A method of high speed countercurrent chromatography(HSCCC) and liquid chromatography was developed for preparation and separation of three major flavonoids from lotus leaf at first.Then,HPLC was used to determine the contents of flavonoids and nuciferine in lotus leaf with different drying methods and harvesting time.Using n-hexane:ethyl acetate: n-butyl alcohol:methanol:0.2% water ice acetic acid =1∶3∶2∶1∶4 (V/V) as two-phase solvent system,elute A and elute B were isolated and prepared from the flavonoids extract from lotus leaf by high speed counter-current chromatography (HSCCC).Elute A was compound 3 with purity of 98.78%.Elute B was further separated by pre-HPLC to get compound 1 with purity of 98.36% and compound 2 with purity of 96.68%.Compounds 1,2 and 3 were identified as hypericin,isoquercitrin and quercetin-3-O-glucuronide respectively by mass spectrometry and NMR.The content order from high to low was quercetin-3-O-glucuronide > hyperoside > isoquercetin.There was no difference in nuciferine content between three drying methods.The content of flavonoids and total content in 60 ℃ oven-dried samples was significantly higher than that in shade-dried and sun-dried samples,while there was no significant difference in the content of nuciferine.The content of each component increased with the drying temperature,and 100 ℃ was the best.There was a significant difference in the content of flavonoids between drying with slice and drying with whole,while there was no difference in the content of nuciferine.From July to September,the content of flavonoids tend to be stable,and the nuciferine tend to rise,August and September are the best harvest time.