly the rhizomes of H. coronarium have been prescribed in traditional Chinese medicine for the treatment of lancinating pain, headache, contusion, inflammation and sharp pain associated with rheumatism (Matsuda et al., 2002a,b; Morikawa et al., 2002; Nakamura et al., 2008). Pharmacological studies have shown that an extract of H. coronarium has antiinflammatory and analgesic effects in an animal model (Shrotriya et al., 2007) and showed in vitro inhibition of 5-lipoxygenase (Braga et al., 2000). Several labdane diterpenes were isolated from this plant and some of them showed cytotoxic activity against V-79 cells (Itokawa et al., 1988a) and inhibitory effects on the increase in vascular permeability induced by acetic acid in mice and nitric oxide production in lipopolysaccharide-activated mouse peritoneal macrophages (Matsuda et al., 2002a,b) and on the release of beta-hexosaminidase from RBL-2H3 cells (Morikawa et al., 2002). Recently, coronarin D, a major component of the rhizomes of this plant, showed inhibition of both constitutive and inducible nuclear factor-KB pathway activation, leading to apoptosis, inhibition of
* Corresponding author at: Chulabhorn Research Institute, Vipavadee Rangsit Road, Bangkok 10210, Thailand. Tel.: +662 5740622x1306; fax: +662 5742027. E-mail addresses: [email protected], [email protected] (S. Techasakul).
invasion, and suppression of osteoclastogenesis (Kunnumakkara et al., 2008). In the course of our investigation on biologically active substance from H. coronarium, we previously reported the isolation of some labdane-type diterpenes (Chimnoi et al., 2008). In the present work, to obtain the minor components, we decided to increase the amount of plant material and reinvestigate the chemical constituents of the rhizomes. One new labdane diterpene, named 7b-hydroxy-(E)-labda-8(17),12-diene-15,16dial (1) (Fig. 1), together with eight known compounds (2–9) (supporting information) were obtained. Among the known compounds, compounds 7–9 were isolated for the first time from this plant. Moreover, the cytotoxicity of some isolated compounds was evaluated against various cancer cell lines. 2. Results and discussion Compound 1 was obtained as colorless gum. The molecular composition of C20H30NaO3 was inferred from high-resolution ESITOF-MS ([M+Na]+ at m/z 341.2085). The IR spectrum of 1 showed absorption bands ascribable to hydroxyl (3425 cm1), aldehydic carbonyl (1725 cm1), a,b-unsaturated aldehydic carbonyl (1679 cm1) and olefinic functional groups (1644 cm1). The 1H NMR spectrum of 1 (see Table 1) displayed three methyl proton signals at dH 0.92 (s, H3-18), 0.83 (s, H3-19), and 0.72 (s, H3-20) and one exomethylene moiety at dH 5.25 (s, H-17) and 4.57 (s, H-17). These are characteristic of the C-8 exomethylene group of labdane
1874-3900/$ – see front matter ß 2009 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2009.06.003
N. Chimnoi et al. / Phytochemistry Letters 2 (2009) 184–187 Table 1 1 H NMR and
13
C NMR spectroscopic data of compounds 1 in CDCl3.
Position
1
1a 1b 2a 2b 3a 3b 4 5 6a 6b 7a 8 9 10
1.04 (ddd, J = 13, 4, 4) 1.69 (br d, J = 12) 1.55a 1.55a 1.19a 1.45 (br d, J = 13)
a
185
H
1.19a 2.12 (m) 1.29 (br t, J = 12) 4.03 (dd, J = 11, 5) 1.83 (br d, J = 10)
13
Position
1
39.0
11
19.2
12
2.40 (ddd, J = 17, 11, 7) 2.53 (ddd, J = 17, 6, 3) 6.74 (t-like, J = 7)
41.7
13
33.5 52.9 33.7
14 15 16
3.40 (d, J = 17)/3.47 (d, J = 17) 9.65 (s) 9.39 (s)
39.3 197.2 193.4
73.6 149.8 54.5 39.2
17 18 19 20
4.57 0.92 0.83 0.72
104.8 33.5 21.6 14.3
C
13
H
C
24.3 159.0 135.1
(s)/5.25 (s) (s) (s) (s)
Overlapping peaks.
diterpenoids (Kimbu et al., 1987). In addition, the analyses of the 1 H NMR spectrum (CDCl3) (Table 1) indicated the existence of two aldehydic groups [dH 9.39 (s, H-16) and 9.65 (s, H-15)] and one oxymethine [dH 4.03 (dd, J = 11, 5 Hz, H-7)]. Using 13C NMR, DEPT and HMQC experiments, 20 resonances were observed and assigned as two aldehydic groups [dC 193.4 (C-16) and 197.2 (C15)], six sp3 methylenes, one sp2 methylene [dC 104.8 (C-17)], two sp3 methines, one oxymethine [dC 73.6 (C-7)], one sp2 methine, three methyls, two sp3 quaternary carbons and two sp2 quaternary carbons. The 1H and 13C NMR (CDCl3) spectra (see Table 1) of the side chain attached to C-9 of compound 1 were comparable with those of (E)-labda-8(17),12-diene-15,16-dial (6) (Morita and Itokawa, 1988). In the 1H–1H COSY spectrum of compound 1 (Fig. 2), the olefinic proton resonating at dH 6.74 (t-like, J = 7 Hz, H12) correlated with an allylic methylene at dH 2.40 (ddd, J = 17, 11, 7 Hz, H-11) and 2.53 (ddd, J = 17, 6, 3 Hz, H-11), which in turn coupled to H-9 at dH 1.83 (br d, J = 10 Hz). In the HMBC spectrum, long-range correlations were observed between the aldehydic proton of H-15 to C-13 and C-14, the aldehydic proton of H-16 to C12 and C-13, the sp2-methine proton of H-12 to C-9, C-11, C-14, and C-16, and the allylic methylene proton of H-11 to C-8, C-9, C12, and C-13 (Fig. 2). These confirmed that the 12-en-15,16-dial moiety was attached to C-11, which in turn was attached to C-9 of the decalin ring and formed the normal six-carbon side chain of labdane diterpenes.
The 1H and 13C NMR of the decalin system of 1 was virtually superposable with that of hedychilactone A (11) (Matsuda et al., 2002a,b). In the 1H–1H COSY spectrum of compound 1 (Fig. 2), a methine group bearing a hydroxyl group at dH 4.03 [dd, J = 11, 5 Hz, H-7] correlated with the methylene protons at dH 2.12 (m, H-6a) and 1.29 (br d, J = 12 Hz, H-6b), which in turn coupled to the signal at dH 1.19 (m, H-5). Long range correlations were established in the HMBC spectra between H-7 and C-6, C-8 and C-17 (Fig. 2). Therefore, it was concluded that a hydroxyl group was attached to C-7 of the decalin system. The configuration of compound 1 was suggested by the correlations observed in a NOESY spectrum (Fig. 3). Correlations were observed from H-7 to H-5, H-6a and H-9. Therefore, the hydroxyl group at C-7 was assigned to be in b-configuration. Moreover, Oh et al. (2006) recently synthesized (+)-hedychilactone A (11) and 7-epi-hedychilactone A. The 1H chemical shift observed for H-7 of (+)-hedychilactone A and 7-epi-hedychilactone A was 3.98 ppm and 4.37 ppm, respectively and H-7 of 1 appeared at d 4.03. In addition, it is worth to remark that the 13C NMR signals of C-17 appeared at dC 104.3 and 109.9 for hedychilactone A (11) and 7-epi-hedychilactone A, respectively (Oh et al., 2006), (dC 104.8 for compound 1). These supported the b-configuration of 7-OH. The correlations in the NOESY spectrum (Fig. 3) between H-16 and H12 and between H-11 and H-14 suggested 12E-configuration of 1. Based on the above evidence, we were able to confirm the skeleton of compound 1 to be 7b-hydroxy-(E)-labda-8(17),12-diene-15,16dial which is a new metabolite. In our previous report (Chimnoi et al., 2008), coronarin D was isolated as a C-15 epimeric mixture. In the present work, coronarin D acetate (10) was prepared to confirm the presence of C-15 epimers of 2. The duplicated signals of 1H and 13C NMR of 10 were clearly seen particularly for the acetyl group on C-15. All of these data suggested that coronarin D (2) was isolated in the form of a C15 epimeric mixture. Additionally, eight known compounds (supporting information), namely C-15 epimers of coronarin D (2) (Chimnoi et al., 2008), coronarin F (3) (Itokawa et al., 1988b), C-14 epimers of
Fig. 2. (a) Key 1H–1H COSY couplings (boldface bonds) of 1 (b) HMBC correlations of 1.
Fig. 3. Key NOESY interactions of compound 1.
Fig. 1. Chemical structure of compound 1.
N. Chimnoi et al. / Phytochemistry Letters 2 (2009) 184–187
186 Table 2 Cytotoxicity data for compounds 1, 2, 4, 5, and 10. Compounds
1 2 4 5 10 Etoposide Doxorubicin
Cell lines (IC50, mg ml1) S102
HuCCA-1
A549
MOLT-3
KB
HeLA
MDA-MB231
T-47D
HL-60
P388
HepG2
NT 21.5 11.5 NT NT NT 5.0
27 4.0 3.4 2.7 3.5 4.0 0.42
47 27.5 5.5 27 NT NT 0.3
2.81 3.91 1.02 0.44 3.48 0.018 NT
NT 3.0 2.8 NT 3.0 0.3 NT
44 4.0 2.9 2.7 3.5 0.5 0.36
NT 2.5 2.3 NT 2.5 0.3 NT
NT 4.8 2.7 NT 4.5 0.05 NT
NT 4.4 2.1 NT 4.6 0.7 NT
NT 1.4 0.68 NT 1.7 0.07 NT
33.5 18.0 5.3 7.75 NT 14 0.28
NT, not test; S102, hepatocellular carcinoma; HuCCA-1, cholangiocarcinoma; A549, lung adenocarcinoma; MOLT-3, T-lymphoblast (acute lymphoblastic leukemia); KB, epidermoid carcinoma; HeLA, cervical carcinoma; MDA-MB231, hormone-independent breast cancer; T-47D, hormone-dependent breast cancer; HL-60, human promyelocytic leukemia cell; P388, mouse lymphoid neoplasm; HepG2, hepatoblastoma.
isocoronarin D (4) (Taveira et al., 2005), coronarin B (5) (Itokawa et al., 1988a), (E)-labda-8(17),12-diene-15,16-dial (6) (Morita and Itokawa, 1988), (E)-15,16-bisnorlabda-8(17),11-dien-13-one (7) (Itokawa et al., 1980c), and a mixture of eicosyl- and docosyl-(E)ferulates (8 and 9) (Jayaprakasam et al., 2006) were isolated from this plant, and their structures were confirmed by the comparison of their spectral data with those in the literature. The in vitro cytotoxicity of labdane 1, 2, 4, 5, and 10 on some cancer cell lines was defined through MTT assays (Mosmann, 1983). The IC50 values are presented in Table 2. Among the tested compounds, compound 4 was shown to be the most cytotoxic in all cancer cell lines, with IC50 at about 4 mg ml1, except for a moderate activity for S102. Compound 2, which is the major component, also displayed pronounced cytotoxicity against most of the cell lines but weak activity against S102, A549, and HepG2. Compound 5 showed remarkable cytotoxicity against HuCCA-1, HeLA, and MOLT-3 cell line but moderate and weak activities against HepG2 and A549, respectively. Coronarin D acetate (10) was revealed to have almost identical cytotoxicity to 2. Nevertheless, it is more stable than coronarin D (2) due mainly to the hemiacetal in 2 being protected by the acetyl group. Compound 1 showed only significant cytotoxicity against MOLT-3 but weak activity against HuCCA-1, A549, HeLA, and HepG2 cell lines. 3. Experimental 3.1. General experimental procedures 1 H, 13C and 2D NMR spectra were recorded in CDCl3 on a Bruker AVANCE 400 (400 MHz for 1H and 100 MHz for 13C). Chemical shifts were given in ppm (d) and referenced to the solvent signals at 7.27 ppm and 77.0 ppm for 1H and 13C NMR, respectively. EI-MS (direct insertion probe, 70 eV) mass spectra were measured using a Finnigan POLARIS Ion Trap Mass Spectrometer. HR-TOF-MS (direct infusion) mass spectra were recorded on a Bruker MicroTOF. IR spectra were determined on a Perkin Elmer Spectrum One (neat sample) and a Perkin Elmer 1760X FT-IR spectrometer (on KBr disc). UV spectra were performed with a Shimadzu UV-1700 Pharmaspec spectrometer. Optical rotation was measured with sodium D line (590 nm) on a JASCO P-1020. Column chromatography was carried out on silica gel and TLC-silica gel 60 F254 obtained from Merck.
3.2. Plant material The rhizomes of H. coronarium were collected from Bangkok, Thailand, in 2005. A voucher specimen (CRI 567) has been deposited at the laboratory of Natural Products, Chulabhorn Research Institute, Bangkok, Thailand. The rhizomes were dried indoors and then extracted with dichloromethane as described below.
3.3. Extraction and isolation The air-dried rhizomes of H. coronarium (2.1 kg) were macerated twice with dichloromethane (15 L 7 days) at room temperature and then concentrated under reduced pressure to produce a dark brown oil (120 g). After which, 60 g of crude extract was subjected to silica gel VLC and eluted with 2%, 5%, 7%, and 10% of ethyl acetate in hexane to yield fractions 1–4, respectively. Each fraction was then subjected to silica gel column chromatography with a gradient of ethyl acetate–hexane system. Compound 7 (5 mg) was obtained from fraction 1. The separation of fraction 2 led to compounds 3 (3 mg), 5 (11 mg) and 6 (3 g). A mixture of compounds 8 and 9 (13 mg) was isolated from fraction 3. Finally, the repeated silica gel column of fraction 4 resulted in the isolation of 1 (22 mg), 2 (17.5 g), and a mixture of 2 and 4 (428 mg). Careful recrystallization of a mixture of 2 and 4 using hexane with a trace amount of dichloromethane provided compound 4 (22 mg) as white needles. 7b-hydroxy-(E)-labda-8(17),12-diene-15,16-dial (1): Color less gum; ½a25 D þ 11:6 (c 0.44, CHCl3); UV (MeOH) lmax (log e): 234 (4.02) nm; IR (neat) nmax 3425, 2927, 2845, 1725, 1679, 1644, 1459, 1389, 1367, 1266, 1163, 1101, 1021, 898, 735 cm1; 1H and 13 C NMR data are given in Table 1; EI-MS m/z 318 ([M]+, 2), 301 ([M–OH]+, 14), 300 ([M–H2O]+, 17), 285 ([M–OH–CH3]+, 9), 271 (15), 257 (13), 239 (10), 231 (14), 207 (13), 194 (47), 189 (17), 176 (33), 123 (100), 109 (32), 105 (38), 95 (34), 91 (43), 81 (40), 79 (49), 77 (26), 67 (39); HRMS (ESI-TOF) m/z 341.2085 (calcd for C20H30NaO3 [M+Na]+ 341.2087). C-15 epimers of coronarin D acetate (10): A solution of compound 2 (500 mg, 1.57 mmol) in pyridine (8 ml) and acetic anhydride (1.5 ml, 15.87 mmol) was stirred at room temperature for 3 h. Pyridine was then removed by partition with dichloromethane and 10% aq. HCl. The dichloromethane layer was combined and concentrated under reduced pressure to dryness. The residue was purified by silica gel column chromatography (10% ethyl acetate in hexane) to yield 10 (355 mg, 63%) as colorless oil. ½a25 D þ 14:0 (c 1.09, CHCl3); UV (MeOH) lmax (log e): 226 (3.79) nm; IR (KBr) nmax 2928, 2868, 1765, 1678, 1645, 1459, 1441, 1367, 1337, 1217, 1166, 1078, 973, 890 cm1; 1H NMR (400 MHz, CDCl3) d: 6.83 (1H, m, H-12), 6.66 (1H, m, H-15), one set of 4.39 (1H, d, J = 1 Hz, H-17) and 4.85 (1H, d, J = 1 Hz, H-17), one set of 4.34 (1H, d, J = 1 Hz, H-17) and 4.83 (1H, d, J = 1 Hz, H-17), 3.13 (1H, m, H-14), 2.82 (1H, br t, J = 14 Hz, H-14), 2.40 (1H, m, H-7b), 2.36 (1H, m, H-11), 2.24 (1H, m, H-11), 2.12/2.11 (3H, s, –OAc), 2.01 (1H, ddd, J = 13, 13, 5 Hz, H-7a), 1.89 (1H, br t, J = 11 Hz, H-9), 1.75 (1H, m, H6a), 1.69 (1H, m, H-1b), 1.59 (1H, m, H-2b), 1.52 (1H, m, H-2a), 1.42 (1H, br d, J = 13 Hz, H-3b), 1.34 (1H, dddd, J = 13, 13, 13, 3 Hz, H-6b), 1.20 (1H, m, H-3a), 1.13 (1H, ddd, J = 12, 5, 3 Hz, H-5), 1.08 (1H, m, H-1a), 0.89 (3H, s, H-18), 0.82 (3H, s, H-19), 0.73 (3H, s, H20); 13C NMR (100 MHz, CDCl3) d: 169.19/169.17 (–OCOCH3), 168.72/168.70 (C-16), 148.03/148.00 (C-8), 144.76/144.67 (C-12),
N. Chimnoi et al. / Phytochemistry Letters 2 (2009) 184–187
122.24/122.19 (C-13), 107.50/107.42 (C-17), 92.34/92.33 (C-15), 56.13/56.12 (C-9), 55.38 (C-5), 41.99/41.98 (C-3), 39.47 (C-10), 39.34/39.32 (C-1), 37.77 (C-7), 33.57 (C-18), 33.55 (C-4), 31.95/ 31.85 (C-14), 25.74/25.71 (C-11), 24.09/24.07 (C-6), 21.71 (C-19), 20.90/20.88 (–OCOCH3), 19.32/19.30 (C-2), 14.37/14.35 (C-20); EIMS m/z 360 ([M]+, 4), 345 ([M–CH3]+, 24), 300 ([M–AcOH]+, 100), 285 ([M–AcOH–CH3]+, 59), 215 (42), 176 (73), 161 (35), 137 (68), 121 (45), 95 (50), 91 (43), 81 (54), 79 (45), 43 (61); HRMS (APCITOF) m/z 361.2375 (calcd for C22H33O4 [M+H]+ 361.2373). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytol.2009.06.003. References Braga, F.C., Wagner, H., Lombardi, J.A., de Oliverira, A.B., 2000. Brazilian plant species for in vitro inhibition of 5-lipoxygenase. Phytomedicine 6, 447–452. Chimnoi, N., Pisutjaroenpong, S., Ngiwsara, L., Dechtrirut, D., Chokchaichamnankit, D., Khunnawutmanotham, N., Mahidol, C., Techasakul, S., 2008. Labdane diterpenes from the rhizomes of Hedychium coronarium. Nat. Prod. Res., Part A 22 (14), 1255–1262. Itokawa, H., Morita, H., Katou, I., Takeya, K., Cavalheiro, A.J., de Oliveira, R.C.B., Ishige, M., Motidome, M., 1988a. Cytotoxic diterpenes from the rhizomes of Hedychium coronarium. Planta Med. 54, 311–315. Itokawa, H., Morita, H., Takeya, K., Motidome, M., 1988b. Diterpenes from rhizomes of Hedychium coronarium. Chem. Pharm. Bull. 36, 2682–2684. Itokawa, H., c Morita, M., Mihashi, S., 1980. Labdane and Bisnorlabdane type Diterpenes from Alpinia speciosa K. SCHUM. Chem. Pharm. Bull. 28, 3452– 3454. Jayaprakasam, B., Vanisree, M., Zhang, Y., Dewitt, D.L., Nair, M.G., 2006. Impact of alkyl esters of caffeic and ferulic acids on tumor cell proliferation, cyclo-
187
oxygenase enzyme, and lipid peroxidation. J. Agric. Food Chem. 54, 5375– 5381. Kimbu, S.F., Ngadjui, B., Sondengam, B.L., Njimi, T.K., Connolly, J.D., Fakunle, C.O., 1987. A new labdane diterpenoid from the seeds of Aframomum daniellii. J. Nat. Prod. 50, 230–231. Kunnumakkara, A.B., Ichikawa, H., Anand, P., Mohankumar, C.J., Hema, P.S., Nair, M.S., Aggarwal, B.B., 2008. Coronarin D, a labdane diterpene, inhibits both constitutive and inducible nuclear factor-KB pathway activation, leading to potentiation of apoptosis, inhibition of invasion, and suppression of osteoclastogenesis. Mol. Cancer Ther. 7, 3306–3317. Matsuda, H., Morikawa, T., Sakamoto, Y., Toguchida, I., Yoshikawa, M., 2002a. Antiinflammatory principles and three new labdane-type diterpenes, hedychilactones A, B and C, from the rhizome of Hedychium coronarium. Heterocycles 56, 45–50. Matsuda, H., Morikawa, T., Sakamoto, Y., Toguchida, I., Yoshikawa, M., 2002b. Labdane-type diterpenes with inhibitory effects on increase in vascular permeability and nitric oxide production from Hedychium coronarium. Bioorg. Med. Chem. 10, 2527–2534. Morikawa, T., Matsuda, H., Sakamoto, Y., Ueda, K., Yoshikawa, M., 2002. New farnesane-type sesquiterpenes, hedychiols A and B 8, 9-diacetate, and inhibitors of degranulation in RBL-2H3 cells from the rhizome of Hedychium coronarium. Chem. Pharm. Bull. 50, 1045–1049. Morita, H., Itokawa, H., 1988. Cytotoxic and antifungal diterpenes from the seeds of Alpinia galangal. Planta Med. 54, 117–120. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Nakamura, S., Okazaki, Y., Ninomiya, K., Morikawa, T., Matsuda, H., Yoshikawa, M., 2008. Medicinal flowers. XXIV. Chemical structures and hepatoprotective effects of constituents from flowers of Hedychium coronarium. Chem. Pharm. Bull. 56, 1704–1709. Oh, S., Jeong, I.H., Shin, W., Wang, Q., Lee, S., 2006. Synthesis and biological activity of (+)-hedychilactone A and its analogs from (+)-sclareolide. Bioorg. Med. Chem. Lett. 16, 1656–1659. Shrotriya, S., Ali, M.S., Saha, A., Bachar, S.C., Islam, M.S., 2007. Anti-inflammatory and analgesic effects of Hedychium coronarium KOEN. Pak. J. Pharm. Sci. 20, 47–51. Taveira, F.N., Oliveira, A.B., Souza Filho, J.D., Braga, F.C., 2005. Epimers of labdane diterpenes from the rhizomes of Hedychium coronarium. J. Koenig. Rev. Bras. Farmacogn. 15, 55–59.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
Phytochemical reinvestigation of labdane-type diterpenes and their cytotoxicity from the rhizomes of Hedychium coronarium Nitirat Chimnoi a, Chonticha Sarasuk b, Nisachon Khunnawutmanotham a, Pakamart Intachote a, Suchada Seangsai a, Busakorn Saimanee a, Somchai Pisutjaroenpong a, Chulabhorn Mahidol a, Supanna Techasakul a,b,* a b
Chulabhorn Research Institute, Vipavadee Rangsit Road, Bangkok 10210, Thailand Chemistry Department, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 April 2009 Received in revised form 18 June 2009 Accepted 18 June 2009 Available online 27 June 2009
A comprehensive reinvestigation of chemical constituents from the rhizomes of Hedychium coronarium resulted in the isolation of one new labdane-type diterpene, together with eight known compounds. Their structures were established by spectroscopic methods. Some of the isolated compounds showed significant cytotoxicity with IC50 values lower than 4 mg ml1. ß 2009 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Hedychium coronarium Zingiberaceae Labdane diterpenes Cytotoxicity
1. Introduction The Zingiberaceae plant Hedychium coronarium is widely used in folk medicine, particularly the rhizomes of H. coronarium have been prescribed in traditional Chinese medicine for the treatment of lancinating pain, headache, contusion, inflammation and sharp pain associated with rheumatism (Matsuda et al., 2002a,b; Morikawa et al., 2002; Nakamura et al., 2008). Pharmacological studies have shown that an extract of H. coronarium has antiinflammatory and analgesic effects in an animal model (Shrotriya et al., 2007) and showed in vitro inhibition of 5-lipoxygenase (Braga et al., 2000). Several labdane diterpenes were isolated from this plant and some of them showed cytotoxic activity against V-79 cells (Itokawa et al., 1988a) and inhibitory effects on the increase in vascular permeability induced by acetic acid in mice and nitric oxide production in lipopolysaccharide-activated mouse peritoneal macrophages (Matsuda et al., 2002a,b) and on the release of beta-hexosaminidase from RBL-2H3 cells (Morikawa et al., 2002). Recently, coronarin D, a major component of the rhizomes of this plant, showed inhibition of both constitutive and inducible nuclear factor-KB pathway activation, leading to apoptosis, inhibition of
* Corresponding author at: Chulabhorn Research Institute, Vipavadee Rangsit Road, Bangkok 10210, Thailand. Tel.: +662 5740622x1306; fax: +662 5742027. E-mail addresses: [email protected], [email protected] (S. Techasakul).
invasion, and suppression of osteoclastogenesis (Kunnumakkara et al., 2008). In the course of our investigation on biologically active substance from H. coronarium, we previously reported the isolation of some labdane-type diterpenes (Chimnoi et al., 2008). In the present work, to obtain the minor components, we decided to increase the amount of plant material and reinvestigate the chemical constituents of the rhizomes. One new labdane diterpene, named 7b-hydroxy-(E)-labda-8(17),12-diene-15,16dial (1) (Fig. 1), together with eight known compounds (2–9) (supporting information) were obtained. Among the known compounds, compounds 7–9 were isolated for the first time from this plant. Moreover, the cytotoxicity of some isolated compounds was evaluated against various cancer cell lines. 2. Results and discussion Compound 1 was obtained as colorless gum. The molecular composition of C20H30NaO3 was inferred from high-resolution ESITOF-MS ([M+Na]+ at m/z 341.2085). The IR spectrum of 1 showed absorption bands ascribable to hydroxyl (3425 cm1), aldehydic carbonyl (1725 cm1), a,b-unsaturated aldehydic carbonyl (1679 cm1) and olefinic functional groups (1644 cm1). The 1H NMR spectrum of 1 (see Table 1) displayed three methyl proton signals at dH 0.92 (s, H3-18), 0.83 (s, H3-19), and 0.72 (s, H3-20) and one exomethylene moiety at dH 5.25 (s, H-17) and 4.57 (s, H-17). These are characteristic of the C-8 exomethylene group of labdane
1874-3900/$ – see front matter ß 2009 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2009.06.003
N. Chimnoi et al. / Phytochemistry Letters 2 (2009) 184–187 Table 1 1 H NMR and
13
C NMR spectroscopic data of compounds 1 in CDCl3.
Position
1
1a 1b 2a 2b 3a 3b 4 5 6a 6b 7a 8 9 10
1.04 (ddd, J = 13, 4, 4) 1.69 (br d, J = 12) 1.55a 1.55a 1.19a 1.45 (br d, J = 13)
a
185
H
1.19a 2.12 (m) 1.29 (br t, J = 12) 4.03 (dd, J = 11, 5) 1.83 (br d, J = 10)
13
Position
1
39.0
11
19.2
12
2.40 (ddd, J = 17, 11, 7) 2.53 (ddd, J = 17, 6, 3) 6.74 (t-like, J = 7)
41.7
13
33.5 52.9 33.7
14 15 16
3.40 (d, J = 17)/3.47 (d, J = 17) 9.65 (s) 9.39 (s)
39.3 197.2 193.4
73.6 149.8 54.5 39.2
17 18 19 20
4.57 0.92 0.83 0.72
104.8 33.5 21.6 14.3
C
13
H
C
24.3 159.0 135.1
(s)/5.25 (s) (s) (s) (s)
Overlapping peaks.
diterpenoids (Kimbu et al., 1987). In addition, the analyses of the 1 H NMR spectrum (CDCl3) (Table 1) indicated the existence of two aldehydic groups [dH 9.39 (s, H-16) and 9.65 (s, H-15)] and one oxymethine [dH 4.03 (dd, J = 11, 5 Hz, H-7)]. Using 13C NMR, DEPT and HMQC experiments, 20 resonances were observed and assigned as two aldehydic groups [dC 193.4 (C-16) and 197.2 (C15)], six sp3 methylenes, one sp2 methylene [dC 104.8 (C-17)], two sp3 methines, one oxymethine [dC 73.6 (C-7)], one sp2 methine, three methyls, two sp3 quaternary carbons and two sp2 quaternary carbons. The 1H and 13C NMR (CDCl3) spectra (see Table 1) of the side chain attached to C-9 of compound 1 were comparable with those of (E)-labda-8(17),12-diene-15,16-dial (6) (Morita and Itokawa, 1988). In the 1H–1H COSY spectrum of compound 1 (Fig. 2), the olefinic proton resonating at dH 6.74 (t-like, J = 7 Hz, H12) correlated with an allylic methylene at dH 2.40 (ddd, J = 17, 11, 7 Hz, H-11) and 2.53 (ddd, J = 17, 6, 3 Hz, H-11), which in turn coupled to H-9 at dH 1.83 (br d, J = 10 Hz). In the HMBC spectrum, long-range correlations were observed between the aldehydic proton of H-15 to C-13 and C-14, the aldehydic proton of H-16 to C12 and C-13, the sp2-methine proton of H-12 to C-9, C-11, C-14, and C-16, and the allylic methylene proton of H-11 to C-8, C-9, C12, and C-13 (Fig. 2). These confirmed that the 12-en-15,16-dial moiety was attached to C-11, which in turn was attached to C-9 of the decalin ring and formed the normal six-carbon side chain of labdane diterpenes.
The 1H and 13C NMR of the decalin system of 1 was virtually superposable with that of hedychilactone A (11) (Matsuda et al., 2002a,b). In the 1H–1H COSY spectrum of compound 1 (Fig. 2), a methine group bearing a hydroxyl group at dH 4.03 [dd, J = 11, 5 Hz, H-7] correlated with the methylene protons at dH 2.12 (m, H-6a) and 1.29 (br d, J = 12 Hz, H-6b), which in turn coupled to the signal at dH 1.19 (m, H-5). Long range correlations were established in the HMBC spectra between H-7 and C-6, C-8 and C-17 (Fig. 2). Therefore, it was concluded that a hydroxyl group was attached to C-7 of the decalin system. The configuration of compound 1 was suggested by the correlations observed in a NOESY spectrum (Fig. 3). Correlations were observed from H-7 to H-5, H-6a and H-9. Therefore, the hydroxyl group at C-7 was assigned to be in b-configuration. Moreover, Oh et al. (2006) recently synthesized (+)-hedychilactone A (11) and 7-epi-hedychilactone A. The 1H chemical shift observed for H-7 of (+)-hedychilactone A and 7-epi-hedychilactone A was 3.98 ppm and 4.37 ppm, respectively and H-7 of 1 appeared at d 4.03. In addition, it is worth to remark that the 13C NMR signals of C-17 appeared at dC 104.3 and 109.9 for hedychilactone A (11) and 7-epi-hedychilactone A, respectively (Oh et al., 2006), (dC 104.8 for compound 1). These supported the b-configuration of 7-OH. The correlations in the NOESY spectrum (Fig. 3) between H-16 and H12 and between H-11 and H-14 suggested 12E-configuration of 1. Based on the above evidence, we were able to confirm the skeleton of compound 1 to be 7b-hydroxy-(E)-labda-8(17),12-diene-15,16dial which is a new metabolite. In our previous report (Chimnoi et al., 2008), coronarin D was isolated as a C-15 epimeric mixture. In the present work, coronarin D acetate (10) was prepared to confirm the presence of C-15 epimers of 2. The duplicated signals of 1H and 13C NMR of 10 were clearly seen particularly for the acetyl group on C-15. All of these data suggested that coronarin D (2) was isolated in the form of a C15 epimeric mixture. Additionally, eight known compounds (supporting information), namely C-15 epimers of coronarin D (2) (Chimnoi et al., 2008), coronarin F (3) (Itokawa et al., 1988b), C-14 epimers of
Fig. 2. (a) Key 1H–1H COSY couplings (boldface bonds) of 1 (b) HMBC correlations of 1.
Fig. 3. Key NOESY interactions of compound 1.
Fig. 1. Chemical structure of compound 1.
N. Chimnoi et al. / Phytochemistry Letters 2 (2009) 184–187
186 Table 2 Cytotoxicity data for compounds 1, 2, 4, 5, and 10. Compounds
1 2 4 5 10 Etoposide Doxorubicin
Cell lines (IC50, mg ml1) S102
HuCCA-1
A549
MOLT-3
KB
HeLA
MDA-MB231
T-47D
HL-60
P388
HepG2
NT 21.5 11.5 NT NT NT 5.0
27 4.0 3.4 2.7 3.5 4.0 0.42
47 27.5 5.5 27 NT NT 0.3
2.81 3.91 1.02 0.44 3.48 0.018 NT
NT 3.0 2.8 NT 3.0 0.3 NT
44 4.0 2.9 2.7 3.5 0.5 0.36
NT 2.5 2.3 NT 2.5 0.3 NT
NT 4.8 2.7 NT 4.5 0.05 NT
NT 4.4 2.1 NT 4.6 0.7 NT
NT 1.4 0.68 NT 1.7 0.07 NT
33.5 18.0 5.3 7.75 NT 14 0.28
NT, not test; S102, hepatocellular carcinoma; HuCCA-1, cholangiocarcinoma; A549, lung adenocarcinoma; MOLT-3, T-lymphoblast (acute lymphoblastic leukemia); KB, epidermoid carcinoma; HeLA, cervical carcinoma; MDA-MB231, hormone-independent breast cancer; T-47D, hormone-dependent breast cancer; HL-60, human promyelocytic leukemia cell; P388, mouse lymphoid neoplasm; HepG2, hepatoblastoma.
isocoronarin D (4) (Taveira et al., 2005), coronarin B (5) (Itokawa et al., 1988a), (E)-labda-8(17),12-diene-15,16-dial (6) (Morita and Itokawa, 1988), (E)-15,16-bisnorlabda-8(17),11-dien-13-one (7) (Itokawa et al., 1980c), and a mixture of eicosyl- and docosyl-(E)ferulates (8 and 9) (Jayaprakasam et al., 2006) were isolated from this plant, and their structures were confirmed by the comparison of their spectral data with those in the literature. The in vitro cytotoxicity of labdane 1, 2, 4, 5, and 10 on some cancer cell lines was defined through MTT assays (Mosmann, 1983). The IC50 values are presented in Table 2. Among the tested compounds, compound 4 was shown to be the most cytotoxic in all cancer cell lines, with IC50 at about 4 mg ml1, except for a moderate activity for S102. Compound 2, which is the major component, also displayed pronounced cytotoxicity against most of the cell lines but weak activity against S102, A549, and HepG2. Compound 5 showed remarkable cytotoxicity against HuCCA-1, HeLA, and MOLT-3 cell line but moderate and weak activities against HepG2 and A549, respectively. Coronarin D acetate (10) was revealed to have almost identical cytotoxicity to 2. Nevertheless, it is more stable than coronarin D (2) due mainly to the hemiacetal in 2 being protected by the acetyl group. Compound 1 showed only significant cytotoxicity against MOLT-3 but weak activity against HuCCA-1, A549, HeLA, and HepG2 cell lines. 3. Experimental 3.1. General experimental procedures 1 H, 13C and 2D NMR spectra were recorded in CDCl3 on a Bruker AVANCE 400 (400 MHz for 1H and 100 MHz for 13C). Chemical shifts were given in ppm (d) and referenced to the solvent signals at 7.27 ppm and 77.0 ppm for 1H and 13C NMR, respectively. EI-MS (direct insertion probe, 70 eV) mass spectra were measured using a Finnigan POLARIS Ion Trap Mass Spectrometer. HR-TOF-MS (direct infusion) mass spectra were recorded on a Bruker MicroTOF. IR spectra were determined on a Perkin Elmer Spectrum One (neat sample) and a Perkin Elmer 1760X FT-IR spectrometer (on KBr disc). UV spectra were performed with a Shimadzu UV-1700 Pharmaspec spectrometer. Optical rotation was measured with sodium D line (590 nm) on a JASCO P-1020. Column chromatography was carried out on silica gel and TLC-silica gel 60 F254 obtained from Merck.
3.2. Plant material The rhizomes of H. coronarium were collected from Bangkok, Thailand, in 2005. A voucher specimen (CRI 567) has been deposited at the laboratory of Natural Products, Chulabhorn Research Institute, Bangkok, Thailand. The rhizomes were dried indoors and then extracted with dichloromethane as described below.
3.3. Extraction and isolation The air-dried rhizomes of H. coronarium (2.1 kg) were macerated twice with dichloromethane (15 L 7 days) at room temperature and then concentrated under reduced pressure to produce a dark brown oil (120 g). After which, 60 g of crude extract was subjected to silica gel VLC and eluted with 2%, 5%, 7%, and 10% of ethyl acetate in hexane to yield fractions 1–4, respectively. Each fraction was then subjected to silica gel column chromatography with a gradient of ethyl acetate–hexane system. Compound 7 (5 mg) was obtained from fraction 1. The separation of fraction 2 led to compounds 3 (3 mg), 5 (11 mg) and 6 (3 g). A mixture of compounds 8 and 9 (13 mg) was isolated from fraction 3. Finally, the repeated silica gel column of fraction 4 resulted in the isolation of 1 (22 mg), 2 (17.5 g), and a mixture of 2 and 4 (428 mg). Careful recrystallization of a mixture of 2 and 4 using hexane with a trace amount of dichloromethane provided compound 4 (22 mg) as white needles. 7b-hydroxy-(E)-labda-8(17),12-diene-15,16-dial (1): Color less gum; ½a25 D þ 11:6 (c 0.44, CHCl3); UV (MeOH) lmax (log e): 234 (4.02) nm; IR (neat) nmax 3425, 2927, 2845, 1725, 1679, 1644, 1459, 1389, 1367, 1266, 1163, 1101, 1021, 898, 735 cm1; 1H and 13 C NMR data are given in Table 1; EI-MS m/z 318 ([M]+, 2), 301 ([M–OH]+, 14), 300 ([M–H2O]+, 17), 285 ([M–OH–CH3]+, 9), 271 (15), 257 (13), 239 (10), 231 (14), 207 (13), 194 (47), 189 (17), 176 (33), 123 (100), 109 (32), 105 (38), 95 (34), 91 (43), 81 (40), 79 (49), 77 (26), 67 (39); HRMS (ESI-TOF) m/z 341.2085 (calcd for C20H30NaO3 [M+Na]+ 341.2087). C-15 epimers of coronarin D acetate (10): A solution of compound 2 (500 mg, 1.57 mmol) in pyridine (8 ml) and acetic anhydride (1.5 ml, 15.87 mmol) was stirred at room temperature for 3 h. Pyridine was then removed by partition with dichloromethane and 10% aq. HCl. The dichloromethane layer was combined and concentrated under reduced pressure to dryness. The residue was purified by silica gel column chromatography (10% ethyl acetate in hexane) to yield 10 (355 mg, 63%) as colorless oil. ½a25 D þ 14:0 (c 1.09, CHCl3); UV (MeOH) lmax (log e): 226 (3.79) nm; IR (KBr) nmax 2928, 2868, 1765, 1678, 1645, 1459, 1441, 1367, 1337, 1217, 1166, 1078, 973, 890 cm1; 1H NMR (400 MHz, CDCl3) d: 6.83 (1H, m, H-12), 6.66 (1H, m, H-15), one set of 4.39 (1H, d, J = 1 Hz, H-17) and 4.85 (1H, d, J = 1 Hz, H-17), one set of 4.34 (1H, d, J = 1 Hz, H-17) and 4.83 (1H, d, J = 1 Hz, H-17), 3.13 (1H, m, H-14), 2.82 (1H, br t, J = 14 Hz, H-14), 2.40 (1H, m, H-7b), 2.36 (1H, m, H-11), 2.24 (1H, m, H-11), 2.12/2.11 (3H, s, –OAc), 2.01 (1H, ddd, J = 13, 13, 5 Hz, H-7a), 1.89 (1H, br t, J = 11 Hz, H-9), 1.75 (1H, m, H6a), 1.69 (1H, m, H-1b), 1.59 (1H, m, H-2b), 1.52 (1H, m, H-2a), 1.42 (1H, br d, J = 13 Hz, H-3b), 1.34 (1H, dddd, J = 13, 13, 13, 3 Hz, H-6b), 1.20 (1H, m, H-3a), 1.13 (1H, ddd, J = 12, 5, 3 Hz, H-5), 1.08 (1H, m, H-1a), 0.89 (3H, s, H-18), 0.82 (3H, s, H-19), 0.73 (3H, s, H20); 13C NMR (100 MHz, CDCl3) d: 169.19/169.17 (–OCOCH3), 168.72/168.70 (C-16), 148.03/148.00 (C-8), 144.76/144.67 (C-12),
N. Chimnoi et al. / Phytochemistry Letters 2 (2009) 184–187
122.24/122.19 (C-13), 107.50/107.42 (C-17), 92.34/92.33 (C-15), 56.13/56.12 (C-9), 55.38 (C-5), 41.99/41.98 (C-3), 39.47 (C-10), 39.34/39.32 (C-1), 37.77 (C-7), 33.57 (C-18), 33.55 (C-4), 31.95/ 31.85 (C-14), 25.74/25.71 (C-11), 24.09/24.07 (C-6), 21.71 (C-19), 20.90/20.88 (–OCOCH3), 19.32/19.30 (C-2), 14.37/14.35 (C-20); EIMS m/z 360 ([M]+, 4), 345 ([M–CH3]+, 24), 300 ([M–AcOH]+, 100), 285 ([M–AcOH–CH3]+, 59), 215 (42), 176 (73), 161 (35), 137 (68), 121 (45), 95 (50), 91 (43), 81 (54), 79 (45), 43 (61); HRMS (APCITOF) m/z 361.2375 (calcd for C22H33O4 [M+H]+ 361.2373). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytol.2009.06.003. References Braga, F.C., Wagner, H., Lombardi, J.A., de Oliverira, A.B., 2000. Brazilian plant species for in vitro inhibition of 5-lipoxygenase. Phytomedicine 6, 447–452. Chimnoi, N., Pisutjaroenpong, S., Ngiwsara, L., Dechtrirut, D., Chokchaichamnankit, D., Khunnawutmanotham, N., Mahidol, C., Techasakul, S., 2008. Labdane diterpenes from the rhizomes of Hedychium coronarium. Nat. Prod. Res., Part A 22 (14), 1255–1262. Itokawa, H., Morita, H., Katou, I., Takeya, K., Cavalheiro, A.J., de Oliveira, R.C.B., Ishige, M., Motidome, M., 1988a. Cytotoxic diterpenes from the rhizomes of Hedychium coronarium. Planta Med. 54, 311–315. Itokawa, H., Morita, H., Takeya, K., Motidome, M., 1988b. Diterpenes from rhizomes of Hedychium coronarium. Chem. Pharm. Bull. 36, 2682–2684. Itokawa, H., c Morita, M., Mihashi, S., 1980. Labdane and Bisnorlabdane type Diterpenes from Alpinia speciosa K. SCHUM. Chem. Pharm. Bull. 28, 3452– 3454. Jayaprakasam, B., Vanisree, M., Zhang, Y., Dewitt, D.L., Nair, M.G., 2006. Impact of alkyl esters of caffeic and ferulic acids on tumor cell proliferation, cyclo-
187
oxygenase enzyme, and lipid peroxidation. J. Agric. Food Chem. 54, 5375– 5381. Kimbu, S.F., Ngadjui, B., Sondengam, B.L., Njimi, T.K., Connolly, J.D., Fakunle, C.O., 1987. A new labdane diterpenoid from the seeds of Aframomum daniellii. J. Nat. Prod. 50, 230–231. Kunnumakkara, A.B., Ichikawa, H., Anand, P., Mohankumar, C.J., Hema, P.S., Nair, M.S., Aggarwal, B.B., 2008. Coronarin D, a labdane diterpene, inhibits both constitutive and inducible nuclear factor-KB pathway activation, leading to potentiation of apoptosis, inhibition of invasion, and suppression of osteoclastogenesis. Mol. Cancer Ther. 7, 3306–3317. Matsuda, H., Morikawa, T., Sakamoto, Y., Toguchida, I., Yoshikawa, M., 2002a. Antiinflammatory principles and three new labdane-type diterpenes, hedychilactones A, B and C, from the rhizome of Hedychium coronarium. Heterocycles 56, 45–50. Matsuda, H., Morikawa, T., Sakamoto, Y., Toguchida, I., Yoshikawa, M., 2002b. Labdane-type diterpenes with inhibitory effects on increase in vascular permeability and nitric oxide production from Hedychium coronarium. Bioorg. Med. Chem. 10, 2527–2534. Morikawa, T., Matsuda, H., Sakamoto, Y., Ueda, K., Yoshikawa, M., 2002. New farnesane-type sesquiterpenes, hedychiols A and B 8, 9-diacetate, and inhibitors of degranulation in RBL-2H3 cells from the rhizome of Hedychium coronarium. Chem. Pharm. Bull. 50, 1045–1049. Morita, H., Itokawa, H., 1988. Cytotoxic and antifungal diterpenes from the seeds of Alpinia galangal. Planta Med. 54, 117–120. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Nakamura, S., Okazaki, Y., Ninomiya, K., Morikawa, T., Matsuda, H., Yoshikawa, M., 2008. Medicinal flowers. XXIV. Chemical structures and hepatoprotective effects of constituents from flowers of Hedychium coronarium. Chem. Pharm. Bull. 56, 1704–1709. Oh, S., Jeong, I.H., Shin, W., Wang, Q., Lee, S., 2006. Synthesis and biological activity of (+)-hedychilactone A and its analogs from (+)-sclareolide. Bioorg. Med. Chem. Lett. 16, 1656–1659. Shrotriya, S., Ali, M.S., Saha, A., Bachar, S.C., Islam, M.S., 2007. Anti-inflammatory and analgesic effects of Hedychium coronarium KOEN. Pak. J. Pharm. Sci. 20, 47–51. Taveira, F.N., Oliveira, A.B., Souza Filho, J.D., Braga, F.C., 2005. Epimers of labdane diterpenes from the rhizomes of Hedychium coronarium. J. Koenig. Rev. Bras. Farmacogn. 15, 55–59.