## 4 Discussion  
In the article, _F. limosus_ crayfish extract (FLE) was characterised for the first time in terms of its many bioactive ingredients (vitamins, carotenoids, glutathione, minerals, amino acids), and it was estimated that it had antioxidant properties comparable to \(\beta\)-carotene. Due to the preparation method (water extraction), FLE contained few fat-soluble ingredients. The main antioxidant substances were phenolic compounds, which probably came from algae consumed by crayfish. Phenols are a broad group of compounds of great importance as antioxidants from natural sources. They come primarily from plant sources, but they can accumulate in the tissues of crustaceans. The antioxidant activity of phenols is based on the transfer of a single electron and hydrogen atom [13]. What distinguishes crustaceans from most plant-based antioxidant sources is their high protein content. The antioxidant effect of the extract was undoubtedly influenced by the relatively high content of GSH. The content of GSH in the extract was 10.76 nmol/mg and was comparable to that of shrimp _Palaemontetes pugio_ embryo (10 embryo stage) [14] and ten times higher than in the water extract of Saposhnikova divarica root [10]. In addition, _F. limosus_ crayfish extract protein has an amino acid composition similar to fish. It is characterised by, among others, relatively high histidine content. Compared to bivalve molluses, _F. limosus_ extract contained more histidine than _A. broughttouiti_ and _S. sachalinensis_, but less than _C. japonica_ tissues. This amino acid is an essential amino acid (EAA) for mammals, poultry and fish. Due to the low supply in the diet, deficiencies are often encountered. Histidine deficiency is associated with impaired antioxidant protection of the body and affects many life processes, e.g., in rats, histidine supplementation provided neuroprotection at an early stage; in fish, it protected against glaucoma; and in humans, it helped to reduce the effects of metabolic syndrome. Moreover, histidine deficiency limited animal growth and milk synthesis in ruminants [15]. The antioxidant properties of FLE were at the level of \(\beta\)-carotene but were significantly lower than those of gallic acid. The lyophiliste from fermented shrimp waste inhibited 40% of the DPPH radical at a concentration of 1 mg/mL; in the reported study, the extract from _F. limosus_ showed similar activity at a 10 times lower concentration [16]. However, many plant extracts have a stronger ability to inhibit the DPPH radical. For example, [PERSON] et al. (2012) studied extracts of popular Greek plants, of which _Eucalyptus globulus_, _Sideritis cretica_, _Origanum vulgare_, _Phlomis cretica_, _Phlomis lanta_, _Nepeta melissifolia_ and _Mentha pulgeium_ had  
Figure 5: The expression of caspase-3, an early/moderate apoptosis marker, in HFFF-2 fibroblasts induced by hydrogen peroxide is shown as a percentage of control values (PBS only). H\({}_{2}\)O\({}_{2}\) only—cells treated with H\({}_{2}\)O\({}_{2}\) without the use of an extract; FLE + H\({}_{2}\)O\({}_{2}\)—cells incubated with 100 \(\mu\)g/mL _F. limosus_ extract and 500 \(\mu\)M hydrogen peroxide. Different letters mean that the values for the bars differ statistically (\(p<0.05\)). higher antiradical activity than FLE [17]. Research on natural products must focus on the various mechanisms that may affect the protective effect against oxidative stress. One of them is probably the action of glutathione, contained in the _F. limosus_ extract. The biological functions of GSH in cells are related to the activity of the hydrosulphide group, thanks to which it participates in antioxidant and detoxification reactions. Numerous studies have demonstrated the function of endogenous glutathione in protective mechanisms against oxidative stress in the body. It has also been shown that GSH supplementation could reduce oxidative stress and inflammation markers in healthy volunteers [18] and HIV-infected patients [19].