1196 Experientia 47 (1991), Birkh/iuser Verlag, CH-4010Basel/Switzerland Research Articles to fish physiology, predominate over inorganic processes in determining trace element concentrations in otolith carbonate. Presumably the reasons for the changes in discrimination by the fish for and against the various ions will be clarified when more is known about seasonal variations in fish physiology, and their precise relation- ship to environmental conditions. Inasmuch as otolith microchemistry provides an environ- mental record, long-lived species and fossil otoliths might provide evidence of past environments, despite the fact that the underlying biological processes are not yet de- fined. In addition, information on the life histories of individual fish, particularly those of migratory habit, should be contained within their otoliths, and it should be possible to reveal this information by microchemical analysis. The work described here has shown the value of FAB-SIMS as a tool for the elemental analysis of intact calcareous samples, giving high spatial resolution. 1 Caflstrom, D., Biol. Bull. 124 (1963) 441. 2 Degens,E. T., Deuser,W.G., and Haedrich,R. L., Mar. Biol.2 (1969) 105. 3 Campana, S. E., and Neilson, J. D., Can. J. Fish. aquat. Sci. 42 (1985) 1014. 4 Summerfelt,R. C., and Hall, G. E., (Eds), Age and Growth of Fish. Iowa State University Press, Ames 1987. 5 Schneider, R. C., and Smith, S. V., Mar. Biol. 66 (1982) 121. 6 Radtke, R. L., Comp. Biochem. Physiol. 92A (1989) 189. 7 Kinsman,D. J. J., and Holland, H. D., Geochim.cosmochim. Acta 33 (i969) 1. 8 Kalish, J. M., J. exp. mar. Biol. Ecol. 132 (1989) 151. 9 Devereux, I., Science 155 (1967) 1684. 10 Seyama, H., and Soma, M., Surf. Interface Anal. 15 (1990) 289. 0014-4754/91/11-12/1193-0451.50+ 0.20/0 9 Birkh/iuser Verlag Basel, 1991 Inhibitory effects of flavonoids on several venom hyaluronidases U. R. Kuppusamy and N. P. Das Laboratory of Flavonoid Research, Department of Biochemistry, Faculty of Medicine, National University of Singapore, Kent Ridge Crescent, Singapore 0511 Received 30 November 1990; accepted 10 May 1991 Abstract. In vitro studies showed that the flavonoid aglycones apigenin, luteolin and kaempferol inhibited the hyaluronidase activity of five different venoms dose-dependently. They were also able to delay the venom action when injected into mice. Naringenin, catechin and flavonoid glycosides had no effect. The flavonoids with unsubstituted hydroxyl groups at C-positions 5, 7 and 4', a double bond between carbons 2 and 3, as well as a ketone group at position 4, exhibited potent inhibitory actions on the venom hyaluronidases. Key words. Flavonoids-hyaluronidase inhibition-venom action. Flavonoids are benzo-~-pyrone derivatives which are ubiquitous in plants 1 and they exhibit a great variety of pharmacological effects in biological systems 2.3. They are also present as important constituents in several preparations used in folk medicine 4. Various plant ex- tracts have been reported to be able to neutralize the lethal activity of snake venoms s, 6. Nevertheless, it must be borne in mind that such effects on the lethal activity do not necessarily correlate with the neutralization of specific pharmacological or enzymatic activities 7. Hyaluronidase (EC 3.2.1.35), an enzyme widely dis- tributed in mammalian testes, leech heads, invasive bac- teria, and venoms of snakes, bees, scorpions and poi- sonous fishes s, 9 depolymerizes hyaluronic acid, a major constituent of animal connective tissues, which is essen- tial for maintaining the integrity of the extracellular ma- trix 1~ Venoms of snake, bee and scorpion contain hyaluronidases with molecular weights ranging from 33,000 to 110,000 11'12 , but little is known about the molecular structures of these enzymes. The activity of venom hyaluronidases shows a broad pH range in vitro, with an optimum pH at 4.0-5.0 9'1~ 12. The end prod- ucts of the action of these enzymes on hyaluronic acid are mainly tetrasaccharide units 9. Tu and Hendon 11 supplied experimental evidence that this enzyme plays a role as a 'spreading factor' in the toxic action of venom. Despite the fact that hyaluronidase in itself is not a toxin 1 a, it may contribute to local or systemic envenomation by accelerating venom absorption and diffusion into the tissues of the victim 14. Therefore, inhibition of this enzyme could play an impor- 9 tant role in the therapy of venom poisoning. Earlier work reported by Rodney et al. 15, and investiga- tions carried out in our laboratory 16, showed that sever- al structurally related flavonoids exhibited potent inhibi- tion of bovine testis hyaluronidase. The present report is an investigation on the effects of a series of 13 flavonoids (table 1) on the hyaluronidase activity present in several commercially available snake, scorpion and bee venoms. In addition, venoms of two different species of snakes, namely the rattlesnake (Crotalus atrox) and the Malayan Cobra (Naja naja sputatrix), were used as models for an