Evaluation of in vitro antioxidant, anti-inflammatory, anticoagulant and antiplatelet potential of Rhus mysorensis

Since ancient times human beings are using plant-based medicines for the treatment of various ailments, especially in the rural areas, due to their availability and affordability. Rhus mysorensis (RM) is widely used as a traditional medicine to treat various ailments. Owing to its potential medicinal value, the present study was designed to explore the in vitro antioxidant, anti-inflammatory, anticoagulant and antiplatelet properties of purified column fraction of RM. The methanol extract of stem bark powder was sequentially fractioned by solvent partitioning. The liquid methanol fraction was further fractionated by column chromatography using gradient elution. Eluted fractions were analyzed using HPLC for percentage purity and yield. The fraction with higher percentage of purity and yield was assessed for in vitro antioxidant activity by measuring SOD and GPx activities, anti-inflammatory activity by the inhibition of nitric oxide (NO) production in LPS induced RAW264.7 cells, anticoagulation by plasma recalcification time and antiplatelet activity by agonists induced platelet aggregation respectively. The antioxidant potency of column fraction (B8) revealed that, highest enzyme activities were recorded at a concentration of 320µg/ml. The enzyme activity was found to be 2.45 U/ml for SOD and 135.75 U/L for GPx respectively. Purified column fraction (B8) of RM significantly reduces the production of NO in LPS stimulated RAW 264.7 cell lines at 320????g/ml concentration with 31.90% of inhibition. The anticoagulant activity of purified fraction was determined in terms of plasma recalcification time. Interestingly, the fraction showed the most potent anticoagulant activity both in PRP and PPP as it prolonged the clotting time. The findings indicate that the stem bark of RM possesses potent antioxidant, anti-inflammatory, anticoagulant and antiplatelet activities, supporting the use of this species for treating oxidative stress-induced inflammatory diseases. Further, bioactivity guided fractionation studies to characterize and identify specific phytochemicals responsible for these biological activities are needed.

Preparation of modified polysulfone material decorated by sulfonated citric chitosan for haemodialysis and its haemocompatibility

Polysulfone (PSF) works potentially in haemodialysis due to its great mechanical and chemical stability, but performs poorly in haemocompatibility. For promoting the unpleasant haemocompatibility, sulfonated citric chitosan (SCACS) with the structure and groups similar to heparin was primarily synthesized by acylation and sulfonation. Furthermore, the chloroacylated PSF was pretreated by electrophilic chloroacetyl chloride to achieve more active sites for further reaction; the following membranes underwent the amination and were named amination polysulfone (AMPSF) membranes. Moreover, SCACS with abundant carboxyl and sulfonic groups was covalently grafted at the surface of pretreated PSF membranes, called PSF-SCACS membranes. The PSF-SCACS membranes were successfully synthesized and characterized by 1 H NMR, ATR-FTIR and XPS. In addition, the water contact angle of PSF-SCACS membranes decreased by 47° and the morphologies of the membranes changed little compared with the unmodified PSF membranes. The haemocompatible testing results, including protein adsorption, platelet adhesion, haemolysis rate, plasma recalcification time, activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT), demonstrated that the PSF-SCACS membranes possessed excellent haemocompatible performances, and SCACS played an important role in the modification.

Study Of The Effect Of Polysaccharides On Hemostasis

The effect of sulfated polysaccharides on the hemostatic system in conditions in vitro. Platelet-rich plasma was obtained by centrifugation at 200 g for 10 minutes. The remaining citrate blood was further centrifuged at 1500 g for 10 min to obtain platelet-poor plasma. The antithrombin activity of the compounds was evaluated in vitro by their effect on the recalcification time, thrombin, and prothrombin time of human blood plasma stabilized with a 3.8% sodium citrate solution in the ratio of 9:1. In studies conducted on the blood plasma of rats, it was found that the studied compounds, to varying degrees, lengthen the APTT, APTT, prothrombin time. At the same time, anticoagulant activity was established to block one of the factors II, V, X. Polysaccharide exhibit a combined anticoagulant effect in the body, due to which they are classified as anticoagulant and antithrombin agents.

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

Background: 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. Objective: To evaluate wound healing potential of crude and partially purified enzyme from Tabernaemontana divaricata (stem and latex). Materials and Methods: 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. Results: 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. Conclusion: The study provides evidence of the procoagulant and thrombolytic activity associated with T. divaricata proteases.

Factor XII-Deficient Chicken Plasma as a Useful Target for Screening of Pro- and Anticoagulant Animal Venom Toxins

The sensitivity of vertebrate citrated plasma to pro- and anticoagulant venom or toxins occurs on a microscale level (micrograms). Although it improves responses to agonists, recalcification triggers a relatively fast thrombin formation process in mammalian plasma. As it has a natural factor XII deficiency, the recalcification time (RT) of chicken plasma (CP) is comparatively long [≥ 1800 seconds (s)]. Our objective was to compare the ability of bee venom phospholipase A2 (bvPLA2) to neutralize clot formation induced by an activator of coagulation (the aPTT clot) in recalcified human and chicken plasmas, through rotational thromboelastometry. The strategy used in this study was to find doses of bvPLA2 that were sufficient enough to prolong the clotting time (CT) of these activated plasmas to values within their normal RT range. The CT of CP was prolonged in a dose-dependent manner by bvPLA2, with 17 ± 2.8 ng (n = 6) being sufficient to displace the CT values of the activated samples to ≥ 1800 s. Only amounts up to 380 ± 41 ng (n = 6) of bvPLA2 induced the same effect in activated human plasma samples. In conclusion, the high sensitivity of CP to agonists and rotational thromboelastometry could be useful. For example, during screening procedures for assaying the effects of toxins in several stages of the coagulation pathway, such as clot initiation, formation, stability, strength, or dissolution.

Heparinized Polyurethane Surface Via a One-Step Photografting Method

Traditional methods using coupling chemistry for surface grafting of heparin onto polyurethane (PU) are disadvantageous due to their generally low efficiency. In order to overcome this problem, a quick one-step photografting method is proposed here. Three heparin derivatives incorporating 0.21, 0.58, and 0.88 wt% pendant aryl azide groups were immobilized onto PU surfaces, leading to similar grafting densities of 1.07, 1.17, and 1.13 μg/cm2, respectively, yet with increasing densities of anchoring points. The most negatively charged surface and the maximum binding ability towards antithrombin III were found for the heparinized PU with the lowest amount of aryl azide/anchor sites. Furthermore, decreasing the density of anchoring points was found to inhibit platelet adhesion to a larger extent and to prolong plasma recalcification time, prothrombin time, thrombin time, and activated partial thromboplastin time to a larger extent. This was also found to enhance the bioactivity of immobilized heparin from 22.9% for raw heparin to 36.9%. This could be explained by the enhanced molecular mobility of immobilized heparin when it is more loosely anchored to the PU surface, as well as a higher surface charge.

Evaluation of hemostatic effect of polyelectrolyte complex-based dressings

The aim of this work was to develop a polyelectrolyte complex-based hemostatic dressing made from chitosan and polygalacturonic acid. Porous dressings were fabricated by ultrasonication of chitosan and alginate solutions followed by freeze-drying. Since chitosan has inherent hemostatic properties, and polygalacturonic acid is anti-inflammatory in nature, it was desired to combine these two polymers to develop an effective hemostatic dressing, which may also promote wound healing. Porous structure of the bandages was observed using field-emission scanning electron microscope. Blood clotting behavior was studied using whole blood clotting assay. Plasma recalcification time, prothrombin time, and activated partial thromboplastin time were also determined to study the mechanism of clotting. The dressings were found to accelerate clotting rates and showed increased thrombin activity with an increase in chitosan concentration.

A simple clot based assay for detection of procoagulant cell-derived microparticles

Background:Cell-derived microparticles (MPs) are important biomarkers in many facets of medicine. However, the MP detection methods used till date are costly and time consuming. The main aim of this study was to standardize an in-house clot based screening method for MP detection which would not only be specific and sensitive, but also inexpensive.Methods:Four different methods of MP assessment were performed and the results correlated. Using the flow cytometry technique as the gold standard, 25 samples with normal phosphatidylserine (PS) expressing MP levels and 25 samples with elevated levels were selected, which was cross checked by the commercial STA Procoag PPL clotting time (CT) assay. A simple recalcification time and an in-house clot assay were the remaining two tests. The in-house test measures the CT after the addition of calcium chloride to MP rich plasma, following incubation with Russell viper venom and phospholipid free plasma.Results:The CT obtained by the in-house assay significantly correlated with the results obtained by flow cytometry (RConclusions:Though preliminary, the in-house assay seems to be efficient, inexpensive and promising. It could definitely be utilized routinely for procoagulant MP assessment in various clinical settings.