Disertasi

Isolasi dan penapisan mikroba endofit tanaman Brucea javanica (I.) Merr serta uji sitotoksik metabolit sekunder terhadap beberapa sel· kanker secara in vitro. = Isolation and screening of endophyte microbes of Brucea javanica (L.) Merr and secondary metobolite cytotoxic assay towards some cancer cells in vitro.

Mikroba endofit merupakan mikroba yang hidup bersimbiosis dengan tanaman inangnya dan dapat menghasilkan metabolit sekunder yang berpotensi seperti enzim, zat pengatur tumbuh, zat anti mikroba, anti fungi dan zat anti kanker. Metabolit ini bersifat bioaktif dan bermanfaat bagi tanaman inangnya dan juga bermanfaat bagi manusia. Di negara berkembang, kanker merupakan penyebab kematian utama disamping penyakit jantung dan serebrovaskuler. Di Indonesia sebagai salah satu negara berkembang, kematian yang disebabkan penyakit kanker menempati urutan ke 6 dan jumlah penderita kanker akan meningkat setiap tahunnya. Keadaan ini mendorong pencarian dan pengembangan obat yang poten dan selektif terhadap sel kanker. Salah satunya dengan menggunakan bahan alam dari tanaman obat. Brucea javanica CL.) Merr dikenal oleh masyarakat dengan nama tanaman buah Makassar. Tanaman ini banyak digunakan oleh masyarakat untuk mengobati kanker leukemia, kanker servik, kanker kulit kanker paru, disamping penggunaan sebagai obat malaria, dan disentri. Penelitian tentang bahan alam dari tanaman telah banyak dilakukan tetapi penelitian mengenai mikroba yang dapat menghasilkan suatu substansi zat anti kanker masih belum banyak dilakukan, oleh karena itu dilakukan penelitian mikroba endofit dari tanaman Bruceajavanica CL.)Merr. Sampel diambil dari 3 lokasi (Bogor, Cianjur dan Tawangmangu) Bagian tanaman yang digunakan adalah ranting, buah dan daun. Tujuan dari penelitian ini untuk mencari mikroba endofit dari tanaman Brucea javanica CL.) Merr yang dapat menghasilkan metabolit sekunder yang berpotensi sebagai zat anti kanker. Metode yang digunakan untuk isolasi mikroba endofit adalah dengan sterilisasi permukaan dan metode tanam langsung. Untuk mendapatkan metabolit sekunder dilakukan fermentasi cair menggunakan medium Potato Dextrose Yeast CPDY) dengan metode goyang selama 14 hari. Untuk uji sitotoksik digunakan sel leukemia L 1210, sel Raji, NS-l, sel HeLa serta sel Vero. Sebagai kontrol positif digunakan Doxorubisin. Pengamatan dilakukan selama 24 jam dan 48 jam dengan menghitung sel hidup menggunakan metode tripan biru. Penghitungan ICso dilakukan secara aritmatikal dengan rumus Reed and Muench. Untuk melihat mekanisme kerja pada proses sitotoksikdilakukan teknik pengecatan DNA menggunakan etidium bromida dan acridine orange. Dari penelitian ini diperoleh 46 bakteri endofit dan 45 kapang endofit. Dapat diidentifikasi 13 spesies bakteri endofit. Isolat kapang endofit 1.2.11 adalah kapang Fusarium chlamydosporum dan isolat kapang 1.2.2 adalah Glomerella sp. Hasil uj i sitotoksik dari 18 kapang endofit terhadap sel Leukemia Ll21O,mempunyai IC so berkisar antara 3,29 - 15,90 ug/ml. Hasil uji sitotoksik isolat 1.2.11. diperoleh nilai ICso. terhadap sel Raji 58,35~g/ml, 88,39 ug/rnl; ICso sel NS-1 162,09 ug/rnl, 66,24 ug/ml; ICso sel He La 361,21 ug/rnl, 219,97 ug/ml. ICso Doxorubisin terhadap sel HeLa 79,14 dan 14,23. Nilai ICso terhadap sel Vero 1075,18 ug/ml, dan 656,82 ug/ml. ICso Doxorubisin terhadap sel Vero 290,77 dan 89,43 ug/ml.Data tersebut masing masing untuk pengamatan 24 jam dan 48 jam. Hasil uji sitotoksik fraksi akhir (F4) terhadap sel leukemia diperoleh nilai ICso 4,29 ug/ml Hasil LC-MS puncak 3 dan 5 diperoleh senyawa senyawa yang diduga mempunyai M (Berat molekul) 487 danM 252 dalton yang mungkin merupakan turunan Bruceosin dan turunan Canthin -6- one Bruceosin dan Canthin -6- one adalah metabolit sekunder dari tanaman Brucea javanica (L.) Merr yang mempunyai efek sitotoksik. Diduga puncak 3 dan 5 kemungkinan merupakan, senyawa derivat dari Bruceonin. Dan Canthin-6-one. Dari hasil penelitian ini dapat disimpulkan kapang dan bakteri endofit dapat diisolasi dari tanaman Brucea javanica (L) Merr Bogor, Cianjur dan Tawangmangu. Isolat kode 1. 2.11 memiliki efek sitotoksik yang selektif terhadap sel kanker. Ada kecenderungan isolat 1.2.11. mempunyai efek sitotoksik terhadap sel NS- 1 melalui mekanisme apoptosis.
Kata kunci : Brucea javanica (L.) Merr, mikroba endofit, uji sitotoksik, isolat kapang 1.2.11, ICso dan apoptosis.



Endophyte microbes are microbes that live on a basis of mutual benefit with its host plant and can produce potential secondary metabolites such as enzymes, growth hormones, anti microbe substances as well as anti fungi and anti cancer substances. These metabolites are bioactive and thus are advantageous for the host plant as well as for human beings. In developing countries cancer is one of the main causes of mortality, besides heart and cerebrovascular diseases. In Indonesia, which is one of the developing countries, cancer is the 6th largest cause of mortality and the number of cancer patients tends to increase every year. This situation makes it necessary for us to find and develop a medicine that is potent and selective to kill cancer cells. One possible way is making use of substances from medicinal plants, such as Brucea javanica (L.) Merr known by laymen as Buah Makassar. This plant has been used for treatment of malaria, dysentery, leukemia, cancer of the cervix, skin and lung. Studies on substances from medicinal plants have been done for many years, but this is not the case for the studies on microbes that can produce an anti cancer substance. For that reason investigation has been made on endophyte microbes from Brucea javanica (L.) Merr. Samples were taken from three locations, Bogor, Cianjur and Tawangmangu. Parts of the plants used as samples were twigs, fruits and leaves. The purpose of this study was to investigate whether endophyte microbes from Brucea javanica (L.) Merr plant can produce secondary metabolites potential as an anti cancer substance. Surface sterilization and direct seed plant were used to isolate endophyte microbes. To obtain secondary metabolites, liquid fermentation with Potato Dextrose Yeast (PDY) broth were carried out followed by incubation over 14 days in shaking incubator. For cytotoxic assay, leukemia cells L1210, Raji cells, NS-l cells, HeLa cells and Vero cells were used, and Doxorubisin was used as positive control. Observations were done for 24 and 48 hours. Viable cells were counted by using Triphan blue method. The ICso was obtained arithmetically using the Reed and Muench standard. Mechanism of action of the cytotoxic was done by DNA staining with etidium bromide and acridine orange. In this study, a total of 46 bacterial and 45 fungal colonies were isolated from the plant samples. Thirteen bacterial and 2 fungal species were successfully identified. The two fungal species identified are Fusarium chlarnydosporum(code 1.2.11.) and Glornerella sp (code 1.2.2.). The cytotoxic assay of 18 fungal isolates showed an ICso of 3.29 to 15.90 ug/ml when incubated with L1210 cells. Furthermore, secondary metabolites produced by these fungi were incubated with Raji, NS-1 and HeLa cells for 24 and 48 hours. The test result showed that fungus 1.2.11 has an ICso of 58.35 ug/rnl and 88.39 ug/ml on Raji cells; 162.09 and 66,24 ug/ml on NS-1 cells and lastly 361.21 ug/rnl to 219.21 ug/ml on Hela cells respectively. ICso of these test substances in Vero cells were 1075.18 ug/ml and 656.82 ug/ml after 24 and 48 h incubation respectively. Doxorubicin (DOX), the positive control was incubated with Hela and Vero cells over the same period of time. After 24 h incubation, it showed ICso values of 79.14 ug/ml and 290.77j.tglml respectively; and after 48 h incubation, ICso dropped to 14.23 ug/ml and 89.43j.tglml respectively. Purification using chromatography and LC-MS were carried out on butanol extract containing these secondary metabolites to yield pure fraction, namely fraction 4 (4). Cytotoxic test of F4 yield ICso of 4.29 ug/ml. LC-MS analysis showed peak 3 and peak 5 has a molecular weight of 487 and 252 respectively. Those substances were likely the derivatives of Bruceocin and canthin - 6 - one, e which are secondary metabolite of Brucea javanica (1.) Merr with a well-known cytotoxic effect. Overall, this study demonstrated clearly that fungal and bacterial endophytes live within the host plant Brucea javanica (L) Merr. The isolated fungus 1.211 belonged to the Fusarium chlamydosporum spp, produced secondary metabolite that have cytotoxic effect and is selective towards cancer cells. This study also showed that these secondary metabolites might cause apoptosis in NS-1 cell.
Key words: Brucea javanica (1.) Merr, endophyte microbes, cytotoxic tests, fungi isolat 1.2.11, ICso and apoptosis.