Melotti L et al. (2023), A Second Life for Seafood Waste: Therapeutical ...

ECB-ART-52538

Antioxidants (Basel) 2023 Sep 07;129:. doi: 10.3390/antiox12091730.

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A Second Life for Seafood Waste: Therapeutical Promises of Polyhydroxynapthoquinones Extracted from Sea Urchin by-Products.

Melotti L , Venerando A , Zivelonghi G , Carolo A , Marzorati S , Martinelli G , Sugni M , Maccatrozzo L , Patruno M .

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Coping with a zero-waste, more sustainable economy represents the biggest challenge for food market nowadays. We have previously demonstrated that by applying smart multidisciplinary waste management strategies to purple sea urchin (Paracentrotus lividus) food waste, it is possible to obtain both a high biocompatible collagen to produce novel skin substitutes and potent antioxidant pigments, namely polyhydroxynapthoquinones (PHNQs). Herein, we have analyzed the biological activities of the PHNQs extract, composed of Spinochrome A and B, on human skin fibroblast cells to explore their future applicability in the treatment of non-healing skin wounds with the objective of overcoming the excessive oxidative stress that hinders wound tissue regeneration. Our results clearly demonstrate that the antioxidant activity of PHNQs is not restricted to their ability to scavenge reactive oxygen species; rather, it can be traced back to an upregulating effect on the expression of superoxide dismutase 1, one of the major components of the endogenous antioxidant enzymes defense system. In addition, the PHNQs extract, in combination with Antimycin A, displayed a synergistic pro-apoptotic effect, envisaging its possible employment against chemoresistance in cancer treatments. Overall, this study highlights the validity of a zero-waste approach in the seafood chain to obtain high-value products, which, in turn, may be exploited for different biomedical applications.

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	Figure 1. Cytotoxicity evaluation of Spinochrome A and B in human skin cells. (a) Chemical structure of the polyhydroxynapthoquinones (PHNQs) extracted from P. lividus food waste used in this study. The percentage of each Spinochrome composing the PHNQs extract is reported. Spinochrome A (2-Acetyl-3,5,6,8-tetrahydroxy-1,4-naphthoquinone), MW = 264.19 g mol−1; Spinochrome B (2,3,5,7-tetrahydroxy-1,4-naphthoquinone), MW = 222.15 g mol−1. (b) Normal human dermal fibroblasts (NHDF) viability after exposure to different concentration of Spinochrome A and B for 24 h was evaluated by MTT assay. Viability is expressed as % of untreated cells. Data are expressed as mean ± SEM of three independent experiments; *** p < 0.001 compared to untreated samples (one-way ANOVA using Bonferroni’s post-test). (c) Representative microphotographs of NHDF exposed to the indicated concentrations of PHNQs extract for 24 (upper panel) and 48 (lower panel) hours. No evident signs of toxicity are present for any of the concentrations tested apart from a limited formation of budding vesicles (indicated by arrows) only visible in the samples treated with 100 μg mL−1 PHNQs at both timepoints.
	Figure 2. Scavenging activity and cytoprotection of PHNQs against oxidative stress. (a) Kinetic evaluation of ROS levels in NHDF pre-treated with different concentrations of PHNQs (1–10 µg mL−1) for 24 h and then exposed to 5 µM AMA. In (b), the ROS levels at the endpoint of 120 min measured in (a) are reported as fold increase with respect to the untreated cells. (c) Cell viability of NHDF pre-treated with PHNQs (1–10 µg mL−1) and exposed to 5 µM AMA for 2 h was assessed using MTT assay. Viability is expressed as % of untreated cells. Means ± SEM of three independent experiments are reported; * p < 0.05, p < 0.01, * p < 0.001 versus AMA-treated samples (yellow column) using one-way ANOVA with Bonferroni’s post-test.
	Figure 3. PHNQs effect on AMA-induced mitochondrial membrane depolarization. NHDF were pre-incubated with PHNQs extract (1–10 µg mL−1) for 24 h and then challenged with 5 µM AMA for an additional 2 h. Mitochondrial membrane potential (ΔΨM values expressed as % of untreated cells) were obtained from the ratio of red fluorescence (aggregates) and green fluorescence (monomers) of the potential-dependent JC-1 fluorescent dye, as described in the Material and Methods section. Data are expressed as mean ± SEM of experiments performed in triplicate; p < 0.01, * p < 0.001 compared to AMA-treated samples (yellow column) using one-way ANOVA with Bonferroni’s post-test.
	Figure 4. PHNQs extract enhances the downregulation of Bcl-2 anti-apoptotic protein induced by Antimycin A treatment. NHDF were pre-treated for 24 h with PHNQs at different concentrations and then challenged with 5 μM AMA for 2 h. (a) Total cell lysate (30 µg proteins/sample) were separated by SDS-PAGE and immunoblotted with antibodies against B-cell lymphoma 2 (Bcl-2) protein and β-actin (loading control). Representative Western blots of three independent experiments are shown. In (b), the densitometric analysis of proteins expression reported as % of untreated cells is shown. Data are expressed as mean ± SEM; p < 0.01, * p < 0.001 compared to untreated cells (one-way ANOVA with Bonferroni’s post-test).
	Figure 5. PHNQs influence on key regulators enzymes of oxidative stress. (a) Representative Western blot analysis of different concentrations of PHNQs tested on NHDF for 24 h. Total cell lysate (30 µg proteins/sample) was separated by SDS-PAGE and immunoblotted with antibodies against the indicated antioxidant enzymes. β-actin was used as loading control. Densitometric analysis of (b) catalase (CAT), (c) glutathione synthetase (GSS), and (d) superoxide dismutase 1 (SOD1). Protein band intensities were normalized to β-actin and expressed as % of untreated cells. Values are reported as mean ± SEM of three independent experiments; * p < 0.05, p < 0.01, * p < 0.001 compared to untreated samples using one-way ANOVA with Bonferroni’s post-test.

References [+] :

Amir, Senescence and Longevity of Sea Urchins. 2020, Pubmed, Echinobase

	Figure 1. Cytotoxicity evaluation of Spinochrome A and B in human skin cells. (a) Chemical structure of the polyhydroxynapthoquinones (PHNQs) extracted from P. lividus food waste used in this study. The percentage of each Spinochrome composing the PHNQs extract is reported. Spinochrome A (2-Acetyl-3,5,6,8-tetrahydroxy-1,4-naphthoquinone), MW = 264.19 g mol−1; Spinochrome B (2,3,5,7-tetrahydroxy-1,4-naphthoquinone), MW = 222.15 g mol−1. (b) Normal human dermal fibroblasts (NHDF) viability after exposure to different concentration of Spinochrome A and B for 24 h was evaluated by MTT assay. Viability is expressed as % of untreated cells. Data are expressed as mean ± SEM of three independent experiments; *** p < 0.001 compared to untreated samples (one-way ANOVA using Bonferroni’s post-test). (c) Representative microphotographs of NHDF exposed to the indicated concentrations of PHNQs extract for 24 (upper panel) and 48 (lower panel) hours. No evident signs of toxicity are present for any of the concentrations tested apart from a limited formation of budding vesicles (indicated by arrows) only visible in the samples treated with 100 μg mL−1 PHNQs at both timepoints.
	Figure 2. Scavenging activity and cytoprotection of PHNQs against oxidative stress. (a) Kinetic evaluation of ROS levels in NHDF pre-treated with different concentrations of PHNQs (1–10 µg mL−1) for 24 h and then exposed to 5 µM AMA. In (b), the ROS levels at the endpoint of 120 min measured in (a) are reported as fold increase with respect to the untreated cells. (c) Cell viability of NHDF pre-treated with PHNQs (1–10 µg mL−1) and exposed to 5 µM AMA for 2 h was assessed using MTT assay. Viability is expressed as % of untreated cells. Means ± SEM of three independent experiments are reported; * p < 0.05, p < 0.01, * p < 0.001 versus AMA-treated samples (yellow column) using one-way ANOVA with Bonferroni’s post-test.
	Figure 3. PHNQs effect on AMA-induced mitochondrial membrane depolarization. NHDF were pre-incubated with PHNQs extract (1–10 µg mL−1) for 24 h and then challenged with 5 µM AMA for an additional 2 h. Mitochondrial membrane potential (ΔΨM values expressed as % of untreated cells) were obtained from the ratio of red fluorescence (aggregates) and green fluorescence (monomers) of the potential-dependent JC-1 fluorescent dye, as described in the Material and Methods section. Data are expressed as mean ± SEM of experiments performed in triplicate; p < 0.01, * p < 0.001 compared to AMA-treated samples (yellow column) using one-way ANOVA with Bonferroni’s post-test.
	Figure 4. PHNQs extract enhances the downregulation of Bcl-2 anti-apoptotic protein induced by Antimycin A treatment. NHDF were pre-treated for 24 h with PHNQs at different concentrations and then challenged with 5 μM AMA for 2 h. (a) Total cell lysate (30 µg proteins/sample) were separated by SDS-PAGE and immunoblotted with antibodies against B-cell lymphoma 2 (Bcl-2) protein and β-actin (loading control). Representative Western blots of three independent experiments are shown. In (b), the densitometric analysis of proteins expression reported as % of untreated cells is shown. Data are expressed as mean ± SEM; p < 0.01, * p < 0.001 compared to untreated cells (one-way ANOVA with Bonferroni’s post-test).
	Figure 5. PHNQs influence on key regulators enzymes of oxidative stress. (a) Representative Western blot analysis of different concentrations of PHNQs tested on NHDF for 24 h. Total cell lysate (30 µg proteins/sample) was separated by SDS-PAGE and immunoblotted with antibodies against the indicated antioxidant enzymes. β-actin was used as loading control. Densitometric analysis of (b) catalase (CAT), (c) glutathione synthetase (GSS), and (d) superoxide dismutase 1 (SOD1). Protein band intensities were normalized to β-actin and expressed as % of untreated cells. Values are reported as mean ± SEM of three independent experiments; * p < 0.05, p < 0.01, * p < 0.001 compared to untreated samples using one-way ANOVA with Bonferroni’s post-test.