Hydrilla verticillata contain some secondary metabolites, such as steroids and triterpenoids. The purpose of this study was to determine antioxidant activity and the toxicity levels of steroids isolates from Hydrilla verticillata chloroform fraction. The biomass of Hydrilla verticillata was dried and then powdered. The powder of Hydrilla verticillata was extracted by maceration methods using ethanol as a solvent. The crude ethanol extracts were hydrolyzed with 2 N HCl before partitioned using chloroform. The steroid compounds were separated by Column Chromatography. Antioxidant activities of Column Chromatography steroid isolates were determined by the 2,2-diphenyl-1-picrylhydrazil (DPPH) method, and the toxicity levels were determined by the Brine Shrimp Lethality Test (BSLT) method. The result showed that extraction by maceration with ethanol produced 2.52 percent yield, whereas the percent yield of the partition with chloroform was 18.48 percent. Separation by column chromatography obtained four steroids isolates B1, B2, B2G1, and G1R1. The steroids isolates of chloroform fraction of Hydrilla verticillata have antioxidant activity and toxicity properties. The EC50 value of the isolates B1, B2, B2G1, and G1R1 were 5375, 179.40, 65.97, and 6.55 ppm, respectively. The LC50 value of B1, B2, B2G1, and G1R1 isolate were 5.99, 3.86, 6.86, and 4.37 ppm, respectively.

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