Kenaf Seed Cysteine Protease (KSCP) Inhibits the Intrinsic Pathway of the Blood Coagulation Cascade and Platelet Aggregation.

BACKGROUND: Thrombosis is the key event that obstructs the flow of blood throughout the circulatory system, leading to stroke, myocardial infarction and severe cardiovascular complications. Currently, available antithrombotic drugs trigger several life-threatening side effects. INTRODUCTION: Antithrombotic agents from natural sources devoid of adverse effects are grabbing high attention. In our previous study, we reported the antioxidant, anticoagulant and antiplatelet properties of kenaf seed protein extract. Therefore, in the current study, purification and characterization of cysteine protease from kenaf seed protein extract responsible for potential antithrombotic activity was undertaken. METHODS: Purification of KSCP (Kenaf Seed Cysteine Protease) was carried out using gel permeation and ion exchange column chromatography. The purity of the enzyme was evaluated by SDS PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis). RP-HPLC (Reverse Phase High-Performance Liquid Chromatography), MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-Of-Flight) and CD (Circular Dichroism techniques) were employed for its characterization. Proteolytic, fibrinolytic and kinetic study was done using spectroscopy. Plasma recalcification time, Prothrombin Time (PT), Thrombin clotting time (TCT), Activated Partial Thromboplastin Time (APTT), bleeding time and platelet aggregation studies were carried out for antithrombotic activity of KSCP. RESULT: A single sharp band of KSCP was observed under both reduced and non-reduced conditions, having a molecular mass of 24.1667kDa. KSCP was found to contain 30.3% helix turns and 69.7% random coils without a beta-pleated sheet. KSCP digested casein and fibrin, and its activity was inhibited by iodoacetic acid (IAA). KSCP was optimally active at pH 6.0 at the temperature of 40°C. KSCP exhibited anticoagulant properties by interfering in the intrinsic pathway of the blood coagulation cascade. Furthermore, KSCP dissolved both whole blood and plasma clots and platelet aggregation. CONCLUSION: KSCP purified from kenaf seed extract showed antithrombotic potential. Hence, it could be a better candidate for the management of thrombotic complications.

Pennisetum glaucum Protein Extract Protects RBC, Liver, Kidney, Small Intestine from Oxidative Damage and Exhibits Anticoagulant, Antiplatelet Activity.

High level of exogenous ROS in the circulation affects RBC membrane integrity which facilitates the generation of endogenous RBC ROS, implicated in series of physiological changes primarily associated with thrombosis and vital tissue damage. Although, Pennisetum glaucum (pearl millet) stores abundance of proteins, their therapeutic potential is least explored. Thus, the purpose of this study is to examine the role of Pennisetum Glaucum Protein Extract (PGE) on oxidative stress induced cell/tissue damage and thrombosis.In this investigation, protein characterization was done by using SDS-PAGE, Native-PAGE, PAS-staining and HPLC. In-vitro oxidative stress was induced in RBC using sodium nitrite. While, in-vivo oxidative stress was induced in experimental rats using diclofenac. Stress markers and biochemical parameters were evaluated. Role of PGE on thrombosis was assessed by using, in-vitro plasma recalcification time, activated partial thromboplastin time, prothrombin time, mouse tail bleeding time (In-vivo) and platelet aggregation.PGE revealed varied range of molecular weight proteins on SDS-PAGE. PGE normalized the sodium nitrite induced oxidative damage of RBC and diclofenac induced oxidative damage in liver, kidney and small intestine. PGE exhibited anticoagulant effect by increasing the coagulation time of both PRP and PPP and mouse tail bleeding time. Furthermore, PGE prolonged the clotting time of only APTT but did not affect PT. PGE inhibited agonists ADP and epinephrine induced platelet aggregation.Our findings suggest, PGE could be a better contender in the management of oxidative stress and its associated diseases. ABBREVIATIONS: PGEPennisetum Glaucum protein ExtractAPPTActivated Partial Thromboplastin TimePTProthrombin TimeROSReactive Oxygen SpeciesPRPPlatelet Rich PlasmaPPPPlatelet Poor PlasmaSDS-PAGESodium Dodecyl Sulfate-Polyacrylamide Gel ElectrophoresisPASPeriodic Acid-schiff StainingODOptical DensityINRInternational Normalized RatioPBSPhosphate Buffered SalineSODSuperoxide DismutaseTCATrichloro Acetatic AcidDTNBDi-Thio-bis-NitroBenzoic acidSGOTSerum Glutamate Oxaloacetate TransaminaseSGPTSerum Glutamate Pyruvate TransaminaseALPAlkaline PhosphataseDFCDiclofenacSylSilymarinMEDMinimum Edema DoseMHDMinimum Hemorrhagic Dose.

Antioxidant and Antithrombotic Activities of Kenaf Seed (Hibiscus cannabinus) Coat Ethanol Extract in Sprague Dawley Rats.

Oxidative stress has been implicated in deadly lifestyle diseases, and antioxidants from plant sources are the primary option in the treatment regime. Kenaf seeds are the storehouse of potential natural antioxidant phytoconstituents. Perhaps, none of the studies documented the phytoconstituents and their antioxidant potential from Kenaf seed coat so far. Thus, the current study focuses on exploring the protective effect of Kenaf Seed Coat Ethanol Extract (KSCEE) against sodium nitrite and diclofenac-induced oxidative stress in vitro (red blood cell and platelets model) and in vivo (female Sprague Dawely rat's model) along with the antithrombotic activity. The infrared spectrophotometry data showed the heterogeneous functional groups (CH, OH, C = C, C = C-C) and aromatic rings. Reverse phase high-performance liquid chromatography and gas chromatography-mass spectrometry chromatogram of KSCEE also evidenced the presence of several phytochemicals. KSCEE displayed about 76% of DPPH scavenging activity with an IC50 value of 34.94 µg/ml. KSCEE significantly (***p < 0.001) normalized the stress markers such as lipid peroxidation, protein carbonyl content, superoxide dismutase, and catalase in sodium nitrite and diclofenac-induced oxidative stress in RBC, platelets, liver, kidney, and small intestine, respectively. Furthermore, KSCEE was found to protect the diclofenac-induced tissue destruction of the liver, kidney, and small intestine obtained from seven groups of female Sprague Dawely rats. KSCEE delayed the clotting time of platelet-rich plasma and platelet-poor plasma and activated partial thromboplastin time, suggesting its anticoagulant property. In addition, KSCEE also exhibited antiplatelet activity by inhibiting both adenosine diphosphate and epinephrine-induced platelet aggregation. In conclusion, KSCEE ameliorates the sodium nitrite and diclofenac-induced oxidative stress in red blood cells, platelets, and experimental animals along with antithrombotic properties.

Sorghum Protein Extract Protects RBC from Sodium Nitrite-Induced Oxidative Stress and Exhibits Anticoagulant and Antiplatelet Activity.

INTRODUCTION: Oxidative stress plays a critical role in the progression of diabetes, arthritis, cancer, eryptosis, cardiovascular disease, and thrombosis. Currently, antioxidants from natural sources are in high demand due to their beneficial role in the management of said diseases. AIM: The purpose of the study was to evaluate the protective effect of sorghum protein buffer extract (SBE) on sodium nitrite-induced oxidative stress and thrombosis. MATERIALS AND METHODS: Protein characterization of SBE was done using SDS-PAGE. Oxidative stress in RBC was induced using sodium nitrite (NaNO2) and the key stress markers such as lipid peroxidation (LPO), protein carbonyl content (PCC), and the level of antioxidant enzymes (SOD and CAT) were measured. The anticoagulant effect of SBE was identified by employing in-vitro plasma recalcification time, activated partial thromboplastin time (APTT), prothrombin time (PT), and in-vivo mouse tail bleeding time. SBE antiplatelet activity was examined using agonist adenosine diphosphate (ADP) and epinephrine-induced platelet aggregation. Non-toxic property of SBE was identified using in-vitro direct haemolytic, haemorrhagic, and edema forming activities using experimental mice. RESULTS: SBE revealed similar protein banding pattern under both reduced and non-reduced conditions on SDS-PAGE. Interestingly, SBE normalized the level of LPO, PCC, SOD, and CAT in stress-inducedRBCs. Furthermore, SBE showed anticoagulant effect in platelet rich plasma by enhancing the clotting time from the control 250 s to 610 s and bleeding time from the control 200 s to more than 500 s (p<0.01) in a dose dependent manner. In addition, SBE prolonged the clot formation process of only APTT but not PT. SBE inhibited the agonists ADP and epinephrine induced platelet aggregation. SBE did not hydrolyze RBC cells, devoid of edema and haemorrhage properties. CONCLUSIONS: This study demonstrates for the first time the anticoagulant, antiplatelet, and antioxidant properties of SBE. Thus, the observed results validate consumption of sorghum as good for health and well-being.

Purification and characterization of non-enzymatic glycoprotein (NEGp) from flax seed buffer extract that exhibits anticoagulant and antiplatelet activity.

The current study deals with the purification and characterization of non-enzymatic glycoprotein (NEGp) from flax seed buffer extract. Sephadex G-100 and DEAE-A25 column chromatography techniques were employed to isolate NEGp. NEGp showed single sharp band at 29 kDa region on 10% SDS-PAGE, and under reduced and non-reduced conditions revealed its monomeric nature. Besides, NEGp taken up the PAS stain at 29 kDa region reveals the presence of carbohydrate moiety. Purity of NEGp was adjudged by RP-HPLC, as it revealed a single sharp peak at the retention time of 3.4 min. The exact molecular mass of NEGp was found to be 26 kDa which was confirmed by MALDI-TOF. Circular di-chromism spectra of NEGp showed 12.0% α-helix, 24.3% α-helix turn and 63.7% random coils without beta pleated sheets. NEGp was found to exhibit anticoagulant activity by extending clotting time of both platelet rich plasma and platelet poor plasma from control 240 s to 1800 s and 280 s to 2100 s respectively at the concentration of 8 µg. NEGp inhibited the agonists such as ADP, epinephrine and arachidonic acid induced platelet aggregation in washed platelets. The percentage of inhibition was found to be 70%, 80% and 60% respectively. While, it did not interfere in thrombin, PAF and collagen induced platelet aggregation. NEGp did not hydrolyse RBC membrane, devoid of haemorrhagic and edema inducing properties in experimental mice.

Caesalpinia crista coat extract protects red blood cell from sodium nitrite-induced oxidative stress and exhibits antiplatelet activity.

: To understand the RBC protecting efficiency and antiplatelet activity of methanolic extract of Caesalpinia crista coat (MECCC). RBC-protecting activity of MECCC was evaluated using assays, such as DPPH, level of lipid peroxidation, protein carbonyl content, superoxide dismutase and catalase as a marker of oxidative stress whereas, platelet aggregation inhibition was performed using human platelet-rich plasma (PRP). MECCC showed about 76% of DPPH-scavenging activity, with an IC50 value of 71.89 µg/ml. The MECCC reduced the level of lipid peroxidation and protein carboxylation in RBC caused by NaNO2 in a dose-dependent manner. In addition, MECCC normalized the levels of superoxide dismutase (SOD) and catalase (CAT) in oxidative stress-induced RBC in a dose-dependent manner. This suggested the protective effect of MECCC on RBC against oxidative stress. Furthermore, MECCC also exhibited mild antiplatelet activity by inhibiting both ADP and epinephrine agonists that induced platelet aggregation. The noticed inhibition percentage was found to be 28 and 23%, respectively at the concentration of 150 µg. Interestingly, MECCC did not hydrolyse the RBC suggesting its nontoxic properties. MECCC possesses protective effect of RBC against NaNO2 (10 mmol/l) induce oxidative stress and inhibits platelet aggregation.