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Evaluation of natural products on inhibition of inducible cyclooxygenase (COX-2) and nitric oxide synthase (iNOS) in cultured mouse macrophage cells

https://doi.org/10.1016/S0378-8741(02)00205-2Get rights and content

Abstract

The inhibitors of prostaglandin biosynthesis and nitric oxide production have been considered as potential anti-inflammatory and cancer chemopreventive agents. In this study, we evaluated approximately 170 methanol extracts of natural products including Korean herbal medicines for the inhibition of prostaglandin E2 production (for COX-2 inhibitors) and nitric oxide formation (for iNOS inhibitors) in lipopolysaccharide (LPS)-induced mouse macrophages RAW264.7 cells. As a result, several extracts such as Aristolochia debilis, Cinnamomum cassia, Cinnamomum loureirii, Curcuma zedoaria, Eugenia caryophyllata, Pterocarpus santalius, Rehmania glutinosa and Tribulus terrestris showed potent inhibition of COX-2 activity (>80% inhibition at the test concentration of 10 μg/ml). In addition, the extracts of A. debilis, Caesalpinia sappan, Curcuma longa, C. zedoaria, Daphne genkwa and Morus alba were also considered as potential inhibitors of iNOS activity (>70% inhibition at the test concentration of 10 μg/ml). These active extracts mediating COX-2 and iNOS inhibitory activities are warranted for further elucidation of active principles for development of new cancer chemopreventive and/or anti-inflammatory agents.

Introduction

A body of evidence suggests that prostaglandins (PGs) and nitric oxide (NO) are involved in various pathophysiological processes including inflammation and carcinogenesis, and inducible isoforms of cyclooxygenase (COX-2) and nitric oxide synthase (iNOS) are mainly responsible for the production of large amounts of these mediators (Schmidt and Walter, 1994, Simon, 1999).

Two isoforms of COX, designated COX-1 and COX-2, have been known, and catalyze the biosynthesis of PGs from arachidonic acid (Vane et al., 1998). COX-1 is constitutively expressed in most tissues and seems to be responsible for housekeeping roles for normal physiological functions. In contrast, COX-2 is not detectable in most normal tissues, but is induced by pro-inflammatory cytokines, growth factors, oncogenes, carcinogens, and tumor promoters, implying a role for COX-2 in both inflammation and control of cell growth (Dean et al., 1991, Oshima et al., 1996, Subbaramaiah et al., 1996). Thus, compounds that inhibit the activity or expression of COX-2 might be an important target for cancer chemoprevention or antiinflammation.

Nitric oxide synthase (NOS) is another important enzyme involved in regulation of inflammation, vascular tone, neurotransmission, tumor cells and other homeostasis of human body. Nitric oxide (NO) is generated via oxidation of the terminal guanidine nitrogen atom of l-arginine by NOS. NO is released during a variety of pathophysiological responses including circulatory shock, inflammation and carcinogenesis (Ohshima et al., 1994, Mordan et al., 1993). NO has also been proposed to be an important mediator of tumor growth. For example, endogenously formed NO appeared to cause the neoplastic transformation of mouse fibroblasts (Mordan et al., 1993). Overexpressed NOS has also been detected in several human tumors (Thomsen et al., 1995, Radomski et al., 1991, Gallo et al., 1998). Thus, along with selective inhibitors of COX-2 or iNOS enzyme activity, a rational enable to address to develop more selective agents for suppression of these genes that might be overexpressed during the inflammation or carcinogenic process. In the present study, therefore, the methanolic extracts of natural products were primarily screened to discover new lead compounds in suppression of corresponding enzyme expression with the assay system of inhibition of prostaglandin E2 accumulation, or inhibition of NO production in LPS-stimulated RAW264.7 cell, a murine macrophage cell line.

Section snippets

Chemicals

Lipopolysaccharide (LPS, E. coli 0111: B4), bovine serum albumin, and sodium bicarbonate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco's modified eagle's medium (DMEM), penicillin–streptomycin and trypsin–EDTA were from GIBCO-BRL (Gaithersburg, MD, USA). Prostaglandin E2 (PGE2) and PGE2-acetylcholineesterase conjugate were from Cayman Co. (Ann Arbor, MI, USA).

Preparation of test materials

Natural products including Korean medicinal plants were purchased from herbal markets

Results and discussion

Several studies have suggested that overexpression of either inducible cyclooxygenase (COX-2) or nitric oxide synthase (iNOS) might be intimately involved in the pathogenesis of diseases including inflammation, cancer, multiple sclerosis, Parkinson's syndrome, and Alzheimer's disease. Thus, there are many efforts to develop enzyme inhibitors or repressors of enzyme formation that are selective for the inducible forms of these enzymes and do not affect the desirable activity of their respective

Acknowledgements

This work was supported in part by the grant of HMP-00-O-21600-0010 funded by the Ministry of Health and Welfare, Korea.

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