ORIGINAL PAPER Structure–function relationships of inhibition of mosquito cytochrome P450 enzymes by flavonoids of Andrographis paniculata Rattanawadee Kotewong & Panida Duangkaew & Ekaruth Srisook & Songklod Sarapusit & Pornpimol Rongnoparut Received: 19 March 2014 /Accepted: 16 June 2014 /Published online: 13 July 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract The cytochrome P450 monooxygenases are known to play a major role in pyrethroid resistance, by means of increased rate of insecticide detoxification as a result of their overexpression. Inhibition of detoxification enzymes may help disrupting insect detoxifying defense system. The Anopheles minimus CYP6AA3 and CYP6P7 have shown pyrethroid degradation activity and been implicated in pyre- throid resistance. In this study inhibition of the extracts and constituents of Andrographis paniculata Nees. leaves and roots was examined against benzyloxyresorufin O- debenzylation (BROD) of CYP6AA3 and CYP6P7. Four purified flavones (5,7,4′-trihydroxyflavone, 5-hydroxy-7,8- dimethoxyflavone, 5-hydroxy-7,8,2′,3′-tetramethoxyflavone, and 5,4′ -dihydroxy-7,8,2′ ,3′ -tetramethoxyflavone), one flavanone (5-hydroxy-7,8-dimethoxyflavanone) and a diterpenoid (14-deoxy-11,12-didehydroandrographolide) containing inhibitory effects toward both enzymes were iso- lated from A. paniculata. Structure–function relationships were observed for modes and kinetics of inhibition among flavones, while diterpenoid and flavanone were inferior to flavones. Docking of flavones onto enzyme homology models reinforced relationships on flavone structures and inhibition modes. Cell-based inhibition assays employing 3-(4,5-di- methylthiazol-2-y-l)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assays revealed that these flavonoids ef- ficiently increased susceptibility of CYP6AA3- and CYP6P7- expressing Spodoptera frugiperda (Sf9) cells to cypermethrin toxicity, due to inhibition effects on mosquito enzymes. Thus synergistic effects on cypermethrin toxicity of A. paniculata compounds as a result of enzyme inhibition could be useful for mosquito vector control and insecticide resistance man- agement in the future. Keywords Anopheles minimus . Andrographis paniculata . Cytochrome P450 . Flavonoids . Inhibition . Synergism Introduction Control of insect vectors by chemical insecticides is an effi- cient strategy in preventing transmission of vector-borne dis- eases including malaria. However, long-term use of synthetic insecticides often leads to insecticide resistance and hinders control of associated diseases, especially resistance to the widely used pyrethroid insecticides has been found wide- spread among mosquito vectors (Hemingway and Ranson 2000; Nkya et al. 2013). Resistance toward insecticides can be due to a number of mechanisms which are reduced sensi- tivity of the target site, reduced penetration of insecticide, and Electronic supplementary material The online version of this article (doi:10.1007/s00436-014-4003-9) contains supplementary material, which is available to authorized users. R. Kotewong : P. Rongnoparut (*) Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Road, Phayatai, Bangkok 10400, Thailand e-mail: pornpimol.ron@mahidol.ac.th P. Duangkaew Faculty of Animal Sciences and Agricultural Technology, Silpakorn University, Petchaburi, IT campus, Sampraya, Chaam, Phetchaburi 76120, Thailand E. Srisook Department of Chemistry, Faculty of Science, Burapha University, Saensook, Muang, Chonburi 20131, Thailand E. Srisook Center for Innovation in Chemistry, Burapha University, Saensook, Muang, Chonburi 20131, Thailand S. Sarapusit Department of Biochemistry, Faculty of Science, Burapha University, Saensook, Muang, Chonburi 20131, Thailand Parasitol Res (2014) 113:3381–3392 DOI 10.1007/s00436-014-4003-9