Tabernaemontana divaricata Stem and Latex Proteases as Haemostatic Agent with Temporally Spaced Intense Fibrinogenolytic and Mild Fibrinolytic Activity

Abstract

Proteases play a crucial role in the pharmacological properties of latex producing plants. Some of them exhibited intervention with fibrinogenolysis and/or fibrinolysis, two crucial wound healing events. To evaluate wound healing potential of crude and partially purified enzyme from Tabernaemontana divaricata (stem and latex). Proteolytic activity, clot inducing/dissolving potential, fibrinogen polymerization, recalcification time, blood clot lysis and Tricine-SDS PAGE for enzyme treated fibrinogen and human plasma clot were performed. Latex PPE exhibited significant proteolytic activity (115.8 ± 0.3 U/ml) compared to that of the stem (28.78 ± 0.2 U/ml). Enzyme preparations exhibited temporally spaced clot inducing and subsequent dissolving properties favoring hemostatic effect, procoagulant effect being dominant and the first event. Significant reduction in fibrinogen absorbance at 540 nm with time, recalcification time and human fibrinogenolytic product analysis on Tricine PAGE substantiated procoagulant effect. Disappearance of Aα and Bβ fibrinopeptides by both stem and latex PPEs in the PAGE was observed. γ subunits were completely hydrolysed by latex PPE, however, it showed comparative resistance to stem PPE. Reduction in blood clot weight and fibrin subunit intensity supported thrombolytic property. The study provides evidence of the procoagulant and thrombolytic activity associated with T. divaricata proteases.