RESUMEN

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.

Asunto(s)

Fibrinolíticos , Petroselinum , Extractos Vegetales , Hojas de la Planta , Animales , Petroselinum/química , Extractos Vegetales/farmacología , Extractos Vegetales/química , Hojas de la Planta/química , Ratas , Masculino , Fibrinolíticos/farmacología , Fibrinolíticos/aislamiento & purificación , Fibrinolíticos/química , Ratas Wistar , Fitoquímicos/farmacología , Fitoquímicos/aislamiento & purificación , Trombosis/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/aislamiento & purificación , Componentes Aéreos de las Plantas/química , Tallos de la Planta/química , Hemostáticos/farmacología , Hemostáticos/aislamiento & purificación , Anticoagulantes/farmacología , Anticoagulantes/aislamiento & purificación , Anticoagulantes/química , Plantas Medicinales/química

RESUMEN

Fucosylated chondroitin sulfates (FCSs) PC and HH were isolated from the sea cucumbers Paracaudina chilensis and Holothuria hilla, respectively. The purification of the polysaccharides was carried out by anion-exchange chromatography on a DEAE-Sephacel column. The structural characterization of the polysaccharides was performed in terms of monosaccharide and sulfate content, as well as using a series of nondestructive NMR spectroscopic methods. Both polysaccharides were shown to contain a chondroitin core [→3)-ß-d-GalNAc (N-acethyl galactosamine)-(1→4)-ß-d-GlcA (glucuronic acid)-(1→]n, bearing sulfated fucosyl branches at O-3 of every GlcA residue in the chain. These fucosyl residues were different in their pattern of sulfation: PC contained Fuc2S4S and Fuc4S in a ratio of 2:1, whereas HH included Fuc2S4S, Fuc3S4S, and Fuc4S in a ratio of 1.5:1:1. Moreover, some GalNAc residues in HH were found to contain an unusual disaccharide branch Fuc4S-(1→2)-Fuc3S4S-(1→ at O-6. Sulfated GalNAc4S6S and GalNAc4S units were found in a ratio of 3:2 in PC and 2:1 in HH. Both polysaccharides demonstrated significant anticoagulant activity in a clotting time assay, which is connected with the ability of these FCSs to potentiate the inhibition of thrombin and factor Xa in the presence of anti-thrombin III (ATIII) and with the direct inhibition of thrombin in the absence of any cofactors.

Asunto(s)

Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Sulfatos de Condroitina/farmacología , Holothuria/metabolismo , Animales , Anticoagulantes/aislamiento & purificación , Antitrombina III/metabolismo , Antitrombinas/aislamiento & purificación , Antitrombinas/farmacología , Sulfatos de Condroitina/aislamiento & purificación , Factor Xa/metabolismo , Inhibidores del Factor Xa/aislamiento & purificación , Inhibidores del Factor Xa/farmacología , Estructura Molecular , Relación Estructura-Actividad , Trombina/antagonistas & inhibidores , Trombina/metabolismo

RESUMEN

Nowadays, pharmaceutical heparin is purified from porcine and bovine intestinal mucosa. In the past decade there has been an ongoing concern about the safety of heparin, since in 2008, adverse effects associated with the presence of an oversulfated chondroitin sulfate (OSCS) were observed in preparations of pharmaceutical porcine heparin, which led to the death of patients, causing a global public health crisis. However, it has not been clarified whether OSCS has been added to the purified heparin preparation, or whether it has already been introduced during the production of the raw heparin. Using a combination of different analytical methods, we investigate both crude and final heparin products and we are able to demonstrate that the sulfated contaminants are intentionally introduced in the initial steps of heparin preparation. Furthermore, the results show that the oversulfated compounds are not structurally homogeneous. In addition, we show that these contaminants are able to bind to cells in using well known heparin binding sites. Together, the data highlights the importance of heparin quality control even at the initial stages of its production.

Asunto(s)

Anticoagulantes/aislamiento & purificación , Sulfatos de Condroitina/aislamiento & purificación , Contaminación de Medicamentos , Heparina/aislamiento & purificación , Animales , Anticoagulantes/química , Bovinos , Sulfatos de Condroitina/química , Heparina/química , Liasa de Heparina/química , Humanos , Hidrólisis , Mucosa Intestinal/química , Espectroscopía de Resonancia Magnética , Polisacárido Liasas/química , Control de Calidad , Porcinos

RESUMEN

Two sulfated polysaccharides (SPs), F2 and F3, isolated from Codium isthmocladum were found to contain galactose, sulfate, and pyruvate. The apparent molecular weights of F2 and F3 were determined to be 62 and 61 kDa, respectively. NMR spectroscopy combined with chemical analysis showed that F2 and F3 have the same structural features. However, F3 showed higher sulfate/sugar ratio (1/2.6) than F2 (1/4). F2 and F3 are essentially (1 → 3)-ß-D-galactans with some branching at C6. Pyruvylation occurs at O3 and O4, forming 3,4-O-(1-carboxyethylidene)-ß-D-Galp residues; some of these pyruvylated residues contain sulfate groups at C6. Some non-branching residues contain sulfate at C4. None of the SPs exhibited antioxidant activity. MTT results indicated that 1 mg/mL of both SPs about 40% of PANC-1 cell viability. At 10 µg/mL, F2 and F3 had 1.7-fold longer clotting times compared to that of Clexane® at the same concentration. The higher sulfate content of F3 is not a determining factor for pharmacological activities of galactans, considering that both F2 and F3 exerted the effects.

Asunto(s)

Anticoagulantes/farmacología , Antioxidantes/farmacología , Chlorophyta/química , Galactanos/farmacología , Algas Marinas/química , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Secuencia de Carbohidratos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Galactanos/química , Galactanos/aislamiento & purificación , Humanos , Piruvatos/química , Piruvatos/aislamiento & purificación , Piruvatos/farmacología , Ésteres del Ácido Sulfúrico/química , Ésteres del Ácido Sulfúrico/aislamiento & purificación , Ésteres del Ácido Sulfúrico/farmacología

RESUMEN

In parasites, cathepsins are implicated in mechanisms related to organism surveillance and host evasion. Some parasite cathepsins have fibrinogenolytic and fibrinolytic activity, suggesting that they may contribute to maintain blood meal fluidity for extended feeding periods. Here, it is shown that BmGTI (Rhipicephalus [Boophilus] microplus Gut Thrombin Inhibitor), a protein previously described as an inhibitor of fibrinogen hydrolysis and platelet aggregation by thrombin, and BmCL1 (Rhipicephalus [Boophilus] microplus Cathepsin-L like 1) are the same protein, hereinafter referred to using the earliest name (BmCL1). To further characterize BmCL1, Rhipicephalus microplus native and recombinant (rBmCL1) proteins were obtained. Native BmCL1 was isolated using thrombin-affinity chromatography, and it displays thrombin inhibition activity. We subsequently investigated rBmCL1 interaction with thrombin. We show that rBmCL1 and thrombin have a dissociation constant (ΚD) of 130.2 ±â€¯11.2 nM, and this interaction likely occurs due to a more electronegative surface of BmCL1 at pH 7.5 than at pH 5.0, which may favor an electrostatic binding to positively charged thrombin exosites. During BmCL1-thrombin interaction, thrombin is not degraded or inhibited. rBmCL1 impairs thrombin-induced fibrinogen clotting via a fibrinogenolytic activity. Fibrinogen degradation by BmCL1 occurs by the hydrolysis of Aα- and Bß-chains, generating products similar to those produced by fibrinogenolytic cathepsins from other organisms. In conclusion, BmCL1 likely has an additional role in R. microplus blood digestion, besides its role in hemoglobin degradation at acid pH. BmCL1 fibrinogenolytic activity indicates a proteolytic activity in the neutral lumen of tick midgut, contributing to maintain the fluidity of the ingested blood, which remains to be confirmed in vivo.

Asunto(s)

Catepsina L/metabolismo , Rhipicephalus/enzimología , Trombina/metabolismo , Secuencia de Aminoácidos , Animales , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Anticoagulantes/metabolismo , Catepsina L/química , Catepsina L/aislamiento & purificación , Bovinos , Cinética , Modelos Moleculares , Proteolisis

RESUMEN

In this paper, carrageenans having distinct sulfation patterns (κ-, ι-, ι/ν-, θ- and λ-carrageenans), were fully or partially oxidized at C-6 of the ß-d-Galp units using 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and trichloroisocyanuric acid (TCCA) in bicarbonate buffer. The modified carrageenans were characterized by mono- and bidimensional 1H and 13C NMR spectroscopy. The influence of the sulfate and carboxyl groups onto anticoagulant activity was evaluated using Activated Partial Thromboplastin Time (aPTT) in vitro assay. The results showed a synergic effect of the carboxyl groups on the anticoagulant activity, which was dependent on the regiochemistry of the sulfate groups in the polysaccharide backbone. Sulfate groups at C2 of the ß-d-GalAp units appeared to positively influence the anticoagulant effect in comparison to C4-sulfate samples. Also, the partially oxidized κ-carrageenan derivative (κLO) showed better anticoagulant effect than the fully oxidized carrageenan (κHO).

Asunto(s)

Anticoagulantes/química , Carragenina/química , Ácidos Urónicos/química , Anticoagulantes/aislamiento & purificación , Carragenina/aislamiento & purificación , Estructura Molecular , Oxidación-Reducción , Tiempo de Tromboplastina Parcial , Rhodophyta/química , Sulfatos/química , Ácidos Urónicos/síntesis química

RESUMEN

Most of the unfractionated and low-molecular-weight heparins available worldwide are produced by Chinese companies from porcine mucosa. China is the world's largest producer of pork and thus has plenty of raw material to produce heparins. However, the deadly African Swine Fever (ASF) outbreaks afflicting China since August 2018 may cause extensive losses to the pig herd, with serious consequences for the global supply of heparins. In 2008, a sudden shortage of heparin's raw material resulting from a viral disease in Chinese pigs prompted adulterations responsible for 80 deaths and hundreds of adverse events. This incident revealed the fragility of such a supply chain, which is mostly based on raw material from a single animal from a single country. A worldwide introduction of bovine mucosa heparins manufactured in different countries certainly is a feasible way to mitigate eventual shortages of these life-saving anticoagulants caused by local veterinary problems such as the ASF threatening China now.

Asunto(s)

Virus de la Fiebre Porcina Africana/patogenicidad , Fiebre Porcina Africana/virología , Anticoagulantes/provisión & distribución , Brotes de Enfermedades/veterinaria , Heparina/provisión & distribución , Mucosa Intestinal/metabolismo , Animales , Anticoagulantes/aislamiento & purificación , China , Heparina/aislamiento & purificación , Sus scrofa , Porcinos

RESUMEN

World fisheries and aquaculture production totaled 167 million tons in 2014. This high fish production generates a lot of waste that could be used as raw material for extraction of substances of pharmacological interest. In this work, we extract and characterize glycosaminoglycans (GAGs) present in the viscera of Nile tilapia (Oreochromis niloticus) and Pacu (Piaractus mesopotamicus), which are among the most vastly produced fishes in inland aquaculture in Brazil. Moreover, the anticoagulant activity of the GAGs fractions was evaluated. GAGs were obtained from total defatted viscera, after proteolysis, precipitation with ethanol, anion exchange chromatography and treatment with chondroitinase. Chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) were identified by agarose gel electrophoresis and NMR analyses. CS, DS and HS were identified in equivalent fractions obtained from both fishes, and all GAGs fractions showed anticoagulant activity.

Asunto(s)

Anticoagulantes/aislamiento & purificación , Anticoagulantes/farmacología , Characiformes/anatomía & histología , Cíclidos/anatomía & histología , Glicosaminoglicanos/aislamiento & purificación , Glicosaminoglicanos/farmacología , Vísceras/química , Animales

RESUMEN

In this study, sulfated polysaccharide-rich extracts were isolated from 22 tropical seaweeds (4 red, 11 brown, and 7 green) found in northeastern Brazil, and evaluated for the role of anticoagulant agents. Fifteen of the extracts showed anticoagulant activity, including all the extracts from green seaweeds. Udotea flabellum (a green seaweed) extract was the most potent, requiring an amount of only 3 µg to double the plasma coagulation time in the activated partial thromboplastin time test. A similar result was obtained with 1 µg of heparin. Two sulfated homogalactans with anticoagulant activity, F-I (130 kDa) and F-II (75 kDa), were isolated from this extract using several bio-guided purification steps. Their anticoagulant activity, as well as properties related to antitumor activity (anti-proliferative, anti-adhesive, and anti-migratory), were accessed. Their anticoagulant activities were close to that of heparin. We found that F-I and F-II (0.5⁻10 µg/mL) were not able to directly inhibit thrombin. In the presence of anti-thrombin, F-I (0.5 µg/mL) was more effective than heparin (0.5 µg/mL) in inhibiting thrombin, while F-II showed similar effects as heparin. F-I and F-II also inhibited B16-F10 (murine melanoma cells) adhesion, migration, and proliferation on a fibronectin-coated surface, but not on laminin- or collagen I-coated surfaces. Except for the antiproliferative activity, the other effects of F-I and F-II were eliminated upon their desulfation (~50%), indicating that the degree of sulfation is not as important for F-I and F-II anti-proliferative activity as the sulfation position. Taken together, the results provide strong evidence for the potential utility of sulfated galactans from U. flabellum, making these compounds an interesting option for future investigations that aim to design new anticoagulant/antitumor agents.

Asunto(s)

Anticoagulantes/farmacología , Antineoplásicos/farmacología , Chlorophyta/química , Extractos Vegetales/farmacología , Algas Marinas/química , Animales , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Brasil , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Galactanos/química , Galactanos/aislamiento & purificación , Galactanos/farmacología , Heparina/farmacología , Ratones , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Sulfatos/química , Sulfatos/aislamiento & purificación , Sulfatos/farmacología , Trombina/antagonistas & inhibidores

RESUMEN

The anticoagulant and antithrombotic properties of three structurally correlated sea urchin-derived 3-linked sulfated α-glycans and their low molecular-weight derivatives were screened comparatively through various in vitro and in vivo methods. These methods include activated partial thromboplastin time, the inhibitory activity of antithrombin over thrombin and factor Xa, venous antithrombosis, the inhibition of platelet aggregation, the activation of factor XII, and bleeding. While the 2-sulfated fucan from Strongylocentrotus franciscanus was observed to be poorly active in most assays, the 4-sulfated fucan from Lytechinus variegatus, the 2-sulfated galactan from Echinometra lucunter and their derivatives showed multiple effects. All marine compounds showed no capacity to activate factor XII and similar low bleeding tendencies regardless of the dose concentrations used to achieve the highest antithrombotic effect observed. The 2-sulfated galactan showed the best combination of results. Our work improves the background about the structure-function relationship of the marine sulfated glycans in anticoagulation and antithrombosis. Besides confirming the negative effect of the 2-sulfated fucose and the positive effect of the 2-sulfated galactose on anticoagulation in vitro, our results also demonstrate the importance of this set of structural requirements on antithrombosis in vivo, and further support the involvement of high-molecular weight and 4-sulfated fucose in both activities.

Asunto(s)

Anticoagulantes/farmacología , Factor XII/metabolismo , Fibrinolíticos/farmacología , Polisacáridos/farmacología , Erizos de Mar/química , Trombosis de la Vena/tratamiento farmacológico , Adulto , Animales , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Anticoagulantes/uso terapéutico , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Factor Xa/metabolismo , Femenino , Fibrinolíticos/química , Fibrinolíticos/aislamiento & purificación , Fibrinolíticos/uso terapéutico , Voluntarios Sanos , Humanos , Masculino , Estructura Molecular , Peso Molecular , Tiempo de Tromboplastina Parcial , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Polisacáridos/uso terapéutico , Conejos , Ratas , Ratas Wistar , Relación Estructura-Actividad , Sulfatos/química , Tromboplastina/administración & dosificación , Trombosis de la Vena/inducido químicamente , Adulto Joven

RESUMEN

Essentials Bovine (HBI) and porcine (HPI) heparins differ in structure and anticoagulant activity. Protamine-neutralization was evaluated on a variety of physical-chemical methods. HBI requires more protamine than HPI to fully neutralize its anticoagulant activity. Protamine preferentially removes higher-sulfated chains of HBI while HPI is evenly precipitated. SUMMARY: Background Protamine neutralization is an essential step for the safe use and inactivation of the unfractionated heparin (UFH) that is widely employed in surgical and non-surgical procedures involving extracorporeal circulation. Objective To compare protamine neutralization of different pharmaceutical-grade UFHs prepared from porcine or bovine intestine (HPI and HBI, respectively). HBI has approximately half the anticoagulant potency of HPI, mostly as consequence of its fraction enriched with N-sulfated α-glucosamine disaccharides. Methods Protamine neutralization of HPI and HBI was evaluated with in vitro, ex vivo and in vivo assays. We also performed in-depth assessments of the complexation of protamine with these distinct UFHs by using nuclear magnetic resonance and mass spectroscopy. Results HPI and HBI interact similarly with protamine on a mass/mass basis; however, HBI requires more protamine than HPI to have its anticoagulant activity fully neutralized, because of its lower potency, which entails the use of higher doses. Nuclear magnetic resonance spectra revealed that HPI precipitates homogeneously with protamine. On the other hand, the low-sulfated fraction of HBI, enriched with N-sulfated α-glucosamine, precipitates at higher concentrations of protamine than the fraction more like HPI, with a preponderance of N,6-disulfated α-glucosamine disaccharides. Finally, mass spectroscopy spectra showed that some of the different peptide components of protamine interact preferentially with the heparins, irrespective of their animal origin. Conclusion Our results have important medical implications, indicating that protamine neutralization of HBI, determined exclusively by point-of-care coagulation assessments, must fail because of its lower-sulfated fraction with reduced anticoagulant activity that could remain in the circulation after the neutralization procedure.

Asunto(s)

Anticoagulantes/farmacología , Antagonistas de Heparina/farmacología , Heparina/farmacología , Protaminas/farmacología , Animales , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Bioensayo , Bovinos , Precipitación Química , Cromatografía de Afinidad , Disacáridos/química , Relación Dosis-Respuesta a Droga , Heparina/química , Heparina/aislamiento & purificación , Mucosa Intestinal/química , Espectrometría de Masas , Resonancia Magnética Nuclear Biomolecular , Tiempo de Tromboplastina Parcial , Protaminas/química , Ratas , Especificidad de la Especie , Azufre/análisis , Porcinos

RESUMEN

The oceans harbor a great diversity of organisms, and have been recognized as an important source of new compounds with nutritional and therapeutic potential. Among these compounds, carbohydrate-based compounds are of particular interest because they exhibit numerous biological functions associated with their chemical diversity. This gives rise to new substances for the development of bioactive products. Many are the known applications of substances with glycosidic domains obtained from marine species. This review covers the structural properties and the current findings on the antioxidant, anti-inflammatory, anticoagulant, antitumor and antimicrobial activities of medium and high molecular-weight carbohydrates or glycosylated compounds extracted from various marine organisms.

Asunto(s)

Organismos Acuáticos/química , Carbohidratos/farmacología , Animales , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Antiinflamatorios/química , Antiinflamatorios/aislamiento & purificación , Antiinflamatorios/farmacología , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Anticoagulantes/farmacología , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Carbohidratos/química , Carbohidratos/aislamiento & purificación , Glicosilación , Estructura Molecular , Océanos y Mares , Relación Estructura-Actividad

RESUMEN

A novel fucosylated chondroitin sulfate (HmG) was isolated from sea cucumber Holothuria mexicana, the structure of which was characterized by monosaccharide composition, disaccharide composition, IR, 1H and 13C NMR spectrum, additionally with two dimensional NMR spectrum of degraded HmG (DHmG). The backbone of HmG was identified as chondroitin 6-O sulfate, while the major O-4 sulfated fucose branches linked to O-3 position of glucuronic acid in almost every disaccharide unit. The anticoagulant activities of HmG and DHmG were assessed and compared with heparin and low molecular weight heparin. The results indicated that HmG and DHmG both could significantly prolong the activated partial thrombo-plastin time, and the properties were well related to its molecular weight. DHmG showed similar anticoagulant properties to low molecular weight heparin with less bleeding risks, making it a safer anticoagulant drug.

Asunto(s)

Anticoagulantes/química , Anticoagulantes/farmacología , Sulfatos de Condroitina/química , Sulfatos de Condroitina/farmacología , Holothuria/química , Animales , Anticoagulantes/aislamiento & purificación , Sulfatos de Condroitina/aislamiento & purificación , Peso Molecular , Monosacáridos/análisis , Relación Estructura-Actividad

RESUMEN

Chondroitin sulfate (CS) is a glycosaminoglycan (GAG) composed of alternating N-acetyl galactosamine and glucuronic acid units within disaccharide building blocks. CS is a key functional component in proteoglycans of cartilaginous tissues. Owing to its numerous biological roles, CS is widely explored in the pharmaceutical market as nutraceutical ingredient commonly utilized against arthritis, osteoarthrosis, and sometimes osteoporosis. Tissues like shark cartilage and bovine trachea are common sources of CS. Nonetheless, a new CS type has been introduced and investigated in the last few decades in what regards its medical potentials. It is named fucosylated chondroitin sulfate (FucCS). This less common CS type is isolated exclusively from the body wall of sea cucumbers. The presence of fucosyl branching units in the holothurian FucCS gives to this unique GAG, therapeutic properties in various pathophysiological systems which are inexistent in the common CS explored in the market. Examples of these systems are coagulation, thrombosis, hemodialysis, atherosclerosis, cellular growth, angiogenesis, fibrosis, tumor growth, inflammation, viral and protozoan infections, hyperglycemia, diabetes-related pathological events and tissue damage. This report aims at describing the medical benefits gained upon fucosylation of CS. Clinical prospects of these medical benefits are also discussed herein.

Asunto(s)

Sulfatos de Condroitina/química , Animales , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Anticoagulantes/uso terapéutico , Aterosclerosis/tratamiento farmacológico , Coagulación Sanguínea/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Sulfatos de Condroitina/aislamiento & purificación , Sulfatos de Condroitina/farmacología , Sulfatos de Condroitina/uso terapéutico , Glicosilación , Humanos , Hiperglucemia/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Infecciones por Protozoos/tratamiento farmacológico , Pepinos de Mar/metabolismo , Trombosis/tratamiento farmacológico , Virosis/tratamiento farmacológico

RESUMEN

Throughout evolution, parasites have adapted in order to successfully intervene in the host defense, producing specific peptides and proteins. Interestingly, these peptides and proteins have been exploited as potential drug candidates against several diseases. Furthermore, biotechnology studies and cDNA libraries have remarkably contributed to identify potentially bioactive molecules. In this regard, herein, a cDNA library of salivary complexes from Haementeria vizottoi leeches was constructed, the transcriptome was characterized and a phylogenetic analysis was performed considering antistasin-like and antiplatelet-like proteins. Hundred twenty three transcripts were identified coding for putative proteins involved in animal feeding (representing about 10% of the expression level). These sequences showed similarities with myohemerythrins, carbonic anhydrases, anticoagulants, antimicrobials, proteases and protease inhibitors. The phylogenetic analysis, regarding antistasin-like and antiplatetlet-like proteins, revealed two main clades in the Rhynchobdellida leeches. As expected, the sequences from H. vizottoi have presented high similarities with those types of proteins. Thus, our findings could be helpful not only to identify new coagulation inhibitors, but also to better understand the biological composition of the salivary complexes.

Asunto(s)

Anticoagulantes/química , Sanguijuelas/química , Inhibidores de Agregación Plaquetaria/química , Glándulas Salivales/química , Animales , Anticoagulantes/aislamiento & purificación , Biología Computacional , Conducta Alimentaria , Perfilación de la Expresión Génica , Biblioteca de Genes , Hemostasis , Sanguijuelas/genética , Sanguijuelas/fisiología , Modelos Moleculares , Filogenia , Inhibidores de Agregación Plaquetaria/aislamiento & purificación , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de Proteína

RESUMEN

Polysaccharides were extracted from the barks of Geoffroea spinosa, purified using anion exchange chromatography and characterized by chemical and methylation analysis, complemented by infrared and NMR spectroscopies. These polysaccharides were tested for their anticoagulant, antithrombotic and antiplatelet activities and also for their effects on bleeding. Unfractionated polysaccharide contains low levels of protein and high levels of carbohydrate (including hexuronic acid). The purified polysaccharides (fractions FII and FIII) are composed of arabinose (Ara), rhamnose (Rha), hexuronic acid, small amounts of galactose, but no sulfate ester. They have highly complex structure, which was partially characterized. NMR and methylation analysis indicate that the polysaccharides have a core of α-Rhap and branches of 5-linked α-Araf. Residues of 4-linked α-GalpA are also found in the structure. The unfractionated (TPL) and fraction FIII, but not fractions FI and FII, prolonged the activated partial thromboplastin time (aPTT). TPL, FII and FIII inhibited the platelet aggregation induced by ADP. More significantly, both unfractionated and purified fractions exhibited potent antithrombotic effect (31-60%) and the fractions did not modify the bleeding tendency. These plant polysaccharides could be alternative source of new anticoagulant, antiplatelet and antithrombotic compounds devoid of the undesirable risk of hemorrhage.

Asunto(s)

Anticoagulantes/química , Fabaceae/química , Fibrinolíticos/química , Polisacáridos/química , Animales , Anticoagulantes/aislamiento & purificación , Anticoagulantes/farmacología , Plaquetas/efectos de los fármacos , Modelos Animales de Enfermedad , Fabaceae/metabolismo , Fibrinolíticos/aislamiento & purificación , Fibrinolíticos/uso terapéutico , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Monosacáridos/química , Monosacáridos/aislamiento & purificación , Monosacáridos/farmacología , Tiempo de Tromboplastina Parcial , Corteza de la Planta/química , Corteza de la Planta/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Polisacáridos/farmacología , Polisacáridos/uso terapéutico , Ratas , Ratas Wistar , Espectroscopía Infrarroja por Transformada de Fourier , Trombosis de la Vena/tratamiento farmacológico

RESUMEN

Sulfated fucans, the complex polysaccharides, exhibit various biological activities. Herein, we purified two fucans from the sea cucumbers Holothuria edulis and Ludwigothurea grisea. Their structures were verified by means of HPGPC, FT-IR, GC-MS and NMR. As a result, a novel structural motif for this type of polymers is reported. The fucans have a unique structure composed of a central core of regular (1→2) and (1→3)-linked tetrasaccharide repeating units. Approximately 50% of the units from L. grisea (100% for H. edulis fucan) contain sides of oligosaccharides formed by nonsulfated fucose units linked to the O-4 position of the central core. Anticoagulant activity assays indicate that the sea cucumber fucans strongly inhibit human blood clotting through the intrinsic pathways of the coagulation cascade. Moreover, the mechanism of anticoagulant action of the fucans is selective inhibition of thrombin activity by heparin cofactor II. The distinctive tetrasaccharide repeating units contribute to the anticoagulant action. Additionally, unlike the fucans from marine alga, although the sea cucumber fucans have great molecular weights and affluent sulfates, they do not induce platelet aggregation. Overall, our results may be helpful in understanding the structure-function relationships of the well-defined polysaccharides from invertebrate as new types of safer anticoagulants.

Asunto(s)

Anticoagulantes/aislamiento & purificación , Descubrimiento de Drogas , Polisacáridos/aislamiento & purificación , Pepinos de Mar/química , Animales , Anticoagulantes/química , Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Brasil , Secuencia de Carbohidratos , Fenómenos Químicos , China , Cofactor II de Heparina/antagonistas & inhibidores , Cofactor II de Heparina/metabolismo , Holothuria/química , Humanos , Cinética , Peso Molecular , Polisacáridos/química , Polisacáridos/farmacología , Pepinos de Mar/crecimiento & desarrollo , Especificidad de la Especie , Relación Estructura-Actividad

RESUMEN

The sulfated polysaccharides (SP) from the edible red seaweed, Gracilaria birdiae, were obtained using five different extraction conditions: Gracilaria birdiae 1 (GB1)-water; GB1s-water/sonication; GB1sp-water/sonication/proteolysis; GB2s-NaOH/sonication; and GB2sp-NaOH/sonication/proteolysis. The yield (g) increased in the following order: GB2sp>GB1sp>GB2s>GB1s>GB1. However, the amount of SP extracted increased in a different way: GB2sp>GB1>GB1sp>GB1s>GB2s. Infrared and electrophoresis analysis showed that all conditions extracted the same SP. In addition, monosaccharide composition showed that ultrasound promotes the extraction of polysaccharides other than SP. In the prothrombin time (PT) test, which evaluates the extrinsic coagulation pathway, none of the samples showed anticoagulant activity. While in the activated partial thromboplastin time (aPTT) test, which evaluates the intrinsic coagulation pathway, all samples showed anticoagulant activity, except GB2s. The aPTT activity decreased in the order of GB1sp>GB2sp>GB1>GB1s>GB2s. The total capacity antioxidant (TCA) of the SP was also affected by extraction condition, since GB2s and GB1 showed lower activity in comparison to the other conditions. In conclusion, the conditions of SP extraction influence their biological activities and chemical composition. The data revealed that NaOH/sonication/proteolysis was the best condition to extract anticoagulant and antioxidant SPs from Gracilaria birdiae.

Asunto(s)

Anticoagulantes , Antioxidantes , Gracilaria/química , Plantas Comestibles/química , Polisacáridos , Proteolisis , Hidróxido de Sodio/química , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Anticoagulantes/farmacología , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Antioxidantes/farmacología , Humanos , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Polisacáridos/farmacología , Sonido

RESUMEN

Snakebites are a serious public health problem due their high morbi-mortality. The main available specific treatment is the antivenom serum therapy, which has some disadvantages, such as poor neutralization of local effects, risk of immunological reactions, high cost and difficult access in some regions. In this context, the search for alternative therapies is relevant. Therefore, the aim of this study was to evaluate the antiophidic properties of Jatropha gossypiifolia, a medicinal plant used in folk medicine to treat snakebites. The aqueous leaf extract of the plant was prepared by decoction and phytochemical analysis revealed the presence of sugars, alkaloids, flavonoids, tannins, terpenes and/or steroids and proteins. The extract was able to inhibit enzymatic and biologic activities induced by Bothrops jararaca snake venom in vitro and in vivo. The blood incoagulability was efficiently inhibited by the extract by oral route. The hemorrhagic and edematogenic local effects were also inhibited, the former by up to 56% and the latter by 100%, in animals treated with extract by oral and intraperitoneal routes, respectively. The inhibition of myotoxic action of B. jararaca reached almost 100%. According to enzymatic tests performed, it is possible to suggest that the antiophidic activity may be due an inhibitory action upon snake venom metalloproteinases (SVMPs) and/or serine proteinases (SVSPs), including fibrinogenolytic enzymes, clotting factors activators and thrombin like enzymes (SVTLEs), as well upon catalytically inactive phospholipases A2 (Lys49 PLA2). Anti-inflammatory activity, at least partially, could also be related to the inhibition of local effects. Additionally, protein precipitating and antioxidant activities may also be important features contributing to the activity presented. In conclusion, the results demonstrate the potential antiophidic activity of J. gossypiifolia extract, including its significant action upon local effects, suggesting that it may be used as a new source of bioactive molecules against bothropic venom.

Asunto(s)

Antídotos/farmacología , Bothrops , Venenos de Crotálidos/antagonistas & inhibidores , Venenos de Crotálidos/farmacología , Euphorbiaceae/química , Extractos Vegetales/farmacología , Adulto , Animales , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Anticoagulantes/farmacología , Antídotos/química , Antídotos/aislamiento & purificación , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Antioxidantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Bothrops/metabolismo , Células HEK293 , Humanos , Ratones , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Hojas de la Planta/química

RESUMEN

Cardiovascular diseases (CDs) are the principal cause of death in the world. Anticoagulation is the commonest therapeutic strategy for treatments of CDs in clinical settings. Although possessed of numerous downsides, heparin is the main clinical anticoagulant/antithrombotic agent used so far. Novel sulfated polysaccharides like the marine dermatan sulfate, sulfated fucans and galactans are also able to block clot and thrombus formation. These relatively new marine glycans call special attention mostly due to their unique structures and distinct mechanisms of action. This structural uniqueness is seen by the peculiar aspect of these polysaccharides being made of clear and regular sulfation patterns. The structures have been reported only in polysaccharides from marine invertebrates like sea urchins and cucumbers. This report intends to prove the promising combination of the triad sea-carbohydrates-clotting in drug discovery of the cardiovascular field.

Asunto(s)

Anticoagulantes/uso terapéutico , Enfermedades Cardiovasculares/tratamiento farmacológico , Dermatán Sulfato/uso terapéutico , Fibrinolíticos/uso terapéutico , Galactanos/uso terapéutico , Polisacáridos/uso terapéutico , Animales , Anticoagulantes/química , Anticoagulantes/aislamiento & purificación , Coagulación Sanguínea/efectos de los fármacos , Enfermedades Cardiovasculares/complicaciones , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Dermatán Sulfato/química , Dermatán Sulfato/aislamiento & purificación , Descubrimiento de Drogas , Fibrinolíticos/química , Fibrinolíticos/aislamiento & purificación , Galactanos/química , Galactanos/aislamiento & purificación , Heparina/química , Heparina/uso terapéutico , Humanos , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Pepinos de Mar/química , Erizos de Mar/química