Isolasi dan karakterisasi komponen antibakteri dari biji atung (Parinarium glaberrimum Hassak)

Abstract

Atung (Parinarium glaberrimum Hassk) merupakan salah satu tanaman tropis yang banyak tumbuh di Daerah Maluku dan sekitarnya. Biji atung telah lama dikenal dan digunakan sebagai bahan pengawet pangan termasuk sebagai obat tradisional khususnya untuk mengobati diare oleh sebagian masyarakat di Maluku. Selanjutnya sejak 1991, beberapa peneliti pendahulu telah melakukan penelitian terhadap biji atung terutama peranannya sebagai pengawet pangan seperti pada udang/ikan dan telah melakukan kajian ekstraksi mengenai pengaruh umur buah, penyimpanan, pelarut-pelarut organik dan metode ekstraksi terhadap aktivitas antibakteri (patogen dan pembusuk) ekstrak biji atung yang dihasilkan. Berdasarkan identifikasi komponen volatil terhadap biji atung, teridentifikasi 17 golongan komponen volatil, terutama: aldehida (34%), alkana (17%), alkena (11 %), alkohol (7%), dan keton (6%). Namun, sebagian besar senyawa volatil dari golongan aldehida, diduga hasil dekomposisi lipid yang terkandung dalam biji atung selama penyimpanan buah atung (4 bulan, -20°C) dan ekstraksi biji atung menggunakan alat Likens Nickerson ( 1 oo0c, 90 men it). Ekstraksi terhadap serbuk biji atung kering menggunakan petroleum eter (PE) 40-60°C (Soxhlet, 8 jam) dan residunya diekstraksi lagi dengan heksana (Soxhlet, 3x8 jam) menghasilkan residu biji atung yang relatif bebas lemak. Selanjutnya residu tersebut diekstraksi dengan metanol (refluks 60°C, 3x3 jam), menghasilkan 11.56% (b.b) ekstrak metanol (EM- 3; coklat tua kehitaman), dan residunya diekstrak lagi dengan etil asetat (refluks 65°C, 3x3 jam}, menghasilkan 1.59% (b.b) ekstrak etil asetat (EE-3; oranye dan bersifat seperti minyak/oi/y), masing-masing dengan diameter zona hambat terhadap S aureus, d'= 99.20 dan 24.23 mm/g biji atung. Ekstraksi pelarut-pelarut terhadap ekstrak EM-3, berturut-turut menggunakan petroleum eter 40-60°C (PE), dan residunya diekstrak dengan PE:etil asetat ( 4: 1, v/v), menghasilkan ekstrak EM-3.6 dan residu biji atung. Kemudian residu tersebut diekstraksi dengan diklorometana, menghasilkan ekstrak EM-3.9 dan rersidunya diekstraksi lagi dengan kloroform, menghasilkan ekstrak EM-3.12. Ketiga ekstrak antibakteri tersebut digabung menjadi ekstrak MT (3.17%, b.b) yang memiliki nilai d' = 391 mm/g ekstrak MT. Ekstrak MT memiliki nilai MIC (minimum inhibitory concentration) dan MBC (minimum bactericidal concentration) terhadap S. aureus, B. cereus, P. fluorescens dan E.coli, masing-masing adalah 1.75/4.99, 2.36/4.63, 3.95/6.49, dan 3.35/5.68 mg/ml. Fraksinasi ekstrak MT dengan kromatografi lapis tipis silika gel 60 F254 menggunakan dua pengembang yang terdiri dari, diklorometana:heksana (90.3:9.7, v/v), kloroform:heksana (68.3:31.7, v/v), dietil eter, masing-masing dengan perbandingan 3:3:4 (v/v/v, Pengembang N) dan 6:3:1 (v/v/v, Pengembang 0), menghasilkan 12 isolat komponen antibakteri biji atung, dengan kemumian komponen tertinggi pada isolat komponen 9 (94% pada 280 nm). Rata-rata aktivitas antibakteri ( S. aureus; metode difusi agar) 12 isolat terse but masih di bawah aktivitas antibakteri ekstrak asal isolat (ekstrak MT). Dari hasil pengujian daya antibakteri pada gabungan fraksi-fraksi antibakteri, diketahui bahwa sebagian besar fraksi antibakteri dalam biji atung bersifat sinergis (saling memperkuat) daya antibakterinya satu sama lain.

Atung (Parinarium g/aberrimum Hassk) is one of tropical trees which grows at Maluku Islands. Atung seeds have been used as a traditional food preservative and for traditional medicines, such as antidiarhea, by many people in Maluku. Based on tentative identification of volatile components from atung seeds, 17-chemical classes of components were identified where the largest component were aldehydes (34%), alkanes (17%), alkenes (11%), alcohols (7%), and ketones (6%). Most aldehydes are most probably derived from decomposition of lipids present in atung seeds formed after storage of the seeds and during hot extraction. The extraction steps of atung seeds were first, extraction using petroleum ether (PE) b.p. 40-60°C (Soxhlet, 8 h), the residue obtained was extracted using hexane (Soxhlet, 3x8 h). The residue obtained after hexane extraction was extracted using methanol (reflux at 60°C, 3x3 h) to give methanol extract, and finally the residue obtained after methanol extraction was extracted using ethyl acetate (reflux at 65°C, 3x3 h) to give ethyl acetate extract. The methanol extract (EM-3 extract, 11.56% w.b) had a growth inhibition activity towards S. aureus (BCC 0007) with an inhibition zone diameter, d' = 99.20 mm/g of atung seeds whereas that of the ethylacetate extract (1.59% w.b) was d' = 24.23 mm/g of atung seeds. Fractionation of the methanol extract using liquid-liquid batch extraction using PE yielded an extract and a residue. The residue was extracted using PE:ethyl acetate (4:1, v/v) to give an EM-3.6 extract. The residue obtained after the extraction using PE:ethyl acetate was extracted further using dichloromethane to give an EM-3.9 extract and the residue obtained was finally extracted using chloroform to give an EM-3.12 extract. The EM.:3.6, EM-3.9, and EM-3.12 extracts were combined together to yield an MT extract. The MT extract (3.17% w.b) had an antibacterial activity towards S. aureus with d' = 391 mm/g MT extract. MIC of MT extract towards S. aureus, B. cereus, and P. fluorescens was 1.75, 2.36, and 3.95 mg/ml, respectively, whereas the MBC of the extract towards the same bacteria was 4.99, 4.63, and 6.46 mg/ml, respectively. Fractionation of the MT extract using TLC system (silica gel 60 F254; Triangle-PRISMA Method) with two suitable mobile phases, each consisted of dichloromethane/hexane (90.3/9.7, v/v): chloroform/hexane (68.3/31.7,v/v): diethyl ether at 3:3:4 (v/v/v, for collecting fractions) and 6:3:1 (v/v/v, for clean-up) were resulted of 12 antibacterial isolates. Isolate-?, 8, and 9 had higher antibacterial activity ( S. aureus; agar difussion method) than the other isolates, but still lower than that of the MT extract (initial extract). This suggested that most antibacterial components of atung seeds have sinergism effects. lsolate-9 (94% purity at 280 nm) showed slightly green fluorescent under long wave UV light. The UV-Vis spectrum of lsolate-9 showed that the Amax (in methanol) was at 213 and 269 nm. IR spectrum of the isolate showed the presence of C-H (2910-2850 cm·1 ) and free O-H (341 0 cm·1)_ MS spectrum of the isolate showed an m/z of 44 (100%) and m/z 57, 69, 83, 97, 115 with low intensity. This suggests that the component is likely to be an aliphatic amine having OH group (from IR spectrum) and two or three conjugated double bonds (from UV spectrum).