In vitro and in vivo anti-inflammatory activities of columbin through the inhibition of cycloxygenase-2 and nitric oxide but not the suppression of NF-κB translocation

Abstract

Columbin, a diterpenoid furanolactone, was isolated purely for the first time from the plant species Tinspora bakis. The anti-inflammatory effects of columbin were studied in vitro, in silico and in vivo. The effect of columbin on nitric oxide was examined on lipopolysaccharide–interferon-gamma (LPS/IFN) induced RAW264.7 macrophages. In vitro and in silico cyclooxygenase-1 and cyclooxygenase-2 inhibitory activities of columbin using biochemical kit and molecular docking, respectively, were investigated. Mechanism of columbin in suppressing NF-kappaB-translocation was tested using Cellomics®NF-κB activation assay and ArrayScan Reader in LPS-stimulated RAW264.7 cells. Moreover, effects of columbin in vivo that were done on carrageenan-induced mice paw-oedema were tested. Lastly, the in vitro and in vivo toxicities of columbin were examined on human liver cells and mice, respectively. Treatment with columbin or N^ω-nitro-l-arginine methyl ester (l-NAME) inhibited LPS/IFN-γ-induced NO production without affecting the viability of RAW264.7. Pre-treatment of stimulated cells with columbin did not inhibit the translocation of NF-κB to the nucleus in LPS-stimulated cells. COX-1 and COX-2 inhibitory activities of columbin were 63.7 ± 6.4% and 18.8 ± 1.5% inhibition at 100 μM, respectively. Molecular docking study further helped in supporting the observed COX-2 selectivity. Whereby, the interaction of columbin with Tyr385 and Arg120 signifies its higher activity in COX-2, as Tyr385 was reported to be involved in the abstraction of hydrogen from C-13 of arachidonate, and Arg120 is critical for high affinity arachidonate binding. Additionally, columbin inhibited oedema formation in mice paw. Lastly, the compound was observed to be safe in vitro and in vivo. This study presents columbin as a potential anti-inflammatory drug.

Introduction

Recent advances in fundamental biomedical research have established an essential role for inflammation in mediating some human afflictions. This list of inflammatory diseases would run to over 100 each of which causes the deterioration of tissue in one or more parts of the body (Kharitonov, 2004, Nathan and Ding, 2010). Therefore, the inflammatory response must be actively ended when no longer needed to prevent unnecessary harmful biological processes. Mechanisms which serve to terminate inflammation include various cellular and immunological responses and could be initiated using potential anti-inflammatory compounds, which work specifically by inhibiting inflammatory components or activating transcription factors (Tak and Firestein, 2001).

Current anti-inflammatory drugs can inhibit inflammation as curative agents. These conventional drugs have not been successful to cure chronic inflammatory disorders. Natural products play a significant role in human health in relation to the prevention and treatment of inflammatory conditions. Therefore, there is a need for new and safe anti-inflammatory agents and one of the on-going research candidates are plant constituents used in herbal and traditional medicine (Nam, 2006, Read, 1995). Tinospora bakis Miers (Menisoermaceae), locally known in Sudan as “Erg-elhagar”, is used traditionally to treat headache and rheumatism (Broun and Massy, 1929, El Ghazali et al., 2003). Recently, Zafinindra et al. (2003) indicated that the aqueous extract of T. bakis roots has shown in vivo antipyretic effects. Alkaloidal extract of this plant have revealed also in vitro antimalarial activity against Plasmodium falciparum chloroquine-resistant strain W2 (Ouattara et al., 2006). Recently our laboratory confirmed that the plant ethanolic crude extract has potent immunosuppressive and antioxidant activities.

Biologically guided phytochemical study in our laboratory led to the isolation of columbin (Fig. 1), a diterpenoid furanolactone, in a bulk amount for the first time from the root of T. bakis (Koko et al., 2008, Koko et al., 2009). This is thus the fourth report of its occurrence in this family. Originally isolated from colombo root (Jateorhiza palmata Miers, Memspermaceae), columbin has more recently been found in seeds of Sphenocentrum Jollyanum (Memspermaceae). It has, however, been found once in an unrelated family, in Melothria maderaspatana Cogn. (Cucurbitaceae), so that it cannot be regarded as being entirely specific to the Menispermaceae family. Nevertheless, the occurrence of several oxides of columbin in another member of the family, Fdxzurru chloroleucu, suggests that this type of diterpenoid bitter principle may be fairly widespread in this plant group (Cava and Soboczenski, 1956, Cava et al., 1959, Chen et al., 1973, Gilbert et al., 1967, Hanuman et al., 1986, Ramstad et al., 1975). The crude drugs Jateorhiza columba, T. capillipes, T. sagittata, Dzoscoreophyllum cummznsiz, Sphenocentrum Jollyanum and M. maderaspatana contain columbin, and were used for traditional healing of various diseases including inflammatory ones (Cava and Soboczenski, 1956, Cava et al., 1959, Chen et al., 1973, Gilbert et al., 1967, Hanuman et al., 1986, Ramstad et al., 1975). However there were few reports on the biological activities of this bitter substance. Therefore, the current study was designed to investigate the mechanistic anti-inflammatory action of columbin in vitro, in silico and in vivo. We have also tested toxicity of the compound on normal mice and human normal liver cells (WRL-68).