10.22376/ijpbs.2019.10.1.p1-12 Volume 6 Issue 3 2015 (July - September) lessThan i greaterThan Millingtonia hortensis lessThan /i greaterThan is widely distributed tree which belongs to Bignoniaceae family, commonly used as a folk medicine, having anti epileptic, antioxidant, antimicrobial, larvicidal and anticancer activities. In the present study, purification and characterization of bioactive compound(s) from methanolic extract of the leaves of lessThan i greaterThan Millingtonia hortensis lessThan /i greaterThan were carried out using silica gel column chromatography, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), FT-IR and mass spectrometry (LC-MS) detection. Structure of the bioactive compound was determined by nuclear magnetic resonance ( lessThan sup greaterThan 13 lessThan /sup greaterThan C and lessThan sup greaterThan 1 lessThan /sup greaterThan H NMR) spectroscopy. The S6 fraction obtained from silica gel column chromatography was identified to have potent antioxidant, antimicrobial and antiproliferative activities with a yield of 2090 mg. The Rf value of the isolated bioactive compound was found to be 0.41 using ethylacetate : methanol (9:1) as mobile phase. The molecular weight of the compound was determined using LC-MS was identified as 301 daltons. The FT-IR spectroscopy showed the presence of phenolic hydroxyl, aromatic and methoxy functional groups. The purified compound was identified as 5,7-dihydroxy-2-(4-hydroxyphenyl)-6-methoxy-4 lessThan i greaterThan H lessThan /i greaterThan -chromen-4-one (Hispidulin) by NMR spectroscopy. The isolated pure bioactive compound was named as MHH. MAHESH KUMAR MVS, VSSL PRASAD TALLURI AND S V RAJAGOPAL Millingtonia hortensis, MHH compound, DPPH, HPLC, FT-IR, LC-MS, 13C and 1H NMR. 348-358