Puerarin Attenuates Ethanol Toxicity in Medaka (Oryzias Latipes) Embryos During Development

The drugs approved by FDA for the treatment of alcoholism are not recommended for the women in pregnancy [1]. Therefore, a drug with anticraving property as well as non-toxic to fetus is required for the treatment of Fetal Alcohol Spectrum Disorder (FASD), a neurobehavioral disorder observed in the babies of alcoholic mothers who consumed alcohol during pregnancy. We have evaluated the potency of Radix puerariae (RP), the root extracts of a wild leguminous creeper kudzu (Pueraria montana), as an alternative natural medicine to prevent FASD using Japanese medaka (Oryzias latipes) embryo-larval development as the model. Previously, we have observed that ethanol was able to induce skeletal dismorphogenesis in medaka by reducing skeletal growth in a dose-dependent manner [2]. In this experiment we have used RP and puerarin (Sigma-Aldrich) as preventive agents of ethanol-induced skeletal dismorphogenesis in medaka. RP was collected from the Lafayette County of Oxford and HPLC analysis indicated that puerarin is the major isoflavone present in the methanolic extract of RP. Fertilized medaka eggs in standard laboratory conditions (16 L: 8D, 25 0C) were exposed to RP extract (0–1.5 mg/ml) for 6 day post fertilization (dpf) and then maintained in 48 well tissue culture plate in hatching solution (one embryo/ml/well). Embryo mortality was observed on 10 dpf. In separate experiments embryos were exposed to RP (0–0.5 mg/ml), Puerarin (0.25–1 mM) with or without ethanol (300 mM) for 2 dpf and then transferred to hatching solution. The calculated IC₅₀ of RP as determined on 10 dpf is 785.3 ± 2.66 µg/ml (n = 5). Hatched embryos on 10 dpf were used for morphometric analysis of skeletal features including the skeleton, cranium, jaw, ethmoid and hypophyseal plate. It was observed that ethanol was able to reduce the growth of all these skeletal features, however, RP or puerarin alone has no effect. When the embryos were treated together with ethanol and RP or puerarin, ethanol-induced skeletal growth reductions were attenuated specifically by puerarin. It is therefore concluded that puerarin, the major flavonoid present in RP, has the potency to prevent ethanol-induced teratogenesis during development and can be used as an alternative natural medicine for the prevention of FASD or other alcohol related disorders. Acknowledgements: This work is supported in part by the United States Department of Agriculture, Agricultural Research Specific Cooperative Agreement No 586408-2-0009, National Center for Natural Product Research, School of Pharmacy, University of Mississippi, National Institute of Alcohol Abuse and Alcoholism (1RO3 AA016915) and from The Center of Research Excellence in Natural products Neurosciences (P20RR021929). References: [1] Williams SH, (2005), Amm Fm Phys, 72: 1775–1780. [2] Wang, et al. (2006), Birth Def Res 77B: 29–39.