Dr. Varuna P. PanickerNIMNA AJAY2024-07-222024-07-222023-03-23https://krishikosh.egranth.ac.in/handle/1/5810212335Collagen, the abundant structural protein in the extracellular matrix has wide application in medical, tissue engineering and pharmaceutical industry. The present study was conducted to characterize and evaluate the wound healing potential of isolated ASC from Daggertooth pike conger (Muraenesox cinereus).Conger eels were collected from Kochi harbour and pre-treatment for removal of fats and non-collagenous proteins. ASC was isolated by treating with 0.5 M acetic acid for 3 days and supernatants were collected. Protein precipitation was carried out by adding NaCl to final concentration of 2.3 M. Precipitated samples were dissolved in 0.5 M acetic acid and dialysed against 0.1 M acetic acid for one day and in distilled water until neutral pH was obtained. Yield of ASC was 12.78 and 31.95 per cent on wet and drymatter basis respectively. ASC on Lowry’s test showed a concentration of 8.8µg/ml. On electrophoretic separation revealed the presence of characteristic α (2 α1 and α2), β, γ subunits similar to that of type I calf collagen. An absorption peak of 221 nm characteristic to triple helix was observed on UV-visible spectroscopy. FTIR spectrum of ASC showed Amide bands similar to that of collagen. Analysis for secondary structure, by CD spectroscopy revealed a CD spectra with positive absorption maximum at 222 nm, and negative at 214 nm. Denaturation temperature of the extract was obtained at 38.2℃ which in turn confirm that collagen can withstand very high temperature. Solubility assay revealed a maximum solubility at low salt concentration and acidic pH. Hydroxyproline concentration in the extract was 9.9g. Invivo wound healing study showed that group treated with paste has increased wound healing compared to gel and control group. Results were confirmed by histopathology, immunohistochemistry and relative quantification of selected growth factors and cytokines. On histopathological evaluation increased fibroblast proliferation, collagen deposit and re-epithelisation were observed on collagen treated group. Difference in the collagen deposition in response to treatment was studied using staining like Picrosirus and Masson’s trichome. Angiogenesis was studied by immunohistochemistry using CD31 antibody. Observations on histopathology and immunohistochemistry could correlate with the gene expression study of TGFβ-1, VEGF, IL-10 and TNF-α. Upregulation of TGFβ-1, VEGF and IL-10 and downregulation of TNF-α was observed in collagen treated groups which might be due to the wound healing potential of collagen.EnglishThesis