Exploring the wound healing potential of Lobostemon fruticosus using in vitro and in vivo bioassays

Ethnopharmacological relevance: Lobostemon fruticosus (L.) H.Buek is a perennial and woody shrub of the Boraginaceae family, found in the Cape region of South Africa. The leaves and twigs are used to treat dermatological conditions such as wounds, burns, ringworm, erysipelas and eczema. Anti-inflammatory, antibacterial, antiviral and anti-proliferative activities of L. fruticosus have been reported. However, there is a void in research which reports on the wound healing properties of this plant. Aim of the study: Aligned with the traditional use of L. fruticosus, our study aimed to use in vitro and in vivo bioassays to confirm the wound healing potential of the plant. Materials and methods: An aqueous methanol extract (80% v/v) of L. fruticosus was prepared using a sample collected from the Western Cape Province of South Africa and chromatographically profiled by ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS). The 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed to determine the non-toxic concentrations of the extract for subsequent use in the in vitro scratch assay. Both the human keratinocyte (HaCaT) and fibroblast (BJ-5ta) cell lines were employed in the in vitro scratch assay. The in vivo caudal fin amputation assay was used to assess the wound healing potential of L. fruticosus, by monitoring fin regeneration in zebrafish larvae treated with the plant extract at various concentrations. Results: Six major compounds were tentatively identified in the L. fruticosus extract namely; globoidnan A, globoidnan B, rutin, rabdosiin, sagerinic acid and rosmarinic acid. The potentially toxic pyrrolizidine alkaloids were also identified and quantitatively confirmed to be present at a low concentration of 119.58 ppm (m/m). Treatment of HaCaT and BJ-5ta cells with the plant extract in the scratch assay resulted in an increase in cell migration, which translates to accelerated wound closure. After 24 hr treatment with 100 mu g/mL of extract, wound closure was recorded to be 91.1 f 5.7% and 94.1 f 1.3% for the HaCaT and BJ-5ta cells, respectively, while the untreated (medium) controls showed 72.3 f 3.3% and 73.0 f 4.3% for the two cell lines, respectively. Complete wound closure was observed between 24 and 36 hr, while the untreated control group did not achieve 100% wound closure by the end of the observation period (48 hr). In vivo, the crude extract at 100 mu g/mL accelerated zebrafish caudal fin regeneration achieving 100.5 f 3.8% regeneration compared to 68.3 f 6.6% in the untreated control at two days post amputation. Conclusions: The study affirms the wound healing properties, as well as low toxicity of L. fruticosus using both in vitro and in vivo assays, which supports the traditional medicinal use. Other in vitro assays that target different mechanisms involved in wound healing should be investigated to support the current findings.