title: Anti-inflammatory activity of a new cyclic peptide, citrusin XI, isolated from the fruits of Citrus unshiu.
abstract: BACKGROUND
  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.
  
  
  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) ((1)H, (13)C, 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 70μM. 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-κB activation by blocking IκBα degradation and NF-κB 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-κB activation associated with IκBα degradation and NF-κB 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.
authors.affiliation: Department of Herbal Crop Research, National Institute of Horticultural & Herbal Science, RDA, Eumseoung 369-873, Republic of Korea.
authors.forename: Hyung Jun
authors.initials: HJ
authors.lastname: Noh
authors.affiliation: Department of Microbiology, College of Natural Sciences, Pukyong National University, Busan 608-737, Republic of Korea.
authors.forename: Dukhyun
authors.initials: D
authors.lastname: Hwang
authors.affiliation: Department of Herbal Crop Research, National Institute of Horticultural & Herbal Science, RDA, Eumseoung 369-873, Republic of Korea.
authors.forename: Eun Suk
authors.initials: ES
authors.lastname: Lee
authors.affiliation: Citrus Research Station, National Institute of Horticultural & Herbal Science, Rural Development Administration, Jeju 697-943, Republic of Korea.
authors.forename: Jae Wook
authors.initials: JW
authors.lastname: Hyun
authors.affiliation: Citrus Research Station, National Institute of Horticultural & Herbal Science, Rural Development Administration, Jeju 697-943, Republic of Korea.
authors.forename: Pyoung Ho
authors.initials: PH
authors.lastname: Yi
authors.affiliation: Department of Herbal Crop Research, National Institute of Horticultural & Herbal Science, RDA, Eumseoung 369-873, Republic of Korea.
authors.forename: Geum Soog
authors.initials: GS
authors.lastname: Kim
authors.affiliation: Department of Herbal Crop Research, National Institute of Horticultural & Herbal Science, RDA, Eumseoung 369-873, Republic of Korea.
authors.forename: Seung Eun
authors.initials: SE
authors.lastname: Lee
authors.affiliation: School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Changhyun
authors.initials: C
authors.lastname: Pang
authors.affiliation: School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Yong Joo
authors.initials: YJ
authors.lastname: Park
authors.affiliation: School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Kyu Hyuck
authors.initials: KH
authors.lastname: Chung
authors.affiliation: Department of Microbiology, College of Natural Sciences, Pukyong National University, Busan 608-737, Republic of Korea. Electronic address: gundokim@pknu.ac.kr.
authors.forename: Gun Do
authors.initials: GD
authors.lastname: Kim
authors.affiliation: School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea. Electronic address: khkim83@skku.edu.
authors.forename: Ki Hyun
authors.initials: KH
authors.lastname: Kim
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000302:isolation & purification / Q000494:pharmacology*; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002957:Citrus; D051546:Cyclooxygenase 2 / Q000378:metabolism; D005638:Fruit / Q000737:chemistry; D006801:Humans; D008070:Lipopolysaccharides; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism; D010456:Peptides, Cyclic / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D000893:Anti-Inflammatory Agents; D008070:Lipopolysaccharides; D016328:NF-kappa B; D010456:Peptides, Cyclic; C000603530:citrusin XI; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2
keywords: Anti-inflammation; Citrus unshiu; Citrusin XI; Glycine (PubChem CID: 750); Peptide; RAW264.7 cells; Rutaceae; d-serine (PubChem CID: 71077); d-tyrosine (PubChem CID: 71098); l-leucine (PubChem CID: 6106); l-proline (PubChem CID: 145742)