ISOLATION AND IDENTIFICATION OF ANTIBACTERIAL COMPOUNDS AGAINST STAPHYLOCOCCUS AUREUS, ESCHERICHIA COLI AND BACILLUS SUBTILIS FROM THE MARINE SPONGE XESTOSPONGIA TESTUDINARIA COLLECTED FROM PROBOLINGGO

Demas Ezra Arianto, Respati Tri Swasono, S.Si., M.Phil., Ph.D. ; Keiichi Konoki, Ph.D.

2026 | Skripsi | KIMIA

Senyawa antibakteri dari spons laut Xestospongia testudinaria yang dikoleksi dari perairan Gili Ketapang, Probolinggo, Jawa Timur, telah diisolasi dan diidentifikasi. Penelitian ini menguji aktivitas antibakteri terhadap Staphylococcus aureus, Escherichia coli, dan Bacillus subtilis, kemudian mengisolasi dan mengidentifikasi senyawa aktif yang bertanggung jawab atas aktivitas tersebut. Ekstraksi dilakukan dengan maserasi menggunakan campuran diklorometana–metanol (1:1 v/v), dilanjutkan partisi cair–cair dengan etil asetat–air. Ekstrak etil asetat dipisahkan melalui tiga tahap: kromatografi kolom silika gel, kromatografi kartrid Sep-Pak C18, dan kromatografi lapis tipis preparatif (PTLC). Uji aktivitas antibakteri metode difusi cakram dilakukan pada setiap tahap, membentuk alur bioassay-guided fractionation. Fraksi aktif dikarakterisasi menggunakan spektroskopi FTIR, LC-HRMS, dan ¹H NMR.

Ekstrak etil asetat kasar diperoleh sebagai pasta cokelat kekuningan (0,2662 g, rendemen 0,02662%) dan tidak menunjukkan aktivitas terhadap S. aureus, hanya aktivitas lemah terhadap E. coli pada 3000 ppm. Fraksinasi bertingkat mengungkap aktivitas antibakteri yang sebelumnya tidak tampak pada ekstrak kasar: fraksi silika gel 5 dan 6 menunjukkan zona hambat sangat kuat (26 mm) terhadap B. subtilis; fraksi Sep-Pak 18 menunjukkan zona hambat kuat (16 mm) terhadap B. subtilis tanpa aktivitas terhadap E. coli; fraksi PTLC-F1 mempertahankan aktivitas kuat (12 mm pada 100 ppm) yang selektif terhadap bakteri Gram-positif. Analisis FTIR terhadap fraksi PTLC-F1 menunjukkan serapan O–H (3397 cm?¹) dan C=O (1739 cm?¹) tanpa serapan C?C (2260–2100 cm?¹), mengindikasikan golongan asam karboksilat/ester non-asetilenik yang sejalan dengan golongan asam lemak terbrominasi atau ester sterol yang telah dilaporkan dari genus Xestospongia. Data ¹H NMR (1D saja, karena keterbatasan massa sampel ?0,1 mg) memberikan dukungan kualitatif terhadap penetapan ini, sementara analisis LC-HRMS pada kedua mode ionisasi terganggu oleh kontaminasi ftalat dan polidimetilsiloksan sehingga rumus molekul definitif belum dapat ditentukan. Identifikasi struktur senyawa aktif dengan demikian baru tercapai pada tingkat golongan fungsional dan memerlukan verifikasi lebih lanjut pada penelitian mendatang.

Antibacterial compounds were isolated and identified from the marine sponge Xestospongia testudinaria, collected from the coastal waters of Gili Ketapang, Probolinggo, East Java, Indonesia. The study evaluated antibacterial activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis, then isolated and identified the active compound(s) responsible. Extraction used maceration with a dichloromethane–methanol mixture (1:1, v/v), followed by liquid–liquid partitioning with ethyl acetate and water. The ethyl acetate extract was fractionated in three stages: silica gel column chromatography, Sep-Pak C18 cartridge chromatography, and preparative thin-layer chromatography (PTLC). Disk diffusion assays were performed at every stage, forming a bioassay-guided fractionation workflow. The active fraction was characterised by ATR-FTIR, LC-HRMS, and ¹H NMR spectroscopy.

The crude ethyl acetate extract was obtained as a yellowish-brown paste (0.2662 g, 0.02662% yield) and showed no activity against S. aureus and only weak activity against E. coli at 3000 ppm. Sequential fractionation unmasked antibacterial activity that was not apparent in the crude extract: silica gel fractions 5 and 6 gave very strong inhibition zones (26 mm) against B. subtilis; Sep-Pak fraction 18 gave a strong zone (16 mm) against B. subtilis with no activity against E. coli; and PTLC fraction F1 retained strong, Gram-positive-selective activity (12 mm at 100 ppm). ATR-FTIR analysis of PTLC-F1 showed O–H (3397 cm?¹) and C=O (1739 cm?¹) absorptions without a C?C stretch (2260–2100 cm?¹), indicating a non-acetylenic carboxylic acid/ester class consistent with the brominated fatty acids or sterol esters previously reported from the genus Xestospongia. ¹H NMR data (1D only, owing to the limited sample mass of ?0.1 mg) gave qualified support to this assignment, while LC-HRMS analysis in both ionisation modes was compromised by phthalate and polydimethylsiloxane contamination, preventing determination of a definitive molecular formula. Structural identification of the active compound was therefore achieved only at the functional-group/class level and requires further verification in future work.

Kata Kunci : Xestospongia testudinaria, antibacterial, bioassay-guided fractionation, FTIR, LC-HRMS