Cosmeceutical Potential Of Extracts Derived From Fishery Industry Residues: Sardine Wastes And Codfish Frames Part 2

3.2. Antibacterial Activity 

Glycoside of cistanche can also increase the activity of SOD in heart and liver tissues, and significantly reduce the content of lipofuscin and MDA in each tissue, effectively scavenging various reactive oxygen radicals (OH-, H₂O₂, etc.) and protecting against DNA damage caused by OH-radicals. Cistanche phenylethanoid glycosides have a strong scavenging ability of free radicals, a higher reducing ability than vitamin C, improve the activity of SOD in sperm suspension, reduce the content of MDA, and have a certain protective effect on sperm membrane function. Cistanche polysaccharides can enhance the activity of SOD and GSH-Px in erythrocytes and lung tissues of experimentally senescent mice caused by D-galactose, as well as reduce the content of MDA and collagen in lung and plasma, and increase the content of elastin, have a good scavenging effect on DPPH, prolong the time of hypoxia in senescent mice, improve the activity of SOD in serum, and delay the physiological degeneration of lung in experimentally senescent mice With cellular morphological degeneration, experiments have shown that Cistanche has the good antioxidant ability and has the potential to be a drug to prevent and treat skin aging diseases. At the same time, echinacoside in Cistanche has a significant ability to scavenge DPPH free radicals and has the ability to scavenge reactive oxygen species and prevent free radical-induced collagen degradation, and also has a good repair effect on thymine free radical anion damage.

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S. aureus and K. pneumoniae were chosen as representative gram-positive and -negative bacteria to evaluate the antimicrobial capacity of the extracts. The antibacterial activity assays performed with codfish frame extracts revealed that all of them were able to affect both bacterial strains’ growth behavior (MIC* results in Tables 3 and 4). However, true growth inhibition (MIC values) did not occur in the presence of any of the extracts at the concentrations tested.

As S1 and S3 were solubilized in DMSO, assays were performed with this solvent to evaluate its influence on the results obtained for the sardine extracts. The results reveal that the maximum DMSO concentration used in the antibacterial activity testing of the different extracts (up to 12.5%) did not affect the growth of both bacterial strains.

In general, all sardine extracts inhibited the bacterial growth of both gram-positive and gram-negative selected strains, but S. aureus was shown to be more susceptible than K. pneumoniae, which is expected since the outer membrane of gram-negative bacteria poses an additional barrier to prevent the interference of different molecules with the cell [44]. S2 was shown to be the extract with the most promising antibacterial potential, indicating that higher extraction temperatures favored the extraction of anti-bacterial compounds. Taking these results into account, S2 was selected to be tested against another gram-positive bacteria, namely C. acnes, an aerotolerant anaerobe linked to the acne skin condition [45]. To evaluate the impact of the defatting process on the inhibition capacity of C. acnes growth,  sample S3 was also selected to be tested in this assay. The results are summarized in Table 5  showing that C. acnes behaves similarly to S. aureus with its growth being affected by both sardine extracts. Between both samples, S2 presented the highest inhibitory effect (lower MIC and MBC values) suggesting that the defatting process could modify or eliminate some anti-microbial compounds from the sardine samples before the extraction process,  especially free fatty acids and lipid oxidation products [22].

Previous studies showed that some amino acid residues present in peptides can lead to different antibacterial activities [46]. Rodrigues et al. showed that protein derivative-rich extracts obtained by extraction with deep eutectic solvents (DES) from sardine processing waste streams have antibacterial activity toward S. aureus and Escherichia coli [47]. However,  the MIC*, MIC, and MBC values of these extracts were lower than those obtained in this work for the same raw material, which could be explained by the synergic and/or additive effect between the extracts’ components and DES, as described previously [29]. Nevertheless, the antimicrobial effect of S2 extract is similar to other extracts obtained by subcritical water extraction, namely using k¯anuka leaves (S. aureus—MIC: 0.9 mg/mL, MBC: 3.8–5 mg/mL; E. coli—MIC: 3.8–7.5 mg/mL, MBC: 4.4–7.5 mg/mL) [48], that are rich in phenolic compounds already recognized as presenting relevant antimicrobial effects [49].

In this study, the highest correlation coefficient obtained was for MIC values of S. aureus and the total glutamic acid content (R2 = 0.6, Table S1).

3.3. Cellular Antioxidant and Anti-Inflammatory Effect

In this work, a human keratinocyte cell line (HaCaT) was used to better understand the bioactivity, namely antioxidant and anti-inflammatory effects, of extracts as some of the processes related to the uptake, distribution, and metabolism of bioactive compounds are better addressed [50]. HaCaT cells are one of the predominant cell types encountered in the skin, being responsible for skin integrity and, when affected by senescence or oxidative stress, can accelerate the skin aging process [51]. Additionally, keratinocytes play an important role in the regulation of skin inflammation, responding to external stimuli, such as bacterial LPS, and actively contributing to inflammation pathways, especially by releasing proinflammatory cytokines or chemokines [31,52].

In the first approach, cytotoxicity assays were performed to evaluate the safety of samples and to select non-toxic concentrations for further bioactivity studies. Table 6  presents the IC50 values of each sample showing that S1 presented the highest cytotoxic effect. In previous studies, these samples showed higher IC50 values in Caco-2, a model for crypt enterocytes, than in HaCaT, which indicates that the samples are more toxic for keratinocytes than intestinal cells [11,22].

The cellular antioxidant activity was then assessed by evaluating the capacity of samples in scavenging intracellular ROS generated by the chemical stressor AAPH. In parallel, anti-inflammatory assays were also performed to investigate the effect of samples in reducing the secretion of IL-8, which has been consistently reported as an important skin inflammation biomarker [53–55], upon pro-inflammatory stimulus with LPS. In these assays, non-cytotoxic concentrations of the extracts were used (0.1875, 0.375, and 0.75 mg/mL for all samples except S1 where 0.75 mg/mL was not tested since this concentration presented a cytotoxic effect, Table 6). In general, all samples inhibited ROS formation in HaCaT, and a dose-dependent effect was observed (Figure 1). Among codfish samples, Cf2 and Cf4 showed the highest cellular antioxidant activities, and amongst sardine samples, S1 showed the highest ROS  percentage reduction relative to the control.

Concerning the anti-inflammatory effect, only Cf1 (0.375 and 0.75 mg/mL), S2 (0.75 mg/mL), and S3 (0.375 and 0.75 mg/mL) revealed the capacity to inhibit IL-8 secretion by HaCaT cells after LPS-induced inflammation (Figure 2). In contrast, the other samples did not reduce IL-8 and, for some concentrations, Cf4 and S1 showed a pro-inflammatory effect. These two extracts were the ones that demonstrated the highest cytotoxic effect in HaCaT cells and thus the concentrations tested, although not leading to cell death, could induce the activation of inflammatory pathways since injured cells can release danger signals that alert other cells to cell death [56].

The extracts with the highest anti-inflammatory effect, namely Cf1, S2, and S3, were further selected to evaluate their capacity in inhibiting IL-6 secretion, a cytokine also recognized as an important biomarker in skin disorders [53]. Results show that Cf1 was the only sample able to significantly reduce IL-6 secretion by 69.4 ± 3.5 and 53.0 ± 14.7% IL-6 (relative to the positive control, p-value < 0.001) at 0.375 and 0.75 mg/mL, respectively,  whereas sardine extracts increased in IL-6 levels of the supernatants (Figure 3). Overall, only Cf1 revealed the capacity to reduce both IL-8 and IL-6 secretion by HaCaT cells, suggesting that this codfish extract could be further explored for anti-inflammatory applications in skin conditions.

The anti-inflammatory activity of codfish extracts was previously evaluated in an intestinal cell line (Caco-2 cells) [11] and the results were in line with the data presented in this study for HaCaT cells. In both cell lines, Cf1 presented the highest anti-inflammatory effect, reinforcing the use of lower temperatures to extract bioactive compounds from codfish frames.

Our results are from previous studies supporting the idea that fish-derived extracts/hydrolysates display a broad spectrum of bioactivities, including antioxidant, antimicrobial, anti-aging, anti-hypertensive, anti-human immunodeficiency virus,  anti-proliferative, or anticoagulant activities [23,57]. Song et al. showed that enzymatic hydrolysates of the marine fish half-fin anchovy contained antibacterial peptide fractions,  with activity against E. coli [58]. Fish skin and collagen hydrolyzed by subcritical water hydrolysis also showed high antioxidant and antimicrobial activity against Bacillus cereus, S. aureus, and Pseudomonas putida [59]. Additionally, Wang and co-workers produced extracts derived from fish side streams of two fish species (rainbow trout and sole) that could inhibit the growth of pathogenic bacteria (S. aureus or Salmonella) and with anti-inflammatory properties [60].

4. Conclusions 

In this work, we applied a platform of in vitro bioassays to evaluate for the first time the cosmeceutical potential of protein extracts derived from fishery wastes, namely sardine residues and codfish frames, obtained by high-pressure technology. We demonstrated that both types of extracts showed antioxidant, anti-aging, and anti-hyperpigmentation potential. Among all, sardine extracts presented the highest anti-bacterial activity, and this effect was more pronounced for samples obtained using higher extraction temperatures (250 ◦C) and without the defatting step. Codfish samples were the most effective anti-inflammatory agents, and in this case, lower temperatures (90 ◦C) favored the extraction of these bioactive compounds. Although further studies are needed to identify which compounds could be responsible for the bioactive effects, total threonine, free valine as well as free and total leucine were identified to highly correlate with the antioxidant activities of samples. Total protein content and free arginine correlated with elastase and tyrosinase inhibition activities, respectively.

This work is a step forward in the development of potential cosmeceutical ingredients with antioxidant, skin whitening, antimicrobial, and anti-inflammatory effects from sardine residues and codfish frames, adding potential high value to these fishery industry wastes.

Author Contributions: Conceptualization, I.C.L., F.B.G., P.S., A.P., R.M., N.F. and A.T.S.; methodology, M.C., A.B., I.C.L., F.B.G., N.F., and A.T.S.; investigation, M.C., I.C.L., F.B.G., M.M., R.M., N.F., and A.T.S.; resources, F.B.G., N.F., and A.T.S.; writing—original draft preparation, M.C.; writing—review and editing, I.C.L., F.B.G., R.M., P.S., N.F. and A.T.S.; supervision, F.B.G., P.S., N.F., and A.T.S.; funding acquisition, F.B.G., P.S., N.F., and A.T.S. All authors have read and agreed to the published version of the manuscript.

Funding: This work was funded by Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through project PTDC/ASPPES/28399/2017  and national funds iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associate Laboratories LS4FUTURE (LA/P/0087/2020) and LAQV (UIDB/QUI/50006/2020). Funding from INTERFACE Programme, through the Innovation, Technology and Circular Economy Fund (FITEC), is gratefully acknowledged. ATS also acknowledges FCT/MCTES for the Individual Grant CEECIND/ 04801/2017.

Acknowledgments: We acknowledge Pascoal and Filhos S.A. (Gafanha da Nazaré, Portugal) and Conservas A Poveira S.A. (Portugal) for providing us, respectively, the Atlantic codfish (Gadus morhua)  frames and Sardine (Sardina pilchardus) waste used in this work.

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