Potensi Senyawa Berat Molekul Rendah dari Streptomyces sp. GMR22 sebagai Antiplasmodium

TARSA RULI TAMBUNAN, Dr. Ir. Jaka Widada, M.P.; Prof. Dr. Mustofa, Apt., M.Kes.

2020 | Tesis | MAGISTER BIOTEKNOLOGI

Senyawa antiplasmodium berat molekul rendah yang diisolasi dari bakteri, khususnya Streptomyces belum banyak dilaporkan. Penelitian ini bertujuan untuk mengetahui potensi senyawa berat molekul rendah dari Streptomyces sp. GMR22 sebagai antiplasmodium. Genome mining dilakukan menggunakan antiSMASH 5.1. Isolasi senyawa berat molekul rendah dari kultur vial dan supernatan kultur fermentasi masing-masing dilakukan dengan menggunakan SPME (fiber DVB/CAR/PDMS) dan pelarut organik n-heksana:etilasetat (EtOAc) (85:15v/v)dan diidentifikasi menggunakan gas chromatography-mass spectrometry (GC-MS). Uji aktivitas antiplasmodium ekstrak n-heksana:EtOAc dilakukan secara in vitro terhadap P. falciparum 3D7. Persentase parasitemia didapatkan melalui pengamatan mikroskopis dan 50% inhibitory concentration (IC50) didapatkan melalui analisis probit. Uji konfirmasi antiplasmodium dilakukan dengan analisis flow cytometry menggunakan SYBR Green I untuk DNA Plasmodium dan antibodi CD235a untuk eritrosit. Drug-likeness senyawa berat molekul rendah diselidiki menggunakan SwissADME. Streptomyces sp. GMR22 diketahui memiliki 65 biosynthetic gene clusters (BGCs), dimana 5 diantaranya merupakan golongan terpena. Empat puluh senyawa berat molekul rendah diidentifikasi dari kultur vial, beberapa merupakan golongan terpena. Ekstrak n-heksana:EtOAc diketahui mengandung 21 senyawa berat molekul rendah berasal dari golongan alkohol, hidrokarbon, ester, aromatik/diester, diester, asam lemak, dan triester dimana memiliki aktivitas antiplasmodium potensi sedang (IC50 = 38,61 ug/mL). Konfirmasi dengan analisis flow cytometry menunjukkan bahwa ekstrak pada konsentrasi 50 ug/mL memiliki aktivitas antiplasmodium berdasarkan penurunan intensitas DNA Plasmodium yang dibandingkan terhadap kontrol. Hasil skrining drug-likeness diperoleh 3 senyawa merupakan drug-likeness, yaitu phenylethyl alcohol, ethyl citrate, dan di-n-butyl phthalate. Streptomyces sp. GMR22 menghasilkan senyawa berat molekul rendah sebagai antiplasmodium, dan diperlukan penelitian lebih lanjut untuk isolasi, identifikasi, dan uji sitotoksisitas senyawa aktif antiplasmodium.

Low molecular weight (LMW) antiplasmodial compounds isolated from bacteria, particularly Streptomyces have not been widely reported. This study aimed to know the potency of the LMW compounds from Streptomyces sp. GMR22 as antiplasmodial. Genome mining was carried out using antiSMASH 5.1. Isolation of LMW compounds from the vial culture and the supernatant of fermentation culture was performed using SPME (DVB/CAR/PDMS fiber) and organic solvent of n-hexane:ethylacetate (EtOAc) (85:15v/v), respectively, and identified using gas chromatography-mass spectrometry (GC-MS). Antiplasmodial assay of n-hexane:EtOAc extract was carried out against P. falciparum (3D7). Parasitemia percentage obtained through microscopic observations and 50% inhibitory concentration (IC50) obtained through probit analysis. The antiplasmodial confirmation test was done by flow cytometry analysis using SYBR Green I for Plasmodium DNA and anti-human CD235a for erythrocyte. The LMW compounds were investigated using SwissADME for drug-likeness. Streptomyces sp. GMR22 possessed 65 biosynthetic gene clusters (BGCs), in which 5 out of 65 were terpene classes. Forty LMW compounds were identified from the vial culture, partially as terpenes. n-Hexane:EtOAc extract contained 21 LMW compounds of alcohol, hydrocarbon, ester, aromatic/diester, diester, fatty acid, and triester classes, which possessed moderate antiplasmodial activity (IC50 = 38.61 ug/mL). The flow cytometry analysis confirmed that the extract at 50 ug/mL exhibited antiplasmodial activity based on a decreased Plasmodium DNA intensity as compared to the control group. The result of drug-likeness screening obtained that 3 LMW compounds were drug-likeness, namely phenylethyl alcohol, ethyl citrate, and di-n-butyl phthalate. Streptomyces sp. GMR22 produced LMW compounds as antiplasmodial, and further study was needed for isolation, identification, and cyctotoxicity test of antiplasmodial active compounds.

Kata Kunci : Senyawa berat molekul rendah, Streptomyces, antiplasmodium, antimalaria