Isolamento, fracionamento e atividade anticoagulante da galactana sulfatada extraída da alga verde Penicillus capitatus

Moreira, Thamyris Almeida; Costa, Bianca Barros da; Celestino, Regina Célia Alves; Faria, Caroline Nogueira; Gianelli, Jéssica Lopes D’Dego; Santos, Gustavo Ramalho Cardoso dos; Glauser, Bianca Fernandes; Soares, Angélica Ribeiro; Mourão, Paulo Antônio de Souza; Rodrigues, Carlos Rangel; Cinelli, Leonardo Paes

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CLINIC AND SURGERY • Cienc. Rural 51 (8) • 2021 • https://doi.org/10.1590/0103-8478cr20200138 copiar

Isolamento, fracionamento e atividade anticoagulante da galactana sulfatada extraída da alga verde Penicillus capitatus

Autoria SCIMAGO INSTITUTIONS RANKINGS

RESUMO:

As algas marinhas são fontes naturais de macromoléculas conhecidas como polissacarídeos sulfatados. Esta classe de compostos atraiu o interesse das Ciências Farmacêuticas devido às suas propriedades farmacológicas como anticoagulante, antiplaquetária e antitrombótica. Portanto, este estudo tem como objetivo avaliar o potencial anticoagulante de polissacarídeos sulfatados extraídos de algas de Penicillus capitatus. Os polissacarídeos sulfatados extraídos foram purificados, parcialmente caracterizados e sua atividade anticoagulante foi avaliada. O processo de extração seguido pela precipitação com etanol resultou em cinco frações. Entre as frações analisadas, F44 foi a maior concentração de polissacarídeos sulfatados. Após a purificação, as frações F23 e F44 mostraram atividade anticoagulante in vitro em um teste de tempo de tromboplastina parcialmente ativada e tempo de protrombina. Seu mecanismo preferencial é baseado nas interações entre trombina e fator Xa. Estudos adicionais sobre a estrutura farmacológica são necessários para testar a viabilidade do uso como agente terapêutico.

Palavras-chave:
polissacarídeo sulfatado; algas marinhas; atividade anticoagulante; Penicillus capitatus

Autoria

Thamyris Almeida Moreira

Grupo de Glicofármacos, Universidade Federal do Rio de Janeiro (UFRJ), 27930-560, Macaé, RJ, Brasil..Universidade Federal do Rio de Janeiro (UFRJ)BrazilMacaé, RJ, BrazilGrupo de Glicofármacos, Universidade Federal do Rio de Janeiro (UFRJ), 27930-560, Macaé, RJ, Brasil..

http://orcid.org/0000-0003-3718-7131

Bianca Barros da Costa

http://orcid.org/0000-0001-8962-2639

Regina Célia Alves Celestino

http://orcid.org/0000-0002-1673-053X

Caroline Nogueira Faria

Jéssica Lopes D’Dego Gianelli

http://orcid.org/0000-0003-4534-9399

Gustavo Ramalho Cardoso dos Santos

Laboratório de Tecido Conjuntivo, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro (UFRJ)BrazilRio de Janeiro, RJ, BrazilLaboratório de Tecido Conjuntivo, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brasil.

Bianca Fernandes Glauser

Angélica Ribeiro Soares

Núcleo em Ecologia e Desenvolvimento Sócioambiental de Macaé (NUPEM), Universidade Federal do Rio de Janeiro (UFRJ), Macaé, RJ, Brasil.Universidade Federal do Rio de Janeiro (UFRJ)BrazilMacaé, RJ, BrazilNúcleo em Ecologia e Desenvolvimento Sócioambiental de Macaé (NUPEM), Universidade Federal do Rio de Janeiro (UFRJ), Macaé, RJ, Brasil.

Paulo Antônio de Souza Mourão

Carlos Rangel Rodrigues

Laboratório ModMolQSAR, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro (UFRJ)BrazilRio de Janeiro, RJ, BrazilLaboratório ModMolQSAR, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brasil.

Leonardo Paes Cinelli ^*E-mail: cinelli@pharma.ufrj.br

E-mail: cinelli@pharma.ufrj.br

DECLARATION OF CONFLICT OF INTERESTS

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

SCIMAGO INSTITUTIONS RANKINGS

Figuras | Tabelas

Figuras (7)
Tabelas (1)

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Figure 1
Extraction process and puriﬁcation of sulfated polyssacharide from the algae Penicillus capitatus.

Thumbnail

Figure 2
SP obtained from sequential increasing ethanol concentrations from P.capitatus were submitted to electrophoresis and the aPTT assay. In (A) precipitations were applied on a 0.5% agarose gel and stained with 0.1% toluidine blue in acetic acid:ethanol:water (0.1:1:5, v/v). (B) The in vitro aPTT clotting assay was performed using normal human plasma at different SP concentrations; PCA F9 (►), PCA F23 (○), PCA F44 (Δ), PCA F60 ( ), PCA F75 (□) and unfractionated heparin standard (UFH) (■).

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Figure 3
Puriﬁcation (A - C) and analysis (B - D) of SP extracted from P. capitatus. In (A) PCA F23 and (C) PCA F44 was applied to a DEAE-cellulose column. The column was run applying a linear gradient of 0  4.0 M NaCl. The fractions were collected from the column and assayed by metachromasia (■) and NaCl concentrations (- - -). PCA F23 P1 and PCA F44 P1 were pooled, dialyzed against distilled water and freeze-dried. (B) Two SP fractions P, including PCA F23 P1 and (D) PCA F44 P1 obtained from the DEAE-cellulose column, were applied on a 0.5% agarose gel and stained with 0.1% toluidine blue in acetic acid:ethanol:water (0.1:1:5, v/v). Legend: unfractionated heparin (UFH); chondroitim sulfate (CS); standard (Std).

Thumbnail

Figure 4
Anticoagulant activity of PCA F23 P1 (A, C) and PCA F44 P1 (B, D) based on the aPTT and PT assays performed with PCA F23 P1 (●), PCA F23 (○), PCA F44 P1 (▲), PCA F44 (Δ) and an unfractionated heparin standard (UFH) (■). A mixture of 100 μL of normal human plasma and increasing concentrations of the SP were incubated with 100 μL of aPTT and PT reagents. After 2 min of incubation at 37 °C, 100 μL of 25 mM CaCl₂ were added to the mixtures and clotting times were recorded.

Thumbnail

Figure 5
Effect of SP on the inactivation of thrombin or factor Xa mediated by antithrombin. Thrombin (0.5 nM) or factor Xa (0.5 nM) inactivation by antithrombin (5 nM) was monitored at increasing heparin concentrations (■), PCA F23 P1 (●) or PCA F44 P1 (▲) in TS/PEG buffer.

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Figure 6
Infrared spectrum (4000 to 200 cm^-1) of the PCA F23 P1 and PCA F44 P1 fractions extracted from the algae P. capitatus. T (%): transmittance; Cm^-1: wave-length.

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Figure 7
Structural analysis of SP F44 P1 extracted from P. capitatus. Monosaccharide composition was evaluated based on the GC-MS analysis of alditol-acetate, identified by its retention time. Gal: Galactose.

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Table 1
Chemical analysis of SP fractions from P. capitatus seaweed.

Figure 1 Extraction process and puriﬁcation of sulfated polyssacharide from the algae Penicillus capitatus.

Figure 2 SP obtained from sequential increasing ethanol concentrations from P.capitatus were submitted to electrophoresis and the aPTT assay. In (A) precipitations were applied on a 0.5% agarose gel and stained with 0.1% toluidine blue in acetic acid:ethanol:water (0.1:1:5, v/v). (B) The in vitro aPTT clotting assay was performed using normal human plasma at different SP concentrations; PCA F9 (►), PCA F23 (○), PCA F44 (Δ), PCA F60 ( ), PCA F75 (□) and unfractionated heparin standard (UFH) (■).

Figure 3 Puriﬁcation (A - C) and analysis (B - D) of SP extracted from P. capitatus. In (A) PCA F23 and (C) PCA F44 was applied to a DEAE-cellulose column. The column was run applying a linear gradient of 0  4.0 M NaCl. The fractions were collected from the column and assayed by metachromasia (■) and NaCl concentrations (- - -). PCA F23 P1 and PCA F44 P1 were pooled, dialyzed against distilled water and freeze-dried. (B) Two SP fractions P, including PCA F23 P1 and (D) PCA F44 P1 obtained from the DEAE-cellulose column, were applied on a 0.5% agarose gel and stained with 0.1% toluidine blue in acetic acid:ethanol:water (0.1:1:5, v/v). Legend: unfractionated heparin (UFH); chondroitim sulfate (CS); standard (Std).

Figure 4 Anticoagulant activity of PCA F23 P1 (A, C) and PCA F44 P1 (B, D) based on the aPTT and PT assays performed with PCA F23 P1 (●), PCA F23 (○), PCA F44 P1 (▲), PCA F44 (Δ) and an unfractionated heparin standard (UFH) (■). A mixture of 100 μL of normal human plasma and increasing concentrations of the SP were incubated with 100 μL of aPTT and PT reagents. After 2 min of incubation at 37 °C, 100 μL of 25 mM CaCl₂ were added to the mixtures and clotting times were recorded.

Figure 5 Effect of SP on the inactivation of thrombin or factor Xa mediated by antithrombin. Thrombin (0.5 nM) or factor Xa (0.5 nM) inactivation by antithrombin (5 nM) was monitored at increasing heparin concentrations (■), PCA F23 P1 (●) or PCA F44 P1 (▲) in TS/PEG buffer.

Figure 6 Infrared spectrum (4000 to 200 cm^-1) of the PCA F23 P1 and PCA F44 P1 fractions extracted from the algae P. capitatus. T (%): transmittance; Cm^-1: wave-length.

Figure 7 Structural analysis of SP F44 P1 extracted from P. capitatus. Monosaccharide composition was evaluated based on the GC-MS analysis of alditol-acetate, identified by its retention time. Gal: Galactose.

Table 1 Chemical analysis of SP fractions from P. capitatus seaweed.

PCA fraction	Yield a (%)	Carbohydrates b (%)	Protein c (%)	Uronic acid d (%)	SP (%) e	Monossacharide f
F9	1.1	53.8	0.34	18.4	0.15	-
F23	3.4	60.3	0.04	9.2	1.51	-
F44	1.8	25.1	0.1	15.6	2.22	Gal
F60	3.0	57.4	N.D.	24.9	1.99	-
F75	5.9	64	0.13	24.2	1.21	-

a Initial weight of the algae was of 22.5g.
b phenol/H₂SO₄.
c protein dosage.
d carbazol dosage.
e barium gelatin method.
f retention time on a gas chromatography/mass spectrometry.
not detected (ND).

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[1] E-mail: cinelli@pharma.ufrj.br