Chethana Ramachandraiaha, Sharath Kumar M. Nandisha, Jayanna Kengaiaha, Chandramma Srinivasa, Ashwini Shivaiaha, Sebastin Santhosh Martinb, Manohar Shindea, Devaraja Sannaningaiaha*
aDepartment of Studies and Research in Biochemistry and Centre for Bioscience and Innovation, Tumkur University, Tumkur, Karnataka, India
bDepartment of Medical Biochemistry and Microbiology (IMBM) Uppsala Biomedical Centre 75237, Sweden
*Address for Corresponding author
Devaraja Sannaningaiah, Ph.D.
Department of Studies and Research in Biochemistry,
Tumkur University, B H road, Tumkur-572103 India.
Objective: The present work demonstrates the anticoagulant and antiplatelet property of Macrotyloma uniflorum Seed Aqueous Extract (MUSAE). Materials and Methods: The protein banding blueprint of MUSAE (100μg) was analyzed on SDS-PAGE. The proteolytic activity of MUSAE was analyzed by means of casein zymography at the concentration of 50 μg. Anticoagulant effect of MUSAE was tested using plasma recalcification time, mouse tail bleeding time, Activated Partial ThromboPlastin Time (APTT) and Prothrombin Time (PT) at concentrations of 0-120μg. Fibrinogen and fibrin clot degrading actions of MUSAE were analyzed on SDS-PAGE under reduced conditions. The non-toxic property of MUSAE was tested by edema, hemorrhage and direct hemolytic activities. Results: MUSAE showed similar protein banding pattern in both reduced and non-reduced conditions on SDS-PAGE. MUSAE exhibited proteolytic activity as it hydrolyzed casein with the specific activity of 0.121units/mg/min. while, the proteolytic activity of MUSAE was totally eradicated by 1, 10-Pheanthroline and PMSF but EDTA and IAA did not; confirms the presence of serine and zinc metallo protease in MUSAE. MUSAE delayed the clotting time of human citrated plasma against the control 184sec to 407sec suggesting its anti-coagulant property. Interestingly, MUSAE delayed the clot formation process of only APTT, suggesting its participation in an intrinsic pathway of blood coagulation cascade. Furthermore, MUSAE hydrolyzed human fibrinogen, fibrin clot without hydrolyzing other plasma proteins. In addition, MUSAE exhibited antiplatelet aggregation property by inhibiting agonists ADP and Epinephrine induced platelet aggregation. The percentage of inhibition was found to be 75% and 72% in PRP. MUSAE was also inhibited the washed platelet aggregation induced by thrombin, ADP, collagen, arachidonic acid and epinephrine in washed platelet. The observed inhibition percentage was found to be 98%, 81%, 54%, 50% and 48% respectively. Moreover, MUSAE was nontoxic as there was no hemolytic, hemorrhagic and edema forming activities were observed. Conclusion: MUSAE exhibited anticoagulant, antiplatelet and clot dissolving properties, hence, it could be promising agent in the management of thrombotic disorders.
Keywords: Macrotyloma uniflorum, Blood coagulation cascade, intrinsic pathway, platelet aggregation
MUSAE Macrotyloma uniflorum seed aqueous extract
PMSF Phenyl Methyl Sulphonyl Fluoride
IAA Iodo Acetic Acid
EDTA Ethylene Di-amine Tetra Acetic Acid
PRP Platelet Rich Plasma
PPP Platelet Poor Plasma