A novel coumarin, (+)-3′-angeloxyloxy-4′-keto-3′,4′-dihydroseselin, isolated from Bupleurum malconense (Chaihu) inhibited NF-κB activity

Huai-Xue Mu,Ai-Ping Lu,Quan-Bin Han,Cheng-Yuan Lin,Da-Jian Yang,Zhao-Xiang Bian,Lin-Fang Huang

doi:10.1186/s13020-016-0077-x

Abstract

BackgroundThis study aims to identify the major anti-inflammatory components in the petroleum ether extract of Bupleurummalconense (Chaihu), by bioassay-guided fractionation, and to investigate the anti-inflammatory mechanisms of active components in lipopolysaccharide (LPS)-stimulated murine macrophage RAW-Blue cells.MethodsA QUANTI-Blue assay was used to guide fractionation of B.malconense root extract. The petroleum ether extract which exerted significant secreted embryonic alkaline phosphatase (SEAP) inhibition effect was purified by silica gel column chromatography and assisted with reverse phase HPLC. The major bioactive compound which significantly inhibited SEAP activity was obtained and its anti-inflammatory effects in LPS-induced RAW-Blue cells were measured by the overproduction of NO (Griess method), gene expression of Il-1β, Tnf-α and iNos (real-time PCR). In parallel, protein expressions of COX-2, iNOS and IκB-α were determined by western blot.ResultsIn bioassay-guided fractionation using LPS-stimulated mouse macrophage RAW-Blue cells, (+)-3′-angeloxyloxy-4′-keto-3′,4′-dihydroseselin (Pd-Ib) was identified by MS and NMR spectral analyses. Pd-Ib (5, 10, 20 μg/mL) suppressed the gene expression of Il-1β (P < 0.0001, P < 0.0001, P < 0.0001 for three respective concentrations), Tnf-α (P = 0.006, P = 0.001, P < 0.0001 for three respective concentrations) and iNos (P = 0.009, P < 0.0001, P < 0.0001 for three respective concentrations) in LPS-stimulated macrophages. The production of cyclooxygenase-2 (P = 0.019, P = 0.002, P < 0.0001), iNOS (P < 0.0001, P < 0.0001, P < 0.0001 for three respective concentrations) and NO (P < 0.0001, P < 0.0001, P < 0.0001 for three respective concentrations) significantly decreased when macrophages were treated with Pd-Ib (5, 10, 20 μg/mL) in the presence of LPS. Pd-Ib (5, 10, 20 μg/mL) suppressed the nuclear activation of NF-κB while it up-regulated the IκB-α level (P = 0.028, P = 0.013, P = 0.005 for three respective concentrations) in LPS-stimulated macrophages.ConclusionsPd-Ib isolated from B.malconense suppressed LPS-induced inflammatory responses in macrophages by inhibiting NF-κB activity and reducing the expression of iNOS, COX-2 as well as pro-inflammatory cytokines.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0077-x) contains supplementary material, which is available to authorized users.

Highlights

This study aims to identify the major anti-inflammatory components in the petroleum ether extract of Bupleurum malconense (Chaihu), by bioassay-guided fractionation, and to investigate the anti-inflammatory mecha‐ nisms of active components in lipopolysaccharide (LPS)-stimulated murine macrophage RAW-Blue cells
Primers for inducible nitric oxide synthase gene, interleukin 1 beta gene (Il-1β) and tumor necrosis factor alpha gene (Tnf-α), beta-actin gene (β-Actin) (Table 1), Trizol reagent, SYBR Green, Dulbecco’s modified Eagle’s medium (DMEM), ECL reagent, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Life Technologies (Carlsbad, CA, USA)
Nuclear factor-kappa B (NF-κB), I-kappa B alpha (IκB-α), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) rabbit antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). β-actin mouse antibody, anti-rabbit IgG and antimouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA)

Summary

Introduction

This study aims to identify the major anti-inflammatory components in the petroleum ether extract of Bupleurum malconense (Chaihu), by bioassay-guided fractionation, and to investigate the anti-inflammatory mecha‐ nisms of active components in lipopolysaccharide (LPS)-stimulated murine macrophage RAW-Blue cells. Bupleurum is used for treatment of inflammation-related diseases, such as autoimmune diseases, inflammatory bowel syndrome and cholecystitis [1,2,3]. Bupleurum species have been extensively studied for their phytochemical characteristics [4]. Saikosaponins are commonly recognized as the main components responsible for the anti-inflammatory activity of the Bupleurum species [7,8,9]. Bupleurum malconense, a species endemic to China, appears to little be known its major bioactive components [10,11,12].

Objectives

Methods

Results