New Cytotoxic Cembranoid from Indonesian Soft Coral Sarcophyton sp.

CONTEXT: Sarcophyton is a soft coral species that contains various secondary metabolites with cytotoxic activity. The production of cytotoxic compounds in soft corals is suggested as their allelochemical to win space competition. Therefore, if a particular soft coral species dominates a reef area, it may suggest to contain interesting bioactive compounds. AIMS: This research aimed to characterize the cytotoxic compounds in dominant soft coral species (Sarcophyton sp.) on the reef at the Western side of Mahengetang Island, Indonesia. SUBJECTS AND METHODS: Isolation of cytotoxic compounds through ethanol extracts had been done with preparative high-performance liquid chromatography and bioassay-guided fractionation by MCF-7 (breast) cancer cell lines. The structures of each cytotoxic compounds were elucidated on the basis of mass and nuclear magnetic resonance spectroscopic studies.
RESULTS: Elucidation through all compounds found a new cembranoid, namely, 2-hydroxy-crassocolide E (1), alongside with 5 known cembranoids; sarcophytoxide (2), sarcrassin E (3), 3,7,11-cembreriene-2,15-diol (4), 11,12-epoxy-Sarcophytol A (5), and sarcophytol A (6). All of these cembranoids were showed to inhibit the growth of MCF-7 (breast) cancer, with 50% inhibition of tumor cell lines growth lower than 30 mg/L.
CONCLUSIONS: Results of this study suggest that a soft coral species which dominate a reef area is a potential source for various bioactive compounds.
SUMMARY: Elucidation of cytotoxic compounds from Sarcophyton sp. that dominate the reef at Mahengetang Island Indonesia revealed a new compound (2-hydroxy-crassocolide E) alongside with 5 known cembranoid compounds. Abbreviations Used: SCUBA: Self-contained underwater breathing apparatus; HPLC: High performance liquid chromatography; NMR: Nuclear magnetic resonance; IT-TOF: Ion trap-time of flight; MTT: 3-(4,5-dimethylthiazol-2-yl)-2; 5-diphenyltetrazolinon bromide; DEPT: Distortionless enhancement by polarisation transfer; COSY: Correlation spectroscopy; HMBC: Heteronuclear multiplebond correlation; NOE: Nuclear overhauser effect; IG50: 50% inhibition growth.

INTRODUCTION

Soft corals are potential sources that contain various bioactive compounds. Diterpenes, sesquiterpenes, furanoditerpenes, terpenoids, and steroids are major secondary metabolites in soft corals which have shown to display a pharmacological activity.[1] Soft corals are known to produce their bioactive compounds as a chemical/defensive weapon in maintaining or win spatial competition at benthic environment.[234] The amount of those bioactive compounds was suggested as a predictor for soft coral invasiveness potential in a coral reef environment.[5] Our previous study has shown a dominant Sarcophyton sp. in the Southern part of Panggang Island, Seribu Islands-Indonesia, which detected to contain high amount of cytotoxic sarcophytoxide.[6] Other studies on soft corals and sponges have also found a similar pattern.[78] Therefore, if a particular soft coral species dominates a reef area, it may suggest that this organism potentially contains an interesting chemical bioactive compounds.

A dominant cover of soft coral Sarcophyton sp. is shown on the reef at the Western part of Mahengetang Island, Indonesia. This condition may suggest to be happened as an impact of natural acidification by huge CO2 seeps from Banua Wuhu underwater volcano. The domination of Sarcophyton sp. in this area is similar to the reef around CO2 hydrothermal vents on the seashore of Iwo-Tori-shima Islands – Japan.[9] Our preliminary study detected higher cytotoxicity of Sarcophyton sp. which was taken from acidification environment on the reef at the Western part of Mahengetang Island compared to normal seawater environment.[10] Therefore, this research aimed to investigate the diversity of bioactive compound in the soft coral Sarcophyton sp. at Mahengetang Island, Indonesia. The study shows Sarcophyton sp. from this area contains 6 cytotoxic cembranoid compounds; a new 2-Hydroxy-crassocolide E cembranoid (1), alongside with known compounds: 11,12-epoxy sarcophytol A (2), sarcopytol A (3), sarcophytoxide (4), 3,7,11-cembreriene-2,15-diol (5), and sarcrassin E (6). This study describes the structural elucidation of 2-Hydroxy-crassocolide E cembranoid and cytotoxic activity of cembranoids from Sarcophyton sp. in Mahengetang Island, Indonesia.

SUBJECTS AND METHODS

General methods

Flash vacuum chromatography was performed using Bulk Phenomenex C18 (50 µm). Preparative high-performance liquid chromatography (HPLC) was performed using a Shimadzu C18 column (250 mm × 21 mm) attached to a Shimadzu Preparative HPLC system with a fraction collector. 1D and 2D-NMR spectra were recorded on Jeol ECS 400 MHz NMR spectrometer, with spectra referenced to 1H and 13C resonances in the deuterated solvents. Accurate mass spectrometric data were analyzed by Shimadzu ion-trap-time-of-flight (IT-TOF) mass spectrometer.

Animal materials

Sarcophyton sp. sample was collected from the Western side of Mahengetang Island, Indonesia, at a depth of 5 m by SCUBA. Soft coral taxonomy was identified by morphological inspection according to Fabricius and Alderslade.[11] Two hundred and fifty grams of sample was immersed in 500 mL ethanol and placed in a cool box containing ice packs for preservation along transportation to the laboratory. The sample was then exhaustively extracted with ethanol, to yield the extract that was used for isolation study. A voucher of sample has been lodged and preserved in Indonesia Research Centre for Marine and Fisheries Product Processing and Biotechnology, Indonesia.

Extraction, isolation, and cytotoxic study

The crude ethanol extract was filtered through a plug of reversed-phase C18 silica using methanol and methylene chloride (1:1) as eluent. The solvent on the extract was then removed with freeze dryer and the resultant dry extract was subjected to a preparative reversed-phase C18 HPLC (15 mL/min in 60 min, gradient elution from 20% acetonitrile: H2O to 100%). Fractions were collected every 30 s. Six fractions were found to be active in cytotoxic testing. Cytotoxic activity of each fraction was analyzed against MCF-7 cell lines, based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolinon bromide (MTT) assay of Zachary.[12] Human breast tumor cell lines (MCF-7) were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 0.5% fungizone, and 2% Penicillin-Streptomycin. The cells were plated, and CO2 incubated at 10,000 cells/well in 37°C temperature for 24 h. After that, the cell-growth medium was removed from each well and leaved the wells containing only with tumor cell attached. The wells were added with tested cembranoid isolates in a doses ranged from 1000 μg/mL to 1 μg/mL (dissolved in RPMI medium) and further CO2 incubation for 24 h. Three kinds of controls were made for final absorbance reading in a microplate reader, which were control of tumor cells, control of medium, and control of samples. After 24 h, the solution was removed from each well, and 100 µl of MTT reagent (500 µg/ml) was added into each well. The samples were then further CO2 incubated. After 4 h of incubation, 100 µl of 10% sodium dodecyl sulfate was added and then the samples were dark-incubated at room temperature (±27°C). After 12 h of incubation, 570 nm absorbance of each well was measured by DYNEX microplate reader and tumor cell lines inhibition percentage was calculated by the absorbance of A (tumor cell control), B (sample), C (sample control), and D (medium control) using the following formula and 50% inhibition of tumor cell lines growth (IG50 value) was gained by the regression plot between samples concentration and % inhibition.

RESULTS

Preparative HPLC separation with bioassay-guided fractionation revealed six cytotoxic fractions. 1H NMR analyses confirmed that fractions number 21 (compound 1, 9.7 mg, 0.004% wet weight sample), 24 (compound 2, 10.9 mg, 0.004%), 26 (compound 3, 11.2 mg, 0.004%), 28 (compound 4, 5.36 mg, 0.002%), 32 (compound 5, 7.4 mg, 0.003%), and 38 (compound 6, 12.2 mg, 0.005%) are isolate of cembranoid compounds. Analyses into mass and NMR spectroscopic data showed that compounds 2-6 are known cembranoids, which are sarcophytoxide (2), Sarcrassin E (3), 3,7,11-cembreriene-2,15-diol (4), 11,12-epoxy sarcophytol A (5), and sarcopytol A (6) that already characterized by previous studies.[1314151617] Further bioassay testing (against human breast tumor cell lines MCF-7) of each compound showed all compounds demonstrated cytotoxic activity with IG50 18.13 ppm (1), 12.22 ppm (2), 24.2 ppm (3), 22.27 ppm (4), 18.88 ppm (5), and 20.041 ppm (6).

Meanwhile, compound 1 was isolated as yellow oil. Molecular mass analysis by IT-TOF spectrometer showed this compound has m/z 333.2216. Deduction into mass and nuclear magnetic resonance spectroscopic data generated the possible structure of 1 as it is shown in Figure 1 (alongside with compounds 2-6). The structure of 1 was elucidated from 2D (COSY, HMBC) and 1D selective NOE NMR data as both are shown in Figure 2. Literature searches for this molecule indicated 1 is a new compound.

Figure 1

Structures of new compound 2-hydroxy-crassocoide E (1) alongside with known cembranoids; sarcophytoxide (2), Sarcrassin E (3), 3,7,11-cembreriene-2,15-diol (4), 11,12-epoxy sarcophytol A (5), and sarcopytol A (6) isolated from the ethanolic extracts of Sarcophyton sp. from Mahengetang Island, Indonesia

Figure 2

COSY, HMBC (a) and selective 1D-NOE (b) of 2-hydroxy-Crassocolide E

Compound 1 1H (CDCl3): δ 2.88 (1H, m, H-1), 5.42 (1H, m, H-2), 5.16 (1H, d, J = 9.62 Hz, H-3), 2.33 (1H, m, H-5α), 2.63 (1H, m, H-5β), 1.65 (1H, n, H-6α); 2.02 (1H, m, H-6β), 5.22 (1H, t, J = 12.36 Hz, H-7), 1.47 (1H, m, H-9α), 1.93 (1H, m, H-9β), 2.17 (1H, m, H-10α), 2.18 (1H, m, H-10β), 2.97 (1H, m, H-11), 2.17 (1H, m, H-13α), 2.36 (1H, m, H-13β), 4.09 (1H, m, H-14), 5.65 (1H, d, J = 2.29 Hz, H-17α), 6.39 (1H, d, J = 2.75 Hz, H-17β), 1.90 (3H, s, Me-18), 1.67 (3H, s, Me-19), 1.28 (3H, s, Me-20);13 C (CDCl3): δ 47.71 (C-1), 73.86 (C-2), 123.83 (C-3), 142.89 (C-4), 36.31 (C-5), 25.21 (C-6), 129.29 (C-7), 130.17 (C-8), 37.03 (C-9), 22.70 (C-10), 62.92 (C-11), 60.40 (C-12), 44.57 (C-13), 71.75 (C-14), 170.13 (C-15), 138.26 (C-16), 122.12 (C-17), 16.98 (Me-18), 16.59 (Me-19), 17.31 (Me-20). The accurate mass determination (positive ion mode) of compound 1 revealed an ion at m/z 333.2216.

DISCUSSION

Mass spectrometer analysis of compound 1 indicated this compound has a molecular formula of C20H28O4, calculated from (M+H)+ for C20H29O4 (m/z 333.2216). This result suggests the molecule has five double bond equivalents (DBEs) of unsaturation.1H and 13C NMR data indicated the molecule contained three C=C double bonds (δC123.83, 142.89, 129.29, 130.17, 138.26, and 122.12). 135 DEPT analysis showed one among these double bond carbons is a CH2 (δC122.12) that attaches to tertiary carbon (δC138.26). Furthermore, DEPT also detected this molecule contained a carbonyl (C=O) double bond (δC170.13), epoxide (δC62.92 and 60.40), and hydroxyl functional group (δC73.86). As there showed two cyclic ring (main 14-cembranoid and epoxide), these deductions lead to another cyclic ring which is a cyclic lactone attaches into the main cembranoid ring (similar to sarcophytoxide) to fulfill the requirement for five DBEs of unsaturation.

1H-1H COSY spectrum detected three continuous chains from H2-13 to H-3, via H-14, H-1, and H-2, respectively, from H2-5 to H-7, via H2-6, and H2-9 to H-11, via H2-10. A long-range 1H-13C HMBC couplings were observed between H3-18 to C-3, C-4, and C-5; H3-19 to C-7, C-8, and C-9; and H3-20 to C-11, C-12, and C-13. From these, it was possible to link all of the main atoms in 14 carbons cembranoid ring. The ring of lactone was generated by the data of 1H-13C couplings between H2-17 to C-16, C-15, and C-1. Unfortunately, coupling between lactone oxygen bridge (H-14 to C-15) was not observed. Interpretation of lactone ring interpreted with a turnaround, with HMBC couplings between C-14 to H-2 and H-2 to C-15. Furthermore, a long range between C-14 to H2-17, a strong coupling between H2-17 to C-15, and spectral data comparison of crassocolides E,[18] confirmed the position of lactone ring in cembranoid structure. The evidence of hydroxyl group is attached in position C-2 is shown by 1H-13C HMBC couplings from H-2 to C-1, C-3, and C-14. Furthermore, it was also detected a long-range 1H-13C coupling from H2-17 to C-2. All of these leading to the planar structure of 1 as it is shown in Figure 2a. Based on literature, inspection to crassocolide E chemical shift and 1D selective NOE of H-2 (δH5.42) that gave rise to signals corresponding to H-3 (δH5.16), the probable geometry of this compound is shown in Figure 2b. Unfortunately, absolute stereochemistry of this compound could not be determined in this study. Literature searches for this molecule indicated 1 is a new compound as a hydroxyl derivative (at C-2) of crassocolide E.[18]

CONCLUSION

Sarcophyton genus is a soft coral organism that has an ability to produce various cembranoid compounds. This study shows soft coral Sarcophyton in acidified environment produced a new type of cembranoid compound. Therefore, it may suggest soft coral produces specific type of cermbranoids as the results of environment characteristics.

Financial support and sponsorship

This study was supported by the Department of Biotechnology, Indonesian Research and Development Center for Marine and Fisheries Products Processing and Biotechnology, Indonesia.

Conflicts of interest

There are no conflicts of interest.

Hedi Indra Januar

ABOUT AUTHOR

First author is a researcher in Indonesian Research and Development for Marine and Fisheries Product Processing and Biotechnology that currently pursuing a PhD study in Department of Marine Science, Faculty of Fisheries and Marine Science, Bogor Agricultural University, Indonesia. The PhD study is supervised by Neviaty Putri Zamani, Dedi Soedharma, and Ekowati Chasanah. His research area is mainly on marine ecology and marine natural products.