Combinatorial Effect of Medicinally Important Plant Extracts for Wound Healing Potentials

Abstract

Wound healing remains as an unmet challenge even with considerable research on advanced wound care globally. Even advanced wound healing therapies possess some disadvantages. These shortcomings necessitated increasing interest in the use of medicinal plants for wound management. Growing interest towards medicinal plants primarily resulted as an outcome of scientific advancement in delineating the principles and molecular mechanisms behind the contribution of various phytoconstituents in them. Twelve medicinal plants with claim on traditional wound treatment were screened for cell viability (WST-1 assay on primary human dermal fibroblasts (HDFs). Plants with viable concentrations less than or equal to 80and#956;g/ml were selected for in vivo wound healing assay. Bacopa monnieri (15and#956;g/ml), Acalypha indica (20and#956;g/ml), and Calotropis gigantea (1and#956;g/ml) showed significantly improved wound closure kinetics compared to vehicle and positive controls (Mupirocin IP 2%). Extract combination (post optimization using Response Surface Methodology) exhibited significant wound closure rate compared to individual plant extracts, vehicle and positive controls. Techniques employed to ascertain the quality of healed tissues are Hematoxylin and Eosin staining, immunohistochemistry of epidermal markers (K5, K1 and Loricrin), proliferation marker (Ki67) and specific staining for collagens I and IV. Results revealed complete re-epithelialization of wounds after extract treatments. Individual plant extracts and their combination did not negatively affect epidermal differentiation (results of K5, K1 and Loricrin) indicating appropriately formed epidermis. Combination did not show significant cell proliferation compared to individual extracts and control indicating nonadditive nature of phytochemical interaction. Significant increase in neovascularization was observed in extract combination compared to individual treatments and control. Combination appeared to reduce collagen I content with improved remodeling indicating a possible antifibrotic contribution compared to individual extracts. This was further validated by Masson s trichrome staining. Plant extracts and control did not show antibacterial properties at tested concentrations on Pseudomonas aeruginosa and Staphylococcus aureus. However, they presented different degree of antiquorum sensing (QS) activity by inhibiting violacein pigment production by Chromobacterium violaceum and biofilm by Pseudomonas aeruginosa and Staphylococcus aureus. LCMS analysis of the extract combination revealed 12 and 9 prominent peaks in positive and negative ion modes respectively. The interaction between these molecules in combination could be contributing to the enhanced wound healing property exhibited when compared to individual extracts. newline