Morin sulfates/glucuronides exert anti-inflammatory activity on activated macrophages and decreased the incidence of septic shock
Introduction
Flavonoids are ubiquitous and abundant in plants and considered very important for preventing a wide variety of diseases, including allergies, cardiovascular disease, certain forms of cancer, hepatic diseases, and inflammation (Middleton et al., 2000). Morin (3, 5, 7, 2′, 4′-pentahydroxyflavone) is a flavonoid constituent of many herbs and fruits. Based on in vitro studies, morin has been reported to show various beneficial activities, including antioxidation Hanasaki et al., 1994, Kok et al., 2000, Ramanathan et al., 1994, anti-mutagenesis Bhattacharya and Firozi, 1988, Francis et al., 1989 and anti-inflammation Kim et al., 1999, Raso et al., 2001. However, there is growing evidence that conjugative metabolism is central to the biological fate of flavonoids (Chao et al., 2002). Therefore, the biological effects of flavonoids could be primarily determined by their conjugated metabolites. A previous study reported that the serum level of conjugated metabolites of morin was much higher than the parent form after oral administration of morin to rabbits (Hsiu et al., 2001). Conjugated metabolites of flavonoids supposedly possess profoundly different physicochemical properties from their parent forms and therefore might show differences regarding to solubility, transport across cell membrane or intrinsic activity. In contrast to previous in vitro studies of morin Kim et al., 1999, Raso et al., 2001, the present investigation is the first report on the anti-inflammatory effects of the major metabolites of morin, morin sulfates/glucuronides. We'll assess the modulation effects of the conjugated metabolites of morin as well as the parent form morin on immune system.
Macrophages play major roles in both innate and acquired immunity. They can be stimulated by cytokines, such as interferon-γ (IFN-γ), or microbial components, such as lipopolysaccharide (LPS) (Adams and Hamilton, 1984). For innate immunity, macrophages phagocytose and kill microbes, and release inflammatory mediators such as nitric oxide (NO) by the action of inducible NO synthase (iNOS). For acquired immunity, macrophages serve as antigen presenting cells and release cytokines such as tumor necrosis factor (TNF-α), interleukin (IL)-1, IL-6, and IL-12 to regulate the functions or development of helper T cells. However, when macrophages are inappropriately activated, an excessive amount of NO would be secreted, which has been implicated in the pathogenesis of various inflammatory disease such as rheumatoid arthritis, and septic shock. Therefore, how to well control the activities of macrophages is an important strategy for the treatment of chronic inflammatory diseases. In this study, we evaluated the anti-inflammatory activities of morin as well as morin sulfates/glucuronides by assessing their effects on the functions of LPS-activated macrophages in vitro. Moreover, in vivo study was also conducted on mice by giving morin orally for two months to investigate the phagocyte activities in the peripheral blood and the protective effects on experimental septic shock.
Section snippets
Mice
BALB/c mice were obtained from the Animal Center of the College of Medicine, National Taiwan University and maintained in the Animal Center of China Medical College. The animal room was on a 12-h light and dark cycle with a constant temperature and humidity. All mice used were 8 wk of age. Four oral doses of morin (0, 50, 100, 200 mg/kg) were given to each group of mice once daily with gastric gavage. All procedures conformed to the Guide for the Care and Use of Laboratory Animals (NRC, USA).
Materials
Effect of morin or morin sulfates/glucuronides on NO produced by the activated murine macrophage cell line and peritoneal excluded macrophages
For investigating the effects of morin or morin sulfates/glucuronides on macrophages, firstly, cell viability was monitored by using MTT assay. The highest concentrations of morin and morin sulfates/glucuronides that would not affect the viability of either macrophage cell line, RAW 264.7, or murine peritoneal macrophages were 25 mM and 25 μM, respectively. The levels of NO were then examined when various concentrations of morin or morin sulfates/glucuronides were co-cultured with
Discussion
This study is the first report on the anti-inflammatory effects of the major metabolites of morin, morin sulfates/glucuronides. Although some studies had reported the in vitro effects of morin Kim et al., 1999, Raso et al., 2001, the previous reports revealed that morin was not promising for inhibiting NO production from RAW 264.7 cells (Kim et al., 1999) and very slightly inhibited iNOS expression only at the concentration of 50 μM in the macrophage cell line J774A.1 (Raso et al., 2001). In
Acknowledgements
This study was supported by NSC 91-2320-B-039-038 granted by National Science Council, R. O. C., CMC-89-M-21 and CMC-90-M-11 by China Medical University.
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