The invention discloses gynostemma pentaphyllum extract compounds (I) with inhibiting actions on human liver cancer HepG2 cells and a preparation method thereof, and application of the compounds in medicine, belonging to the field of phytochemistry. The preparation method of the compounds disclosed as Formula (I) comprises the following steps: carrying out high-temperature high-pressure treatment on whole gynostemma pentaphyllum, extracting with ethanol under reflux, and concentrating the extracting solution under reduced pressure to obtain an extractum; adding distilled water into the extractum to form a suspensoid, and extracting sequentially with dichloromethane and ethyl acetate; concentrating under reduced pressure to obtain an ethyl acetate extract; passing the extract through an HP20 resin, eluting sequentially with 20%, 50% and 70% ethanol, and concentrating the 70% ethanol eluate under reduced pressure to obtain an elution product; and treating the elution product with silica gel column chromatography, eluting with dichloromethane / methanol in a ratio of 20:1-10:1 (v / v), sectionally collecting the eluates to obtain 6 components, and eluting the component 3 with acetonitrile / water in a ratio of 53:47 (v / v) at the flow rate of 3 mL / minute in a semipreparative chromatographic column (250*10mm, 5 mu m). The pharmacodynamical test indicates that the compounds disclosed as Formula (I) have obvious activities for inhibiting cancer cell proliferation.