PJB-2022-254
Investigation of the biocompatibility and in vivo wound healing effect of Cotinus coggyria extracts
Esra ARAT
Abstract
This study aimed to evaluate Cotinus coggyria, a plant species known to have beneficial effects in wound healing, for its phenolic contents, cytotoxicity/proliferation, hemolytic, antimicrobial, genotoxic, apoptotic necrotic activities and in vivo wound healing effects. The methanol extract of C. Coggyria was analyzed by TOF/LC-MS to determine phenolic compounds and flavonoids. Cytotoxicity was analyzed via the WST-1 test exposing L929 fibroblasts to aqueous extracts of methanol extracts. The double staining method were used to reveal apoptotic and necrotic L929 fibroblasts. A real time cell analysis system, xCELLigence, was used to evaluate cell proliferation. The micronucleus test was employed to examine genotoxicity on Chinese Hamster Ovary (CHO) cells. The extracts were applied at two different concentrations 0.6 mg/ml and 1.2 mg/ml on L929 fibroblast and CHO cells. The hemolysis test was conducted with the water extract to evaluate hemocompatibility in part. The disc diffusion method was used to evaluate antimicrobial activity of the aqueous and methanol extracts against E.coli, S.aureus and C.albicans. Expressions of glycosaminoglycans in L929 fibroblast cells, to which extracts were applied with dimethyl methylene blue, were determined. To determine the expression of collagen type I, type IV, fibronectin and elastin involved in wound healing, immunocytochemistry was performed in L929 fibroblast cells to which the extracts were applied. In order to investigate the wound healing effect of C.coggyria extracts, burn wounds were created in rats and healing rates were followed for 21 days. The TOF-LC/MS analyses revealed that phenolic compounds and flavonoids in the methanol extract of C.coggyria leaf and stem were as follows: gallic acid, catechin, protocatechic acid, vanillic acid, ellagic acid, rosmarinic acid, quercetin, 4-hydroxybenzoic acid, chlorogenic acid, routine and apigenin 7-glycoside. The aqueous and methanol extracts obtained from Cotinus coggyria caused neither cytotoxicity nor genotoxicity (p>0.05) on L929 fibroblasts with a negligible apoptotic and necrotic effects. The aqueous extract of C. coggyria had no hemolytic effect. The strongest antibacterial activity of the methanol and aqueous extracts were observed against E.coli with a zone diameters of 9 mm and 8 mm, respectively. It was determined that GAG expression increased in cells to which C.coggyria stem extract was applied compared to the control group. Immunocytochemical staining revealed collagen type I, type IV, fibronectin and elastin. In the in vivo burn model, the wound diameter was measured as 1.8 cm at day 0. At the end of the 21st day, the wound diameter was 0.8 cm in animals treated with leaf extract, 0.9 cm in animals treated with stem extract, and 1.3 cm in the control group. As a result, Cotinus coggyria has antibacterial properties especially against E.coli with no cytotoxic, genotoxic, hemolytic, apoptotic and necrotic activities and C.coggyria was effective in wound healing by in vitro and in vivo studies.
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