ABSTRACT
A protocol was established to produce bioactive compounds in a callus culture of Ageratina pichinchensis by using 1 mg L-1 NAA with 0.1 mg L-1 KIN. The phytochemical study of the EtOAc extract obtained from the callus biomass, allowed the isolation and characterization of eleven secondary metabolites, of which dihydrobenzofuran (5) and 3-epilupeol (7), showed important anti-inflammatory activity. Compound 5 inhibits in vitro the secretion of NO (IC50 = 36.96 ± 1.06 µM), IL-6 (IC50 = 73.71 ± 3.21 µM), and TNF-α (IC50 = 73.20 ± 5.99 µM) in RAW (Murine macrophage cells) 264.7 macrophages, as well as the activation of NF-κB (40% at 150 µM) in RAW-blue macrophages, while compound 7 has been described that inhibit the in vivo TPA-induced ear edema, and the in vitro production of NO, and the PLA2 enzyme activity. In addition, quantitative GC-MS analysis showed that the anti-inflammatory metabolites 5 and 7 were not detected in the wild plant. Overall, our results indicated that A. pichinchensis can be used as an alternative biotechnological resource for obtaining anti-inflammatory compounds. This is the first report of the anti-inflammatory activity of compound 5 and its production in a callus culture of A. pichinchensis.
Subject(s)
Ageratina/chemistry , Anti-Inflammatory Agents/pharmacology , Benzofurans/pharmacology , Edema/drug therapy , Pentacyclic Triterpenes/pharmacology , Animals , Anti-Inflammatory Agents/isolation & purification , Benzofurans/isolation & purification , Culture Techniques , Ear , Edema/chemically induced , Edema/immunology , Edema/pathology , Ethanol/chemistry , Interleukin-6/antagonists & inhibitors , Interleukin-6/biosynthesis , Kinetin/pharmacology , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/pharmacology , Male , Mice , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Naphthaleneacetic Acids/pharmacology , Nitric Oxide/antagonists & inhibitors , Nitric Oxide/biosynthesis , Pentacyclic Triterpenes/isolation & purification , Phospholipases A2/metabolism , Plant Extracts/chemistry , Plant Leaves/chemistry , RAW 264.7 Cells , Secondary Metabolism/drug effects , Solvents/chemistry , Tetradecanoylphorbol Acetate/administration & dosage , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
BACKGROUND: Bursera copallifera (Burseraceae) releases a resin known as "copal ancho" which has been used, since pre-Colombian times, as ceremonially burned incense and to treat tooth ache, tumors, arthritis, cold, cough, and various inflammatory conditions; however, its anti-inflammatory potential is poorly studied. The aim of the present study was to isolate, quantify, and to investigate the anti-inflammatory activity of triterpene compounds isolated from the copal resin of B. copallifera. METHODS: The constituents present in the total resin of B. copallifera were obtained by successive chromatographic procedures, and quantitative analysis was performed by High Performance Liquid Chromatography (HPLC). Anti-inflammatory effects of the isolated triterpenes were investigated to determine their inhibitory effects on phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced edema in mice, viability and nitric oxide (NO) production inhibition on lipopolysaccharide (LPS)-activated RAW 264.7 macrophages, and inhibition of cyclooxygenase (COX)-1, COX-2 and secretory Phospholipase A2 (sPLA2) activities in vitro. RESULTS: Quantitative phytochemical analysis of the copal resin showed the presence of six pentacyclic triterpenes of which, 3-epilupeol (59.75 % yield) and α-amyrin (21.1 % yield) are the most abundant. Among the isolated triterpenes, 3-epilupeol formiate (Inhibitory Concentration 50 % (IC50) = 0.96 µmol), α.amyrin acetate (IC50 = 1.17 µmol), lupenone (IC50 = 1.05 µmol), and 3-epilupeol (IC50 = 0.83 µmol) showed marked inhibition of the edema induced by TPA in mice. α-amyrin acetate and 3-epilupeol acetate, at 70 µM, also inhibited the activity of COX-2 by 62.85 and 73.28 % respectively, while α-amyrin and 3-epilupeol were the best inhibitors of the production of NO in LPS-activated RAW 264.7 cells with IC50 values of 15.5 and 8.98 µM respectively, and did not affected its viability. All compounds moderately inhibited the activity of PLA2. CONCLUSIONS: This work supports the folk use of B. copallifera and provides the basis for future investigations about the therapeutic use of this resin in treating inflammation.
