1.0 INTRODUCTION:
Inflammation is a body response to injury, infection, or destruction characterized by heat, redness, pain, swelling, and disturbed physiological functions, caused by physical trauma, noxious chemical, or microbial agents. It is the body's response to inactivate or destroy the invading organisms, remove the irritants, and set the stage for tissue repair. It is triggered by the release of chemical mediators from injured tissue and migrating cells. The migration of leukocytes from the venous systems to the site of damage, and the release of cytokines, are known to play a crucial role in the inflammatory response. These chemicals cause the widening of blood capillaries (vasodilation) and the permeability of the capillaries. This will lead to increased blood flow to the injured site.
2.0 METHODOLOGY:
· Preparation Of Erythrocyte Suspension:
· 3 ml of fresh whole blood will be collected intravenously from a healthy volunteer who had not taken any NSAIDs for two weeks prior to the experiment.
· The blood sample will be collected into a heparinized vacutainer to prevent coagulation.
· Packed blood cells will be obtained by washing the collected blood sample three times with 0.9% saline and centrifuging for 10 min at 3000 revolutions/minute.
· A stock solution of 10% v/v suspension of human red blood cells (HRBCs) will be prepared using isotonic phosphate buffer (154 mM NaCl) in 10 mM sodium phosphate buffer (pH 7.4).
· Heat-Induced Hemolysis
· The test Sample (2ml) contains 1.0 ml of 10% HRBC and 1.0 ml of various concentrations (10, 30, and 100μg/ml) will be added to each test tube and gently mixed.
· The positive control contains 1.0 ml of HRBC and 1.0 ml of various concentrations of diclofenac sodium (10, 30, 100μg/ml).
· The negative control consisted of 1.0 ml of 10% erythrocyte suspension and 1.0 ml of normal saline alone.
· The experiment will be performed in triplicates.
· The resulting solution will be heated at 56°C for 30 min and cooled to room temperature and centrifuged at 2500 rpm for 10 min.
· The supernatant will be collected and the absorbance of each solution will be measured on a 560nm using a spectrophotometer as an indicator of the degree of hemolysis.
· The percentage inhibition of hemolysis was calculated using the formula
(%) Percentage inhibition = Ac – At/Ac × 100
Where At is the absorbance of the test and Ac is the absorbance of the control.
3.0 ENDPOINT PARAMETER(S):
· % Percentage inhibition activity
4.0 REFERENCE(S):
4.1 Douglas Bosco Aidoo, Daniels Konja, Isaac Tabiri Henneh, and Martins Ekor. Protective Effect of Bergapten against Human Erythrocyte Hemolysis and Protein Denaturation In Vitro. International Journal of Inflammation Volume 2021, Article ID 1279359, 7 pages https://doi.org/10.1155/2021/1279359
4.2 Yesmin, S., Paul, A., Naz, T. et al. Membrane stabilization as a mechanism of the anti-inflammatory activity of ethanolic root extract of Choi (Piper chaba). Clin Phytosci 6, 59 (2020). https://doi.org/10.1186/s40816-020-00207-7
4.3 K. D. P. P. Gunathilake, K. K. D. S. Ranaweera, and H. P. Vasantha Rupasinghe. In Vitro Anti-Inflammatory Properties of Selected Green Leafy Vegetables. Biomedicines 2018, 6, 107; doi: 10.3390/biomedicines6040107.
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