Flavonoids,
coumarins and other polyphenolic compounds are powerful
antioxidants both in hydrophilic and lipophylic environments with diverse pharmacological properties including anti-inflammatory activity. Despite being widely used as powerful therapeutic agents for
blood coagulation disorders, more specifically to control some
serine protease enzymes, the mechanism of anti-inflammatory activity of
coumarins is unknown, unlike that of
flavonoids. Although their controlling effect on
serine proteases is well acknowledged, their action on
secretory phospholipase A2 (
sPLA2) remains obscure. The present study describes the interaction between
umbelliferone (7-HOC) and the
sPLA2 from Crotalus durissus collilineatus
venom. In vitro inhibition of
sPLA2 enzymatic activity by 7-HOC was estimated using 4N3OBA as substrate, resulting in an irreversible decrease in such activity proportional to 7-HOC concentration. The biophysical interaction between 7-HOC and
sPLA2 was examined by fluorescent spectral analysis and circular dichroism studies. Results from both techniques clearly showed that 7-HOC strongly modified the secondary structure of this
enzyme and CD spectra revealed that it strongly decreased
sPLA2 alpha-helical conformation. In addition, two-dimensional electrophoresis indicated an evident difference between HPLC-purified native and 7-HOC-treated
sPLA2s, which were used in pharmacological experiments to compare their
biological activities. In vivo anti-inflammatory activity was assessed by the sPLA2-induced mouse paw
edema model, in which 7-HOC presented an effect similar to those of
dexamethasone and
cyproheptadine against the pro-inflammatory effect induced by native
sPLA2 on the mouse paw
edema, mast cell degranulation and skin
edema. On the other hand, 7-HOC exhibited a more potent inhibitory effect on
sPLA2 than that of
p-bromophenacyl bromide (p-BPB). Our data suggest that 7-HOC interacts with
sPLA2 and causes some structural modifications that lead to a sharp decrease or inhibition of the edematogenic and myotoxic activities of this
enzyme, indicating its potential use to suppress
inflammation induced by
sPLA2 from the
snake venom.