The effect of three Ghanaian plants on fibroblast migration, inflammation and bacterial growth in vitro

Abstract

Medical conditions which cause morbidity and mortality, such as chronic wounds and infection, lead to significant medical costs. Much of the world's population is dependent on alternative medicine, of which herbal medicine forms a crucial part. In Ghana and other countries around the world, an estimated 70% of the population rely on alternative treatments like herbs for management of various forms of disease. However, even though medicinal plants are widely used for treatment, most have not been scientifically proven to be safe and efficacious. Hence there is need to assess the biological activity of these plants which may be a potential lead in drug development. This study assessed the wound-related biological activities of three commonly used medicinal plants in Ghana (Aspilia africana, Boerhavia diffusa, and Erythrina senegalensis). Sequential extracts were prepared from the three plants using hexane, ethyl acetate, methanol, and water as solvents, in increasing polarity. Also, ethnomedicinal extracts were obtained with water as solvent, in accordance with the method used by traditional healers. Extracts were screened for phytochemical components using thin layer chromatography (TLC), and phytochemical fingerprinting performed with ultra performance liquid chromatography in tandem with time-of-flight mass spectrometry (UPLC-qTOF-MS). Cytotoxic potential of the extracts in SC-1 fibroblasts, C2C12-myoblasts, and differentiated THP-1-macrophages was determined using the sulforhodamine B staining assay, and cells morphologically assessed with phase contrast, PlasDIC, and live/dead staining microscopy. Acellular antioxidant activity was conducted by exploring the 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, whilst ability to protect against cellular oxidative stress was assessed using 2',7'-dichlorofluorescin diacetate as marker. Anti-inflammatory potential of extracts was evaluated using xanthine oxidase activity. Also, the ability of extracts to alter closure of artificially generated wounds in fibroblast and myoblast monocultures was determined using the scratch assay. The extracts were also assessed for their antibacterial effect using the disk diffusion and microdilution assays. Extracts with a minimum inhibitory concentration (MIC) above or equal to 1 mg/mL were evaluated for their ability to inhibit bacterial biofilms. Analysis using TLC indicated that alkaloids, flavonoids, and phenols were the major groups present. The UPLC-qTOF-MS analysis led to the verification of previously identified, as well as tentative identification of already-described phytochemical compounds or their derivatives in the plants. In addition to the already reported ascorbic acid, quercetin was also identified in A. africana. Furthermore, kaempferol, quercetin, and rutin were identified in the extracts of B. diffusa, the latter compound being identified for the first time in this plant. Also, for the first time, kaempferol, rotenone and rutin, were identified in extracts of E. senegalensis. Apart from these three compounds, neobavaisoflavone was also detected. All the extracts recorded a half maximal inhibitory concentration (IC50) above 100 _g/mL in all three cell-lines. The most cytotoxic extracts to the myoblasts, fibroblasts, and macrophages, were the methanol and ethnomedicinal extracts of A. africana, and the hexane extract of E. senegalensis, with a maximum of 38.8% reduction in cell density respectively. The minimal cytotoxic potential of the extracts was further confirmed by the absence of morphological differences between treated and untreated cells. Most of the extracts exhibited good ABTS radical scavenging activity (IC50 < 100 _g/mL). The strongest effect against the free radical was observed with the ethyl acetate extract of B. diffusa (IC50 = 21.23 _g/mL). On the contrary, most of the extracts recorded poor ability to scavenge the DPPH free radical. Only the methanol extracts of A. africana (IC50 = 278 _g/mL) and E. senegalensis (IC50 = 291 _g/mL) yielded IC50 values below the maximum tested concentration (320 _g/mL). This could possibly be ascribed to the differences in the stereoselectivity between the two free radicals, and the poor DPPH scavenging ability of hydrophilic antioxidants. The effect of the extracts against AAPH-induced oxidation in the cells correlated with the antioxidant potential of the extracts. Whilst most of the extracts with good antioxidant potential suppressed AAPH-induced oxidative stress, the most profound effect was observed with pre-treatment of macrophages with the ethyl acetate extract of A. africana. The extract caused a 1.74-fold decrease in intracellular reactive oxygen species (ROS) concentration after 120 min following pre-treatment with 100 _g/mL, when compared with the AAPH control. This was comparable to the 1.89-fold reduction caused by the positive control compound, 5 _g/mL Trolox. The ethnomedicinal extracts of B. diffusa and E. senegalensis exhibited a dose-dependent increase in intracellular ROS in fibroblasts, with intracellular ROS concentration upon treatment with the extracts at 100 _g/mL being at least 23% higher than the negative controls. This suggest that the extracts could exhibit a possible pro-oxidant effect at higher concentrations. Quercetin, a compound with pro-oxidant effects at higher concentrations, was detected in the ethyl acetate extract of B. diffusa, which may describe this effect. However, none of the extracts used in the current study demonstrated the ability to significantly inhibit xanthine oxidase activity. The strongest activity against the enzyme (maximum of 15% inhibition) was exhibited by extracts of E. senegalensis. The hexane extract of A. africana and the water extracts of B. diffusa increased migration of myoblast cells by 44.4% and 39.4%, respectively. This indicates a possible role of the extracts in enhancing collagen deposition and wound remodelling, two processes with myoblast involvement. On the other hand, six of the extracts decreased fibroblast migration, and therefore could have negative effects on wound healing processes such as collagen and matrix metalloproteinase synthesis. Further analysis would be required to ascertain the extent to which the extracts could impact activity of the cells. Also, the methanol extract of E. senegalensis (MIC = 0.5 mg/mL in E. coli) was the most effective against the micro-organisms tested. All the other extracts had MICs above 1 mg/mL. None of the extracts showed activity against Pseudomonas aeruginosa, Staphylococcus epidermidis and bacterial biofilms. In conclusion, this study has scientifically demonstrated that the three plants may assist wound healing at different stages in the healing process. This could be achieved through their antioxidant effects, ability to suppress oxidative stress, antibacterial activity, and ability to enhance activity of fibroblasts and myoblasts. Practitioners should be cautioned against using high concentrations because of possible cytotoxicity.