ISOLASI DAN UJI AKTIVITAS SENYAWA ANTIMIKOBAKTERIUM DARI ACTINOMYCETES INDONESIA

MAYA DIAN R, Prof. Dr. Mustofa, M.Kes., Apt; Prof. dr. Tri Wibawa, Sp.MK(K)., Ph.D

2020 | Disertasi | DOKTOR ILMU KEDOKTERAN DAN KESEHATAN

Latar Belakang Kenaikan kasus Multi Drug Resistant/Rifampicin Resistant-Tuberculosis (MDR/RR-TB) menyebabkan adanya kebutuhan obat antimikobakterium baru. Indonesia merupakan negara dengan biodiversitas yang besar, namun ekplorasi Actinomycetes Indonesia untuk isolasi senyawa antimikobakterium masih belum banyak dilakukan. Metode Penelitian ini melakukan eksplorasi sistematik terhadap Actinomycetes Indonesia untuk menghasilkan isolasi senyawa antimikobakterium baru. Eksplorasi tersebut meliputi penggunaan dereplikasi biologi, dereplikasi kimia, serta optimasi fermentasi untuk meniadakan re-isolasi senyawa metabolit sekunder. Penelitian juga dilanjutkan dengan melakukan proses bioassay guided isolation untuk mendapatkan senyawa yang mempunyai aktivitas antimikobakterium dari InaCC A758. Hasil Berdasarkan hasil skrining, didapatkan isolat Actinomycetes InaCC A758 yang mampu menghambat pertumbuhan M. tuberculosis strain H37Rv. Berdasarkan analisis taksonomi, InaCC A758 diketahui merupakan Actinomycetes yang pernah ditemukan dan dieksplorasi sebelumnya (mempunyai kedekatan homologi dengan S. parvus strain NBRC 14599 (99,64%)). Selain itu, gen NRPS dan PKS InaCC A758 diketahui mempunyai kedekatan dengan gen NRPS milik S. parvulus (93%), serta borrelidin type-PKS seperti halnya S. rochei dan S. parvulus (99%). Sementara itu, hasil dereplikasi kimia menggunakan HR-MS, diketahui bahwa InaCC A758 berpotensi untuk menghasilkan senyawa baru. Selanjutnya, berdasarkan hasil analisis multivariat, didapatkan hasil bahwa terdapat peningkatan potensi keragaman profil metabolit sekunder dari ekstrak InaCC A758 hasil optimasi fermentasi dan co-kultur. Namun, berdasarkan hasil bioassay guided isolation, didapatkan dua senyawa re-isolasi yang berpotensi sebagai antimikobakterium dari InaCC A758, yaitu 1) A758p1 dengan aktivitas hambatan pertumbuhan M. tuberculosis strain H37Rv sebesar 0,78 µg/mL. Senyawa tersebut berdasarkan hasil pemeriksaan LC-MS, HR-MS, UV-Vis, FTIR, dan NMR teridentifikasi sebagai actinomycin D (C62H86N12O16; m/z 1255,6426) dan Actinomycin V (C62H84N12O17; m/z 1269,6131), dan 2) A758p4 dengan aktivitas hambatan pertumbuhan M. tuberculosis strain H37Rv sebesar 100 µg/mL. Senyawa tersebut berdasarkan hasil pemeriksaan LC-MS dan HR-MS diduga adalah (a) (2-chlorophenyl){2-[(-methoxyphenyl) imino] -4-methylidene-3-thia-1-azaspiro[4,5]dec-1-1yl} methanone (C12H9N3OS; m/z 243,04573 / C23H23ClN2O2S; m/z 426,959), (b) dimethenamid (C12H18ClNO2S; m/z 276,07228), atau (c) timoprazole/orbencarb (C13H11N3OS; m/z 257,06151). Kesimpulan Metode eksplorasi sistematik menggunakan dereplikasi biologi, dereplikasi kimia, dan optimasi fermentasi terhadap Actinomycetes Indonesia dapat berpotensi untuk meningkatkan keragaman metabolit sekunder dari InaCC A758. Namun metode sistematik tesebut masih belum dapat menghasilkan isolasi senyawa baru yang mempunyai potensi sebagai antimikobakterium.

Backgrounds The increase in Multidrug Resistant/Rifampicin Resistant-Tuberculosis (MDR/RR-TB) cases has led to the need for new antimycobacterial drugs. Indonesia is a country with a large biodiversity, but the exploration of Indonesian Actinomycetes as an antimycobacterial is still rarely done. Methods This study carried out a systematic exploration of Indonesian Actinomycetes to produce new antimycobacterial. Exploration carried out by the use of biological dereplication, chemical dereplication, and optimization of fermentation to eliminate re-isolation of secondary metabolites. The research also continued by conducting bioassay guided isolation to obtain compounds that have antimycobacterial activity from InaCC A758. Results Based on screening results, Actinomycetes InaCC A758 was able to inhibit the growth of M. tuberculosis strain H37Rv. InaCC A758 is known to be an Actinomycetes that has been found and explored before (homology closeness with S. parvus strain NBRC 14599 (99.64%)). In addition, InaCC A758 isolates are known to be close to the NRPS gene of S. parvulus (93%), as well as PKS-type borrelidin of S. rochei and S. parvulus (99%). The result of chemical dereplication using HR-MS states that InaCC A758 isolate has the potential to produce new compounds. Furthermore, based on the results of multivariate analysis, it was found that there was an increase in the chemical diversity of the secondary metabolite profile of the InaCC A758 extract resulting from optimization of fermentation and co-culture. However, based on the results of bioassay guided isolation, two re-isolation compounds that have potential as antimycobacterial obtained from InaCC A758, namely 1) A758p1 with activity to inhibit the growth of M. tuberculosis strain H37Rv, MIC value 0,78 µg/mL. The compound based on LC-MS, HR-MS, UV-Vis, FTIR, and NMR data is identified as actinomycin D (C62H86N12O16; m/z 1255,6426) and actinomycin V (C62H84N12O17; m/z 1269,6131), and 2) A758p4 with activity to inhibit the growth of M. tuberculosis strain H37Rv, MIC value 100 µg/mL. The compound based on LC-MS and HR-MS data is predicted as (a) (2-chlorophenyl){2-[(-methoxyphenyl) imino] -4-methylidene-3-thia-1-azaspiro[4,5]dec-1-1yl} methanone (C12H9N3OS; m/z 243,04573 / C23H23ClN2O2S; m/z 426,959), (b) dimethenamid (C12H18ClNO2S; m/z 276,07228), or (c) timoprazole/orbencarb (C13H11N3OS; m/z 257,06151). Conclusions Systemic exploration methods using biological dereplication, chemical dereplication, and optimization of fermentation of Indonesian Actinomycetes can potentially increase the chemical diversity of secondary metabolites from InaCC A758. However, the systematic method still cannot produce isolation of new compounds that have potential as antimycobacterial.

Kata Kunci : Antimycobacterial, Indonesian Actinomycetes, Actinomycin, Secondary Metabolite of Streptomyces sp.