Cytotoxic Labdane Diterpenes from Hedychium ellipticum Buch.-Ham. ex Sm.

In order to reveal the constituents and their biological activities, we carried out a phytochemical study on Hedychium ellipticum Buch.-Ham. ex Sm. (Zingiberaceae). Ten labdane diterpenoids (1–10) were isolated from the rhizomes of H. ellipticum for the first time. Their structures were identified on the basis of spectroscopic analyses including two-dimensional NMR and comparison with literature data. All of these compounds were evaluated for their antimycobacterial activity against Mycobacterium tuberculosis and cytotoxicity against KB, MCF7, NCI-H187 and Vero cells. The result showed that compounds 1 and 7 exhibited moderate activity against Mycobacterium tuberculosis and compounds 4, 6 and 7 displayed remarkable cytotoxic activity. This is the first report on the presence of all compounds in H. ellipticum and the first time that their structure activity relationship has been discussed.


Introduction
Several species of Hedychium are used widely in traditional medicines for treatment of skin diseases, stomach ailments, cough and apoplexy, and as an analgesic, anti-inflammatory, antimicrobial and anti-rheumatic treatment [1,2]. Previous phytochemical studies on Hedychium species revealed the presence of diarylheptanoids, sesquiterpenes and diterpenes, some of which showed hepatoprotective effects [3], antiinflammatory activities [4] and cytotoxicity against various cancer cells [5][6][7][8][9][10][11][12]. Hedychium ellipticum Buch.-Ham. ex Sm. (Zingiberaceae), cultivation from Doi Suthep overlooking Chiang Mai in Thailand has bright green rolled bracts. No detailed phytochemical and pharmacological investigation on H. ellipticum has been reported, except for a report on the chemical composition of leave and rhizome essential oils, which showed that (E)-nerolidol and 1,8-cineole were the major constituents [13,14]. The methanolic extracts of rhizomes showed potent activity to inhibit the biosynthesis of leukotriene B 4 in bovine polymorphonuclear leukocytes and the essential oils exhibited moderate-to-good Fe 2+ chelating activity [15]. In our preliminary investigation on the bioactivities of Hedychium plants, we found that the crude n-hexane and CH 2 Cl 2 extracts of H. ellipticum rhizomes showed moderated antimycobacterial activity against Mycobacterium tuberculosis and cytotoxic activity against human cancer cell lines. Therefore, the aim of the present work is to determine the chemical constituents from the rhizomes of H. ellipticum and to evaluate their antimycobacterial activity against Mycobacterium tuberculosis and cytotoxicity against KB, MCF7, NCI-H187 and Vero cells. The structure-activity relationships of all isolated compounds were also reported.

Results
Phytochemical investigation of the rhizomes of H. ellipticum resulted in the isolation of 10 labdane-type diterpenes: coronarin E (1) [10], (E)-15,16-bisnorlabda-8(17),11-dien-13-one (2) [16], (E)-14,15,16-trinorlabda-8(17),11-dien-13-oic acid (3) [3], villosin (4) [10], (E)-labda-8(17),12-dien-15,16dial (5) [3], 15-methoxylabda-8(17),11,13-trien-15,16-olide (6) [17], 16-hydroxylabda-8(17),11,13-trien-15,16-olide (7) [17], coronarin D (8) [8], zerumin A (9) and zerumin B (10) [18] (Figure 1). Their structures were determined by spectroscopic methods and by comparison of their spectral data with those reported in the literature. These compounds are known but they were isolated from this plant for the first time. This phytochemical investigation of H. ellipticum has shown that its chemistry differs from other species of Hedychium. Compounds 1, 4, 5, 6 and 8 were isolated from the rhizomes of Hedychium coronarium J. Koenig [3][4][5]8,12], Hedychium villosum Wall. [1] and Hedychium gardnerianum Sheppard ex Ker Gawl. [10]. Interestingly, compounds 2, 3, 7, 9 and 10 have not been reported from any species of the Hedychium and could be regarded as an important chemotaxanomic marker. All isolated compounds were evaluated for their antimycobacterial activity against Mycobacterium tuberculosis and cytotoxicity against three human tumor cell lines, human oral cavity cancer cell (KB), human breast cancer cell (MCF7) and human small cell lung cancer cell (NCI-H18) and non-cancerous (Vero) cell lines. The results of the antimycobacterial activity against Mycobacterium tuberculosis of all compounds are given in Table 1. Compounds 7 and 1 exhibited moderate activity against Mycobacterium tuberculosis with MIC 6.25 and 12.5 μg/mL, respectively, while compounds 2, 3-6, and 8-10 were inactive. All compounds exhibited over 20-fold inferior than standard drug streptomycin. To the best of our knowledge, this is the first report for the antimycobacterial activity of all compounds. In a comparison of all compounds, furan ring or α,β-unsaturated γ-lactone ring is essential for the antimycobacterial activity. Change in an α,β-unsaturated γ-lactone ring did affect the activity as indicated in the case of compounds 4, 6, 7 and 10. The hydroxyl group at C-16 significantly enhanced the activity of compound 7, which suggested that the relative hydrophilicity of this part of the molecule contributes to the activity. However, lack in activity of compound 10, implying that the orientation of cyclic hemiacetal α,β-unsaturated γ-lactone is also influenced the activity. Although the structure-activity relationships of these compounds were not conclusively determined, this finding provided a useful starting point for antimycobacterial activity of labdan-type diterpenoids.   In previous studies, some labdane diterpenes from the species of Hedychium were found to show cytotoxicity. Therefore, compounds 1-10 were also evaluated for cytotoxic activities against three cancer cell lines: human oral cavity cancer cell (KB), human breast cancer cell (MCF7) and human small cell lung cancer cell (NCI-H18) ( Table 1). All of the 10 labdane-type diterpenoids exhibited generally stronger cytotoxic effect against NCI-H187 cell than those against KB and MCF-7 cells. Results revealed that compounds 4, 6 and 7 possessed potent cytotoxic activity against the NCI-H187 cell with an IC 50 value of 0.12, 0.90 and 0.72 µg/mL, respectively. In addition, compound 4 showed higher cytotoxicity than a positive control, ellipticine (IC 50 0.44 µg/mL) which agree with previous reported [10]. Its toxicity was undetectable at concentration less than 50 µg/mL and very selective for NCI-H187 cell. Furthermore, compound 7 was also possessed potent cytotoxic activity against the KB cell and moderate activity against the MCF7 cell with an IC 50 value of with an IC 50 value of 0.92 and 2.89 µg/mL, respectively. However, compound 7 was very toxic to Vero cells (IC 50 5.37 µg/mL) and not selective for tested cell lines. Compounds 2, 3, 5, 8 and 9 showed weak to no effect, while 1 and 10 showed moderate to weak effect on all tested cell lines. It should be noted that compound 1 has been reported to exhibit moderate cytotoxicity against P388, B16 and SNU-1 cells with IC 50 10.0, 18.8 and 8.0 µg/mL [19] but weak cytotoxicity against NCI-H187 cell with IC 50 49.73 µM [10]. Compounds 5 and 8 have been reported to exhibit cytotoxic activity against V-79 cell with IC 50 18.5 and 17.0 µg/mL, respectively [5,12]. Compound 8 exhibited weaker cytotoxic activity against KB (40.01 µg/mL) and NCI-H187 (25.72 µg/mL) cells than previous reported which showed moderate activities with IC 50 14.7 and 9.8 µg/mL [8]. However, it has been reported to displayed potent cytotoxic activity against A-549 cell line with the LC 50 value of 14.0 µM and weak cytotoxicity against SK-N-SH, MCF-7 and HeLa cells with LC 50 32.0, 21.9 and 60.6 µM, respectively [12]. To the best of our knowledge, this is the first report for the cytotoxic activities of compounds 2, 3, 6, 7, 9 and 10. On comparing the cytotoxic activity of compounds 1-10, drastic decrease or lack in cytotoxicity of compounds 1-3, 5, 8 and 9 against tested cell lines, implying that an α,β-unsaturated γ-lactone ring plays a crucial role in mediating cytotoxic activity. Furthermore, change in the lactone moiety did affect the cytotoxicity. In a comparison of compound 4 with 6 and 10, the hydroxyl or methoxyl substitution at C-15 significantly reduced the cytotoxicity against all the tested cells. Nevertheless, among the compounds 7 and 10, the orientation of cyclic hemiacetal α,β-unsaturated γ-lactone also influenced the cytotoxicity against all the tested cells. This is similar to their cytotoxicities against K562 cancer cell lines which was reported previously [6]. It is noteworthy to mention that the hydroxyl group at C-16 significantly enhanced the cytotoxicity of compound 7 against KB and MCF-7 cell lines, which suggested that the relative hydrophilicity of this part of the molecule contributes to the cytotoxicity. However, the lack of trans double bond conjugated with the lactone ring seemed to decrease cytotoxic activity, as indicated in the case of compound 10.

Plant Material
The rhizomes of H. ellipticum were collected from Chiang Mai province of Thailand, in January 2007. A voucher specimen (06-609 J.F. Maxwell) was deposited with the CMU Herbarium, Department of Biology, Faculty of Science, Chiang Mai University, Thailand.

Extraction and Isolation
Air-dried powdered rhizome (920 g) of H. ellipticum was successively extracted with n-hexane and CH 2 Cl 2 (4 Lˆ3) at room temperature. The solvents were combined and evaporated to dryness under reduced pressure to yield a brownish residue of n-hexane (44.26 g) and CH 2 Cl 2 (39.50 g) extracts.

Cytotoxic Activity
The cytotoxicity against human tumor cells was determined by resazurin microplate assay (REMA) which was a modified method of the use of a fluorescent dye for mammalian cell cytotoxicity [21]. The cytotoxicity against non-cancerous cells, African green monkey kidney (Vero) cell line was determined by the green fluorescent protein (GFP) detection [22]. Briefly, cells at a logarithmic growth phase were harvested and diluted to 9ˆ10 4 cells/mL in fresh medium. Successively, 5 µL of test sample, diluted in 10% DMSO and 45 µL of cells suspension were added to 384-well plates. Plates were incubated at 37˝C in 5% CO 2 incubator for 5 days. After that, 12.5 µL of 62.5 µg/mL resazurin solution was added to each well and the plates were then incubated at 37˝C for 4 h. Fluorescence signal was measured using SpectraMax M5 multidetection microplate reader at dual wavelengths of 530 and 590 nm. For non-cancerous cells such as the African green monkey kidney (Vero) cell line, the cytotoxicity was determined by the green fluorescent protein (GFP) detection [22]. Briefly, cells at a logarithmic growth phase were harvested and diluted to 3.3ˆ10 4 cells/mL in fresh medium. Successively, 5 µL of test sample, diluted in 0.5% DMSO and 45 µL of cells suspension were added to 384-well plates. Plates were incubated at 37˝C in 5% CO 2 incubator for 4 days. Fluorescence signals were measured on day zero, for subsequent background subtraction, and on day 4 using SpectraMax M5 multi-detection microplate reader in the bottom-reading mode with excitation and emission wavelengths of 485 and 535 nm. IC 50 values were derived from dose-response curves, using six concentrations of 2-fold serially diluted samples, by the SOFTMax Pro software (Molecular device). The standard drugs ellipticine exhibited IC 50 values against KB, NCI-H187 and Vero cell at 0.302, 0.440 and 1.345 µg/mL and doxorubicin exhibited IC 50 values against MCF7 at 0.663 µg/mL. The selectivity index corresponded to the IC 50 value determined for cytotoxicity of isolated compounds on Vero cells divided by the IC 50 determined for cancer cells.