The in vitro inhibition of genotoxicity by plant extracts and the isolation and characterization of antimutagenic compounds from Combretum microphyllum (Combretaceae)

Abstract

The possibility of moderating the response of cells to a particular mutagen by phytomedicines opens new horizons in cancer prevention. On this basis, the search for antimutagens presents many possibilities for the discovery of new anticarcinogenic compounds. Determination of the antimutagenic potential of plant extracts is an important step in the discovery of new effective cancer chemopreventive agents. The main aim of this study was to isolate and characterize antimutagenic compounds active against 4-nitroquinoline 1-oxide (4NQO), mitomycin-C (MMC) and ethyl methanesulfonate (EMS) in vitro. Antioxidant compounds play a preventive role against mutation related diseases and thus may have potential antimutagenic activity. It was for this reason that methanol leaf extracts of 120 plant species from the existing plant material collection of tree leaves in the Phytomedicine Programme of the University of Pretoria were assayed for qualitative antioxidant activity. Almost 98% of the extracts (117) had well defined antioxidant compounds. From these 117 species, 31 were selected for investigation of qualitative antioxidant activity, total phenolic content, mutagenic and antimutagenic activity. Methanol extracts of the selected 31 species effectively reduced the DPPH free radical with EC50 values ranging from 1.20 ± 0.22 to 19.07 ± 1.50 μg/ml and total phenolic content measured in gallic acid equivalents (GAE) ranged from 5.17 ± 0.97 to 18.65 ± 3.86 mgGAE/mg extract. In some instances, the plant extracts had better antioxidant activity than the positive control, L-ascorbic acid (vitamin C) with EC50 value of 2.28 ± 0.02 μg/ml. Only one plant (Halleria liucida) extract was mutagenic in the Ames test using Salmonella typhimurium TA98 and TA100. Upon investigating antimutagenicity, the percentage inhibition of 4-NQO in the Ames test ranged from 8.8 ± 2.4 to 76.7 ± 4.7% in S. typhimurium TA98 and from 0.8 ± 6.9 to 99.00 ± 2.9% in TA100. There was a direct correlation between the presence of antioxidant activity and antimutagenic activity of the plant extracts confirming the initial hypothesis of the study. Some of the plant extracts had a comutagenic effect as they potentiated the mutagenic effects of 4-NQO. From the 31 plant species investigated, 4 species (2 antimutagenic and 2 comutagenic) were selected for in-depth genotoxicity (mutagenicity and antimutagenicity) studies using the Ames test, cytokinesis block micronucleus/cytome assay and alkaline single-cell gel electrophoresis/comet assay. These species were: Combretum microphyllum Klotzsch (Combretaceae), Leucospermum erubescens Rourke (Proteaceae), Kirkia wilmsii Engl (Simaroubaceae) and Thespisia acutiloba (Baker f) Exell & Mendonça (Malvaceae). No plant extract was mutagenic in the Ames test and micronucleus/cytome assay. However, some extracts were slightly mutagenic in the comet assay and this may be attributed to cytotoxicity rather than genotoxic effects. The extracts of C. microphyllum and L. erubescens inhibited the mutagenic effects of 4-NQO (S. typhimurium TA98 and TA100) and MMC (S. typhimurium TA102) with values from 10% to more than 30% in the Ames test. However, extracts of K. wilmsii and T. acutiloba enhanced the mutagenic effects of 4-NQO and MMC in all tester strains. Extracts of C. microphyllum and L. erubescens prevented micronuclei induction by up to 65.9%, chromosomal rearrangements by 51.9% and gene amplification by 86.1% in the micronucleus/cytome assay. In the comet assay, there was a clear dose dependent decrease in comet tail length. Based on the preliminary screening results, in depth genotoxicity investigation results and availability of plant material, C. microphyllum was selected for the isolation of antimutagenic compounds. Bioassay-guided fractionation of the crude methanol leaf extract, using the Ames test (S. typhimurium TA98, TA100 and TA102) as an indicator of antimutagenicity was used for the isolation of antimutagenic compounds. To simplify the isolation of the antimutagenic compounds, the methanol leaf extract of C. microphyllum was first separated into four fractions based on polarity using a solvent-solvent fractionation procedure. The solvents used were: hexane, ethyl acetate, water and butanol. The fractions were subjected to antimutagenicity testing in the Ames test. The ethyl acetate fraction was the most active in all three tester strains i.e. S. typhimurium TA98, 100 and 102 with percentage antimutagenicity of up to 32.7 ± 2.1, 30.6 ± 3.8% and 21.4 ± 3.1% respectively at the highest concentration (5 mg/ml) assayed. The activity was dose dependent. Bioactivity-guided fractionation of the ethyl acetate fraction by open column chromatography led to the isolation of three compounds. The structures of the compounds were determined using NMR and were identified as n-tetracosanol (C1), eicosanoic acid (C2) and olean-12-ene-28-oic acid (arjunolic acid) (C3). The antimutagenic activity in the Ames test using S. typhimurium TA98, 100 and 102, and the cytotoxicity on C3A human hepatocarcinoma cell line of the isolated compounds were determined. In the Ames test, the compounds were assayed at concentrations 10 times lower than the concentrations used for the crude extract and fraction. Arjunolic acid was more active in all three tester strains with percentage antimutagenicity of up to 41.9 ± 9.6%, 35.8 ± 1.5% and 43.8 ± 0.18% in S. typhimurium TA98, 100 and 102 respectively, followed by eicosanoic acid and lastly n-tetracosanol. Overall, the compounds had much higher antimutagenic activity than the crude extract and the fractions. The quantities of the isolated compounds were not sufficient to allow testing in the micronucleus/cytome assay and comet assay. The compounds were not cytotoxic at the highest concentration tested i.e. 200 μg/ml. n-Tetracosanol and eicosanoic acid had LC50 values > 200 μg/ml (with percentage cell viability of 59.7 ± 7.2and 50.1 ± 6.2% at the highest concentration respectively) and arjunolic acid had LC50 value of 106.4 ± 5.1 μg/ml. Arjunolic acid was the only compound with pronounced antioxidant activity with an EC50 value of 6.3 ±0.3 μg/ml. This was moderate antioxidant activity compared to that of vitamin C. n-Tetracosanol and eicosanoic acid did not have antioxidant activity. The antimutagenic activity of arjunolic acid at least in part may be attributed to its antioxidant activity resulting in the detoxification of reactive oxygen species produced during mutagenesis, but other mechanisms were probably involved with the other compounds. These results also show that it would not have worked by just isolating antioxidant compounds and testing these for genotoxicity. Combretum microphyllum has potential antimutagenic activity and protective effects against cancer since the crude extract of this plant species effectively inhibits the genotoxic end-points induced by 4-nitroquinoline 1-oxide (4NQO), mitomycin-C (MMC) and ethyl methanesulfonate (EMS) in vitro. This conclusion is supported by the fact that chromosomal biomarkers of genomic instability are relevant to cancer and that genotoxicity involving gene mutations, chromosomal aberrations and rearrangements and DNA strand breakages play a major role in cancer initiation. Pure compounds isolated from the ethyl acetate fraction of this extract had antimutagenic effects in the Ames test using S. typhimurium TA98, 100 and 102. The compounds had better activity compared to the crude extract and fractions at concentrations 10 times lower. The compounds were not cytotoxic against C3A human hepatocarcinoma cells at the highest concentration tested of 200 μg/ml. Overall, these types of studies on plant extracts may provide leads to the discovery of chemopreventive agents that can be used to develop pharmacologically active agents for prevention of chronic degenerative diseases. The compounds isolated in this study have been previously isolated from other plant species and are known to possess numerous biological activities, including amongst others: antioxidant activity, antimicrobial activity, antitumor effects and anticholinesterase activity. Even when new chemical structures are not found in medicinal plant research studies and drug discovery, known compounds with new biological activities can provide important drug leads Some of the extracts, fractions or compounds may be used as elements in nutraceuticals, functional foods and other applications as antimutagens to limit the possibility of mutations. Antimutagens and anticarcinogens play a major role in the primary prevention of mutations and cancer development by lowering the frequency or rate of mutations. This is the first report of the isolation of n-tetracosanol, eicosanoic acid and arjunolic acid from C. microphyllum. We also report for the first time the potential antigenotoxic effects of the crude extract, fractions and the compounds isolated from C. microphyllum.