ISOLASI DAN UJI AKTIVITAS ANTIPLASMODIUM In Vitro SENYAWA AKTIF DARI UMBI HATI TANAH (Angiopteris evecta)

ARNIDA, Prof. Dr. Wahyono, SU., Apt; Prof. Dr. Mustofa, M.Kes., Apt; Prof. Dr. Ratna Asmah Susidarti, MS., Apt

2015 | Disertasi | S3 Ilmu Farmasi

Resistensi Plasmodium terhadap obat malaria mengakibatkan kegagalan pengobatan. Hal ini merupakan ancaman terlebih belum ditemukannya obat alternatif yang efektif untuk melawan resistensi. Oleh karena itu ketersediaan antimalaria baru sangat diperlukan untuk melawan resistensi. Pencarian obat baru terus dilakukan melalui berbagai cara termasuk eksplorasi dan pengembangan bahan alam. Pemilihan bahan alam biasanya berdasarkan penggunaan secara empiris oleh masyarakat. Tanaman hati tanah (Angiopteris evecta) digunakan oleh masyarakat di Palangkaraya Kalimantan Tengah untuk mengobati malaria. Ekstrak etanol umbi A. evecta mempunyai aktivitas antiplasmodium in vitro tergolong sangat potensial dengan IC50 2,86 mikrogram/mL SE 0,27. Berdasarkan hal tersebut perlu dilakukan penelitian lebih lanjut untuk melakukan isolasi dan penentuan struktur kimia dari senyawa aktif yang memiliki aktivitas antiplasmodium in vitro. Perlu juga mengetahui aktivitas antiplasmodium in vitro senyawa aktif pada stadium intraeritrosit kultur P. falciparum strain FCR3 serta mekanisme aksi penghambatan polimerisasi hem. Isolasi senyawa dilakukan berdasarkan bioassay guided fractionation pada umbi A.evecta terhadap kultur P. falciparum strain FCR3. Senyawa aktif yang murni dielusidasi dan diidentifikasi struktur kimianya berdasarkan data spektra UV-Vis, FT-IR, Massa, 1H-NMR, dan 13C-NMR serta NMR dua dimensi (COSY, HMQC, dan HMBC). Uji aktivitas antiplasmodium in vitro dilakukan menurut metode candle jar yang dimodifikasi. Parameter yang digunakan untuk menyatakan aktivitas antiplasmodium in vitro adalah nilai IC50. Hasil fraksinasi yang dilakukan dengan triturasi diperoleh 3 fraksi yaitu FA (n-heksana), FB (etil asetat), dan FC (tidak larut etil asetat) dengan nilai IC50 masing-masing 37,93 mikrogram/mL SE 1,19 mikrogram/mL; 5,16 mikrogram/mL SE 0,11; > 250 mikrogram/mL. Fraksi FB mempunyai aktivitas antiplasmodium in vitro yang paling baik diantara fraksi lainnya. Isolasi FB menggunakan KCV dihasilkan 5 fraksi yaitu B1, B2, B3, B4, dan B5. Aktivitas antiplasmodium in vitro dari B1, B2, B3, B4, dan B5 ditunjukkan dengan nilai IC50 masing-masing fraksi adalah berturut-turut > 200; 37,13 mikrogram/mL SE 0,22; 4,48 mikrogram/mL SE 0,01; > 200; dan > 200 mikrogram/mL. Fraksi B3 diisolasi dan dimurnikan sehingga diperoleh isolat B3b. Dari analisis spektrum FT-IR, LC-MS, DEPT, dan NMR satu dan dua dimensi, serta membandingkan dengan data spektra NMR dari literatur, mengidentifikasikan bahwa isolat B3b memiliki kesesuaian data dengan senyawa Angiopterisida. Aktivitas antiplasmodium in vitro dari senyawa B3b tergolong sangat aktif dengan nilai IC50 0,85 mikrogram/mL SE 0,42 mikrogram/mL. Aktivitas antiplasmodium in vitro pada stadium perkembangan intraeritrosit dari senyawa B3b adalah menghambat perkembangan tahap tropozoit menjadi skizon serta mencegah terjadinya invasi merosoit ke eritrosit. Terjadi penghambatan polimerisasi hem senyawa B3b berdasarkan nilai IC50 yang diperoleh yakni 368,77 mikrogram/mL SE 12,33.

The resistance of Plasmodium to antimalarial drugs resulted in treatment failure. It posed a threat in the absence of alternative effective drug for the resistance. Therefore, the availability of new antimalarial drug was highly required to overcome this problem. Exploration of new antimalarial drugs was conducted using various methods, including exploration and development of natural substance. The selection of the natural substance was usually conducted on the basis of empirical use by people. Angiopteris evecta was used by people living in Palangkaraya of Central Kalimantan to treat malaria. The ethanol extract of the tubers of the A. evecta had in vitro antiplasmodial activity that was considered to be very potential with IC50 2,85 microgram/mL SE 0.27. Based on the fact it was necessary to further study by insulating and determining the chemical structure of the active compound with the in vitro antiplasmodial activities. It was also necessary to investigate the in vitro antiplasmodial activity of the active compound at intraerythrocytic development cycle of P. falciparum strain FCR3 culture and the inhibiting action mechanism of heme polymerization. The isolation of the compound was conducted using bioassay guided fractionation of the tubers of the A. evecta and the P. falciparum strain FCR3 culture. The pure active compound was elucidated and its chemical structure was identified on the basis of spectra data of UV-Vis, FT-IR, 1H-NMR, 13C-NMR one and two dimensional NMR (i.e., COSY, HMQC, and HMBC), and mass spectra. The test of the in vitro antiplasmodial activity was carried out using modified candle jar method. The parameter used to identify the in vitro antiplasmodial activity was IC50 value. The results of the fractionation conducted using trituration method were three fractions, which were FA (n-hexane), FB (ethyl acetate), and FC (insoluble in ethyl acetate) with the IC50 value of each 37.93 microgram/mL SE 1.19; 5,16 microgram/mL SE 0.11 ; > 250 microgram/mL. The fraction FB had better in vitro antiplasmodial activity among other fractions. The fractionation of FB using VLC gave 5 fractions, which were B1, B2, B3, B4 and B5. The in vitro antiplasmodial assay of the B1, B2, B3, B4 and B5 indicated the IC50 value of each of the fractions, were > 200; 37.13 microgram/mL SE 0.22; 4.48 microgram/mL SE 0.01; > 200; dan > 200 µg/mL, respectively. The fraction B3 was purified to give B3b isolate. The results of the analysis of the spectrum of FT-IR, LC-MS, DEPT and NMR one and two dimensional and the results of the comparison of them and the NMR spectra data found in literature showed that the data of the B3b isolate fitted angioptericide compound. The in vitro antiplasmodial activity of the B3b was considered to be very active with the IC50 value of 0.85 microgram/mL SE 0,42. The in vitro antiplasmodial activity at the intraerythrocytic development cycle of the B3b compound was to inhibit the development of trophozoite stadium into schizone and prevented the invasion of merozoit into erythrocyte. The inhibition of the heme polymerization of the B3b compound took place based on the resulting IC50 value, which was 368.7 microgram/mL SE 12.33.

Kata Kunci : Angiopteris evecta, Plasmodium falciparum, IC50, Malaria, Angiopterisida