Tesis

Karakterisasi enzim malat:kinon oksidoreduktase dari Plasmodium falciparum (PfMQO; EC: 1.1.5.4) dan penghambatan aktivitasnya oleh α-mangostin sebagai kandidat antimalaria = Characterization of malate:quinone oxidoreductase enzyme from Plasmodium falciparum (PfMQO; EC: 1.1.5.4) and activity inhibition by α-mangostin as an antimalaria candidate.

Latar belakang: Plasmodium falciparum merupakan salah satu parasit penyebab penyakit malaria yang menyerang manusia. Mitokondria P. falciparum memiliki perbedaan komponen dengan manusia terutama jenis enzim yang terlibat dalam rantai transpor elektron. Malat: kinon oksidoreduktase (MQO) adalah enzim fungsional yang bekerja dalam mengkatalisis konversi malat menjadi oksaloasetat yang dibutuhkan pada siklus asam sitrat. Elektron yang dihasilkan akan dimanfaatkan untuk pembentukan ATP melalui fosforilasi oksidatif. -mangostin merupakan senyawa xanton utama yang berasal dari manggis, Garcinia mangostana Linn. Ekstrak kulit buah manggis dan -mangostin diketahui memiliki efek antiplasmodium yang dibuktikan melalui penelitian in vitro. Metode: Enzim rekombinan PfMQO diekspresikan pada bakteri Eschericia coli BL21star(DE3). Fraksi membran E.coli yang mengikat enzim diisolasi menggunakan sentrifugasi kecepatan 104.000xg. Karakteristik enzim PfMQO ditentukan secara spektrofotometri dengan mengikuti kinetika reaksi enzim terhadap substrat malat dan ubikinon pada 600 nm. Aktivitas penghambatan αmangostin terhadap PfMQO diuji dengan mengikuti reduksi ubikinon pada panjang gelombang 278 nm. Uji konfirmasi aktivitas inhibisi α-mangostin dilakukan terhadap kultur P. falciparum in vitro dan sel limfosit manusia. Hasil: PfMQO bekerja pada kondisi optimal di suhu 37ºC dan pH netral. Enzim PfMQO yang terikat pada fraksi membran bakteri memiliki karakter nilai aktivitas spesifik sebesar 13,3890 μmol/menit/mg, konstanta Michaelis-Menten (K ) untuk ubikinon sebesar (6,2090 ± 0,6486 μM) dan V sebesar (16,9600 ± 0,5866 μmol/menit/mg). Nilai konstanta Michaelis-Menten (K max ) untuk malat sebesar (5,9960 ± 0,3440 mM) dan V max m sebesar (16,4000 ± 0,3838 μmol/menit/mg). mangostin memiliki aktivitas penghambatan terhadap enzim PfMQO dengan nilai sebesar 1,7390 ± 0,0077 µM. Penghambatan -mangostin terhadap enzim PfMQO di situs pengikatan malat dan ubikinon melalui mekanisme campuran dengan nilai konstanta inhibisi (Ki) masing-masing sebesar 2,3260 mM dan 1,6720 μM. -mangostin memiliki aktivitas penghambatan pertumbuhan terhadap IC50 = 5,7060 ± 1,0976 µM). Uji toksisitas -mangostin terhadap sel limfosit manusia memberikan nilai hambatan (CCkultur P. falciparum (IC 50 ) sebesar 11,3800 μM. Evaluasi toksisitas α-mangostin melalui nilai perhitungan SI didapatkan SI terhadap PfMQO sebesar 6,5440 dan kultur P. falciparum 1,9944. Kesimpulan: Enzim MQO dalam tubuh parasit P. falciparum dapat ditetapkan 50 sebagai target pengobatan malaria dan -mangostin berpotensi untuk dikembangkan sebagai antimalaria namun masih bersifat toksik bagi sel manusia.
Kata kunci: Plasmodium falciparum, malat:kinon oksidoreduktase, -mangostin


Background: Plasmodium falciparum is one of parasite causing malaria disease that attacks human. Mitochondria of P. falciparum has a different component with human especially enzyme that involve in electron transport chain. Malate: quinone oxidoreductase (MQO) is functional enzyme which catalyze conversion of malate to oxaloacetate which needed in citric acid cycle. Generated electron will be utilized to form ATP through oxidative phosphorylation. -mangostin is a main xanton of mangosteen, Garcinia mangostana Linn. Mangosteen pericarp extract and -mangostin have been known in having antiplasmodial effect by in vitro study. Method: PfMQO recombinant enzyme was expressed in bacteria Eschericia coli BL21star(DE3). E.coli membrane fraction that expressed enzyme were isolated using centrifugation 104.000 x g. Characterization of PfMQO were determined by spectrophotometry with following kinetic reaction of enzyme to malate and ubiquinone as substrate at 600 nm. Inhibition activity α-mangostin against PfMQO were assayed by following ubiquinone reduction at 278 nm. Confirmation test of α-mangostin inhibition activity were conducted againts Plasmodium falciparum culture in vitro and human lymphocyte cell. Results: PfMQO has an optimum condition at 37ºC and netural pH. PfMQO enzyme which bind on bacterial membrane fraction has a specific activity 13.3890 μmol/minutes/mg, Michaelis-Menten value (K ) for ubiquinone is (6.2090 ± 0.6486 μM) and V max m is (16.9600 ± 0.5866 μmol/minutes/mg). Michaelis-Menten value (K m ) for malate is (5.9960 ± 0.3440 mM) and V is (16.4000 ± 0.3838 μmol/minutes/mg). α-mangostin has inhibition activity against PfMQO enzyme with inhibition concentration (IC 50 max ) value of (1.7390 ± 0.0077) µM. Inhibition of α-mangostin to PfMQO at malate and ubiquinone binding site by mixed-type inhibition with inhibition constant (Ki) values are 2.3260 mM and 1.6720 μM, respectively. α-mangostin has inhibition activity to P. falciparum growth with IC 50 value of 5.7060 ± 1.0976µM. Toxicity value (CC ) to human lymphocyte cells is 11.3800 μM. Evaluation of α-mangostin toxicity based on SI with SI value to PfMQO of 6,5440 and to P. falciparum culture of 1,9944. Conclusion: PfMQO enzyme in parasite P. falciparum body could be determined 50 as malaria drug target and -mangostin is potential to be developed as antimalarial agent but still toxic for human cell.
Key words: Plasmodium falciparum, malate:quinone oxidoreductase, -mangostin