Abstract: Ultrasonic extraction conditions for total flavonoids from Humulus lupulus L. were optimized by single factor experiments and orthogonal design with the aim of establishing a sample preparation method for determination of the content of total flavonoids in Humulus lupulus L. by NaNO2-Al(NO3)3-NaOH colorimetric method using rutin as the reference. The optimal extraction process parameters were material-to-liquid ratio of 1:50, extraction duration of 1 h and repeated extraction number of 2. The content of total flavonoids from Humulus lupulus L. was 69.98 mg/g under the optimal extraction conditions, which was much higher than that of residues and beer. The linear range was between 10.2μg/mL and 40.8μg/mL with a correlation coefficient of 0.9985. The average recovery rate was 99.33% with a relative standard deviation of 3.60%. The extraction and determination methods were simple and feasible, which is suitable for the preparation and quality control of flavonoids extracted from Humulus lupulus L.

Key words: Humulus lupulus L., total flavonoids, extraction, determination