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Our  previous  studies demonstrated  the  preventive effect  of  Nigella  sativa  extract  on asthma and water-soluble substances of this extract, especially its methanol fraction were responsible for this relaxation on contracted tracheal chains of guinea pigs. For the first time, the present study has been conducted to determine the main constituents of its methanolic extract. Four constituents of 20%-methanolic fraction, consisting of two flavonoids (20-20% and 21-20% fractions) and two polysaccharides (1-20% and 2-20% fractions), were purified by analytical and  preparative  HPLC.  The  relaxant  effects  of  four  cumulative  concentrations  of  each constituent (50, 100, 150 and 200 mg/lit) in comparison with saline (1 ml) as negative control and four cumulative concentrations of theophylline (0.2, 0.4, 0.6 and 0.8 mM) as positive control were examined on methacholine-precontracted guinea pig tracheal chains (n=6). All concentrations  of theophylline and most concentrations  of 20-20, 21-20 fractions showed significant relaxant effects compared to  that  of saline. 20-20 fraction (Comferol diglucoside) was the most potent bronchodilator. Their relaxant effects were lower than that of theophylline. Polysaccharides (1-20, 2-20 fractions) did not  have any relaxant effects compared to that of saline. These results revealed that two flavonoids of 20%-methanolic fraction of Nigella  sativa were its main relaxant constituents.