Abstract
Secretions derived from amphibian skin glands serve as a potential reservoir of various valuable active molecules. Currently, the multiple substances with diverse therapeutic activities among the components of glandular secretions of different species of amphibians have been found. It has been proven that they have antibacterial, antifungal, antiprotozoal, antidiabetic, antineoplastic, analgesic, and sleep-inducing properties. Taking this into consideration, to get the basic knowledge about the properties of the components of skin secretions of some Anura species that inhabit the territory of Ukraine is crucial for further investigation of the most potential ones. The red blood cell hemolysis assay is a prevalent test to study the cytotoxicity of studied samples. The aim of the present study was to analyze the hemolytic activity of skin secretions of Bombina bombina, Bombina variegata, Bufotes viridis, Rana temporaria, Pelophylax ridibundus, and Pelobates fuscus, and to obtain the primary data on the possible mechanism of their toxicological action on the blood cells membranes. The skin secretions of six amphibian species mentioned above were incubated with erythrocyte suspension in different concentrations. Eminently active B.variegata skin secretions, having the HD HD50 value at 0.5 µg/ml, were taken for the subsequent researches, where the effects of osmotic protectants, divalent cations, antioxidants, chelating agent, and serine protease inhibitor on the cell lysis ability of B. variegata skin secretions was studied. All studied cations inhibited the hemolytic activity of B. variegata secretions in a dose-depend manner. While the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), markedly decreased the hemolytic activity of studied skin secretions. We can assume that the bioactive peptides in these skin secretions have an enzymatic mechanism of action.
Highlights
The Amphibians skin provides a rich source of bioactive molecules that have some effective therapeutic potencies, such as: antibacterial, antifungal, antiprotozoal, antidiabetic, antineoplastic, analgesic and sleep-inducing properties [1]
The most active B. variegata skin secretion, which had HD50 value 0.5 μg/ml, was used for further research to establish basic information on its toxicological properties. For this purpose we investigated the effects of different factors on the skin secretion hemolytic activity, including osmotic protectants – 25 mM D-glucose and 25 mM D-lactose; divalent cations, such as Mn2+, Mg2+, Ca2+, Zn2+ in concentration range from 1 to 100 mM, Fe2+ in concentration range from 10 to 50 μM, and Cu2+ in concentration range from 1 to 100 μM; antioxidants – 2 mM ascorbic acid and 2 mM cysteine; chelating agent – 2 mM EDTA; serine protease inhibitor – 2 mM phenylmethylsulfonyl fluoride (PMSF)
Granular glands of amphibian skin produce a large variety of bioactive substances, including antimicrobial peptides [8], neurotoxic peptides [9], gastric disturbance peptides [10], and alkaloids [11]
Summary
The Amphibians skin provides a rich source of bioactive molecules (peptides, proteins, steroids, alkaloids, opiods) that have some effective therapeutic potencies, such as: antibacterial, antifungal, antiprotozoal, antidiabetic, antineoplastic, analgesic and sleep-inducing properties [1]. The composition and the mechanism of action of biologically active compounds from amphibian skin secretions are not fully investigated by this time. Toxicity of an active molecule is a key factor during drug design, and the hemolytic activity represents a useful starting point in this regard. It provides the primary information on the interaction between molecules and biological entities at cellular level. The ESRD burden on health care is caused largely due to the lack of an effective anti-fibrotic agents that can target CKD. Little success has been made over the past decade in developing agents or therapies that can prevent renal fibrosis to slow the progression of CKD to ESRD [23].
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