ABSTRACT
Hemostasis is a tightly regulated process which maintains a fluid state of blood within the vasculature and provides thrombotic response upon tissue injury. Various scientific studies have implicated the role of plant latex proteases in hemostasis using in vitro experiments. However, in vivo models substantiate their role in hemostasis. Therefore, in the present study, the effect of plant latex thrombin-like proteases (PTLPs) on hemostasis was investigated systematically using mice tail bleeding as a preclinical model. In this direction, latex protease fractions (LPFs), which showed potent thrombin-like activity, were selected as they act directly on fibrinogen to form clot and quickly stop bleeding. Thrombin-like activity was exhibited mainly by cysteine proteases. Calotropis gigantea, Carica papaya, Jatropha curcas, Oxystelma esculentum, Tabernaemontana divaricata, and Vallaris solanacea LPFs and papain from C. papaya latex significantly reduced bleeding on a topical application in normal and aspirin administered mice. In addition, PTLPs accelerated the clotting of factor VIII deficient plasma, while, papain brought back the clotting time to normal levels acting like a bypassing agent. Further, papain failed to show activity in the presence of specific cysteine protease inhibitor iodoacetic acid; confirming protease role in all the activities exhibited. At the tested dose, PTLPs except C. gigantea did not show toxicity. Further, structural and sequence comparison between PTLPs and human thrombin revealed structural and sequence dissimilarity indicating their unique nature. The findings of the present study may open up a new avenue for considering PTLPs including papain in the treatment of bleeding wounds.
Subject(s)
Aspirin/adverse effects , Cysteine Endopeptidases/administration & dosage , Factor VIII/metabolism , Hemorrhage/drug therapy , Latex/chemistry , Animals , Asclepias/chemistry , Calotropis/chemistry , Carica , Cysteine Endopeptidases/pharmacology , Disease Models, Animal , Hemorrhage/chemically induced , Hemorrhage/metabolism , Homeostasis , Humans , Jatropha/chemistry , Mice , Papain/administration & dosage , Papain/pharmacology , Plant Proteins/administration & dosage , Plant Proteins/pharmacology , Tabernaemontana/chemistryABSTRACT
Snakebite is a global health problem affecting millions of people. According to WHO, India has the highest mortality and/or morbidity due to snakebite. In spite of commendable research on Indian BIG FOUR venomous species; Naja naja and Bungarus caeruleus (elapid); Daboia russelii and Echis carinatus (viperid), no significant progress has been achieved in terms of diagnosis and management of biting species with appropriate anti-snake venom. Major hurdle is identification of offending species. Present study aims at differentiation of Indian BIG FOUR snake venoms based on their distinguish action on rodent blood coagulation. Assessment of coagulation alterations by elapid venoms showed negligible effect on re-calcification time, prothrombin time, activated partial thromboplastin time and factors assay (I, II, V, VIII and X) both in vitro and in vivo. However, viperid venoms demonstrated significant anticoagulant status due to their remarkable fibrinogen degradation potentials as supported by fibrinogenolytic activity, fibrinogen zymography and rotational thromboelastometry. Though results provide hint on probable alterations of Indian BIG FOUR snake venoms on blood coagulation, the study however needs validation from human victim's samples to ascertain its reliability for identification of biting snake species.
Subject(s)
Blood Coagulation/drug effects , Elapid Venoms/toxicity , Snake Venoms/toxicity , Viper Venoms/toxicity , Animals , Blood Coagulation Tests , Bungarus , Dose-Response Relationship, Drug , Elapidae , Fibrinolysis/drug effects , Freeze Drying , India , Kinetics , Lethal Dose 50 , Mice , Random Allocation , Rats, Wistar , ViperidaeABSTRACT
A novel, simple, specific, sensitive and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of abiraterone (ART) in rat plasma. The analytical procedure involves extraction of ART and diclofenac (internal standard, IS) from rat plasma with a simple liquid-liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system with a Betasil C(18) column maintained at ambient room temperature and an isocratic mobile phase [acetonitrile-water-10 mm potassium dihydrogen phosphate (pH 3.0), 55:5:40, v/v/v] at a flow rate of 1.00 mL/min with a total run time of 10 min. The eluate was monitored using an UV detector set at 255 nm. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 93.4-3251 ng/mL (r(2) = 0.997). The intra- and inter-day precisions were 0.56-4.98 and 3.03-7.18, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study of ART in rats.
