Study On Isolation & Purification Process Of Total Flavonoids Of Trifolium Pretense L.

Objective: To establish isolation and purification technical process for total flavonoids of Trifolium pretense L. to heighten the content of total flavonoids from 17.29% to 50%.Method:â‘ HPLC was used to determine the content of formonone and biochanin in crude extract of Trifolium pretense L. The chromatographic column was ODS C₁₈ column, the mobile phase consisted of methanol and water ( 60∶40 ), the eluate was detected at 260nm, formonone and biochanin was determinated in external standard method.â‘¡70% ethanol were used to extract the crude extract, and then use acetoacetate to extract the extractant of ethanol, then base solution was used to extract the acetoacetate , separate the base solution , add hydrochloric acid to adjust the pH 5～6 to precipitate the flavonoids. Each process was optimized by orthogonal experiment design. Results:â‘ The total flavonoids content was 17.29%, formonone and biochanin was good linear in the range of 0.13μg -1.2μg, the regression equation of each one was: formonone Y=36.93X- 0.101 (r=0.9998) biochanin Y=34.40X - 0.105 ( r=0.9999 ) .â‘¡The optimum ethanol extraction process was that adding 40,20,20 times of 70% ethanol for extracting three times (1.5 hour each time) at 80℃; The optimum acetoacetate extraction process was that 30,20,10 times of acetoacetate, extracting temperature was 80℃and extracted three times ,1.5 hours at a time; The optimum alkali solution extraction process was that acetoacetate solution was concentrated to 2/3 volume and adding 5% Na₂CO₃ solution extracted for 3 times ,ratio of acetoacetate (mL) to 5% Na₂CO₃ solution (mL) was 1-1.6 one time.Conclusion: The technical process of isolation and purification total flavonoids of Trifolium pretense L. was suitable to modern industrialization and it can produce the product with total flavonoids higher than 50%.