INTEGRATING BETANIN AND THEOBROMINE FOR DUAL PATHWAY ACTIVATION IN WOUND HEALING

Abstract

Background: Wound healing is a complex physiological phenomenon that involves the regulation of oxidative stress, cellular proliferation, and the restructuring of the extracellular matrix. Natural bioactive compounds such as Betanin and Theobromine show antioxidant and mitogenic effects; however, their joint effectiveness in improving wound repair has not been explored so far. Objective: The goal of this research is to recommend the synergetic power of betanin and theobromine in 1:1 ratio at various concentrations (25, 50, and 100 µg/mL) in vitro and in vivo models, with primary focus on the modulation of two pathways: SOD1 antioxidant defense and TGF-β/SMAD signaling. Methods: For the in vitro trials, periodontal ligament fibroblasts were used in both MTT and scratch tests. The in vivo wound recovery examinations were conducted on zebrafish, which were observed for 14 days in total. With the help of qRT-PCR, gene expression analysis was performed for sod1, tgfb1, smad2, smad3a, smad4, col1a1a, and mmp9. Histopathological evaluations and imaging of the wound closure were performed to corroborate subjective morphological assessments.

 Results: The Betanin and Theobromine mixture at 100 µg/mL are more effective than any other combination to promote the multi nuclei cell activity in vitro balance. Moreover, in the live models, this mix boosted wound healing and epithelium restoration quite drastically. Gene expression tests confirmed clearly both sod1 and the TGF-β/SMAD elements upregulating. Thus, col1a1a and mmp9 were found more than double and this was related mostly to collagen and ECM remodeling. Conclusion: The 1:1 blend of Betanin and Theobromine at 100 µg/mL not only enhances wound healing by dual pathways antioxidant and regenerative molecular pathways but also seeks to a new development as a topical agent or used in scaffold-integrated wound healing strategy.