DIRECT CHARACTERIZATION OF CAFFEOYL ESTERS WITH ANTIHYALURONIDASE ACTIVITY IN CRUDE EXTRACTS FROM ECHINACEA-ANGUSTIFOLIA ROOTS BY FAST-ATOM-BOMBARDMENT TANDEM MASS-SPECTROMETRY

Fast atom bombardment (FAB-MS) and fast atom bombardment tandem mass s pectrometry (FAB-MS/MS) techniques (negative ions) have been successfu lly applied for identification of the constituents responsible for the antihyaluronidase activity of Echinacea angustifolia roots, whose ext racts are widely employed for the adjuvant therapy of chronic inflamma tory diseases. Crude extracts from different solvents were tested for antihyaluronidase activity, and those with the greatest inhibitory act ion (the ethylacetate, butylacetate and chloroform fractions, IC50 0.4 4, 0.50 e 0.62 mg/ml) were directly analyzed by MS. Full scan mass spe ctra produced intense molecular anions: collisional activation of thes e resulted in tandem mass spectra rich in significant product ions. Fo ur main caffeoyl conjugates were detected and identified by tandem mas s spectrometry (daughter and parent ion mode): 2,3-O-dicaffeoyltartari c acid (chicoric acid) and 5-O-dicaffeoylquinic acid (cynarine) and 2- O-caffeoyltartaric acid (caffaric acid) in the ethylacetate fraction. Among these caffeoyl conjugates, chicoric and caftaric acids had the g reatest antihyaluronidase activity: IC50 = 0.42 and 0.61 mM, while the IC50 of cynarine and chlorogenic acid were 1.85 and 2.25 mM.