Isolation of antimicrobial and antioxidant compounds from two mistletoes (Viscum rotundifolium and tapinanthus oleifolius) and synergistic effects with their host

Abstract

The aim of the study was to isolate and characterise the antibacterial and antioxidant compounds from the leaf extracts of the two mistletoes (Viscum rotundifolium and Tapinanthus oleifolius) and to determine the synergistic effects of the plants with their hosts (Mystroxylon aethiopicum and Dichrostachys cinerea). The leaves of the selected plants were collected, dried and ground into fine powder. The powdered plant leaves were extracted using n-hexane, ethyl acetate, acetone, methanol and water. The qualitative phytochemical analysis was done using standard chemical tests and thin layer chromatography. The total phenolic, tannin and flavonoid content were estimated using spectrophotometric methods. The qualitative antioxidant activity was determined using 2, 2-Diphenyl-1-pycrylhydryzyl (DPPH) free radical scavenging assay on thin layer chromatography and quantitative antioxidant activity was determined using colorimetric DPPH assay and ferric reducing power assay. The antibacterial activity of extracts was tested against S. aureus, E. faecalis, E. coli and P. aeruginosa using bioautography and serial broth micro-dilution assay. The cytotoxic effects of the plant extracts were determined using cell viability assay. The active compounds were extracted using serial exhaustive extraction and isolated using the bioassay-guided fractionation and then purified using thin layer chromatography and open column chromatography. The pure compound was identified using the NMR and mass spectroscopy. Methanol was the best extractant with the highest percentage yield. The distinct fluorescing compounds were observed on the CEF and EMW mobile phase. The non-fluorescing compounds detected with vanallin-sulphuric acid spray reagent showed that V. rotundifolium, T. oleifolius and D. cinerea have more polar compounds while M. aethiopicum have more non-polar compounds. All the plants revealed the presence of terpenoids, flavonoids, phlobatannin, tannins steroids and cardiac-glycosides and the absence of alkaloids and saponins. The n-hexane extract of T. oleifolius was significantly high in flavonoid content (34.801±0.798 mgQE/g of extract) and tannin content (15.367±0.320 mgGAE/g of extract) whereas the ethyl acetate extract of M. aethiopicum was high in phenolic content (893.210±3.016 mgGAE/g of extract). The results indicate that the compounds that exhibit antioxidant activity are non-polar to polar, which was confirmed by quantitative tests. M. aethiopicum showed activity against all tested bacteria while V. rotundifolium only had activity against E. faecalis whereas T. oleifolius and D. cinerea did not have any activity. The quantitative antibacterial test confirmed the activity of the plant extracts where the MIC values ranged from 0.04-2.5 mg/mL. The combination of V. rotundifolium and M. aethiopicum (n-hexane, ethyl acetate and acetone extracts) and T. oleifolius and D. cinerea (n-hexane, acetone and methanol extracts) showed synergistic effects in inhibiting the growth of S. aureus whereas the methanol extract of T. oleifolius and D. cinerea showed antagonistic effects in inhibiting the growth of all tested bacteria. The cell viability assay indicated that acetone extracts of all plants were non-toxic on the human liver (C3A) cells. M. aethiopicum was selected for isolation and purification of bioactive compounds. The bioassay-guided fractionation led to the isolation of oleanolic acid acetate. This study demonstrated that the selected plants have antibacterial potential that is ascribed to the phytochemicals present. Further studies including in vivo assays are needed in order to support their use in traditional medicine