Anti-inflammatory mechanisms of neovestitol from Brazilian red propolis in LPS-activated macrophages Bruno Bueno-Silva a,1 , Pedro L. Rosalen b , Severino M. Alencar c , Marcia P.A. Mayer a,⇑ a Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Zip-code: 05508-900, São Paulo, SP, Brazil b Piracicaba Dental School, University of Campinas – UNICAMP, Department of Physiologic Science, C.P. 52, Zip-code: 13414-903, Piracicaba, SP, Brazil c College of Agriculture ‘‘Luiz de Queiroz” (ESALQ), University of São Paulo, C.P. 9, Zip-code: 13418-900, Piracicaba, SP, Brazil article info Article history: Received 27 March 2017 Received in revised form 14 June 2017 Accepted 10 July 2017 Chemical compounds studied in this article: Neovestitol (PubChem CID44257510) Keywords: Natural products Propolis Cytokines Inflammation Nutraceutical abstract Neovestitol is considered one of the main bioactive components of Brazilian red propolis. Neovestitol’s antimicrobial and antioxidant effects have already been demonstrated. Here, neovestitol immune mod- ulatory effects were investigated on LPS activated macrophages. RAW264.7 murine macrophages acti- vated with LPS were treated with neovestitol and NO production, cell viability and cytokines profile were determined. Activation of inflammatory signaling pathways and macrophage polarization were determined by RT-qPCR and Western blot. Neovestitol at 0.22 mM inhibited NO production by 60% with- out affecting cell viability and reduced GM-CSF, IFN-c, IL-1b, IL-4, TNF-a and IL-6 levels, whereas increased IL-10 production. These cytokines profile changes were associated with the downregulation of transcription of genes involved in nitric oxide production, NF-jB, IL-1b, and TNF-a signaling pathways. NF-jB and MAPK signaling pathways inhibition and decreased levels of TIRAP were further confirmed by Western blot. Neovestitol, as a nutraceutical, is a potential candidate to modulate chronic inflammation in humans. Ó 2017 Published by Elsevier Ltd. 1. Introduction Brazilian red propolis (BRP) is a resinous natural product col- lected by Apis mellifera bees in Alagoas state (Northeast region, Bra- zil) (Silva et al., 2008). Natural products are proven rich sources of bioactive compounds with distinct therapeutic properties (Cragg & Newman, 2013). These agents can be used as nutraceuticals as long as they lack toxic effects and provide physiological benefits or pro- tection against chronic diseases (Massaro, Scoditti, Carluccio, & De Caterina, 2010; Nicoletti, 2012). BRP presents pharmacological properties such as antimicrobial (Bueno-Silva, Marsola, Ikegaki, Alencar, & Rosalen, 2017; Silva et al., 2008), antibiofilm, anticaries (Bueno-Silva, Koo, et al., 2013), antioxidant (Oldoni et al., 2011) and anti-inflammatory (Bueno-Silva, Alencar, et al., 2013; Bueno-Silva et al., 2016). We have recently shown that BRP is able to inhibit lipopolysaccharide (LPS) signaling in macrophages (Bueno-Silva et al., 2015), although the bioactive component associated with this activity was not determined. Neovestitol is an isoflavonoid shown to be a major bioactive component (Bueno-Silva, Alencar, et al., 2013; Bueno-Silva, Koo, et al., 2013; Inui et al., 2014; Righi et al., 2011) of BRP. It was iso- lated for the first time in 1976 from fungus-infected stems of Cajanuscajan, an African leguminosae plant (Ingham, 1976) and remained unnoticed until few years ago, when chemical studies revealed the presence of neovestitol in the Cuban red propolis (Campo Fernandez et al., 2008; Cuesta-Rubio et al., 2007). Our group has demonstrated that BRP from the Northeast region is a very unique propolis with isoflavonoids in its chemical profile, such as neovestitol, a major bioactive constituent. Some of the BRP therapeutic properties are shared by neovestitol, indicating its potential use in new drugs or alimentary formulations (Bueno-Silva, Alencar, et al., 2013; Bueno-Silva, Koo, et al., 2013; Franchin et al., 2016). Exacerbated inflammatory responses are common features in several human diseases, such as diabetes, cardiovascular diseases http://dx.doi.org/10.1016/j.jff.2017.07.029 1756-4646/Ó 2017 Published by Elsevier Ltd. Abbreviations: BRP, Brazilian red propolis; DAMP, danger-associated molecular patterns; MAMP, microbe-associate molecular patterns; TIRAP, Toll-Interleukin 1 Receptor; TIR, Domain Containing Adaptor Protein. ⇑ Corresponding author at: Lineu Prestes, 1374 Cidade Universitária, São Paulo, SP, Brazil. E-mail address: mpamayer@icb.usp.br (M.P.A. Mayer). 1 Present address: Dental Research Division, Guarulhos University, Guarulhos, Zip-code: 07023-070, São Paulo, SP, Brazil. Journal of Functional Foods 36 (2017) 440–447 Contents lists available at ScienceDirect Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff