Abstract

Marine glycosaminoglycans (GAG) isolated from different invertebrates, such as molluscs, starfish or jellyfish, have been described as unique molecules with important pharmacological applications. Scarce information is available on GAG extract from Rapana venosa marine snail. The aim of this study was to isolate a GAG extract from R. venosa marine snail and to investigate its physicochemical, antioxidant and antiproliferative properties for further biomedical use. The morphology, chemical and elemental composition of the extract were established as well as the sulfate content and N- to O-sulfation ratio. Fourier transform infrared (FTIR) spectra indicated that GAG extract presented similar structural characteristics to bovine heparan sulfate and chondroitin sulfate. The pattern of extract migration in agarose gel electrophoresis and specific digestion with chondroitinase ABC and heparinase III indicated the presence of a mixture of chondroitin sulfate-type GAG, as main component, and heparan sulfate-type GAG. Free radical scavenging and ferric ion reducing assays showed that GAG extract had high antioxidant activity, which slightly decreased after enzymatic treatment. In vitro MTT and Live/Dead assays showed that GAG extract had the ability to inhibit cell proliferation in human Hep-2 cell cultures, at cytocompatible concentrations in normal NCTC clone L929 fibroblasts. This capacity decreased after enzymatic digestion, in accordance to the antioxidant activity of the products. Tumoral cell migration was also inhibited by GAG extract and its digestion products. Overall, GAG extract from R. venosa marine snail exhibited antioxidant and antiproliferative activities, suggesting its potential use as novel bioactive compound for biomedical applications.

Abstract

Silver carp (H. molitrix) is one of the most popular species in fish farms around the world. In this paper, the bioactive properties of four protein hydrolysates from silver carp residues obtained with papain, flavourzyme, alcalase and combination of flavourzyme & alcalase treatment were analyzed. Physicochemical characterization showed that the alcalase extract had the highest extraction yield of 52.07%, presented 90.33% protein content and the highest degree of hydrolysis (76.23%). Gel electrophoresis pattern indicated that hydrolysate obtained with alcalase contains most peptides with a molecular weight below 15 kDa, while those present in the hydrolysate obtained with flavourzyme & alcalase had a molecular weight between 10-15 kDa. The effect of protein hydrolysates on DPPH free radicals inhibition varied between 47.37-50.14%, the highest value of antioxidant activity being recorded for hydrolysate obtained with papain. The hydrolysates presented antihypertensive potential determined as inhibition of angiotensin-converting enzyme, with the highest activity in the case of flavourzyme & alcalase extract. The fish protein hydrolysates were cytocompatible in a normal fibroblast culture, NCTC cell line showing cell viability over 80% for all variants. When cultivated in Hep-2 cancer cells at 10 mg/ml, the protein hydrolysates obtained by papain and flavourzyme & alcalase mixture decreased the cell viability, indication antitumoral potential. In conclusion, the fish protein hydrolysates demonstrated important bioactive properties, including antioxidant, antihypertensive and antiproliferative activity.

Abstract

Research background. Various protocols for enzymatic hydrolysis of fish by-products are increasingly tested to ensure value-added products with functional and biological properties important for food, cosmetic and medical applications. In addition, they attempt to minimize waste from industrial processing and environmental requirements. This study aims to establish an efficient protocol based on two-step enzymatic hydrolysis of freshwater fish skin and to evaluate the effect of resulting bioactive peptides on free radical scavenging, redox balance and regulation of fibroblast proliferation and migration. Experimental approach. Pepsin-soluble collagen extracted from silver carp (Hypophthalmichthys molitrix) skin was hydrolyzed by proteinase K at specific sites under controlled conditions. The molecular mass of ultrafiltration permeate was determined by gradient electrophoresis and gel filtration chromatography. The biological activity of intermediate and small size bioactive peptides was evaluated in experimental models in vitro mimicking oxidative stress and skin wound conditions. Results and conclusions. Extracted fish collagen was hydrolysed using proteinase K, the most efficient enzyme for the cleavage of the primary structure of the molecule, as previously found in silico. Established optimal conditions increased the enzyme specificity and the process yield. Bioactive peptides exerted significantly higher scavenging activity on free stable radicals and hydroxyl radicals often found in vivo, compared to fish collagen. They stimulated fibroblast metabolism in a dose-dependent manner and up-regulated cell migration in a scratch wound model. Pretreatment of fibroblasts with induced oxidative stress using optimal concentrations of fish peptides prevented the increase of reactive oxygen species production. In conclusion, bioactive peptides from carp skin demonstrated valuable properties of maintaining redox balance and skin wound healing process improvement, which indicated further potential applications in the development of pharmaceutical and nutraceutical formulations. Novelty and scientific contribution. In this study the enzymatic hydrolysis was applied to isolated protein, in contrast to previous studies using waste tissue with variable composition. Recovered bioactive peptides acted not only as antioxidant agents, but also as regulators of oxidative stress and wound healing processes in skin cell models. Their nutritional and cosmetic application is recommended in novel formulations fighting skin ageing phenomena.

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