Bioassay Guided Fractionation of Marine Streptomyces sp. GMY01 and Antiplasmodial Assay using Microscopic and Flow Cytometry Method

Marine-derived Streptomyces sp. GMY01 is a potential actinobacteria
which have anticancer activities. In previous studies, several anticancer
agents exhibited antiplasmodial activity. This study aimed to evaluate
antiplasmodial activity of GMY01 extract using bioassay guided fractionation.
Crude extract of ethyl acetate was obtained from supernatant of 11 days
fermentation in starch nitrate medium. Crude extract was fractionated using
n-hexane and methanol solvent. Re-fractionation was applied using flash
chromatography and column chromatography. Antiplasmodial assay was
performed on Plasmodium falciparum FCR3 and P. falciparum 3D7 by
microscopic method using thin blood smear + Giemsa stain, and was
confirmed by flow cytometry method using SYBR Green I stain. Toxicity assay
was performed on Vero cells line (normal cells). Main constituent of active
fraction was analyzed using LCMS/MS. The result of the study showed that
ethyl acetate-methanol fraction has high antiplasmodial activity
(IC50=3.96μg/mL) with very low toxicity on Vero cells (IC50=30,072μg/mL).
Bioassay guided fractionation resulted F4.7 which has the highest
Plasmodium inhibition (94.3% at 5μg/mL). Main constituent analysis showed
C10H13NO (163.09971 Da) and C11H15NO13 (209.10519 Da) as two mayor
compounds in F4.7. This study guided the isolation and structure elucidation
of potential compound in further research.
Key words: Actinomycetes, drug discovery, malaria, antiplasmodial assay,
SYBR Green I