Heparin sodium

The anticoagulant activity was determined by measuring the activated partial thromboplastin time (APTT) and prothrombin time (PT) using heparin as a positive control, as per the method described by Song et al. (2019) with slight modifications [37 (link)]. In brief, different concentrations of the EPS were prepared (50, 100, and 500 μg/mL) by dissolving the required amount of the EPS in 0.9% NaCl. Human blood was collected from healthy donors in a tube containing 2.5% sodium citrate (9:1 v/v) and centrifuged at 3000 rpm for 20 min to separate the plasma from blood cells at 4 °C. For the APTT assay, the citrated plasma (100 μL) was mixed with 100 μL of EPS and incubated at 37 °C for 1.0 min. The APTT reagent (100 μL) was added to the mixture and incubated for 3.0 min at 37 °C. The clotting time was determined after adding 50 μL of 0.025 mM CaCl₂ solution. For the PT assay, 100 μL of citrated plasma was mixed with 100 μL of EPS and incubated at 37 °C for 1 min, and then, 200 μL of the pre-warmed PT assay reagent was added and the clotting time was recorded using a coagulometer. Heparin sodium was purchased from HiMedia Laboratories, Mumbai, India. The assays were performed in triplicates and the results represent the mean values ± standard deviations.

Sodium nitrate, calcium chloride dehydrates, magnesium sulfate heptahydrate, potassium phosphate, monopotassium phosphate, sodium chloride, boric acid, EDTA, zinc sulfate heptahydrate, chloride tetrahydrate, molybdenum trioxide, copper (II) sulfate pentahydrate and cobalt (II) nitrate hexahydrate were obtained from Sigma-Aldrich, Dorset, UK. 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), 1,1-diphenyl-2 picrylhydrazyl (DPPH), were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Commercial kits for aPTT and PT tests were purchased from Fisher Scientific, Canada. Heparin sodium was obtained from Hi-Media Laboratories, Mumbai, India.