Octacosanoic acid has been isolated from the marine sponges Xestospongia sp. collected from Bidong Island, Terengganu. The structure was elucidated using various spectroscopic techniques (IR, MS, 1 H NMR and 13 C NMR). The structure and bioactivities of this long chains saturated fatty acid compound were reviewed

Abas, Faridah

Ali, Abdul Manaf

Mohamad, Habsah

Mohamad, Khamsah Suryati

Wan Abdul Jamil, Wan Ainur Najmiah

Universiti Putra Malaysia Institutional Repository

Pertanika J. Trop. Agric. Sci. 32(1): 51 - 55 (2009) ISSN: 1511-3701    ©Universiti Putra Malaysia PressReceived: 20 May 2008Accepted: 8 October 2008*Corresponding AuthorOctacosanoic Acid, Long Chains Saturated Fatty Acid from  the Marine Sponges Xestospongia sp.Habsah Mohamad1*, Wan Ainur Najmiah Wan Abdul Jamil1, Faridah Abas2,  Khamsah Suryati Mohamad1 and Abdul Manaf Ali31Department of Chemistry, Faculty of Science and Technology,Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia2Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia,3Faculty of Agrobiotechnology, Universiti Darul Iman Malaysia, 20400 Kuala Terengganu, Terengganu, Malaysia*E-mail: habsah@umt.edu.myABSTRACTOctacosanoic acid has been isolated from the marine sponges Xestospongia sp. collected from Bidong Island, Terengganu. The structure was elucidated using various spectroscopic techniques (IR, MS, 1H NMR and 13C NMR). The structure and bioactivities of this long chains saturated fatty acid compound were reviewed. Keywords: Marine sponges, Xestospongia sp., Bidong Islands, bioactivities, long chains saturated fatty acidINTRODUCTIONFatty acids are widely distributed in nature and are important as nutritional substances and metabolites in living organisms. In marine organisms, particularly sponges, fatty acids have provided some of the most interesting structural varieties. Many of these marine fatty acids originate from unusual biosynthetic pathways. In recent years, excellent reviews have appeared on the fatty acid structural types which are present in these organisms, their possible roles in membranes, and their biogenesis. However, little is known about or has been reviewed on the biomedical potential of these unusual sponge fatty acids, specifically about the differences which exist in their bioactivity, as compared to what have been reported for more common fatty acids (Carballeira, 2008). In addition to their wide range of natural product contents, sponges turn out to be a rich source of unusual lipids which play a primary structural and functional role in their plasma membranes. Since the early studies by Litchfield et al. (1976), several investigations have proven the presence of long-chain, unsaturated carboxylic acids in a variety of sponges from different marine environments, all of which are members of the Demospongiae (Thiel et al., 1999). These compounds display the characteristic of unsaturated patterns and may in some cases, exhibit terminal as well as mid-chain branching. They may occur as mono-, di- and trienoic compounds (tetraenoic and pentaenoic exceptionally) and cover a relatively broad carbon-number range, typically between C24 and C30. Other organisms are apparently lacking these characteristic compounds which have thus been introduced as “demospongic” acids into the literature (Thiel et al., 1999).  The total FA content of a sponge is the sum of FA of the own sponge cells and symbiont bacteria, cynobacteria and/or algal species. As a result, the total mixture of sponge FA is very complex, while the number of components 52 Pertanika J. Trop. Agric. Sci. Vol. 32(1) 2009Habsah Mohamad et al. exceeds 100. A lot of sponge FA is specific for the symbiont types in the sponge tissue. Some lipids isolated from marine sponges showed a cytotoxic and anti-cancer activity; therefore, these sponges may be a good source of bioactive compounds with a unique chemical structure (Imbs and Rodkina,  2004). This study reports on the isolation and structure elucidation of the new fatty acid metabolites and its bioassays.MATERIALS AND METHODSSample Preparation and ExtractionSamples of Xestospongia sp. were collected via SCUBA diving at the depth of 5-10 meters off Kapas Island, in Terengganu. The sponges were directly frozen after the collection and transferred into a –20°C freezer for storage. One part of the fresh sample was preserved in methanol as a taxonomic voucher. Specimens were also deposited at the Biodiversity Museum, Oceanography Institute, Universiti Malaysia Terengganu (Voucer speciman no: B01/002/04). The remaining sponges were cleaned, chopped and dried in an air grafied oven (40°C) prior to extraction. The dried sponges were macerated in methanol 99.7%. The extracts were filtered and dried under reduced pressure using a rotary evaporator. After that these dried extracts were de-salted and kept under –20°C prior to analysis.IsolationThe concentrated hexane extract was chromatographed over silica gel with hexane/CHCl3/EtOAc to give the oily cream fractions. Further purification of the fractions by repeated colum chromatography and recrystalized from methanol yielded compound 1 (30mg, fraction 12-17 from B01/002/04). Free Radical-Scavenging Activity (1, 1-diphenyl-2-picrylhydrazyl)The scavenging activity of DPPH free radical of 1 was done according to the method reported by Oktay et al. (2003) with some modifications. The compound (50 µl) was mixed with 1.95 ml of 0.1 mM DPPH-methanol solution. Methanol was used as a blank sample in this experiment. After 30 min. of incubation at room temperature in the dark, the reduction of the DPPH free radical was measured by reading the UV absorbance at 517 nm. Butylated hydroxyanisole (BHA) and Quercetin were used as positive controls. In this study, the scavenging activity (%) was calculated using the following equation:Free radical scavenging activity (%) = {(abscontrol – abssample)/abscontrol} x 100. The scavenging activity (SA) of each test sample was classified into four ranks as follows: strongly active (SA≥70%); moderately active (70%>SA≥50%); weakly active (50%>SA≥30%) and inactive (SA<30%).Micro-organisms and MediaAntibacterial activity was determined against certified strains of three micro-organisms, namely Bacillus cereuss (Gram-positive), Escherichia coli and Klebsiella pneumoniae (Gram-negative), which were cultured in the appropriated broths at 30°C for overnight. They were obtained from the Pathology Centre, Sultanah Nur Zahirah Hospital, Kuala Terengganu, Terengganu. The bacterial stock cultures were maintained on the nutrient agar (NA) respectively, which were stored at 28°C.Antibacterial Activity AssayThe agar cultures of the test micro-organisms were prepared as described previously by Mackeen et al. (1997). For an initial screening, 1 mg of compound was loaded onto each Whatman No.1 filter paper disc (∅ 6 mm) and placed on the previously inoculated agar. The plates were inverted and incubated for 24 hour at 30°C. For this purpose, streptomycin and gentamycin were used as positive controls. A stock solution of compound was made in DMSO. The presence of antibacterial activity would be indicated by the occurrence of clear inhibition zones around the disc. The assay was carried out in triplicates. The strength of the activity was classified as strong for inhibition zone diameters (i.d.) >16.0 mm, moderate (good) for diameters ranging from 11 to 16 mm, weak for diameters 7 to 11 mm and no activity for diameters < 7 mm. The diameters (mm) of the growth inhibition halos were measured using a ruler. Cytotoxic AssayCytotoxic assay was carried out against Human Caucassian Promyelocytic leukaemia cells (HL-60 cell line) obtained from the National Cancer Institute, USA. The cells were maintained in 53Pertanika J. Trop. Agric. Sci. Vol. 32(1) 2009Octacosanoic Acid, Long Chains Saturated Fatty Acid from the Marine Sponges Xestospongia sp. RMPI-1640 medium supplemented with 10% foetal calf serum, 100 IU/ml penicillin and 100 µg/ml streptomycin. The cytotoxic effect of the compound was assessed using the MTT Assay. Briefly, 100µl of exponentially growing HL-60 cells, at the concentration of 1x105 cells/ml, were seeded into 96-flat bottom microwell plate in the presence of various concentrations of compound and incubated for 3 days. A volume of 20µl MTT solution (5mg/ml in PBS) was added to each well. After 4 hours of incubation, the medium was replaced with 100µl of DMSO. The MTT-formazan product, dissolved in DMSO, was estimated by measuring the absorbance at 570nm in the ELISA plate reader (BIO-TECH INSTRUMENT, INC). The cytotoxicity was expressed as inhibition concentration fifty percent (IC50), i.e. the concentration to reduce the absorbance of the treated cells by 50% with reference to the control (untreated cells).RESULTS AND DISCUSSIONSHomogenized Xestospongia sp. (8 kg, dry weight) was extracted with methanol. The extract (394 g) was then subjected to solvent partitioning, i.e., aq. MeOH against hexane, ethyl acetate and butanol. The extracted hexane (76.34 g) was chromatographed using silica gel Merck Kieselgel 60 PF254 Art no. 7749, eluted with hexane/CHCl3/EtOAc. Sequential CC on silica gel led to the isolation of fatty acid compound, octacosanoic acid (Fig. 1).  Octacosanoic acid (Fig. 1) was isolated as a colourless amorphous solid and showed a molecular ion peak at m/z 424 (M)+ in the EIMS. The presence of 28 carbon signals in the 13C NMR spectrum was consistent with the molecular formula of C28H52O2. The data of 1H NMR and 13C NMR are summarized in Table 1. The characterization of 1 was as follows:Octacosanoic acid (1). IR (CCl4) 3649, 2918, 2848, 1702, 1464, 1432, 1410, 1297 cm-1; 1H-NMR (CDCl3) 0.864 (brt, J =, 3H, -CH3), 1.265 (brs, 26H, -CH2-), 1.644 (m, 2H), 2.358 (t, 2H); MS, m/e (relative intensity) 424 (M+), 410 (19), 354 (7), 326 (17), 311 (24), 297 (36), 283 (31), 269 (21), 255 (21), 241 (33), 227 (29), 213 (24), 199 (32), 185 (51), 171 (43), 157 (49), 143 (44), 129 (71), 87 (100).Position COctacosanoic acidδH δC1 178.5722 2.358 (2H, t) 33.9653 1.644 (2H, m) 24.9294-21 1.265 (26H, br, s) 29.926-30.41222 32.19423 22.92924 27.32325 37.33626 32.98827 19.95928 0.864 (3H, t) 14.351TABLE 11H-(400MHz) and 13C-(100 MHz) NMR chemical shifts (CDCl3) of octacosanoic acidFig. 1: The octacosanoic acid54 Pertanika J. Trop. Agric. Sci. Vol. 32(1) 2009Habsah Mohamad et al.  Three new brominated fatty acids, (5E, 11E, 15E, 19E)-20-bromoeicosa-5,11,15,19-tetraene-9,17-diynoic acid, (5Z, 11E, 15E, 19E)-6,20-dibromoeicosa-5,11,15,19-tetraene-9,17-diynoic acid and (Z E)-14,14-dibromo-4,6,13-tetradecatrienoate (characterized as the methyl ester) were isolated from Xestospongia sp. from the Indian Ocean (Brantley et al., 1995). The bioassay results for the isolated compound are shown in Table 2. The octacosanoic acid was inactive in free radical-scavenging activity  exhibited only 10.03% inhibition. The growth-inhibition, induced in cancer cells after exposure to the compound, was expressed as the percentage of the cell viability observed in the treated cells versus untreated HL-60. The results are summarized in Table 2. When tested in vitro against Human Caucassian Promyelocytic Leukaemia (HL-60) cell line, octacosanoic acid demonstrated cytotoxicity at IC50 2.2 µg/ml, respectively.  In this study, octacosanoic acid was found to give a weak activity against Bacillus cereuss and Escherichia coli when tested for antimicrobial activity. Only Klebsiella pneumoniae inhibited significantly moderate activity by this isolated compound. These results are also presented in Table 2.TABLE 2Activities of pure compound isolated from Xestospongia sp. Specimen aAO (%)  bCytotoxicc* AntibacterialB. cereus E. coli K. pneumoniaeCompound Octacosanoic acid 10.03 2.2 + + ++Control Gentamycin +++ +++ +++ Streptomycin +++ +++ +++* B.cereus: Bacillus cereuss; E.coli: Escherichia coli; K. pneumoniae: Klebsiella pneumoniae(-) No activity, (+) weak activity (7-11 mm halo), (++) moderate activity (11-16 mm halo), (+++)   high activity (>16 mm halo).The experiments were run in triplicates. a The reduction of the DPPH free radical was measured by reading the UV absorbance at 517 nm. bThe cytotoxic effect of compound human caucassian promyelocytic leukaemia cells (HL-60 cell line) was assessed by MTT Assay. cAntibacterial activity of Octacosanoic acid as measured by the disc diffusion method. The inhibition zones were measured in mm.CONCLUSIONSThe isolation of hexane extract, from Xestospongia sp., yielded a long chain saturated fatty acid compound, i.e. octacosanoic acid, which might not have been reported previously in Xestospongia. For its bioactivities, the compound was found to be inactive against antioxidant activity (DPPH), moderate activity against Klebsiella pneumoniae in antibacterial activity and strong cytotoxicity against Human Caucassian Promyelocytic Leukaemia (HL-60) cell line. This showed that the Malaysian water is a potential source of sponge secondary metabolites worthy as a lead compound for developing a therapeutic agent for various kind of diseases.ACKNOWLEDGEMENTSThe authors acknowledge the support from the Ministry of Science, Technology and Innovation for the fund provided (IRPA 01-02-12-0068-EA 10707) and FRGS (vote: 59004). The authors also thank the Department of National Marine Parks for the sampling permit.REFERENCES Biavatti, M.W., vieira, P.C., Silva, M.F.G.F., FernandeS, J.B., alBuquerque, S., MaGalhãeS, C.M.i. and PaGnoCCa, F.C. (2001). Chemistry and bioactivity of Raulinoa echinata Cowan, an endemic Brazilian Rutaceae species. Phytomedicine, 8, 121-124.55Pertanika J. Trop. Agric. Sci. Vol. 32(1) 2009Octacosanoic Acid, Long Chains Saturated Fatty Acid from the Marine Sponges Xestospongia sp. Brantley, S.e., MolinSki, t.F., PreSton, C.M. and delonG, e.F. (1995). Brominated acetylenic fatty acids from Xestospongia sp., a marine spongebacteria association.Tetrahedron, 51, 7667-7672.CarBalleira, n.M. (2008). New advances in fatty acids as antimalarial, antimycobacterial and antifungal agents. Progress in Lipid Research, 40, 50-61. iMBS a.B. and rodkina S.a. (2004). Isolation of 2-methyl branched unsaturated very long fatty acids from marine sponge Halichondria panicea and identification of them by GC–MS and NMR. Chemistry and Physics of Lipids, 129, 173-181.litChField, C., GreenBerG, a.J., noto, G. and MoraleS, r.W. (1976). Unusually high levels of C24–C30 fatty acids in sponges of the class Demospongiae. Lipids, 11, 567–570.MaCkeen, M.M., ali, a.M., el-SharkaWy, S.h., ManaP, M.y., Salleh, k.M., laJiS, n.h. and kaWazu, k. (1997). Antimicrobial and Cytotoxic properties of some Malaysian traditional vegetables. International Journal of Pharmacognosiy, 35, 174-178.Munro, M.h.G., duMdei, J.W., hiCkFord, e.J., lill, S.J., ShanGxiao, r.e., BatterShill, C.n. and duCkWorth, a.r. (1999). The discovery and development of marine compounds with pharmaceutical potential, J. Biotech., 70, 15-25.oktay, M., GulCin, i. and kuFrevioGlu, o. (2003). Determination of in vitro antioxidant activity of fennel (Foeniculum vulgare) seeds extract. Lebensmittel-Wissenschaft and Technology, 36, 263-271.thiel, v., JeniSCh, a., Wörheide, G., löWenBerG, a., reitner, J. and MiChaeliS, W. (1999). Mid-chain branched alkanoic acids from “living fossil” demosponges: a link to ancient sedimentary lipids? Organic Geochemistry, 30, 1-14.

Octacosanoic acid, long chains saturated fatty acid from the marine sponges Xestospongia sp.

http://psasir.upm.edu.my/17978/1/Octacosanoic%20Acid%2C%20Long%20Chains%20Saturated%20Fatty%20Acid%20from%20the%20Marine%20Sponges%20Xestospongia%20sp..pdf

Octacosanoic acid, long chains saturated fatty acid from the marine sponges Xestospongia sp.

Abstract

Similar works

Full text

Available Versions

Universiti Putra Malaysia Institutional Repository