Abstract: Six kinds of extraction methods,including 60% ethanol at room temperature,60% ethanol at 70 ℃ water-bath,95% ethanol at room temperature,95% ethanol at 70 ℃ water-bath,95% methanol at room temperature,95% methanol at 70 ℃ water-bath were used for the extraction of Usnea diffracta Vain fraction.The crude extracts were then further extracted by 80% ethanol,and the ethanol extracts were finally fractionated by chloroform to obtain sample A,B,C,D,E and F,respectively.The yield,antitumor activity and HPLC chromatograms of 6 samples were then compared.The HPLC chromatograms of 2 purified bioactive compounds(compound a and b)were used for the identification of bioactive peaks.Our results showed that ethanol,not methanol,can effectively extract bioactive compounds a and b.Sample B showed the highest inhibitory rates both against Hela and T-24 tumor cell lines(81.17% and 88.56% at 40 μg/mL,respectively).Present study indicated that extraction method and extraction solvent significantly influenced the bioactivity of sample.60% ethanol at 70 ℃ waterbath was the optimal method for the extraction of the antitumor bioactive fraction among the 6 methods.

Key words: extraction method, antitumor activity, bioactive fraction, high performance liquid chromatography