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Abstract
Rosmarinic acid has been reported to inhibit complement activation in vivo as well
as in vitro. Previous studies suggested that the inhibitory effect was due to inhibition
of C3/C5 convertases, but inhibition of C3b attachment would yield the same results.
Recent work in our laboratory demonstrated that compounds with polyhydroxylated phenyl
rings are highly reactive with the thioester bond in nascent C3b. These compounds
block complement activation by preventing attachment of C3b to the activating surface.
Because rosmarinic acid contains two 3,4-dihydroxyphenyl groups, the current study
was undertaken to re-examine the mechanism of inhibition by analyzing the effect of
rosmarinic acid on C3b attachment. In assays using purified complement proteins, rosmarinic
acid inhibited covalent attachment of C3b to cells with an 1C50 = 34 microM. Inhibition
of C5 convertase activity required 1500 microM rosmarinic acid, and no significant
inhibition of the C3 convertase enzyme, which produces C3b from C3, was observed at
10,000 microM. In hemolytic assays using human serum, rosmarinic acid was shown to
inhibit activation of both the classical (IC50 = 180 microM) and the alternative (IC50
= 160 microM) pathways of complement. Rosmarinic acid concentrations up to 10,000
microM did not cause direct inactivation of C3. Radioiodination of rosmarinic acid
was used to demonstrate covalent activation-dependent incorporation of rosmarinic
acid specifically into the thioester-containing alpha'-chain of nascent C3b. These
findings indicate that inhibition of complement activation by rosmarinic acid is due
to the reaction of rosmarinic acid with the activated thioester of metastable C3b,
resulting in covalent attachment of the inhibitor to the protein.