Anti-inflammatory activity of a new cyclic peptide, citrusin XI, isolated from the fruits of Citrus unshiu

Ethnopharmacological relevance: Citrus unshiu (Rutaceae) is an easy-peeling citrus fruit, which has been used as a traditional Korean medicine for improving skin elasticity, relieving fatigue and cough, and preventing bronchitis, flu, and various cancers. However, its active components associated with anti-inflammation and underlying mechanisms remain unknown. In this study, we investigated the active constituents from the fruits of Citrus unshiu and evaluated the anti-inflammatory activity in order to support the traditional usage of Citrus unshiu. Material and methods: Repeated column chromatography, together with a semi-preparative HPLC purification was used to separate the bioactive constituent from the EtOAc soluble fraction of the EtOH extract of Citrus unshiu fruits. Anti-inflammatory effects of the isolated compounds on lipopolysaccharide (LPS)-induced production of pro-inflammatory mediators were examined using RAW264.7 macrophage cells. Results: A new cyclic peptide, citrusin XI (1), was isolated and identified from the fruits of Citrus unshiu. The structure of compound 1 was elucidated by spectroscopic analysis, including 1D and 2D nuclear magnetic resonance (NMR) (H-1, C-13, COSY, HMQC and HMBC experiments), and high resolution (HR)-mass spectrometry, and its absolute configurations were further confirmed by the Marfey's method. Compound 1 decreased NO production in LPS-stimulated RAW264.7 cells in a dose-dependent manner with an IC50 value of 70 mu M. Compound 1 suppressed NO production by decreasing iNOS expression but COX-2 expression was slightly associated with the reduction by compound 1 in LPS-induced RAW264.7 cells. Furthermore, compound 1 inhibited NF-kappa B activation by blocking I kappa B alpha degradation and NF-kappa B phosphorylation in LPS-stimulated RAW264.7 cells. Conclusions: These results indicate that a new cyclic peptide, citrusin XI, from Citrus unshiu fruits has anti-inflammatory properties that inhibit the release of pro-inflammatory mediators. Compound 1 decreases NO production by decreasing iNOS expression and NF-kappa B activation associated with I kappa B alpha degradation and NF-kappa B phosphorylation in LPS-induced RAW264.7 cells. This is the first study to clarify the underlying mechanism of the anti-inflammatory effect exerted by a pure isolated compound from Citrus unshiu in LPS-stimulated RAW264.7 macrophage cells. The phytochemical, citrusin XI of Citrus unshiu may serve as lead compound in the design of new agents for preventing and treating inflammatory diseases. (C) 2015 Elsevier Ireland Ltd. All rights reserved.