In vitro and in vivo anti-inflammatory potential of Cryptolepis buchanani
Introduction
Cryptolepis buchanani Roem. & Schult (Asclepiadaceae), a climbing tree, is widely used in folk medicine in southeast Asia. In Thailand, the alcoholic extract of stem of this plant is commonly used for the treatment of inflammatory conditions such as arthritis, and muscle and joint pain (Panthong et al., 1986, Laupattarakasem et al., 2003). A weak inhibition of eicosanoid generation from rat leukocytes was previously reported but this was not dose-dependent (Laupattarakasem et al., 2003). Therefore, we designed experiments to re-evaluate the anti-inflammatory potential of this plant in both acute and chronic inflammation using several in vitro and in vivo experimental models. An acute toxicity test was included in this preliminary toxicity study.
Inflammation is a process involving multiple factors acting in a complex network. The ingress of leukocytes into the site of inflammation is crucial for the pathogenesis of inflammatory conditions (Colditz, 1985, Kasama et al., 1995). Neutrophils and macrophages are known to recruit and play pivotal roles in acute and chronic inflammation, respectively (Kasama et al., 1993). At the inflamed site, the recruited cells are activated to release many inflammatory mediators which elicit the initiation and maintenance of an inflammatory response, causing a change from the acute phase to the chronic phase of inflammation. Therefore, inhibition of the cellular reactions is one of the targets that are generally used as an in vitro model for anti-inflammatory testing. For in vivo tests, inflammation can be induced in animals by many substances. Rat paw edema is the most commonly used model for acute inflammation while subcutaneous implantation of biomaterial is usually used for the chronic inflammatory model. In the present study, evaluation of the anti-inflammatory potential of 50% ethanol extract of Cryptolepis buchanani was performed using both in vitro and in vivo tests. Inhibition of eicosanoid generation from rat neutrophils and pro-inflammatory cytokine (e.g., tumor necrosis factor-α, TNF-α) release from macrophages are used as markers for in vitro tests for acute and chronic inflammation, respectively. Carrageenan-induced rat paw edema and cotton thread-induced granulation in rats were used as the model for acute and chronic inflammation.
Section snippets
Chemicals
Calcium ionophore (A23187), oyster glycogen (Type II), indomethacin (IDM), 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-p-benzoquinone (AA 861), acetylsalicylic acid (ASA), ethyl acetate, dimethylsulfoxide (DMSO), carrageenan, phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS) from E.coli 026:B6 were obtained from Sigma–Aldrich (St. Louis, MO, USA). RPMI-1640 medium, fetal bovine serum (FBS), penicillin and streptomycin, human endothelial SFM medium, medium 199, trypsin-EDTA
Statistical analysis
The statistical significance of the results was analyzed by Student's t-test for unpaired observations. The results were expressed as the mean ± standard error of mean (S.E.M.), the values of statistical significance being set at *P ≤ 0.05 and **P ≤ 0.001 levels.
Effects of plant extract on eicosanoid generation
Addition of 1 μM calcium ionophore A23187 to the suspension of rat mixed leukocytes (PMN neutrophils and mononuclear cells) caused the generation of eicosanoids via cyclooxygenase (COX) and 5-lipooxygenase (5-LO) pathways, resulting in the release of TXB2 and LTB4, respectively (Fig. 1). Two compounds, known to be potent and specific inhibitors, were used as reference drugs. Pre-treatment of cells with indomethacin (COX inhibitor, 5 μM) caused suppression of TXB2 level while LTB4 suppression was
Discussion and conclusion
Our results provide evidence that a 50% ethanol extract of Cryptolepis buchanani stems possesses anti-inflammatory activity. The extract was tested with the standard models for both acute and chronic inflammation. For the acute inflammatory model, the in vitro test was performed using the calcium ionophore A23187-stimulated rat leukocyte model. It was found that the extract inhibited both TXB2 and LTB4, the products of arachidonic acid mediated by COX and 5-LO pathways, respectively. The COX
Acknowledgements
This work was partly supported by the Invitation Research Fund, Faculty of Medicine, Khon Kaen University. The authors would like to thank Dr. Arunporn Itharat, Department of Pharmacognosy and Pharmaceutical Botany, Prince of Songkla University, Hat Yai, Thailand, for plant identification and supply of plant materials. The authors would like to thank Prof. Keith Godfrey and Prof. Peter Houghton for their valuable suggestions.
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