Thrombolytic Activity Research Articles

GC-MS Analysis and Thrombolytic Property of Methanolic Leaf Extracts of Terminalia pallida Brandis against Carrageenan Instigated Tail Thrombosis Model in Mice

Pharmacognosy Research,2022,14,1,53-60.DOI:10.5530/pres.14.1.9Published:January 2021Type:Research ArticleAuthors:Sarvan Kumar Guguloth, Narender Malothu, and Prasanth DSNBK Author(s) affiliations:Sarvan Kumar Guguloth1,2, Narender Malothu1,*, Prasanth DSNBK1 1Department of Pharmaceutical Chemistry, KL College of Pharmacy, Koneru Lakshmaiah Education Foundation, Vaddeswaram, Guntur, Andhra Pradesh, INDIA. 2Department of Pharmacology, Vijaya College of Pharmacy, Munaganoor, Hayathnagar, Ranga Reddy, Telangana, INDIA. Abstract:Background: Several plants of Terminalia species are being reported as medicinally useful. Terminalia pallida Brandis is one of the plants of the Combretaceae family, which constitutes several pharmacologically active substances. Present work explored leaf extract’s thrombolytic property, which could serve as the superior choice for currently using drugs. Objectives: The present study primarily focused on investigating the phyto-constituents such as phenolic and flavonoid compounds in methanolic leaf extract followed by GC-MS analysis and determining the in vitro thrombin inhibition and in vivo thrombolysis activity. Materials and Methods: Swiss albino mice (total = 36) were randomly distributed into six groups (n=6). Thrombosis was instigated by injecting 40 μL of 1% carrageenan (Type-I) via the subplantar region of the right hind paw of each mouse. The plant extract was screened for its in vitro thrombin inhibitory potency. In animal studies, the size of the blood clots in mouse tails was recorded by administering the plant extracts at 100, 200 and 300 mg/kg body weight for every 24, 48 and 72 hr, respectively. Results: A dose of 200 and 300 mg/kg of the extract showed significant thrombolytic activity at 24, 48, and 72 hr (p< 0.001) in a concentration-dependent manner when correlated with the control group. A reduction in the length of blood clots (p< 0.01) was observed at 48 and 72 hr. In acute oral toxicity study, administration of leaf extract showed no mortality and no significant behavioral changes up to 2000 mg/kg dose. The GC-MS analysis explored the occurrence of about 16 eluted compounds; among these, few have been reported as medicinally useful, which accounts for the therapeutic importance of the plant. Conclusion: In conclusion, the methanolic extract of T. pallida Brandis leaves at 200, and 300 mg/kg showed significant inhibition of the Thrombosis in carrageenan instigated model of mice and in vitro thrombin activity. Keywords:Carrageenan, Heparin, Quercetin., Terminallia pallida Brandis, ThrombosisView:PDF (4.38 MB) Full Text

Thrombolytic Potentials of Some Medicinal Plants Used by the Local People for Cardiovascular Diseases in Bangladesh

Cardiovascular diseases (CVDs) are one of the major causes of death in the world. Medicinal plants with thrombolytic properties may be used as an alternative to modern medicines for CVDs. The present study was aimed to evaluate the thrombolytic potential of six medicinal plants available in Bangladesh using an in vitro clot lysis method where streptokinase and ethanol were used as a positive and negative control, respectively. Ethanolic extract at a dose of 10 mg/ml of Arjun tree (Terminalia arjuna), Garlic (Allium sativum), Elephant apple (Dillenia indica), Amla (Phyllanthus emblica), Yellow mombin (Spondias pinnata) and Burmese grape (Baccaurea ramiflora) showed 14.18 ± 1.23%, 10.72 ± 0.78%, 8.25 ± 0.42%, 7.08 ± 0.64%, 5.42 ± 0.47% and 2.47 ± 0.19% clot lysis, respectively, whereas the standard drug streptokinase lysed 41.11±0.31% clot at a dose of 30,000 IU. From the data, it is evident that ethanolic extracts of six selected medicinal plants possess a moderate to insignificant thrombolytic activities. Among these plants, Arjun tree and Garlic exhibited the highest thrombolytic activity and the Burmese grape showed the lowest thrombolytic activity. Through our study, it could be concluded that Arjun tree, Garlic, and Elephant apple might be used as traditional healing purposes of CVDs. However, further animal studies will prove the scientific justification of their uses. Conservation efforts should be given for Arjun tree, Elephant apple, Yellow mombin, Burmese grape, and Amla to save these plants from extinction in nature. Bangladesh J. Plant Taxon. 28(2): 405-412, 2021 (December)

Assessment of Cytotoxic, Antioxidant, Thrombolytic, Anti Inflammatory and Antimicrobial Activity of Curcuma longa Linn, Cissus quadrangularis and Boerhaavia diffusa Herbal Mixture - An In vitro Study

Background and Aim: Plants play an important role in drug research, and the pharmaceutical industry is heavily reliant on natural products for new drug development. Curcumin, a natural compound contained in the rhizomes of the plant Curcuma longa Linn., has been shown to have anti-inflammatory properties in scientific studies. Cissus quadrangularis L. is a fleshy plant that can be found all over the world, especially in Asia, Africa, and a few other warm tropical areas. Boerhaavia diffusa L. (Nyctaginaceae) is present in the tropical regions of India, South America, and Africa. B. diffusa roots are commonly used in Ayurveda to treat various ailments. The aim of the study is to assess the cytotoxic, antioxidant and thrombolytic, antiinflammatory and antimicrobial properties of the aqua-alcoholic extract of the herbal mixture (Curcuma longa Linn +Cissus quadrangularis +Boerhaavia diffusa )&#x0D; Methods: Aqua-alcoholic extract of mixture of Curcuma longa Linn, Cissus quadrangularis and Boerhaavia diffusa was prepared. Phytochemical in-vitro studies were done using the CCB mixture cytotoxicity by brine shrimp lethality assay, antioxidant activity by DPPH assay, anti inflammatory activity by albumin denaturation assay, antimicrobial activity against Staphylococcus aureus, Streptococcus mutans, Escherichia coli and Candida albicans by agar well diffusion method, thrombolytic activity by clot lysis assay.&#x0D; Results: The CCB mixture showed good cytotoxic activity at varying concentration in brine shrimp lethality assay, increasing antioxidant activity with DPPH assay, increasing thrombolytic activity, increasing anti inflammatory activity with increasing concentration, the anti microbial activity was not efficient as positive controls.&#x0D; Conclusion: In this in-vitro study the CCB mixture shows improved cytotoxic, antioxidant, thrombolytic, anti-inflammatory and antimicrobial activity. Further studies on invivo animal and human clinical trials needs to be done.

Sustainable valorization of papaya peels for thrombolytic cysteine protease isolation by ultrasound assisted disruptive liquid phase microextraction with task specific switchable natural deep eutectic solvents

The present investigation deals with task-specific purification of thrombolytic cysteine protease from Carica papaya peels using switchable eutectic solvent-based ultrasound-assisted dispersive liquid-phase microextraction. Four switchable polarity natural deep eutectic solvents were synthesized and their thermo-physical profile was recorded as a function of temperature. Ultrasound-assisted liquid phase microextraction was performed yielding 33.2 mg/g of papaya protease. Kinetic and thermodynamic modeling of the extraction process signifies that first-order rate laws describe the extraction process with an optimal time of 15 min. Ultrapure fractions were obtained from copper immobilized metal affinity chromatography resulting in a purity fold of 48.6 and specific enzyme activity of 112.5. The thrombolytic activity of the resulting ultrapure protease fraction was determined by the blood clot digestion assay yielding 4126 U/mg of thrombolytic papain.

Aktivitas Agen Trombolitik Daun Pegagan (Centella asiatica) Hasil Fermentasi Oleh Acetobacter aceti FNCC 0016 Dengan Metode Clot Lysis Secara In-Vitro

Thrombolytic agents are plasminogen activators which can dissolve blood clot that caused cardiovascular disease (CVD). Thrombolytic agents can be obtained from microorganism such as Acetobacter aceti FNCC 0016 and from plants such as Centella asiatica, these thrombolytic agents are combined using fermentation method. Fermentation can improves the pharmacological properties of plants through modification of their metabolites. Fermentation mediated by microorganism has been shown to enhance the therapeutic efficacies of some plants. This research aimed to determine the increase of thrombolytic activity from the fermentation product of Centella asiatica by Acetobacter aceti FNCC 0016. Fermentation was carried out at various times (0 h, 24 h, 48 h, 72 h) at a temperature of 30±1°C. The thrombolytic activity was determined using in-vitro clot lysis method. The results showed a significant increase in thrombolytic activity after 72 hours of fermentation with thrombolytic index of 91,49 compared to Centella asiatica extract (42.93) and Acetobacter aceti FNCC 0016 (19.99). Conclusion: Thus, Centella asiatica that has undergone a fermentation process with Acetobacter aceti FNCC 0016 can increase the thrombolytic activity.

In Vitro study of Thrombolytic activity from the different parts of Carica papaya plant on COVID-19 patients.

Background: Carica papaya plant has been used for medicinal purpose throughout the world because it have anti-inflammatory, anti-oxidant, antiviral, anti-cancer and wound healing like properties.&#x0D; Objective: The main object of this research was to evaluate the thrombolytic activity of Carica papaya plant in normal healthy individuals and COVID-19 patients to determine either parts of the plant (roots, seeds and leaves) exhibit more activity in normal persons or in COVID-19 patients.&#x0D; Material and method: For this study, total 20 blood samples were taken, 10 for normal individuals and 10 for positive COVID-19 patients. We used two different solvents i.e. autoclaved distilled water and concentrated methanol to prepare the 10% root, seed and leaf extracts of Carica papaya plant. For the thrombolytic activity of these plant extracts, samples were arranged as a triplicate for the accuracy of results percentage. &#x0D; Results: Our results evaluated that in normal individuals, distilled water root extract and methanolic seed extract exhibits maximum thrombolytic activity. The mean value with distilled water (root extract) and methanol (seed extract) is 36.9% and 32.9% respectively. While in covid-19 patients, distilled water leaf extract and methanolic leaf extract reveal maximum thrombolytic activity. In patients, the mean value with distilled water and methanol (leaf extract) is 21.8% and 23% respectively.&#x0D; Conclusion: In our study, we have observed that normal persons showed highly significant results as compared to COVID-19 patients. Because in COVID-19 disease, plasminogen activator inhibitor 1 (PAI-1), thrombin activatable fibrinolysis inhibitor and tissue plasminogen activator (tPA) factors elevate which results in hypo-fibrinolysis.

Rescuing ischemic stroke by biomimetic nanovesicles through accelerated thrombolysis and sequential ischemia-reperfusion protection

Rational design of nanomedicine to accelerate thrombolysis and sequentially avoid thrombolysis-mediated reperfusion injury is still a challenge. Here, we develop a biomimetic nanovesicle (tPA/MNP@PM, tMP) by simple encapsulating melanin nanoparticles (MNP) and tPA with a platelet membrane vesicle (PM), which integrates the thrombus targeting property of PM, the photothermal conversion performance and free radical scavenging property of natural melanin for cascaded ischemic stroke treatment. Benefiting from natural thrombus-targeted adhesion capability of PM, nanovesicles could efficiently target thrombus site. Then near-infrared (NIR) mediated photothermal of MNP could lead to rupture of nanovesicles, thus achieving precise release of tPA in thrombus. Interestingly, local hyperthermia also increases the activity of tPA for accelerating thrombolysis. Afterwards, site specific released MNP (4.5 nm) accompanied by hemoperfusion can cross the BBB and accumulate in cerebral ischemia site, scavenging various free radicals and suppressing inflammation- and immune response–induced injury to achieve neuroprotection after thrombolysis. In addition, the biomimetic nanovesicle could block tPA-induced brain hemorrhage after stroke to improve thrombolytic therapy. The evaluation in ischemic stroke mice confirmed that the simple-prepared nanomedicine with cascaded thrombus targeting, precise thrombolysis and ischemia-reperfusion protection properties can significantly enhance the treatment effect of ischemic stroke. Statement of significanceIschemic stroke is recognized as a leading cause of death and disability in the world. Rational design of nanomedicine to accelerate thrombolysis and sequentially avoid thrombolysis-mediated reperfusion injury is still a challenge. Herein, a biomimetic nanovesicle (tMP) was developed for sequential ischemic stroke treatment. It could overcome the drawbacks of free tPA for safe thrombolysis: i) platelet membrane biomimetic coating significantly increases thrombus targeting; ii) NIR-mediated photothermal of natural melanin precise controlled release of tPA in thrombus in situ, and local hyperthermia also increases the thrombolytic activity of tPA. Notably, released melanin nanoparticles (4.5 nm) accompanied by hemoperfusion can across BBB and avoid ischemia-reperfusion injury through free radical scavenging and inflammation/immune response suppression.

Zinc(II) Induced Neurological Thrombolytic Activities for COVID-19 Thrombus Prevention, Inflammation, Fibrin Degradation, Fibrinolysis of Dissolving Blood Clots and Blood Flow Reperfusion after Thrombolysis

Zinc(II) Induced Neurological Thrombolytic Activities for COVID-19 Thrombus Prevention, Inflammation, Fibrin Degradation, Fibrinolysis of Dissolving Blood Clots and Blood Flow Reperfusion after Thrombolysis

Antioxidant and Thrombolytic Activities of Methanolic Extracts of Achyranthes aspera Linn.

Background: Plants have been used since a long time for the therapeutic use and precursors of therapeutic agents. People depend upon the herbal and traditional medicine to satisfy their health care needs. Achyranthes aspera L. is a multifunctional plant used to treat various kinds of diseases. It possesses various type of activities such as antioxidant, anti-inflammatory, antimicrobial and thrombolytic activity. Study of various biological activities helps to provide evidence to the traditional knowledge. Therefore, antioxidant and thrombolytic activities of its stem-leaves were aimed to explore in the present study. Methods: Quantitative experimental method was applied to its antioxidant and thrombolytic activities. Stem-leaves of Achyranthes aspera collected from Thankot, Kathmandu was subjected for methanolic extraction by maceration method. The extract was screened for the antioxidant activity by using DPPH radical scavenging assay as well as thrombolytic activity through the clot lysis assay in human blood. Results: The methanolic extractive value by using maceration was found to be 6.12%. Extract showed their antioxidant and thrombolytic potentialities. The concentration of antioxidants needed to decrease the initial DPPH concentration by 50% (IC50) was found to be 30.5μg/ml for its extract and it was almost 5-fold lesser than ascorbic acid, 6.62 μg/ml. Similarly, the% clot lysis was found to be 7.29 % and 15.35 % in 10 mg/ml and 25 mg/ml concentration respectively. Conclusion: The study suggests that stem-leaves of Achyranthes asperaare the possible sources of natural radical scavenger as well as thrombolytic agents. Thus, they could be used as natural antioxidants as well antithrombosis in the beverage, functional food and pharmaceutical industries that need further wide range in vivo studies.

Molecular target prediction and docking of anti-thrombosis compounds and its activation on tissue-plasminogen activator to treat stroke

The main aim of this study is to analyze effective thrombolytic drugs of natural origin for the treatment of stroke. Thrombolytic activity of natural sources has been reported and active molecules have been isolated and characterized. In this study, a total of ten compounds from nature were selected for docking studies. Caesalpinine c, caesalpinine a, vanillylamine, terpinen-4-Ol, dihydrocapsaicin and 3-carene showed fibrinolytic properties on analysis with PASS server. The present work is based on computer aided molecular modeling and the strength of the ligand was validated using binding energy. Caesalpinine c exhibited good docking score and this compound showed potent thrombolytic activity than other compounds. The drug likeliness varied based on the chemical and physical properties of the ligand. Lipinski’s rule of five was accepted by all of the selected compounds. Pfizer’s rule, and GSK rule were accepted in caesalpinine C. The drug likeliness properties of the all ten selected ligands were accepted in most of the cases and few rejections were observed mostly in Golden Triangle rule. However further in vitro or in vivo trials are required to validate thrombolytic potential of caesalpinine c and vanillylamine.

Robust thrombolytic and anti-inflammatory action of a constitutively active ADAMTS13 variant in murine stroke models

AbstractAdvances in our understanding of ADAMTS13 structure, and the conformation changes required for full activity, have rejuvenated the possibility of its use as a thrombolytic therapy. We have tested a novel Ala1144Val ADAMTS13 variant (constitutively active [ca] ADAMTS13) that exhibits constitutive activity, characterized using in vitro assays of ADAMTS13 activity, and greatly enhanced thrombolytic activity in 2 murine models of ischemic stroke, the distal FeCl3 middle cerebral artery occlusion (MCAo) model and transient middle cerebral artery occlusion (tMCAO) with systemic inflammation and ischemia/reperfusion injury. The primary measure of efficacy in both models was restoration of regional cerebral blood flow (rCBF) to the MCA territory, which was determined using laser speckle contrast imaging. The caADAMTS13 variant exhibited a constitutively active conformation and a fivefold enhanced activity against fluorescence resonance energy transfer substrate von Willebrand factor 73 (FRETS-VWF73) compared with wild-type (wt) ADAMTS13. Moreover, caADAMTS13 inhibited VWF-mediated platelet capture at subphysiological concentrations and enhanced t-PA/plasmin lysis of fibrin(ogen), neither of which were observed with wtADAMTS13. Significant restoration of rCBF and reduced lesion volume was observed in animals treated with caADAMTS13. When administered 1 hour after FeCl3 MCAo, the caADAMTS13 variant significantly reduced residual VWF and fibrin deposits in the MCA, platelet aggregate formation, and neutrophil recruitment. When administered 4 hours after reperfusion in the tMCAo model, the caADAMTS13 variant induced a significant dissolution of platelet aggregates and a reduction in the resulting tissue hypoperfusion. The caADAMTS13 variant represents a potentially viable therapeutic option for the treatment of acute ischemic stroke, among other thrombotic indications, due to its enhanced in vitro and in vivo activities that result from its constitutively active conformation.

Modulation of hemostasis by inhibiting enzymes with the extract of Averrhoa carambola leaves

Herbal medicines represent an advantageous alternative for the prevention and treatment of several diseases when compared to allopathic medicines. Averrhoa carambola (Oxalidaceae) is a plant rich in phenolic compounds and popularly known for its medicinal properties such as anti-inflammatory, antioxidant, and hypoglycemic. Different enzymes of the human organism participate in physiological processes which involve hemostasis, inflammation, and formation of new tissue. These enzymes are highlighted as pharmaceutical targets for the treatment of numerous pathologies. The present work evaluated the aqueous and ethanolic extracts from A. carambola leaves on phospholipase, hemolytic, caseinolytic, thrombolytic, coagulant, and fibrinogenolytic activities induced by phospholipases A2 and proteases. Phenolic compounds and total flavonoids were quantified in the aqueous and ethanolic extracts of the leaves of Averrhoa carambola. These extracts were evaluated, in vitro, on phospholipase, proteolytic, hemolytic, thrombolytic and fibrinogenolytic activities induced by snake venoms. The results confirm the pharmacological potential of A. carambola since the extracts were able to modulate all evaluated activities related to hemostasis through inhibitions or potentiation of the enzymatic activities (phospholipases A2 and proteases). The constituents of A. carambola may act interfering in processes such as coagulation, thrombus dissolution, and fibrinogenolysis.

Effect of Fermentation Parameters on Natto and Its Thrombolytic Property.

Natto is a popular food because it contains a variety of active compounds, including nattokinase. Previously, we discovered that fermenting natto with the combination of Bacillus subtilis GUTU09 and Bifidobacterium animalis subsp. lactis BZ25 resulted in a dramatically better sensory and functional quality of natto. The current study further explored the effects of different fermentation parameters on the quality of natto fermented with Bacillus subtilis GUTU09 and Bifidobacterium BZ25, using Plackett–Burman design and response surface methodology. Fermentation temperature, time, and inoculation amount significantly affected the sensory and functional qualities of natto fermented with mixed bacteria. The optimal conditions were obtained as follows: soybean 50 g/250 mL, triangle container, 1% sucrose, Bacillus subtilis GUTU09 to Bifidobacterium BZ25 ratio of 1:1, inoculation 7%, fermentation temperature 35.5 °C, and fermentation time 24 h. Under these conditions, nattokinase activity, free amino nitrogen content, and sensory score were increased compared to those before optimization. They were 144.83 ± 2.66 FU/g, 7.02 ± 0.69 mg/Kg and 82.43 ± 5.40, respectively. The plate thrombolytic area and nattokinase activity both increased significantly as fermentation time was increased, indicating that the natto exhibited strong thrombolytic action. Hence, mixed-bacteria fermentation improves the taste, flavor, nattokinase activity, and thrombolysis of natto. This research set the groundwork for the ultimate manufacturing of natto with high nattokinase activity and free amino nitrogen content, as well as good sensory and thrombolytic properties.

Designing rt-PA Analogs to Release its Trapped Thrombolytic Activity

Recombinant tissue-type plasminogen activator (rt-PA, alteplase) is an FDA-approved thrombolytic drug. Designing rt-PA analogs to suppress its inhibition by plasminogen activator inhibitor-1 (PAI-1) without compromising its pharmacological activity has been a continued effort because rt-PA is rapidly inactivated by endogenous PAI-1, leading to the thrombolytic activity of rt-PA being trapped by endogenous PAI-1. Here, incorporating currently available structure of the rt-PA-PAI-1 Michaelis complex structures, this paper uncovers the interstructural biophysics underlying the rt-PA-PAI-1 binding interface, and puts forward a structural biophysical basis for the design of a previously reported rt-PA analogue (T103N, N117Q, KHRR (296-299) AAAA). In addition, this paper proposes a set of rt-PA analogs with lower or higher affinity to PAI-1, including rt-PA (alanine scanning for E_60A, D_189 and R_39), rt-PA (Q60E) and rt-PA (Q60E-F150H-Y151K), towards the release of the trapped thrombolytic activity of rt-PA, and also in the hope that there is still room for the improvement of the efficacy-safety balance of rt-PA in future.

Thrombolytic and anticoagulant effects of a recombinant staphylokinase-hirudin fusion protein

A pure recombinant staphylokinase-hirudin fusion protein (SFH) was obtained by recombinant genetic engineering and purification techniques. The thrombolytic and anticoagulant activities of SFH were investigated using in vitro coagulation models and chromogenic assays. The results showed that intact SFH had targeted thrombolytic activity, and gained anticoagulant activity when cleaved by FXa. In addition, we investigated the pharmacodynamics of SFH in vivo using a variety of animal models, including a rat inferior vena cava thrombosis model, a rat coronary thrombosis model, a rabbit carotid artery thrombosis model and a canine coronary thrombosis model. We found that SFH had an obvious thrombolytic effect and could prevent and reduce re-embolization after thrombolysis and reduce the serious bleeding side effects caused by the combination of thrombolytic and anticoagulant drugs. The results suggest that SFH can be used for thrombolytic therapy in thromboembolic diseases.

Prediction of endogenous thrombolytic activity in patients with coronary artery disease

Abstract Background Endogenous thrombolytic activity (ETA) has been suggested as an essential factor related to the acute coronary syndrome. However, there have been little data regarding clinical characteristics of ETA in East Asians. Method As an interim study of the entire cohort (n=2,000), we analyzed a total of 278 patients who underwent percutaneous coronary intervention (PCI) due to coronary artery disease (CAD). Informed consent was obtained from all research subjects. Blood samples of patients were brought before the procedure. The Global Thrombosis Test (GTT, Thromboquest, UK), a novel test for examining ETA, was used. Lysis time (LT), which means the time interval between blood flow occlusion and restart, was used as an indicator for ETA. Clinical, laboratory and angiographic characteristics were obtained. LT=3000 seconds was used as a cut-off value to divide patients into two groups. P value&amp;lt;0.05 was regarded as significant. Results LT of Korean CAD patients showed bimodal distribution. Median value was 1695 [IQR: 1099, 5932] and it was higher than previous data from Europeans (Figure 1). Patients with impaired ETA (LT&amp;gt;3000) were older and more diabetic. They showed higher creatinine, aPTT, fibrinogen, D-dimer, c-reactive protein, and proBNP values. Moreover, they had lower hemoglobin and platelet levels. Intracoronary thrombus was more frequently observed in LT&amp;gt;3000 group. In the multivariable regression analysis, hemoglobin (per g/dL, odds ratio 0.766, 95% confidence interval (CI) 0.632–0.928) and fibrinogen level(per 10mg/L, odds ratio 1.054, 95% CI 1.015–1.095) could significantly predict impaired ETA. Conclusion East Asian patients showed a right-shifted distribution of ETA compared to that of Europeans. Patients with impaired ETA had different clinical, laboratory and angiographic characteristics from those with intact ETA. Hemoglobin and fibrinogen level were significantly associated with impaired ETA. Further studies are warranted to confirm causal relationship among these factors. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Research Foundation, Republic of Korea

Thrombolytic Effect of Nattokinase and Its Mechanism

Objective: Exploring the anti-thrombotic effect and mechanism of nattokinase in vitro and vivo. Methods: The thrombolytic effect of NK on old thrombus was studied by blood clot lysis method in vitro, the rat carotid artery thrombosis model induced by ferric chloride and the rat venous thrombosis model induced by inferior vena cava ligation method were used to study the thrombolytic effect of NK in vivo. TXB2, 6-k, PGE2, t-PA, \begin{document}$\cdot {\rm{O}}_2^- $\end{document} , ·OH, XOD and other indicators were detected to explore the antithrombotic mechanism of NK. Results: Nattokinase could significantly dissolve old thrombus (P \begin{document}$\cdot {\rm{O}}_2^- $\end{document} , ·OH and XOD. The values of TXB2, PEG2, \begin{document}$\cdot {\rm{O}}_2^- $\end{document} , ·OH and XOD were increased significantly, while the values of 6-K-PGF1 α and t-PA were decreased significantly. Conclusion : NK had strong thrombolytic activity in vivo and vitro, and had effect on fresh thrombus and old thrombus. NK could inhibit thrombosis by inhibiting platelet aggregation, reduce oxidative damage and reducing the level of coagulation factors. NK could also play a direct thrombolytic role by stimulating the production of t-PA, dissolving fibrin, and might play an indirect thrombolytic role by inhibiting the production of PAI-1.

Chemical Structure and Anticoagulant Property of a Novel Sulfated Polysaccharide from the Green Alga Cladophora oligoclada

Marine macroalgae are efficient producers of sulfated polysaccharides. The algal sulfated polysaccharides possess diverse bioactivities and peculiar chemical structures, and represent a great potential source to be explored. In the present study, a heparinoid-active sulfated polysaccharide was isolated from the green alga Cladophora oligoclada. Results of chemical and spectroscopic analyses indicated that the sulfated polysaccharide was composed of →6)-β-d-Galp-(1→, β-d-Galp-(1→, →6)-α-d-Glcp-(1→ and →3)-β-d-Galp-(1→ units with sulfate esters at C-2/C-4 of →6)-β-d-Galp-(1→, C-6 of →3)-β-d-Galp-(1→ and C-3 of →6)-α-d-Glcp-(1→ units. The branches consisting of β-d-Galp-(1→ and →6)-β-d-Galp-(1→ units were located in C-3 of →6)-β-d-Galp-(1→ units. The sulfated polysaccharide exhibited potent anticoagulant activity in vitro and in vivo as evaluated by activated partial thromboplastin time (APTT), thrombin time, and the fibrinogen level. For the APTT, the signal for clotting time was more than 200 s at 100 μg/mL in vitro and at 15 mg/kg in vivo. The obvious thrombolytic activity of the sulfated polysaccharide in vitro was also found. The mechanism analysis of anticoagulant action demonstrated that the sulfated polysaccharide significantly inhibited the activities of all intrinsic coagulation factors, which were less than 1.0% at 50 μg/mL, but selectively inhibited common coagulation factors. Furthermore, the sulfated polysaccharide strongly stimulated the inhibition of thrombin by potentiating antithrombin-III (AT-III) or heparin cofactor-II, and it also largely promoted the inhibition of factor Xa mediated by AT-III. These results revealed that the sulfated polysaccharide from C. oligoclada had potential to become an anticoagulant agent for prevention and therapy of thrombotic diseases.

Chemical, biological and protein-receptor binding profiling of Bauhinia scandens L. stems provide new insights into the management of pain, inflammation, pyrexia and thrombosis

Bauhinia scandens L. (Family, Fabaceae) is a medicinal plant used for conventional and societal medication in Ayurveda. The present study has been conducted to screen the chemical, pharmacological and biochemical potentiality of the methanol extracts of B. scandens stems (MEBS) along with its related fractions including carbon tetrachloride (CTBS), di-chloromethane (DMBS) and n-butanol (BTBS). UPLC-QTOF–MS has been implemented to analyze the chemical compounds of the methanol extracts of Bauhinia scandens stems. Additionally, antinociceptive and anti-inflammatory effects were performed by following the acetic acid-induced writhing test and formalin-mediated paw licking test in the mice model. The antipyretic investigation was performed by Brewer Yeast induced pyrexia method. The clot lysis method was implemented to screen the thrombolytic activity in human serum. Besides, the in silico study was performed for the five selected chemical compounds of Bauhinia scandens, found by UPLC-QTOF–MS By using Discover Studio 2020, UCSF Chimera, PyRx autodock vina and online tools. The MEBS and its fractions exhibited remarkable inhibition in dose dependant manner in the antinociceptive and antiinflammatory investigations. The antipyretic results of MEBS and DMBS were close to the standard drug indomethacin. Investigation of the thrombolytic effect of MEBS, CTBS, DMBS, and BTBS revealed notable clot-lytic potentials. Besides, the phenolic compounds of the plant extracts revealed strong binding affinity to the COX-1, COX-2, mPGES-1 and plasminogen activator enzymes. To recapitulate, based on the research work, Bauhinia scandens L. stem and its phytochemicals can be considered as prospective wellsprings for novel drug development and discovery by future researchers.

Evaluation on In Vitro Blood Clot Dissolving Potential of Aqueous Extract of Sida acuta Burm. F. Leaves

Clotting of blood is the vital processes and a perplexing interaction of various mechanisms of circulatory system due of failure of which is sometimes considered as a concern within the circulatory system causing acute myocardial or cerebral infarction which might cause demise. Sida acuta burm. f (Malvaceae) is abundantly growing small perennial shrub utilized by natives for diuretic, anthelmintic, calmative and wound healing properties, and are utilized in treating disorders like blood, bile, liver, nervous, urinary diseases and rheumatism. The present study was intended to evaluate the blood clot dissolving potential of Sida acuta leaf aqueous extract in vitro. The plant material as leaves were locally collected and subjected to phytochemical extraction with distilled water. The preliminary phytochemical tests total phenolic content was estimated by Folin-Ciocalteu’s method. In vitro thrombolytic activity of 3 different concentrations of aqueous extract was estimated on goat blood clot compared to the activity of streptokinase. The aqueous extract of S. acuta leaves are reported to be rich in alkaloids, flavonoids, tannins, terpenoids and glycosides while the total phenolic content was estimated to be 17.48 % in extract which are mostly responsible for any pharmacological activity. Compared to the thrombolytic activity of standard streptokinase which was 73 %, the aqueous leaf extract of S. acuta displayed considerable blood clot dissolving activity at concentration 10 mg/100µl, 5.0 mg/100µl, and 2.5 mg/100µl as 41 %, 34 % and 12 % respectively. This property of plant extract is promising which could be could be exploited in development of new biopharmaceutical and therapeutic agents after stringent further physiological compatibility and in vivo pharmacological studies.&#x0D; Keywords: Sida acuta, phytochemical extract, thrombolytic activity, streptokinase