رسائل ماجيستير
English
2022
Effect of Ricinus communis, phenytoin and metformin and their combination on wound healing: An in vitro study
انتصار جواد حمد المختار, دنيا عبد الحسين شاكر ناجي
كلية الطب-جامعة بابل · العراق
Wound is a physical injury of the body that is described by disruption of normal structures of the body. A wound may cause damage to superficial skin layers and reach structures underlying the skin. Wound healing is an important physiological process to maintain the integrity of skin after trauma, either by accident or by intentional procedure. The normal wound healing involves three successive but overlapping phases, including hemostasis/inflammatory phase, proliferative phase, and remodeling phase.
This study aimed to investigate the effect of Ricinus communis leaf extracts (ethanolic and aqueous), metformin, and phenytoin alone and in combination on healing wounds induced on the normal Madin-Darby Canine Kidney (MDCK) cell line.
This study includes two parts:
Part One: In order to determine the safety of tested agents (ethanolic and aqueous plant extracts, metformin, and phenytoin), a pilot study was performed to investigate their cytotoxicity on the MDCK normal cell line and accordingly a range of concentrations to be used for the wound healing assay was chosen. In the cytotoxicity assay, the MDCK cell line was seeded in tissue culture 96-well plates and treated with each ethanolic and aqueous Ricinus communis leaf extract with different concentrations (31.25, 62.5, 125, 250, 500, 1000 μg/ml). The same experiment was done using different concentrations of metformin (3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500, 1000, 2000 μg/ml) or phenytoin (62.5, 125, 250, 500, 1000, 2000 μg/ml). After that, all treated cells were incubated for 24 hours at 37°C, then the MTT assay was performed.
Part Two: A vertical wound was made by using a 200 μl sterile plastic micropipette tip pressed firmly against the cell monolayer of the tissue culture plate. Then the wounded cells were exposed to different concentrations of each tested agent obtained from part one of this study. Six experiments were conducted as follows:
Treatment of the MDCK wound model with (15.6, 31.25, 62.5, 125, 250, 500 μg/ml) of ethanolic leaf Ricinus communis extract.
Treatment of the MDCK wound model with (15.6, 31.25, 62.5, 125, 250, 500 μg/ml) of aqueous leaf Ricinus communis extract.
Treatment of the MDCK wound model with (3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500, 1000 μg/ml) of metformin.
Treatment of the MDCK wound model with a combination of metformin serial dilution (125, 250, 500, 1000 μg/ml) plus either 250 or 500 μg/ml of Ricinus communis leaf ethanolic extract.
Treatment of the MDCK wound model with (1.9, 3.9, 7.8, 15.6, 31.25, 62.5 μg/ml) of phenytoin.
Treatment of the MDCK wound model with a combination of phenytoin serial dilution (1.9, 3.9, 7.8, 15.6, 31.25, 62.5 μg/ml) plus either 250 or 500 μg/ml of Ricinus communis leaf ethanolic extract.
After that, the wound areas were digitally photographed every 3 hours up to 12 hours.
The cytotoxicity results showed that the concentration (125 μg/ml) of Ricinus communis leaf aqueous extract significantly increased (p ≤ 0.05) the viability of the MDCK cell line, while no significant differences were found (p > 0.05) after treatment with the ethanolic leaf extract in comparison to the control group. The results showed that the viability of the MDCK cell line was significantly increased (p ≤ 0.05) after treatment with the concentration (62.5 μg/ml) of each metformin and phenytoin, while at high concentrations of metformin (2000 μg/ml) and phenytoin (2000, 1000, 500, 250 μg/ml), the viability was significantly decreased (p < 0.001).
Wound healing results showed that all concentrations except 500 μg/ml of Ricinus communis leaf ethanolic extract caused highly significant decrease (p ≤ 0.001) in the wound diameter, while all concentrations of Ricinus communis leaf aqueous extract caused highly significant decrease (p ≤ 0.001) in the wound diameter with complete healing in comparison to the control group. Also, results showed that all concentrations of metformin except (15.6, 31.25, 62.5 μg/ml) and all concentrations of phenytoin except the lowest one (1.9 μg/ml) caused highly significant decrease (p ≤ 0.001) in the diameter of the wound with complete healing.
Regarding the combined effect of Ricinus communis leaf ethanolic extract plus either metformin or phenytoin, results showed that both combinations caused highly significant decrease (p ≤ 0.001) in wound diameter. The healing results showed incomplete healing of wounds treated with all concentrations of metformin plus Ricinus communis leaf ethanolic extract except 250 μg/ml of plant extract plus 125 and 250 μg/ml of metformin, and complete healing with all concentrations of phenytoin plus Ricinus communis leaf ethanolic extract except those containing 500 μg/ml of plant extract plus 31.25 and 62.5 μg/ml of phenytoin, which caused incomplete wound healing.
In conclusion: Ricinus communis (ethanolic and aqueous) leaf extracts, metformin, and phenytoin have a healing effect on the MDCK cell line wound model when used alone, better than when used in combination, except for the combination of phenytoin plus 250 μg/ml of plant extract, which resulted in faster wound healing.