Caracterização estrutural e propriedades anti-inflamatória e antiviral de polissacarídeos sulfatados de algas marinhas do genêro Hypnea

Abstract

Marine seaweeds fave demonstrated to be a importante source of bioactive molecules. Algae from the genus Hypnea are known for the production carrageenan polysaccharides, a hidrocolloid used in pharmaceutical, cosmetic, food and biotechnological industries. Due to the morphological simplicity of these organisms, the identification of these organisms based on anatomic and morphological characters has led to mistakes. To overcome this problem, several studies have proposed the use of gene markers to aid in the identification of these organisms. In this work, specimens of the species Hypnea pseudomusciformis variety. “musciformis” “and “nigrescens” and Hypnea cervicornis, collected on the coast of Ceará, São Paulo and Bahia were studied. The identification of H. pseudomusciformis was made through morphologial characters. Though, for H. cervicornis a morphological analysis followed by the sequence of two gene markers (COI-5P and rbcL) were used. Later, sulfated polysaccharides were isolated from each group. The chemical structure of these polymers was characterized by elemental microanalysis, FT-IT, NMR and High Performance Size Exclusion Chromatography. The polysaccharide isolated from H. pseudomusciformis var. “musciformis” (HmP) showed a degree of substitution for sulfate groups (DS sulfate) of 0.88, while the variety nigrescens presented a DS sulfate = 1.05. The polysaccharide from H. cervicornis (HcP) had a DS sulfate of 0.79. The peak molar mass of HmP was 123.6 kDa. To HnP, the value observed was 71.4 kDa, while HcP peak molar mass was estimated in 70.2 kDa. FT-IR spectra of HmP, HnP and HcP revealed the presence of bands with similiar wavenumbers, characteristic of kappa-carrageenan. NMR spectra of HmP and HnP revealed that 7.3% of HmP and 9.2% of HnP were ι-carrageenan. NMR also demonstrated that 95% HcP had 95% of kappa-carrageenan and only 5% as ι-carrageenan. HcP was also evaluated regarding its effect on classic models of inflammation in mice. HcP (30 mg/kg; i.p.) reduced paw edema induced by carrageenin in 69% and the migration of inflammatory cells, as seen by mieloperoxidase (MPO) counting reduction of 53%. HcP also reduced the migration of neutrophils to the peritoneal cavity induced by carrageenin and fMLP. Though, HcP did not reduced the osmotic/vascular inflammatory response induced by histamine or 48/80 compound. Yet, HcP (30 mg/kg; i.p.) exerted antinociceptive effect in mice when inflammation was induced by formalin injection in the animal paw. The licking time was significantly reduced in the second phase, which is known to involve inflammatoy response. Though, no effect was detected in the first phase, regarded to be neurogenic. The analgesic effect of HcP was confirmed by a hyperssensitivity test (Von Frey assay). The polysaccharides isolated from H. pseudomusciformis were evaluated regarding their potential for neutralizing virus, especially HIV. In a first step it was verified the bond capacity of HnP and HmP against several viral proteins by ELISA. Both polysaccharides bound to te glycoproteins gp120/gp140 from HIV, S2-rGP, S2-GPe and ZEBOV rGP from Ebola virus, Dengue III, Chikungunya E1 and E2. Then, kinetics and affinity of HnP binding to the glyprotein gp140 from HIV was evaluated by Surface Plasmon Resonance in a gold CM-5 chip. The affinity constanto (KD =1,450 x 10-9) observed for HnP binding to gp120 was determined. Later, it was analysed HnP neutralizing effect against HIV cultivated in Tzmbl cells expressing luciferase. It was tested one virus of clade B in a live form called HIV Bal (IC50 = 73,51 μg/mL; IC90 = 661,59 μg/mL) and the four HIV pseudoviruses of clades B and C as follows: Bal pseudovirus (IC50 = 54,84 μg/mL; IC90 = 493,56 μg/mL), PCAAN S342 (IC50 = 36,67 μg/mL; IC90 = 330,03 μg/mL), SC422-661.8 (IC50 = 25,79 μg/mL; IC90 = 232,11 μg/mL) and ZM53M.PB12 SVPC11 (IC50 = 70,85 μg/mL; IC90 = 637,65 μg/mL). In addition, HnP and HmP effect on Rabies virus cultivated in BHK21 cells. HnP neutralized Rabies CVS11 pseudovirus with IC50 = 71,04 μg/mL and IC90 = 639,36 μg/mL. Though, HmP effect against Rabies CVS11 pseudovirus was significantly higher (IC50 = 10,99 μg/mL; IC90 = 98,91 μg/mL). It was also evaluated HnP and HmP cytotoxicity against the two cell lines used for viral cultivation by the Lactate dehydrogenase assay (LDH). No HnP toxicity was found against Tzmbl and BHK21 cells until 375 μg/mL when incubated for 2h and until 750 μg/mL when incubated for 9h. Similar result was found was found to Tzmbl and BHK21 cells incubated with HmP. Finally, it is possible to infer that HcP revealed and important anti-inflammatory potential and that HnP and HmP demonstrated a potent antiviral effect and could be used in the development of new therapeutic approaches.