Structure identification of cytotoxic of isolated compound from Kaliapsis sp. sponge collected from North west Bali sea was conducted. The identity of the structure was analyzed based on physical and spectral data, namely, ultraviolet, MS, one- and two-dimensional 1H-NMR and 13C-NMR and comparison to published values. The isolated compound was confirmed as Theonellapeptolide Id.
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Sponge is the lowest rank multicellular organism (metazoa) and considered as the oldest multicellular organism on earth. Sponge is belonging to Porifera phylum. Porifera is a Latin word for phorus (small porous matter) and ferre (to bear). Therefore, Porifera means organism having porous main organ part (Castro and Huber, 1997; Leys et al., 2005).
Almost 98% of sponges grow and are found in the sea from equator to south and north pole, either in deep or shallow seas. As compared to metazoa other members, sponge does not have any special organs for reproduction, digestion, respiration, sensory or excretory. The response from environment is individual. Self defense against parasite organism or microbe is done based on secondary metabolites resulted by sponge (Hooper and Soest, 2002; Colin and Anderson, 1995).
In this study, Kaliapsis sp. (Kaliapsis, Bowerbank) sponge was collected from the sea around Menjangan island, West Bali, Indonesia. Kaliapsis sp. sponge is sticky and elastic. Megasclere consists of desmas tetraclone (tetracrepidial), phyllotriaenes, Short shafe triane, Orthotriaene and Triaeniform and mikrosclere consists of Microstrongyle, Microxea, Amphiaster streptaster. Sponge belongs to Animalia Kingdom, Porifera phylum, Class of Demospongiae, Ordo Lithisda, Sub-ordo Triaenosina, Family of Theonellidae and Genus of Kaliapsis, Bowerbank, 1868 (Hooper, 1997).
By bioactivity guided fractionation and isolation, various cytotoxic constituents we have isolated (Setyowati et al., 2007a, b). In vitro cytotoxic assay of further isolated compound 5M74 using Myeloma cells showed its IC50 of 10.3 μg mL-1. On this occasion we are reporting the structure identification of 5M74.
MATERIALS AND METHOD
Materials: Kaliapsis sp. sponge (collected from Menjangan Island, West Bali National Park at 20 m bellow sea surface, on October 15, 2004). Sample specimen was deposited at Gadjah Mada University Laboratory.
Analytical apparatus: Infra red spectrometer (FTIR 8201 PC Shimadzu), EIMS (Electron Impact Mass Spectroscopy) dari INCOS 50 (Finigan MT). Nuclear Magnetic Resonance (NMR) 500 MHz (Jeol) with radiofrequency strength for 13C at 125 MHz. Ultraviolet spectrometer (UV) (Milton Roy 3000).
Method for identification of bioactive compound: Identification of bioactive compound was conducted based on its physical, spectral data (IR, UV and MS, one- and two-dimension 1H-13C NMR) and comparison to published values.
Isolation procedure: The bioactivity guided extraction, fractionation and isolation of the active isolate were conducted based on standard procedure as reported previously (Setyowati et al., 2007b; Houssen and Jaspars, 2005).
Cytotoxic evaluation: The cytotoxic evaluation was conducted based on standard protocols (Doyle and Griffiths, 1998).
RESULTS AND DISCUSSION
Isolated 5M74, white orthorhomic crystals, mp 67-169 °C, was chloroform and methanol soluble. On TLC plate (its Rf value of 0.57 solvent system methanol:ethyl acetate 7:2 v/v), showed blue color at UV λ 366 nm and orange color on iodium vapor.
Its infra red spectrum, 5M74 showed having functional group of OH or amide (v 3448 cm-1). Functional of alkyl groups were representated of its peaks at v 2800 cm-1, 2962, 2873.9, 1465.8 and 1419.5 cm-1 for its stretching vibration. The ether functional groups were indicated by v 1732-1740, 1253.6 and 1199.6 cm-1. Infrared absorption of v 1624.5 cm-1 was specific for amide carbonyl functional group. The secondary amide was represented by v 1546.8 cm-1. From above data the isolate 5M74 might have -OH, NH, -N-C=O-, HO-C=O functional groups (Silverstein and Webster, 2000).
Mass spectra of 5M74 was obtained from Maldi Tof High Resolution Electrospray Ionisation (HRESI-MS) LCMS. Its fragment ions were m/z 1404.5565 (M++1)(30%); 711.3475 (8%), 704.9012 (8%), 702.9031 (100%, base peak), 463.5227 (1%), 179.8647 (1%). The molecular ion was found at m/z 1404.5565 indicating its molecular weight. Therefore, the molecular formula of 5M74 was C70H125N13O16 calculated for 1403.9374.
UV spectra of 5M74 showed maximum absorption at λ 205 nm in methanol.
Figure 1 showed the 1H-NMR spectrum of 5M74. The proton resonances at δ 5, 4-δH 3,6 ppm indicated the alfa proton of an aminoacid. The aliphatic protons were at the highfield area of δH 1.5-2.5 ppm. The methyl groups were showed at < δH 1, 5 ppm.
13C-NMR spectra were indicating 5M74 (Fig. 2a) had 69 atom carbons, in which the down field carbonyls being 13 atoms of quaternary carbonyls from δC 176.3 ppm to δC 168.5 ppm (Fig. 2b).
The expansion of certain areas of 13C-NMR spectra of 5M74 were showed at Fig. 2c-e. The Distortionless Enhancement by Polarization Transfer (DEPT) result, showed that 5M74 had 14 quaternary, 17 methine, 16 methylene and 22 methyl carbons. Table 1 showed the chemical shifts of 13C-NMR spectra of 5M74 resulted from DEPT experiment.
Application of two-dimensional NMR, HMQC (Heteronuclear Multiple Quantum Coherence) (Fig. 3) will resolved the proton carbon chemical shifts correlation of 5M74. Table 2 showed that the NMR data of 5M74 were indeed in accordance with those of Theonellapeptolide 1d (Roy et al., 2000).
Long-range proton-carbon correlation of 5M74 were analyzed using HMBC (Heteronuclear Multiple Bond Coherence) spectrum (Fig. 4). Figure 5 showed the proton-carbon long-range correlation, deducted from HMBC spectrum.
|Fig. 1:||1HNMR spectra of 5M74|
|Fig. 2a:||13C-NMR spectra of 5M74|
|Fig. 2b:||13C-NMR spectra of 5M74 at the carbonyl area|
|Fig. 2c:||13C-NMR spectra of 5M74 from δ 48-60 ppm|
|Fig. 2d:||13C-NMR spectra of 5M74 at aliphatic (upfield) areas|
|Fig. 2e:||13C-NMR spectra of 5M74 at aliphatic areas (continued)|
|Table 1:||13C-NMR of 5M74 in CDCl3|
|Table 2:||NMR (CDCL3) data of 5M74|
|Fig. 3:||HMQC spectra of 5M74|
The 1H-1H COSY (Correlation Spectroscopy) gave information on proton-proton correlation either geminal (2J) or vicinal (3J) in a molecule (Jenie et al., 2006; Silverstein and Webster, 2000). The proton-proton network of 5M74 was showed at COSY spectrum (Fig. 6).
The bold numbers indicated proton correlations of amino acid moiety of 5M74 (Fig. 6). As an example, number 1 showed the L-Thr (L-threonin; Table 2) amino acid which showed the proton correlation of δH 8.32 ppm (2NH) to H-2 proton (δH 4.37 ppm) and H-3 proton (δH 5.12 ppm).
From all above data, it was concluded that 5M74 was Theonellapeptolide 1d as showed in Fig. 7.
The 5M74, a tridecapeptide lactone, was characterized with its a number of D-amino acid, N-methyl amino acid and β-amino acid. From above data it was concluded that 5M74 was Theonellapeptolide 1d that had been isolated from Theonella swinhoe (Roy et al., 2000). This compound showed various activities, namely, cytotoxic, Na+, K+ ions transport and inhibitory activity on ATP ase (Roy et al., 2000). On this occassion we are reporting the Theonellapeptolide 1d from Kaliapsis sp. The cytotoxic data on this experiment using various cancer cell lines were following (Table 3).
|Fig. 4:||HMBC spectrum for proton-carbon long-range correlation of 5M74|
|Fig. 5:||HMBC long-range correlation of 5M74|
|Fig. 6:||1H-1H COSY spectrum of 5M74|
|Fig. 7:||Structure of 5M74 (Theonellapeptolide Id )|
|Table 3:||Cytotoxic effect of 5M74 against several cell lines|
This data showed that marine organisms are rich in bioactive compounds, including those are potential for anticancer.
The researchers (EPS) would like to acknowledge the funding support from Indonesian Ministry of National Education (Hibah Bersaing Grant No. XV/1/2007). Mass spectra and NMR were provided by LIPI Research Center for Chemistry Serpong Indonesia. The staff is acknowledged.
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