Oleanolic acid acetate (OAA), a major triterpenoid compound of Vigna angularis (azuki bean, V. angularis), has been shown to downregulate inflammatory responses in macrophages. Here, we show the molecular basis for the effect of OAA on Toll-like receptor (TLR) downstream signaling. OAA treatment significantly inhibited the secretion of embryonic alkaline phosphatase (SEAP) induced by polyinosinic acid (poly(I), TLR3 ligand) in a dose-dependent manner and without cytotoxicity in THP1-XBlue cells. In addition, OAA downregulated the gene expression of poly(I) induced pro-inflammatory cytokines and chemokines genes such as MCP-1, IL-1β, IL-8, VCAM-1 and ICAM-1. Furthermore, we found that the inhibition activity of OAA was accompanied by decreased activation of not only nuclear factor-kappa B (NF-κB) signaling but also mitogen-activated protein kinase (MAPK) signaling upon stimulation with the TLR3 agonist. Interestingly, the interaction of OAA with IκB kinase α/β (IKKα/β) strongly attenuated the production of certain proteins and inflammatory cytokines in the TLR3 signaling pathway, such as nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IkBα), extracellular regulated kinases (ERK), and p38, in an in vitro model. The action of OAA was regulated by TLR3, demonstrating that TLR3 plays a critical role in mediating the physiologically-relevant anti-inflammatory action of OAA and that the interaction with IKKα/β is modulated through TLR3. These results reveal new insight into the understanding of the regulatory mechanisms of the downstream TLR3 signaling pathway and consequent inflammatory responses that are involved in the development and progression of inflammatory diseases.

Abstract. This study examined the effect of norkurarinol on the toll-like receptor 3 (TLR3)-mediated signaling pathways and rotavirus replication. Norkurarinol, a lavandulylated flavanone, was isolated from the roots of Sophora flavescens, which has been shown to have anti-inflammatory activity. Norkurarinol suppressed the NF-κB and AP-1 inducible secreted embryonic alkaline phosphatase (SEAP) activity induced by poly(I:C), TLR3 ligand, in THP1-Blue-CD14 cells with IC 50 values of 20.9 μM. Norkurarinol also significantly suppressed the mRNA expression of proinflammatory and adhesive molecules induced by poly(I:C) and rotavirus infection. Pretreatment of norkurarinol blocked the NF-κB and AP-1 signaling pathway and the phosphorylation of MAPKs induced by poly(I:C). On the other hand, norkurarinol increased the level of IRF3 phosphorylation and IFNβ expression in a dose-dependent manner. Moreover, norkurarinol inhibited the rotavirus-induced cytopathic effects. These results suggest that norkurarinol can modulate the TLR3-mediated inflammatory responses and rotavirus replication.

A reproducible analytical method using reverse-phase high liquid performance chromatography combined with UV detecting was developed for the quantitative determination of four compounds isolated from the ethanol extract of Phaseolus angularis seeds (PASE): oleanolic acid (1), oleanolic acid acetate (2), stigmasterol (3) and β-sitosterol (4). This method was fully validated in terms of linearity (r 2 > 0.999), accuracy (98.5%-100.8%), precision (<0.92%), LOD (<0.0035 mg/mL), and LOQ (<0.0115 mg/mL). The effects of the PASE and isolated compounds 1-4 on TLR4 activation were tested in THP1-Blue cells. Among the tested substances, compound 2 showed potent inhibitory activity with an IC 50 value of 3.89 ± 0.17 µM.