Brazil holds a significant part of the world's plant diversity and of tropical biomes, such as the savannah area also called "Cerrado". Therefore, it's biodiversity represents a great chemical space for the discovery of new active natural products1. Despite of the extensive phytochemical and biological investigations of plant extracts from the Brazilian "Cerrado", there is still lack of isolation and purification of active compounds from complex mixtures in amounts sufficient for further pharmacological assays that clearly relates the medicinal properties with a particular active principle1,2. In the present study, an in vivo bio-guided fractionation approach was performed using an intraperitoneal glucose tolerance test (ipGTT) and preparative medium pressure liquid chromatography (MPLC) to identify compounds responsible of the hypoglycemic activity of native plant specie from Brazilian Cerrado. The obtained extract was first dereplicated via UPLC-TOF-HRMS and then fractionated using vacuum liquid chromatography (VLC) followed by medium pressure chromatography (MPLC) bioguided by in vivo glucose tolerance test (GTT) in diabetic mice. The analytical HPLC (15−25 μm, 250 × 4.6 mm i.d.) conditions were first optimized and transferred to the MPLC (15−25 μm, 460 × 49 mm i.d.) with a gradient transfer method3 for the target isolation of the compounds from the extract. The structure of the isolated compounds was elucidated by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry and the absolute configurations were elucidated by means of electronic circular dichroism computations. The active compounds present in the extract were quantified by NMR. The major compound was purified in high amounts in one step and submitted to the ipGTT in diabetic mice for 28 consecutive days to determine their hypoglycemic potential in comparison with Metformin®. This approach led to the isolation and identification of eleven active compounds and also highlighted the great hypoglycemic potential of the major compound.