Blood Clot Lysis Research Articles

Small leucine zipper protein negatively regulates liver fibrosis by suppressing the expression of plasminogen activator inhibitor-1

Liver fibrosis, which is caused by viral infection, toxic exposure, and autoimmune diseases, is a chronic liver disease. Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor of tissue-type plasminogen activator (tPA) and urokinase plasminogen activator, which convert plasminogen into plasmin. Therefore, PAI-1 suppresses fibrinolysis by blocking plasmin synthesis and is involved in liver fibrosis via extracellular matrix deposition. Small leucine zipper protein (sLZIP) acts as a transcription factor and plays critical roles in many cellular processes. However, the role of sLZIP in liver fibrosis remains unclear. In this study, we investigated the role of sLZIP in regulating PAI-1 transcription and liver fibrosis. sLZIP knockdown enhanced the expression of PAI-1 at the mRNA and protein levels. sLZIP knockdown also increased PAI-1 secretion and suppressed blood clot lysis by blocking tPA activity. Moreover, conditioned medium derived from sLZIP knockdown cells downregulated the expression of matrix metalloprotease (MMP)-2 and MMP-9 in the presence of tPA in hepatic stellate cells (HSCs). Liver-specific sLZIP knockout mice showed deteriorated liver fibrosis compared to control mice in a bile duct ligation-induced fibrosis model. These findings demonstrate that sLZIP functions as a negative regulator of liver fibrosis by suppressing PAI-1 transcription and HSC activation.

In vivo and in silico studies of membrane-stabilizing and clot lysis activities of Trachyspermum ammi

Trachyspermum ammi contains many important phytochemicals, including thymol, which is used to treat many diseases. This study aimed to check the membrane-stabilizing and clot-lysis capacity of the ethanol extract of T. ammi (ETA) and its main chemical component, thymol (THY). For this, we used hypotonic solution-induced erythrocyte lysing and egg-albumin protein denaturation for membrane-stabilizing capacity and human blood clot lysis for anti-atherombosis capacity. Further, we observed in silico study of THY and ntric oxide synthase (NOSs) isoform. The stabilizing effects of ETA and THY on the membrane changed depending on the concentration. At the highest concentration (160 μg/mL), they stopped the most bleeding, while the standard drug, acetylsalicylic acid, stopped less bleeding at the same concentration. In the erythrocyte lysing test, ETA showed 102.60 ± 0.01 and 75.83 ± 1.44% membrane protection. In the egg-albumin denaturation test, they showed 27.62 ± 0.02% and 56.71 ± 0.02% membrane protection, respectively. In our clot lysis experiment, ETA and THY also showed significant and concentration-dependent clot lysis capacity. The IC50 and EC50 values of THY were lower than ETA when it came to keeping the membrane stable and breaking up clots, respectively. In our in silico investigation, THY and NOSs showed significant binding interaction. Taken together, ETA and its major component, THY, exhibited potent membrane stabilizing activity and clot lysis activity in in vitro studies. Further studies are required to elucidate its other active principles and their biological effects, along with possible mechanisms.

In vitro Biological Activities and Fatty Acid Composition Analysis of Musa paradisiaca L. Inflorescence

Musa paradisiaca L. is native to tropical climate. Inflorescences of this herb are used as a treatment of digestive distresses. This study involved analysis of the fatty acid contents and some in-vitro pharmacological activities of the inflorescence. Modified Kupchan method was used for extraction in solvents of varying polarity. Fatty acid content analysis was conducted on n-hexane extract; Palmitoleic acid was found to be the highest in proportion in both bound (34.40%) and free (37.80%) forms. n-hexane and dichloromethane extracts exhibited cytotoxicity against carcinoma cell (HeLa cell line). Ethyl acetate extract possessed significant amount of free radical (DPPH) scavenging capability (IC50 = 66.92±0.3065 μg/ml). The n-Hexane extract was found to be rich in phenolics (26.40 ± 0.033 mg GE/g) and flavonoids (83.40± 0.099 mg QE/g). Dichloromethane fraction exhibited the highest zone of inhibition (15 mm) against the bacterium Shigella dysenteriae. Ethyl acetate fraction showed blood clot lysis activity of 29.35% (standard streptokinase 63.05%). Presence of potent bioactivities imply the prospects of this sample as a source of lead compounds for effective formulations against chronic digestive diseases. Dhaka Univ. J. Pharm. Sci. 23(1): 69-76, 2024 (June)

Membrane-stabilizing and clot lysis activities of (±) citronellal: In-vitro studies

Numerous plants and their phytochemicals have been identified as having potential medicinal values and are used to treat different ailments worldwide. (±) Citronellal (CTL) is a monoterpene phytochemical with potential therapeutic benefits including anti-inflammatory, antioxidant, antifungal, and antibacterial actions. AimThe present study aimed to evaluate the membrane-stabilizing and clot-lysis activities of CTL through in vitro studies. For this, we performed hypotonic solution-induced erythrocyte lysing and human blood clot lysis methods to check the membrane-stabilizing and clot lysis capacities of CTL using acetylsalicylic acid (ASA) and streptokinase (STK) as standards, respectively. CTL exhibited concentration-dependent membrane-stabilizing activity with an IC50 = 28.83 ± 1.19 µg/mL. In the clot lysis test, CTL also showed a concentration-dependent clot lysing capacity, where it exhibited 50.46 ± 3.81 % clot lysis at a concentration of 160 μg/mL. In the latter case, the IC50 value was I158.22 ± 2.21 µg/mL. CTL exhibit potent membrane-stabilizing and moderate clot-lysis activity. We suppose that CTL may exert these effects, possibly through its capacity to inhibit inflammatory mediators. Further in vivo and in silico studies are required to elucidate CTL’s exact molecular mechanisms behind these biological effects.

Phytochemical analysis and biological investigation of Cheilanthes tenuifolia (Burm.f.) Swartz.

Introduction: Cheilanthes tenuifolia is an evergreen ornamental small fern, belonging to the family Pteridaceae, that grows in warm and rocky regions worldwide. Many species of Cheilanthes genus are evidently endowed with important phytochemicals and bioactivities. This study aimed to perform a preliminary phytochemical analysis of Cheilanthes tenuifolia leaves alongside an evaluation of free radical scavenging, anti-inflammatory, antimicrobial, and clot lysis activities of extract fractions. Materials and methods: A preliminary phytochemical analysis was done after fractionation of ethanolic extract (ECT) with n-hexane (HCT) and chloroform (CCT). Then, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, egg albumin and RBC membrane stabilization tests, disc diffusion, and human blood clot lysis assays were performed. Results: Phytochemical investigations suggested that the plant is rich in alkaloids, glycosides, tannins, and flavonoids. All obtained fractions exhibited concentration-dependent radical scavenging, inhibition of egg protein denaturation and RBC membrane lysis capacities. Except for antifungal tests, ECT exhibited better DPPH radical scavenging, anti-inflammatory, antibacterial, and clot lysis capacities than HCT and CCT fractions. However, all fractions exhibited a mild anti-inflammatory activity. Conclusion: C. tenuifolia might be a good source of antioxidant, anti-microbial, and anti-atherothrombotic agents. Further studies are required to isolate and characterize the active principles liable for each bioactivity, along with possible molecular interactions.

Chemico-pharmacological evaluation of the methanolic leaf extract of Catharanthus ovalis: GC-MS/MS, in vivo, in vitro, and in silico approaches.

Introduction: Natural plant-based medicines have gained popularity recently as a major source of inventive, risk-free, and more potent secondary bioactive compounds with medicinal potential. Catharanthus ovalis is a perennial shrub containing various indole alkaloids cultivated extensively for local medical purposes. Methods: This research is conducted to identify the phytocompounds present in the leaves of C. ovalis and its central and peripheral analgesic, thrombolytic, and membrane-stabilizing activities through tail immersion, acetic acid-induced writhing, human blood clot lysis, and erythrocyte lysis by heat and hypotonic solution methods, respectively. Results and discussion: A total of 39 compounds were identified using GC-MS/MS techniques, including hexadecanoic acid, methyl ester (56.749%), methyl stearate (29.782%), carvacrol and its TBDMS derivative (12.586%), and 9-octadecenoic acid, methyl ester, (E)-] (9.297%) presented in high quantity. The highest tail immersion latency was observed for the 600mg/kg extract of C. ovalis crude extract. Both 400 and 600mg/kg doses of C. ovalis crude extract exhibited prominent peripheral analgesic activity. The maximum thrombolytic effect was observed by DCM soluble fraction extract by inhibiting 54.87% of the clot. However, the aqueous-soluble fraction of this extract manifested an excellent membrane-stabilizing effect by showing 73.98% and 87.51% hemolysis against heat- and hypotonic-induced hemolysis, respectively. Some of the compounds were identified as active agents against different receptors related to these diseases, which supported the findings of in vitro and in vivo tests. Conclusion: Further investigation needs to be conducted to specify and identify the exact mechanism of action of these compounds.

The Inhibition of Serine Proteases by Serpins Is Augmented by Negatively Charged Heparin: A Concise Review of Some Clinically Relevant Interactions.

Serine proteases are members of a large family of hydrolytic enzymes in which a particular serine residue in the active site performs an essential role as a nucleophile, which is required for their proteolytic cleavage function. The array of functions performed by serine proteases is vast and includes, among others, the following: (i) the ability to fight infections; (ii) the activation of blood coagulation or blood clot lysis systems; (iii) the activation of digestive enzymes; and (iv) reproduction. Serine protease activity is highly regulated by multiple families of protease inhibitors, known collectively as the SERine Protease INhibitor (SERPIN). The serpins use a conformational change mechanism to inhibit proteases in an irreversible way. The unusual conformational change required for serpin function provides an elegant opportunity for allosteric regulation by the binding of cofactors, of which the most well-studied is heparin. The goal of this review is to discuss some of the clinically relevant serine protease-serpin interactions that may be enhanced by heparin or other negatively charged polysaccharides. The paired serine protease-serpin in the framework of heparin that we review includes the following: thrombin-antithrombin III, plasmin-anti-plasmin, C1 esterase/kallikrein-C1 esterase inhibitor, and furin/TMPRSS2 (serine protease Transmembrane Protease 2)-alpha-1-antitrypsin, with the latter in the context of COVID-19 and prostate cancer.

Evaluation of Phytochemical Screening, Antioxidant, and Thrombolytic Activity of Methanolic Extract of Phlogacanthus thyrsiflorus

Objective: The main objectives of the study are preliminary phytochemical screening, antioxidant activity and in vitro thrombolytic activity evaluation of extract Phlogacanthus thyrsiflorus. Methods: Preliminary phytochemical analysis was done by using standard methods. Human blood clot lysis was used to evaluate the thrombolytic activities. The antioxidant activity is evaluated by free radical scavenging activity of DPPH (1,1-diphenyl-2-picrylhydrazyl) method and total phenolic content. Results: Preliminary phytochemical screening revealed the presence of various bioactive components like Tannin, Saponins, Phenol and Flavonoids. In thrombolytic assay, a significant clot lysis was observed plant extract compared to the negative control. The plant could be beneficial in synthetic drug formulation by virtue of the presence of antioxidant activity. The crude extract of plant were found to have Antioxidant activity with the concentration of 20 µg/ml, 40 µg/ml, 60 µg/ml, 80 µg/ml, 100 µg/ml respectively compared to control and standard. In DPPH scavenging assay, IC50 values of extract and ascorbic acid were found to be 31.87 µg/ml and 52.04 µg/ml respectively. Total phenol content was 10.5 mg. Conclusion: In conclusion, with this study it could be concluded that the phytochemical, antioxidant assay and thrombolytic activity of methanol extract Phlogacanthus thyrsiflorus would give valuable information for further characterization and exploitation of this important medicinal plant.

Ultrasound combined with urokinase under key-shaped bone window enhances blood clot lysis in an in vitro model of spontaneous intracerebral hemorrhage.

Minimally invasive surgery for spontaneous intracerebral hemorrhage is impeded by inadequate lysis of the target blood clot. Ultrasound is thought to expedite intravascular thrombolysis, thereby facilitating vascular recanalization. However, the impact of ultrasound on intracerebral blood clot lysis remains uncertain. This study aimed to explore the feasibility of combining ultrasound with urokinase to enhance blood clot lysis in an in vitro model of spontaneous intracerebral hemorrhage. The blood clots were divided into four groups: control group, ultrasound group, urokinase group, and ultrasound + urokinase group. Using our experimental setup, which included a key-shaped bone window, we simulated a minimally invasive puncture and drainage procedure for spontaneous intracerebral hemorrhage. The blood clot was then irradiated using ultrasound. Blood clot lysis was assessed by weighing the blood clot before and after the experiment. Potential adverse effects were evaluated by measuring the temperature variation around the blood clot in the ultrasound + urokinase group. A total of 40 blood clots were observed, with 10 in each experimental group. The blood clot lysis rate in the ultrasound group, urokinase group, and ultrasound + urokinase group (24.83 ± 4.67%, 47.85 ± 7.09%, 61.13 ± 4.06%) was significantly higher than that in the control group (16.11 ± 3.42%) (p = 0.02, p < 0.001, p < 0.001). The blood clot lysis rate in the ultrasound + urokinase group (61.13 ± 4.06%) was significantly higher than that in the ultrasound group (24.83 ± 4.67%) (p < 0.001) or urokinase group (47.85 ± 7.09%) (p < 0.001). In the ultrasound + urokinase group, the mean increase in temperature around the blood clot was 0.26 ± 0.15°C, with a maximum increase of 0.38 ± 0.09°C. There was no significant difference in the increase in temperature regarding the main effect of time interval (F = 0.705, p = 0.620), the main effect of distance (F = 0.788, p = 0.563), or the multiplication interaction between time interval and distance (F = 1.100, p = 0.342). Our study provides evidence supporting the enhancement of blood clot lysis in an in vitro model of spontaneous intracerebral hemorrhage through the combined use of ultrasound and urokinase. Further animal experiments are necessary to validate the experimental methods and results.

Применение офтальмологической композиции «Альтимезатон» в переднюю камеру глаза для лечения посттравматической гифемы

The aim of the study is to introduce into veterinary clinical practice an effective ophthalmic composition under the working name «Altimezaton» with fibrinolytic and mydriatic properties; to establish its effectiveness in the lysis of blood clots in the anterior chamber of the eye, as well as the prevention and treatment of synechiae after blunt trauma of the eye with the occurrence of posttraumatic hyphema; to work out a method of introducing this composition into the anterior chamber of the eye.

CLOT LYSIS AND MEMBRANE PROTECTION POTENTIALS OF CHEILANTHES TENUIFOLIA METHANOLIC LEAF EXTRACT

Cheilanthes tenuifolia is a little perennial fern that falls within the taxonomic classification of the Pteridaceae botanical family. The plant has a diverse array of phytochemical substances, including alkaloids, phenolic compounds, flavonoids, saponins, steroids, and triterpenoids, which have demonstrated promising medicinal properties. This study aimed to evaluate the in-vitro membrane-stabilizing and clot lysis activities of a methanol leaf extract of C. tenuifolia (MCT). For this, we performed hypotonic solution-induced erythrocyte lysing and human blood clot lysis methods to check the membrane stabilizing and clot lysis capacities of MCT using acetylsalicylic acid and streptokinase as standards, respectively. Additionally, we also checked its phytochemical groups. The results of a preliminary phytochemical screening indicate the presence of alkaloids, glycosides, tannins, flavonoids, and saponins in the plant. MCT inhibited hemolysis in a concentration-dependent manner and inhibited 78.93 ± 0.01% hemolysis (IC50 = 46 ± 2.11 µg/ml) at the higher concentration (160 μg/ml), whereas the standard drug, acetylsalicylic acid (IC50 = 64.10 ± 2.08 µg/ml) inhibited 97.71 ± 0.01% at the same concentration. It also exhibited clot lysis in a concentration-dependent manner, where the maximum percentage of clot lysis was observed at 160 μg/100 ml where the IC50 value was 198.41 ± 1.87 µg. The standard drug streptokinase showed 77.51 ± 0.01% clot lysis. C. tenuifolia possesses various important secondary metabolites and shows membrane stabilizing and clot lysis capacity. Further studies are required to elucidate its active principles and their biological effects.

Unveiling the Potent Fibrino(geno)lytic, Anticoagulant, and Antithrombotic Effects of Papain, a Cysteine Protease from Carica papaya Latex Using κ-Carrageenan Rat Tail Thrombosis Model

While fibrinolytic enzymes and thrombolytic agents offer assistance in treating cardiovascular diseases, the existing options are associated with a range of adverse effects. In our previous research, we successfully identified ficin, a naturally occurring cysteine protease that possesses unique fibrin and fibrinogenolytic enzymes, making it suitable for both preventing and treating cardiovascular disorders linked to thrombosis. Papain is a prominent cysteine protease derived from the latex of Carica papaya. The potential role of papain in preventing fibrino(geno)lytic, anticoagulant, and antithrombotic activities has not yet been investigated. Therefore, we examined how papain influences fibrinogen and the process of blood coagulation. Papain is highly stable at pH 4–11 and 37–60 °C via azocasein assay. In addition, SDS gel separation electrophoresis, zymography, and fibrin plate assays were used to determine fibrinogen and fibrinolysis activity. Papain has a molecular weight of around 37 kDa, and is highly effective in degrading fibrin, with a molecular weight of over 75 kDa. Furthermore, papain-based hemostatic performance was confirmed in blood coagulation tests, a blood clot lysis assay, and a κ-carrageenan rat tail thrombosis model, highlighting its strong efficacy in blood coagulation. Papain shows dose-dependent blood clot lysis activity, cleaves fibrinogen chains of Aα, Bβ, and γ-bands, and significantly extends prothrombin time (PT) and activated partial thromboplastin time (aPTT). Moreover, the mean length of the infarcted regions in the tails of Sprague–Dawley rats with κ-carrageenan was shorter in rats administered 10 U/kg of papain than in streptokinase-treated rats. Thus, papain, a cysteine protease, has distinct fibrin and fibrinogenolytic properties, suggesting its potential for preventing or treating cardiovascular issues and thrombosis-related diseases.

Computational modeling of blood clot lysis considering the effect of vessel wall and pulsatile blood flow.

Stroke is one of the major causes of global death, which can occur due to blockage in a blood vessel by a clot. The immediate dissolving of the clot is essential to restore the blood flow and prevent tissue necrosis. Clot dissolution can be achieved via thrombolytic therapy using plasminogen activators. In this study, a clot dissolution model is developed for a three-dimensional patient-specific carotid artery that investigates the effect of different vessel wall models on clot dissolution. The lysis pattern of the clot and hemodynamics of blood flow are evaluated using three different models of the vessel wall, namely, rigid, linear elastic, and Mooney-Rivlin hyperelastic. The effect of flow condition is considered by solving the Navier-Stokes equationsfor the free flow domain and the Brinkman equationfor the clot domain with the same pressure and velocity fields. This will result in continuous pressure and velocity over the interfaces of the free flow and clot domains. The blood inflow is assumed to be pulsatile. In addition, the species transport driven by diffusion and convection is considered to be different in the porous medium and plasma. The obtained results show that in all models, the starting time of clot volume decrease is almost the same and the clot starts dissolving from the inner curvature of the artery. However, in the hyperelastic model, dissolving the clot takes longer compared to the other two models. By monitoring the vessel wall deformation, the exact time of vessel recanalization is determined.

Potensi Ekstrak Enzim Daun Pepaya (Carica papaya L.) Sebagai Agen Fibrinolitik Dengan Metode Clot Lysis in Vitro

Stroke is a disease caused due to blockage of blood vessels. Health department in 2013 stated that 15,4% of people died from strokes. One of the treatment therapies that can be used is fibrinolytic. Fibrinolytic agents have a role in lysing blood clots, these agents can be obtained from microbes, plants and animals. This study aims to determine the protein content contained in papaya leaf enzyme extract (Carica papaya L.), and to determine the potential of the enzyme extract at certain concentrations as an alternative to natural fibrinolytic drugs. The study began with plant determination, sampling, enzyme extraction using a blender, purification of the extract by precipitation using ammonium sulphate, protein content determination using the Lowry method, and testing the fibrinolytic activity of the clot lysis method in vitro. Three series of test concentrations were made, namely 20, 40, and 80%. The results of the research on papaya leaf extract showed that the protein content in purification pellet 1 was 196,22 µg/ml and purification pellet 2 was 306,33 µg/ml. In papaya leaves, fibrinolytic activity was proven with the optimal percentage of blood clot lysis in purification pellet 2 with a concentration of 80% which was 79%.

PRELIMINARY PHYTOCHEMICAL SCREENING AND BIOACTIVITY INVESTIGATIONS OF AVORRHEA CARAMBOLA L.

Averrhoa carambola L. (A. carambola) is particularly well-known for its distinctive star-shaped fruit (Star fruit), cultivated in Southeast Asia, the Indian subcontinent, and northern and southern America. Historically, it has been used for a long time to cure many ailments, including vomiting, fever, arthralgia, cough, hypertension, hangovers, chronic paroxysmal headache, hyperglycemia, and diabetic nephropathy. This study aimed to examine its preliminary phytochemical and some biological properties. For this, fresh leaves of A. carambola were extracted (hot and cold) with ethanol, which was then fractionated with n-hexane and chloroform. Following a preliminary investigation of phytochemicals, radical scavenging, membrane stabilizing, alpha-glucosidase inhibitory, and clot lysis effects were evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, egg albumin model, alpha-amylase assay, and human blood clot lysis assay, respectively using suitable reference standards. The results suggest that A. carambola contains alkaloids, glycosides, steroids, tannins, flavonoids, saponins, and reducing sugars. All the organic fractions demonstrated significant (p &lt;0.05) radical scavenging, membrane stabilizing, alpha-glucosidase inhibitory, and clot lysis activities in a concentration-dependent approach. Except for DPPH radical scavenging, the ethanol fraction showed better activities than the n-hexane and chloroform fractions. In summary, A. carambola might be a potential source of antioxidant, anti-inflammatory, hypoglycemic, and atherothrombolytic agents. Further studies are required to isolate, characterize, and establish molecular mechanisms for each component’s biological effects.

Potensi Ekstrak Kasar Enzim dari Tempe Kedelai Hitam (Glycine soja (L.) Merr.) sebagai Obat Fibrinolitik Alami dengan Metode Clot Lysis In Vitro

Fibrinolytic enzymes are enzymes that can be used to break down fibrin. In thrombus therapy (cardiovascular disease), this enzyme has an activity similar to plasmin, which is able to reduce fibrin by hydrolyzing fibrin into dissolved products and inhibiting the formation of fibrin clots that can trigger cardiovascular disease. Black soybean tempeh has various benefits, one of which is to lower the risk of cardiovascular disease and reduce the risk of blood clots. Due to the content of the enzyme protease in black soybeans which is able to degrade fibrin threads. The purpose of this study is to determine the potential of black soybean tempeh (Glycine soja (L.) Merr) can produce enzyme extracts as natural fibrinolytic drugs. This study was conducted by taking ingredients, crude extraction of enzymes from black soybean tempeh by centrifugation, purification of crude extracts of enzymes using ammonium sulfate salts of 80%, determination of enzyme protein levels with the Lowry method and testing of fibrinolytic potential with the clot lysis method in vitro. The variation in the concentration of the extract used in the test was 12.5; 25; 50 and 100%. The results showed that crude extracts of black soybean tempeh enzymes were able to lyse blood clots. The protein content of the black soybean tempeh extract sample after purification using ammonium sulfate was 245.76 ?g/mL. The optimum percentage of blood clot lysis at a concentration of 100% of the sample after purification was 59%.

Clot Imaging Using Photostable Nanodiamond.

While thrombosis is the leading cause of morbidity and mortality in the United States, an understanding of its triggers, progression, and response to anticoagulant therapy is lacking. Intravital fluorescence microscopy has advanced the study of thrombus formation by providing targeted, multi-color contrast. However, photodegradation of fluorophores limits the application in longitudinal studies (e.g., clot progression and/or dissolution). Fluorescent nanodiamond (FND) is a fluorophore which utilizes intrinsic fluorescence of chromogenic centers within and protected by the diamond crystalline lattice. Recent developments in diamond processing have allowed for the controlled production of nanodiamonds emitting in green or red. Here, the use of FND to label blood clots and/or clot lysis is demonstrated and compared to commonly used organic fluorophores. Model ex vivo clots were formed with incorporated labeled fibrinogen to allow imaging. FND was shown to match the morphology of organic fluorophore labels absent of photobleaching over time. The addition of tissue plasminogen activator (tPa) allowed visualization of the clot lysis stage, which is vital to studies of both DVT and pulmonary embolism resolution.

Production of Fibrinolytic Enzyme by Soil Actinobacteria

Thrombotic diseases are increasingly becoming among the prominent causes of death across the globe. Researchers are now turning attention towards fibrinolytic enzymes as potential alternative treatment for thrombolytic diseases. This present study focused on the production of extracellular fibrinolytic enzyme from soil Actinobacteria and evaluation of its hydrolytic activity on blood clot. The Actinobacteria was isolated from compost soil of semi-arid climate in Nigeria. Bacterial isolation was achieved using starch casein agar. Potent fibrinolytic enzyme producing Actinobacteria were identified and subjected to enzyme production using submerged fermentation method. The interactive effects of incubation time, temperature, pH and media components on enzyme production were analysed. Extracellular fibrinolytic enzyme produced by the selected Actinobacteria was partially purified by ammonium sulphate precipitation and subsequently assayed for blood clot lysis activity. Results of these studies indicated that fibrinolytic enzyme was produced optimally at pH 8 and temperature of 40 oC after 72 hour of fermentation. Partially purified fibrinolytic enzyme was able to degrade blood clot comparable to the positive control. These results shows that soil Actinobacteria of unexplored semi-arid climate of Nigeria present a prospect in search of novel microorganisms with potentials in the production of fibrinolytic enzyme that can serve as an alternative blood clot buster in treating thrombolytic diseases.

Antioxidant, Anti-inflammatory, Antimicrobial and Thrombolytic Activities of Eclipta alba L. Growing in Bangladesh

Eclipta alba L. (Family: Asteraceae) is an evergreen herb of ethnomedicinal significance in the Indian subcontinent. The present study was carried out to determine the in vitro antioxidant, anti-inflammatory, antimicrobial and thrombolytic activities of n-hexane, chloroform and aqueous soluble fractions of ethanol crude extract of E. alba whole plant. Preliminary phytochemical screening was performed by qualitative tests which revealed the presence of alkaloids, steroids, glycosides, tannins, flavonoids, saponins and amides in E. alba. During the DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay, the aqueous soluble fraction of E. alba at a dose of 100 μg/ml showed maximum 87.5% inhibition in scavenging the DPPH radical with IC50 value of 7.86 μg/ml. Anti-inflammatory activity was evaluated by egg albumin denaturation technique and RBC membrane stabilizing method. In both experiments, all the test samples of E. alba showed a concentration-dependent anti-inflammatory potential while the aqueous soluble fraction at the dose of 500 μg/ml demonstrated the maximum 90.12% inhibition of egg albumin denaturation and 78.42% inhibition of RBC membrane hemolysis against hypotonic solution. Antimicrobial sensitivity was evaluated by disc diffusion assay against several bacterial and fungal species. All the test samples at a concentration of 500 μg/disc exhibited noticeable antibacterial (zone of inhibition = 11-23 mm) and antifungal (zone of inhibition = 10-21 mm) activities when compared to the respective standard drug. The aqueous soluble fraction showed maximum activity against Lactobacillus casai (zone of inhibition = 23 mm). During antifungal test, the maximum activity was exhibited by the n-hexane fraction against Pityrosporum ovale (zone of inhibition = 21 mm). During in vitro thrombolytic assay, the aqueous soluble fraction revealed 56.84% lysis of blood clot, as compared to the standard streptokinase (84.60%). The results of our study suggest that E. alba can be considered for further investigation in order to discover the pharmacologically active natural products. Bangladesh Pharmaceutical Journal 26(1): 20-27, 2023 (January)

In vitro Antioxidant, Thrombolytic and Membrane Stabilizing Activities of the Rourea minor Leaves

Rourea minor is a species which belongs to the family Connaraceae. Rourea minor reported previously for antibiotic, antibacterial, hemostatic and wound-healing activities. In this study, we evaluated the antioxidant, thrombolytic and membrane stabilizing activities of the crude methanolic extract of the leaves of Rourea minor and its different organic soluble fractions. We measured the total phenolic contents and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity of the extracts to observe their antioxidant potential. The thrombolytic activity was determined by human blood clot lysis. Anti-inflammatory activity was evaluated by human red blood cell (HRBC) membrane stabilization based on hypotonic solution- and heat-induced haemolysis. The total phenolic content ranged from 9.4 to 57.4 mg GAE/g of the dry extracts and IC50 values of the different extracts (Crude methanol, petroleum ether, ethyl acetate and aqueous) were between 20.38 and 269.34 μg/ml. All the extracts showed mild to moderate thrombolytic potential inhibiting the lysis of clot by 3.47% - 18.43%. The extracts also exhibited potential membrane stabilizing activity in both hypotonic solution- and heatinduced haemolysis indicating their anti-inflammatory activity. Thus, it can be concluded that the methanolic extract from the leaves of Rourea minor and its different fractions possess mild to moderate antioxidant, thrombolytic activities, and membrane stabilizing potential. Bangladesh Pharmaceutical Journal 26(1): 51-55, 2023 (January)