Bioactive Molecules from the Exoskeleton of Procambarus clarkii: Reducing Capacity, Radical Scavenger, and Antitumor and Anti-Inflammatory Activities

Abstract

This study evaluates, for the first time, the reducing capacity, radical scavenger activity, and in vitro antitumor and anti-inflammatory effects of chitosan, astaxanthin, and bio-phenols extracted from the exoskeleton of Sicilian Procambarus clarkii, the most widespread species of invasive crayfish in the Mediterranean region. Among the extracted compounds, astaxanthin exhibited the highest antioxidant activity in all assays. Chitosan and polyphenols demonstrated reducing and radical scavenging activity; chitosan showed significant ferric ion reducing capacity in the FRAP test, while bio-phenolic compounds displayed notable radical scavenging activity in the DPPH and ABTS assays. Both astaxanthin and polyphenols showed dose-dependent cytotoxicity on two different cancer cell lines, with IC50 values of 1.45 µg/mL (phenolic extract) and 4.28 µg/mL (astaxanthin extract) for HepG2 cells and 2.45 µg/mL (phenolic extract) and 4.57 µg/mL (astaxanthin extract) for CaCo-2 cells. The bio-phenolic extract also showed potential anti-inflammatory effects in vitro by inhibiting nitric oxide production in inflamed RAW 264.7 macrophages, reducing the treated/control NO ratio to 77% and 74% at concentrations of 1.25 and 1.5 μg/mL, respectively. These results suggest that P. clarkii exoskeletons could be a valuable source of bioactive molecules for biomedical, pharmaceutical, and nutraceutical application while contributing to the sustainable management of this invasive species.

Keywords: antioxidant; astaxanthin; chitosan; crustacean; freshwater; invertebrates; phenolic compounds.

Figure 1

IR spectrum of chitosan extracted from P. clarkii.

Figure 3

Dose–response effect of phenolic extract from P. clarkii exoskeleton on the viability of HepG2 and CaCo-2 cells after 24 h of exposure. The error bars correspond to the standard error of the mean (s.e.m.) of three independent measurements. p values comparing viability ratios to controls were <0.05 for every measurement.

Figure 4

Dose–response effect of 24 h and 7 days of extraction of astaxanthin extract from P. clarkii exoskeleton on the viability of HepG2 (A) and CaCo-2 cells (B) after 24 h of exposure. The error bars correspond to the standard error of the mean (s.e.m.) of three independent measurements. p values comparing viability ratios to controls were <0.05 for every measurement.

Figure 5

Effect of astaxanthin (A) and polyphenol extract (B) from P. clarkii exoskeleton on the viability of RAW 264.7 macrophages after 24 h of exposure. The error bars correspond to the standard error of the mean (s.e.m.) of three independent measurements. * p < 0.05.

Figure 6

Effect of astaxanthin (A) and polyphenol extract (B) from P. clarkii exoskeleton on NO production by RAW 264.7 macrophages after 24 h of co-exposure with LPS compared with LPS alone. The error bars correspond to the standard error of the mean (s.e.m.) of three independent measurements. * p < 0.05.

Figure 2

HPLC/MS chromatogram and MS/MS spectrum of the peak 13.6 min from the ethanolic P. clarkii extract.