Nanoemulsion of flavonoid-enriched oil palm (Elaeis guineensis Jacq.) leaf extract enhances wound healing in zebrafish

Background: Oil palm (Elaeis guineensis Jacq.) leaf (OPL) is reported to possess excellent wound healing properties, which have been related to the naturally high amounts of flavonoids it contains. Purpose: In this present investigation, we developed a flavonoid enriched extract of OPL (OPL-FEE) and further incorporated it into a nanoemulsion system (OPL-FEE-NE), for evaluation of its toxicity, wound healing properties and transcriptional analysis, using zebrafish (Danio rerio) model. Methods: OPL-FEE-NE was prepared using high pressure homogenization at different cycles and their physicochemical properties were characterized. The toxicity profiles of OPL-FEE and OPL-FEE-NE were studied in three different models, i.e in 3T3 fibroblast cells, embryonic and adult zebrafish. Biopsy punch injury was induced in adult zebrafish and the dermal wounds were treated with pre-optimized doses of OPL-FEE and OPL-FEE-NE, over 30-days using immersion technique. The wound healing effects were evaluated from the progression of wound closure and expressions of the wound healing related genes i.e. tgfβ1, mmp9, mmp13, timp2b, il1β, tnfα, sod1 and cat. Results: The result of IC50 of OPL-FEE and its loaded nanoemulsion was more than 900 mg/l, when tested on dermal cells. However, when tested on zebrafish model, the LC50 of OPL-FEE was within 36-114 mg/l while OPL-FEE-NE gave LC50 in the range of 850-1012 mg/l, suggesting zebrafish toxicity was a more sensitive test than cytotoxicity. At the safe and effective doses, both samples showed comparable wound healing efficacy as compared to positive control drug, allantoin. Nevertheless, OPL-FEE-NE (0.5 g/l) with particle size (< 100 nm) exhibited faster and better wound healing activity, compared to the unencapsulated OPL-FEE (6.25 mg/l). Both OPL-FEE and OPL-FEE-NE expressed their anti-inflammatory and antioxidant properties during the wound repair process, particularly at the early post-wounding stages. Conclusion: The rapidity of the wound healing, as shown in the zebrafish model, suggested that OPL-FEE has potential for further development into a botanical-based wound healing agent and incorporation of OPL-FEE into nanoemulsion systems could be an efficient approach to enhance its wound healing effect towards more effective applications in wound care management.