Chemical and biochemical Studies of an Antidote Active Compound against Hemorrhagic Snake Venom from Argusia argentea

Argusia argentea (Boraginaceae) is locally used in Okinawa as an antidote for jellyfish toxin and sea snakes venom poisoning. The crude methanol extract of the twigs and leaves of Argusia argentea was fractionated with ethyl acetate, 1-butanol and water successively. An acidic compound, rosmarinic acid RA (1), was isolated and purified from the ethyl acetate fraction by silica gel column chromatography, HPLC with ODS column and Cosmosil Cholester column. Snake venom neutralization activities of the active compound were examined by pharmacological assay methods. The results revealed that rosmarinic acid effectively inhibited venom-induced fibrinogenolytic, cytotoxic, edema, lethality, digestion of type IV collagen activity. Moreover, rosmarinic acid (1) inhibited both hemorrhage and neutrophil infiltrations caused by protobothrops flavoviridis venom in pathology sections. In this study, we investigated the structural requirements necessary for inhibition of snake venom activity through the use of compounds, which are structurally related rosmarinic acid (1). By examining anti-hemorrhagic activity of cinnamic acid analogs against protobothrops flavoviridis venom, it was revealed that the presence of the E-enoic acid moiety (-CH=CH-COOH) was critical. Furthermore, we examined the effect of benzenepolycarboxylic acids and substituted benzoic acids against protobothrops flavoviridis venom-induced hemorrhage. Pyromelltic acid (1,2,4,5- benzenetetra carboxylic acid) was found to be a potent inhibitor of hemorrhage, with an IC50 value of 0.35 PM