RESUMO
Thrombosis is currently among the major causes of morbidity and mortality in the World. New prevention and therapy alternatives have been increasingly sought in medicinal plants. In this context, we have been investigating parsley, Petroselinum crispum (Mill.) Nym, an aromatic herb with two leaf varieties. We report here the in vitro, in vivo, and ex vivo anti-hemostatic and antithrombotic activities of a parsley curly-leaf variety. Aqueous extracts of aerial parts (PCC-AP), stems (PCC-S), and leaves (PCC-L) showed significant in vitro antiplatelet activity. PCC-AP extract exhibited the highest activity (IC50 2.92 mg/mL) when using ADP and collagen as agonists. All extracts also presented in vitro anticoagulant activity (APTT and PT) and anti-thrombogenic activity. PCC-S was the most active, with more significant interference in the factors of the intrinsic coagulation pathway. The oral administration of PCC-AP extract in rats caused a greater inhibitory activity in the deep vein thrombi (50%; 65 mg/kg) than in arterial thrombi formation (50%; 200 mg/kg), without cumulative effect after consecutive five-day administration. PCC-AP extract was safe in the induced bleeding time test. Its anti-aggregating profile was similar in ex vivo and in vitro conditions but was more effective in the extrinsic pathway when compared to in vitro results. Apiin and coumaric acid derivatives are the main compounds in PCC-AP according to the HPLC-DAD-ESI-MS/MS profile. We demonstrated for the first time that extracts from different parts of curly parsley have significant antiplatelet, anticoagulant, and antithrombotic activity without inducing hemorrhage, proving its potential as a source of antithrombotic compounds.
Assuntos
Fibrinolíticos , Petroselinum , Extratos Vegetais , Folhas de Planta , Animais , Petroselinum/química , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Folhas de Planta/química , Ratos , Masculino , Fibrinolíticos/farmacologia , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/química , Ratos Wistar , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/isolamento & purificação , Trombose/tratamento farmacológico , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/isolamento & purificação , Componentes Aéreos da Planta/química , Caules de Planta/química , Hemostáticos/farmacologia , Hemostáticos/isolamento & purificação , Anticoagulantes/farmacologia , Anticoagulantes/isolamento & purificação , Anticoagulantes/química , Plantas Medicinais/químicaRESUMO
There is a clinical need to develop safe therapeutic strategies to mitigate bleeding. Previously, we found that a novel zymogen-like factor Xa variant (FXa-I16L) was effective in correcting the coagulation defect in hemophilic mice. Here we expand the mutational framework to tune the FX(a) zymogen-like state. Alteration of FXa zymogenicity yields variants (V17M, I16L, I16M, V17T, V17S, and I16T) with a wide range (≤1000-fold) of reduced function toward physiologic substrates and inhibitors. The extent of zymogen-like character, including resistance to antithrombin III, correlates well with plasma half-life (<2 minutes to >4 hours). Importantly, biologic function, including that of the most zymogen-like variant (FXa-I16T), was greatly enhanced when bound to FVa membranes. This resulted in improvement of clotting times and thrombin generation in hemophilic plasma. The FXa variants were remarkably effective in mouse injury models. In these systems, the data show that the more active the protease, the more difficult it is to overcome the protective mechanism of circulating inhibitors to achieve a therapeutic benefit. Depending on the treatment situation, the more zymogen-like variants (V17S and I16T) were most useful when given before injury whereas variants exhibiting greater activity but shorter half-lives (I16L and I16M) were most effective when administered after injury. This new class of FXa variants provides a useful and flexible platform for selectively bioengineering biologic function and half-life to target different clinical bleeding scenarios.
Assuntos
Precursores Enzimáticos , Fator Xa , Hemostáticos/isolamento & purificação , Animais , Coagulação Sanguínea/efeitos dos fármacos , Domínio Catalítico , Análise Mutacional de DNA , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Fator Xa/química , Fator Xa/genética , Fator Xa/metabolismo , Meia-Vida , Hemofilia A/sangue , Hemofilia A/tratamento farmacológico , Hemostáticos/síntese química , Hemostáticos/química , Hemostáticos/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/uso terapêutico , Protrombina/metabolismo , Tromboplastina/químicaRESUMO
Snake venom metalloproteinases (SVMPs) are major components in most viperid venoms that induce disturbances in the hemostatic system and tissues of animals envenomated by snakes. These disturbances are involved in human pathology of snake bites and appear to be essential for the capture and digestion of snake's prey and avoidance of predators. SVMPs are a versatile family of venom toxins acting on different hemostatic targets which are present in venoms in distinct structural forms. However, the reason why a large number of different SVMPs are expressed in some venoms is still unclear. In this study, we evaluated the interference of five isolated SVMPs in blood coagulation of humans, birds and small rodents. P-III class SVMPs (fractions Ic, IIb and IIc) possess gelatinolytic and hemorrhagic activities, and, of these, two also show fibrinolytic activity. P-I class SVMPs (fractions IVa and IVb) are only fibrinolytic. P-III class SVMPs reduced clotting time of human plasma. Fraction IIc was characterized as prothrombin activator and fraction Ic as factor X activator. In the absence of Ca2+, a firm clot was observed in chicken blood samples with fractions Ic, IIb and partially with fraction IIc. In contrast, without Ca2+, only fraction IIc was able to induce a firm clot in rat blood. In conclusion, functionally distinct forms of SVMPs were found in B. neuwiedi venom that affect distinct mechanisms in the coagulation system of humans, birds and small rodents. Distinct SVMPs appear to be more specialized to rat or chicken blood, strengthening the current hypothesis that toxin diversity enhances the possibilities of the snakes for hunting different prey or evading different predators. This functional diversity also impacts the complexity of human envenoming since different hemostatic mechanisms will be targeted by SVMPs accounting for the complexity of the response of humans to venoms.
Assuntos
Venenos de Crotalídeos/enzimologia , Hemostáticos/química , Metaloproteases/química , Proteínas de Répteis/química , Adaptação Biológica , Sequência de Aminoácidos , Animais , Bothrops , Galinhas , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Fator X/química , Feminino , Hemostáticos/isolamento & purificação , Hemostáticos/farmacologia , Humanos , Masculino , Metaloproteases/isolamento & purificação , Metaloproteases/farmacologia , Camundongos , Proteólise , Protrombina/química , Ratos , Proteínas de Répteis/isolamento & purificação , Proteínas de Répteis/farmacologia , Mordeduras de SerpentesRESUMO
Bellucia dichotoma Cogn. (Melastomataceae) is one of various plant species used in folk medicine in the west of the state of Pará, Brazil, to treat snake bites. Many studies have been carried out to evaluate the effectiveness of anti-snake bite plants, but few of these use the same preparation methods and doses as those traditionally used by the local populations. This study therefore compared inhibition of the main local effects of B. atrox venom (BaV) by aqueous extract of B. dichotoma (AEBd) administered according to traditional methods and pre-incubated with BaV). The concentrations of phenolic compounds (tannins and flavonoids) in AEBd were determined by colorimetric assays. The effectiveness of AEBd in inhibiting the hemorrhagic and edematogenic activities of BaV was evaluated in mice in four different experimental in vivo protocols: (1) pre-incubation (venom:extract, w/w); (2) pre-treatment (p.o.); (3) post-treatment (p.o.); and (4) AEBd (p.o.) in combination with Bothrops antivenom (BA) (i.v.). To assess in vitro inhibition of BaV phospholipase A2 activity, the pre-incubation method or incorporation of AEBd or BA in agarose gels were used. The effect of AEBd on BaV was determined by SDS-PAGE, zymography and Western blot. Colorimetric assays revealed higher concentrations of (condensed and hydrolyzable) tannins than flavonoids in AEBd. Hemorrhagic activity was completely inhibited using the pre-incubation protocol. However, with pre-treatment there was no significant inhibition for the concentrations tested, and with the post-treatment only the 725 mg/kg dose of AEBd was able to inhibit 40.5% (p = 0.001) of the hemorrhagic activity of BaV. Phospholipase A2 activity was only inhibited when AEBd was pre-incubated with BaV. BaV-induced edema was completely inhibited with pre-incubation (p < 0.05) and significantly reduced (p < 0.05) with pre- and post-treatment (p.o.) for the concentrations tested. The reduction in local edema was even greater when AEBd was administered in combination with BA. The SDS-PAGE profiles showed that several of the BaV protein (SDS-PAGE) and enzyme (zymography) bands were not detected when the venom was pre-incubated, and Western blot revealed that this was not caused by the AEBd enzymes observed in the zymogram. The "pseudo inhibition" observed after pre-incubation in this study may be due to the presence of tannins in the extract, which could act as chelating agents, removing metalloproteins and Ca²âº ions and thus inhibiting hemorrhagin and PLA2 activity. However, when administered according to traditional methods, B. dichotoma extract was effective in blocking BaV-induced edematogenic activity and had an additional effect on inhibition of this activity by BA.
Assuntos
Antivenenos/uso terapêutico , Bothrops , Venenos de Crotalídeos/antagonistas & inibidores , Melastomataceae/química , Casca de Planta/química , Extratos Vegetais/uso terapêutico , Mordeduras de Serpentes/tratamento farmacológico , Animais , Antivenenos/química , Antivenenos/isolamento & purificação , Antivenenos/farmacologia , Brasil , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/toxicidade , Edema/etiologia , Edema/prevenção & controle , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Etnofarmacologia , Fosfolipases A2 do Grupo II/antagonistas & inibidores , Fosfolipases A2 do Grupo II/metabolismo , Hemorragia/etiologia , Hemorragia/prevenção & controle , Hemostáticos/química , Hemostáticos/isolamento & purificação , Hemostáticos/farmacologia , Hemostáticos/uso terapêutico , Masculino , Medicina Tradicional , Camundongos , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/toxicidade , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Proteínas de Répteis/antagonistas & inibidores , Proteínas de Répteis/metabolismo , Pele/efeitos dos fármacos , Mordeduras de Serpentes/fisiopatologia , Taninos/análise , Taninos/farmacologia , Taninos/uso terapêuticoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Qualea parviflora Mart. is a medicinal species commonly found in the Brazilian Cerrado biome. AIM OF THE STUDY: Based on ethnopharmacological data, methanolic extract from Qualea parviflora (QP) bark was evaluated for its antiulcer, analgesic, anti-hemorrhagic, mutagenic and anti-Helicobacter pylori activities. MATERIAL AND METHODS: The gastroprotective action of the extract was evaluated in rodent experimental models (HCl/ethanol, ethanol or NSAID). We also evaluated mutagenic effect (Ames assay), anti-Helicobacter pylori, anti-hemorrhagic action, analgesic and inflammatory effects (hot-plate test and carrageenin-induced hind paw edema) of methanolic extract from Qualea parviflora. RESULTS: QP (500 mg/kg, p.o.) was able to protect gastric mucosa against HCl/ethanol solution (77%), absolute ethanol (97%), and also against injurious effect of NSAID (36%). When QP was challenged with sulfhydryl depletor compound, the gastroprotective action of extract was abolished. QP treatment was able to maintain the GSH level and show a concentration-dependent inhibition effect on the lipid peroxidation. QP present anti-Helicobacter pylori effect (MIC=75 microg/mL), anti-hemorrhagic and antidiarrheal action but not present analgesic or anti-inflammatory effect. CONCLUSION: methanolic extract from Qualea parviflora had gastroprotective effect related to the increase of gastric mucosa defensive factors such PGE(2) levels and maintain the basal gastric glutathione levels. The methanolic extract also showed anti-Helicobacter pylori activity, anti-hemorrhagic effect and antioxidant action, but absence of analgesic, mutagenic and toxic effects, a profile that adds safety to its use.
Assuntos
Antiulcerosos/uso terapêutico , Antidiarreicos/uso terapêutico , Mucosa Gástrica/efeitos dos fármacos , Hemostáticos/uso terapêutico , Extratos Vegetais/uso terapêutico , Plantas Medicinais , Animais , Antiulcerosos/isolamento & purificação , Antiulcerosos/farmacologia , Antidiarreicos/isolamento & purificação , Antidiarreicos/farmacologia , Feminino , Mucosa Gástrica/patologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/fisiologia , Hemostáticos/isolamento & purificação , Hemostáticos/farmacologia , Masculino , Camundongos , Testes de Mutagenicidade/métodos , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Ratos , Ratos WistarRESUMO
Fibrin sealant, a widely available tissue adhesive, has been used since 1940 in a variety of clinical applications. Commercially available fibrin sealant products are synthesized from bovine thrombin and human fibrinogen, which may transmit infectious diseases, and recipients may also develop antibodies against bovine thrombin. Bearing these disadvantages in mind, a new fibrin sealant was developed in 1989 by a group of researchers from the Center for the Study of Venoms and Venomous Animals, in Sao Paulo State, Brazil. The main purpose was to produce an adhesive fibrin without using human blood, to avoid transmitting infectious diseases. The components of this novel sealant were extracted from large animals and a serine proteinase extracted from Crotalus durissus terrificus snake venom. The applicability of this sealant was tested in animals and humans with beneficial results. The new fibrin sealant can be a useful tool clinically due to its flexibility and diversity of applications. This sealant is a biological and biodegradable product that (1) does not produce adverse reactions, (1) contains no human blood, (3) has a good adhesive capacity, (4) gives no transmission of infectious diseases, and (5) may be used as an adjuvant in conventional suture procedures. The effectiveness of this new fibrin sealant is reviewed and its development and employment are described.
Assuntos
Venenos de Crotalídeos/uso terapêutico , Crotalus , Adesivo Tecidual de Fibrina/uso terapêutico , Hemostáticos/uso terapêutico , Adesivos Teciduais/uso terapêutico , Animais , Venenos de Crotalídeos/efeitos adversos , Venenos de Crotalídeos/isolamento & purificação , Adesivo Tecidual de Fibrina/efeitos adversos , Adesivo Tecidual de Fibrina/isolamento & purificação , Hemostáticos/efeitos adversos , Hemostáticos/isolamento & purificação , Humanos , Procedimentos Cirúrgicos Operatórios , Adesivos Teciduais/efeitos adversos , Adesivos Teciduais/isolamento & purificaçãoRESUMO
The venom of Bothrops insularis snake, known in Brazil as jararaca ilhoa, contains a variety of proteolytic enzymes such as a thrombin-like substance that is responsible for various pharmacological effects. B. insularis venom chromatography profile showed an elution of seven main fractions. The thrombin-like activity was detected in fractions I and III, the latter being subjected to two other chromatographic procedures, so to say DEAE and Hi Trap Benzamidine. The purity degree of this fraction was confirmed by analytical reverse phase HPLC, which displayed only one main fraction confirmed by SDS-PAGE constituting fraction III. About 5 microg of fraction III protein potentiated the secretion of insulin induced by 2.8 mM of glucose in rats isolated pancreatic beta-cells treated; the increase being around 3-fold higher than its respective control. B. insularis lectin (BiLec; 10 microg/mL) was also studied as to its effect on the renal function of isolated perfused rat kidneys with the use of six Wistar rats. BiLec increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa+) and chloride tubular reabsorption (%TCl-) decreased at 120 min, without alteration in potassium transport. In conclusion, the thrombin-like substance isolated from B. insularis venom induced an increase in insulin secretion, in vitro, and transiently altered vascular, glomerular and tubular parameters in the isolated rat kidney.