Isolation and characterization of the compounds responsible for the antimutagenic activity of Combretum microphyllum (Combretaceae) leaf extracts

BMC Complement Altern Med. 2017 Sep 6;17(1):446. doi: 10.1186/s12906-017-1935-5. Makhafola TJ1, Elgorashi EE2,3, McGaw LJ1, Awouafack MD1, Verschaeve L4,5, Eloff JN6. Author information 1 Phytomedicine Programme, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa. 2 Toxicology and Ethnoveterinary Medicine, Food, Feed and Veterinary Public Health, ARC-Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort, 0110, South Africa. 3 Department of Paraclinical Sciences, Faculty of Veterinary Science, Private Bag X04, Onderstepoort, 0110, South Africa. 4 Scientific Institute of Public Health, Rue Juliette Wytsmanstreet 14, 1050, Brussels, Belgium. 5 Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Belgium. 6 Phytomedicine Programme, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa. kobus.eloff@up.ac.za. Abstract BACKGROUND: Mutations play a major role in the pathogenesis and development of several chronic degenerative diseases including cancer. It follows, therefore that antimutagenic compound may inhibit the pathological process resulting from exposure to mutagens. Investigation of the antimutagenic potential of traditional medicinal plants and compounds isolated from plant extracts provides one of the tools that can be used to identify compounds with potential cancer chemopreventive properties. The aim of this study was to isolate and characterise the compounds responsible for the antimutagenic activity of Combretum microphyllum. METHODS: The methanol leaf extract of C. microphyllum was evaluated for antimutagenicity in the Ames/microsome assay using Salmonella typhimurium TA98. TA100 and TA102. Solvent-solvent fractionation was used to partition the extracts and by using bioassay-guided fractionation, three compounds were isolated. The antimutagenic activity of the three compounds were determined in the Ames test using Salmonella typhimurium TA98, TA100 and TA102. The antioxidant activity of the three compounds were determined by the quantitative 2,2-diphenyl-1-picrylhydrazyl (DPPH)-free radical scavenging method. The cytotoxicity was determined in the MTT assay using human hepatocytes. RESULTS: A bioassay-guided fractionation of the crude extracts for antimutagenic activity led to the isolation of three compounds; n-tetracosanol, eicosanoic acid and arjunolic acid. Arjunolic acid was the most active in all three tested strains with a antimutagenicity of 42 ± 9.6%, 36 ± 1.5% and 44 ± 0.18% in S. typhimurium TA98, TA100 and TA102 respectively at the highest concentration (500 μg/ml) tested, followed by eicosanoic acid and n-tetracosanol. The antioxidant activity of the compounds were determined using the quantitative 2,2 diphenyl-1-picryhydrazyl (DPPH)-free radical scavenging method. Only arjunolic acid had pronounced antioxidant activity (measured as DPPH-free scavenging activity) with an EC50 value of 0.51 μg/ml. The cytotoxicity of the isolated compounds were determined in the MTT assay using human hepatocytes. The compounds had low cytotoxicity at the highest concentration tested with LC50 values >200 μg/ml for n-tetracosanol and eicosanoic acid and 106.39 μg/ml for arjunolic acid. CONCLUSIONS: Based on findings from this study, compounds in leaf extracts of C. microphyllum protected against 4-NQO and MMC induced mutations as evident in the Ames test. The antimutagenic activity of arjunolic acid may, at least in part, be attributed to its antioxidant activity resulting in the detoxification of reactive oxygen species produced during mutagenesis. KEYWORDS: Antimutagenicity; Antioxidant activity; Arjunolic acid; Combretum microphyllum; Cytotoxicity; Eicosanoic acid; n-Tetracosanol PMID: 28874162 PMCID: PMC5585923 DOI: 10.1186/s12906-017-1935-5 Free PMC Article