Isolasi dan karakterisasi komponen antibakteri dari biji atung (Parinarium glaberrimum Hassak)
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Date
2002Author
Murhadi
Soekarto, Soewarno T.
Jenie, Betty Sri Laksmi
Apriyanto, Anton
Yasni, Sedarnawati
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Show full item recordAbstract
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).