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Uji Toksisitas Ekstrak Kulit Batang Pulai Basung ( Alstonia Spatulata Bl) Dengan Metode Brine Shrimp Lethality Test

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Uji Toksisitas Ekstrak Kulit Batang Pulai Basung ( Alstonia Spatulata Bl) Dengan Metode Brine Shrimp Lethality Test ISSN 2085-0050 UJI TOKSISITAS EKSTRAK KULIT BATANG PULAI BASUNG ( ALSTONIA SPATULATA BL) DENGAN METODE BRINE SHRIMP LETHALITY TEST Haiyul Fadhli, Hilwan Yuda Teruna* dan Christine Jose Jurusan Kimia Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Riau Kampus Binawidya KM 12,5 Simpang Baru Pekanbaru, 28293, Indonesia E-mail: [email protected] Abstract An investigation on the toxicity of pulai basung ( Alstonia spatulata Bl) extract has been done using brine shrimp lethality test (BSLT) method. The extract was obtained by macerating the stem barks of A. spatulata using n-hexane and methanol respectively. The methanol extract was fractionated based on a method for isolation of alkaloid compounds to afford a crude alkaloid extract. Since a trace of alkaloids was detected in the remaining aqueous solution, this solution was then partitioned with n-butanol to give a n-butanol extract. An effect of toxicity from those extracts were identified for their death persentage of Artemia salina nauplii and counted for their LC 50 by probit analysis. The result showed that the crude alkaloid extract was more toxic than other extracts with LC 50 163 ppm. Keywords : Alstonia spatulata Bl, Artemia salina Leach, BSLT, LC 50 PENDAHULUAN (back to nature ) semakin tinggi dengan lebih memilih menggunakan obat-obatan tradisional. Salah satu tumbuhan yang mengandung Sejak lama penyakit kanker menjadi senyawa kimia aktif adalah pulai basung. Pulai salah satu penyakit yang menakutkan bagi basung merupakan spesies dari Apocynaceae banyak orang. Hal ini disebabkan karena dengan nama latin Alstonia spatulata Bl . kanker merupakan penyakit penyebab kemati- Penelitian pada tanaman dengan tingkatan an kedua di dunia setelah penyakit jantung dan taksonomi yang sama, yaitu pada famili pembuluh darah (Tjay & Rahardja, 2007). Apocynaceae seperti Catharanthus roseus dan Kanker payudara merupakan penyebab utama Camptotheca acuminata (Guirimand et al., kematian pada wanita di berbagai belahan 2010) dan genus Alstonia ( A. yunnanensis dan dunia. Kanker payudara banyak diderita kaum A. scholaris ) menunjukkan potensi kandungan wanita di Indonesia setelah kanker leher rahim zat sitotoksik (Jagetia et al., 2005; Jossang et (Tjindarbumi & Mangunkusumo, 2002). al., 1998). Metode terapi yang lazim dilakukan Penelitian tumbuhan A. spatulata ini selama ini untuk mengatasi kanker adalah pernah dilakukan di Universitas Riau ( Gambar pembedahan, radiasi dan kemoterapi 1) . Bagian tumbuhan yang diteliti tersebut (Nafrinaldi & Gan, 1995). Pembedahan umum- adalah daun, kulit batang dan kulit akar. Hasil nya tidak efektif lagi untuk sel yang telah penelitian menyatakan bahwa daun, kulit mengalami metastasis, penyinaran seringkali batang dan kulit akar tumbuhan A. spatulata tidak efektif dan tidak aman untuk sel-sel yang positif mengandung senyawa alkaloid (Rahmi, normal. Sedangkan penggunaan kemoterapi 1998; Teruna et al., 2011), pada kulit akar belum memberikan hasil yang optimal karena tumbuhan A. spatulata ditemukan alkaloid obat tersebut bekerja tidak spesifik sehingga echitamine (Teruna et al. , 2011), sedangkan perlu dikembangkan upaya penemuan obat pada daun tumbuhan A. spatulata ditemukan baru dari alam (Pamilih, 2009; Tjay & alkaloid vincamine (Teruna & Zamri, 2001). Rahardja, 2007). Senyawa vincamine ini telah pernah dilaporkan Obat kanker umumnya merupakan obat yang diisolasi dari tumbuhan genus Vinca , hasil sintesis dengan harga relatif mahal dan namun belum pernah dilaporkan dari genus memiliki efek samping. Sehingga kecen- Alstonia . derungan masyarakat untuk kembali ke alam 10 J. Ind.Che.Acta Vol. 3 (1) November 2012 Uji Toksisitas Ekstrak Kulit Batang Pulai Basung ( Alstonia Spatulata Bl) diperoleh ekstrak kental metanol dan ditimbang beratnya. Isolasi alkaloid dilakukan dengan metoda ekstraksi asam-basa(Cordell, 1981). Ekstrak metanol yang diperoleh diasamkan dengan asam tartarat 2% sampai pH 3-4, selanjutnya lapisan asam dibasakan dengan larutan amonia sampai pH 9-10, partisi dengan diklorometana. Kemudianlapisan air partisi dengan butanol untuk menarik alkaloid kuarterner sampai lapisan air tersebut negatif terhadap pereaksi Dragendorff. Masing-masing lapisan diklorometana dan butanol yang Gambar 1. Tumbuhan Alstonia spatulata Bl mengandung alkaloid dikeringkan dengan natrium sulfat anhidrat kemudian dipisahkan, Di Indonesia pemanfaatan tumbuhan A. lapisan diklorometana dan butanol masing- spatulata hanya sebatas pemanfaatan lang- masing diuapkan dengan rotary evaporator sung seperti bahan mebel, bahan bakar, dan sehingga diperoleh ekstrak diklorometana akhir-akhir ini merupakan salah satu bahan (alkaloid kasar) dan ekstrak butanol (alkaloid industri kayu lapis yang memiliki nilai ekonomi kuarterner). yang cukup tinggi (Sutomo & Mukaromah, 2006). Sementara disisi lain komponen bioaktif b. Uji Toksisitas dan potensi antikanker yang terkandung dalam Uji toksisitas dilakukan berdasarkan tumbuhan A. spatulata itu sendiri belum di- metoda BSLT (Carballo et al., 2002). Timbang ketahui secara pasti. Melalui penelitian ini ekstrak total n-heksana, metanol, dikloro- diharapkan dapat mengetahui kandungan dan metana dan butanol seperlunya untuk mem- potensi antikanker dari tumbuhan A. spatulata peroleh ekstrak uji dengan konsentrasi 1.000 sehingga tumbuhan A. spatulata ini bermanfaat µg/ml, 100 µg/ml dan10 µg/ml. Masing-masing dalam bidang pengobatan sehingga dapat ekstrak di dalam vial uji dibiarkan menguap dikembangkan untuk kepentingan masyarakat kemudian dilarutkan kembali dengan 50 µl banyak. DMSO, selanjutnya air laut ditambahkan hampir mencapai batas kalibrasi. Larva udang dimasukkan sebanyak 10 BAHAN DAN METODA ekor kedalam masing-masing vial yang telah berisi air laut. Air laut ditambahkan lagi Tumbuhan Alstonia spatulata Bl diambil beberapa tetes sampai batas kalibrasi, dari daerah Pekanbaru, Provinsi Riau. Kulit kemudian kematian larva udang diamati batang tumbuhanyang telah dibersihkan dari setelah 24 jam. Data yang diperoleh dihitung kotoran yang melekat dikeringkan tanpa sinar LC dengan metoda probit dengan membuat matahari langsung, hingga berat konstan. 50 kurva hubungan antara persen penghambatan Setelah itu dihaluskan hingga diperoleh serbuk dengan dosis. halus dan kemudian siap untuk diekstraksi. a. Ekstraksi HASIL DAN PEMBAHASAN Sampel kulit batang dari tumbuhan A. spatulata yang telah dihaluskan direndam Ekstraksi kulit batang Alstonia spatulata dengan pelarut n-heksan selama 24 jam, Bl. kemudian sampel diultrasonikasi selama 30 Sebanyak 1,5 kg serbuk kering kulit menit. Langkah ini dilakukan sampai 5 kali. batang A. spatulata dimaserasi dengan n- Kemudian maserat diuapkan dengan alat heksana sebanyak 2 kali pengulangan masing- rotary evaporator hingga diperoleh ekstrak masing selama 24 jam. Hal ini dilakukan untuk kental n-heksana dan ditimbang beratnya. membebaskan lemak dan fraksi non-polar Residu dari maserasi n-heksana yang yang terdapat pada serbuk sampel. Sebelum telah bebas lemak direndam dengan metanol maserat disaring terlebih dahulu dilakukan selama 24 jam, kemudian diultrasonikasi ultrasonikasi yang bertujuan untuk menambah selama 30 menit. Perendaman dengan kelarutan senyawa dalam pelarut yang di- metanol dilakukan berulang hingga maserat gunakan. Maserat yang sudah disaring di- terakhir memberikan hasil negatif terhadap pekatkan menggunakan rotary evaporator , reagen uji alkaloid. Kemudian maserat sehingga diperoleh ekstrak total n-heksan diuapkan dengan alat rotary evaporator hingga berwarna kuning sebanyak 41,46 gram. 11 J. Ind.Che.Acta Vol. 3 (1) November 2012 Uji Toksisitas Ekstrak Kulit Batang Pulai Basung ( Alstonia Spatulata Bl) Serbuk sisa perendaman dengan n- alkaloid yang larut dalam air. Untuk mem- heksana dikeringkan dan kemudian dilakukan bebaskan alkaloidnya dilakukan dengan cara perendaman kembali dengan pelarut metanol penambahan amonia. Selanjutnya alkaloidnya sebanyak3 kali pengulangan masing-masing dipartisi dengan menggunakan diklorometan. selama 24 jam. Kemudian dilakukan ultra- Fraksi diklorometan yang masih mengandung sonikasi dan rotary evaporator , diperoleh air dihilangkan airnya dengan menggunakan ekstrak kental metanol sebanyak 71,09 gram natrium sulfat anhidrat, kemudian disaring. yang berwarna coklat kehitaman. Fraksi diklorometan diuapkan pelarutnya se- Sebanyak 50 gram ekstrak metanol hingga diperoleh ekstrak kental diklorometan diasamkan dengan asam tartarat 2%, se- sebanyak 1,822 gram. lanjutnya lapisan asam dibasakan dengan Uji toksisitas yang dilakukan dengan larutan amonia. Larutan air dipartisi meng- metoda Brine Shrimps Lethality Test (BSLT) gunakan diklorometan diperoleh fraksi dikloro- menggunakan larva Artemia salina Leach metan, maka diperoleh alkaloid kasar basa terhadap ekstrak total n-heksana, metanol, kuat. Sisa larutan air yang telah difraksi diklorometana dan butanol menghasilkan suatu diklorometan kemudian dipartisi kembali data (Tabel 1) yang kemudian diolah dengan dengan butanol untuk menarik alkaloid metoda probit untuk menentukan nilai LC 50 . kuarterner. Selanjutnya tiap-tiap fraksi di- Hasil analisis data diperoleh nilai LC 50 untuk tambahkan natrium sulfat anhidrat untuk meng- ekstrak total n-heksan >1000 ppm, ekstrak ikat air yang terbawa dan dilakukan rotary total metanol >1000 ppm, fraksi diklorometana evaporator menghasilkan ekstrak kental di- 136 ppm dan fraksi butanol >1000 ppm. Hal ini klorometan yang berwarna coklat kehitaman menunjukkan bahwa fraksi diklorometana sebanyak 1,822 gram dan ekstrak butanol mempunyai sifat sangat toksik dan ekstrak sebanyak 1,523
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