Photoprotective and Anti-Aging Properties of the Apical Frond Extracts from the Mediterranean Seaweed Ericaria amentacea

Abstract

There is a growing interest in using brown algal extracts thanks to the bioactive substances they produce for adaptation to the marine benthic environment. We evaluated the anti-aging and photoprotective properties of two types of extracts (50%-ethanol and DMSO) obtained from different portions, i.e., apices and thalli, of the brown seaweed, Ericaria amentacea. The apices of this alga, which grow and develop reproductive structures during summer when solar radiation is at its peak, were postulated to be rich in antioxidant compounds. We determined the chemical composition and pharmacological effects of their extracts and compared them to the thallus-derived extracts. All the extracts contained polyphenols, flavonoids and antioxidants and showed significant biological activities. The hydroalcoholic apices extracts demonstrated the highest pharmacological potential, likely due to the higher content of meroditerpene molecular species. They blocked toxicity in UV-exposed HaCaT keratinocytes and L929 fibroblasts and abated the oxidative stress and the production of pro-inflammatory cytokines, typically released after sunburns. Furthermore, the extracts showed anti-tyrosinase and anti-hydrolytic skin enzyme activity, counteracting the collagenase and hyaluronidase degrading activities and possibly slowing down the formation of uneven pigmentation and wrinkles in aging skin. In conclusion, the E. amentacea apices derivatives constitute ideal components for counteracting sunburn symptoms and for cosmetic anti-aging lotions.

Keywords: Cystoseira amentacea; Ericaria amentacea; anti-collagenase; anti-hyaluronidase; anti-tyrosinase; antioxidant; inflammation; meroditerpenes; polyphenols.

Scheme 1

Schematic representation of E. amentacea seaweed body parts.

Figure 1

Antioxidant activity of E. amentacea extracts in spectrophotometric tests. (A) ROS scavenging activity by the DPPH assay. Data are expressed as a percentage of scavenging of the DPPH radical with respect to the absorbance of the DPPH radical alone (control). Symbols indicate significance in paired Tukey test (ANOVA p < 0.0001; Tukey of DMSO apex vs. DMSO thallus’ respective concentration, * p < 0.05; Tukey of EtOH apex vs. EtOH thallus’ respective concentration, $ p < 0.05) (B) Fe (III)-reducing power assay, measured by the potassium ferricyanide method. Data are expressed as a percentage of reducing power compared to the Fe reduction of ascorbic acid (AA, concentration 30 μg/mL) positive control. Symbols indicate significance in paired Tukey test (ANOVA p < 0.000001; Tukey of DMSO apex vs. DMSO thallus’ respective concentration, * p < 0.05; Tukey of EtOH apex vs. EtOH thallus’ respective concentration, $ p < 0.005). (C) OH radical scavenging activity by Mohr’s salt assay. Data are expressed as a percentage of inhibition of OH radical production with respect to the absorbance of the OH radical-producing solution alone (Control). Symbols indicate significance in paired Tukey (ANOVA p < 0.0001; Tukey of DMSO apex vs. DMSO thallus’ respective concentration, * p < 0.05; Tukey of EtOH apex vs. EtOH thallus’ respective concentration, $ p < 0.05). (D) NO radical scavenging activity was measured by the sodium nitroprusside method coupled with the Griess assay. Data are expressed as a percentage of inhibition of NO production with respect to the absorbance of the NO radical-producing solution alone (Control).

Figure 1

Antioxidant activity of E. amentacea extracts in spectrophotometric tests. (A) ROS scavenging activity by the DPPH assay. Data are expressed as a percentage of scavenging of the DPPH radical with respect to the absorbance of the DPPH radical alone (control). Symbols indicate significance in paired Tukey test (ANOVA p < 0.0001; Tukey of DMSO apex vs. DMSO thallus’ respective concentration, * p < 0.05; Tukey of EtOH apex vs. EtOH thallus’ respective concentration, $ p < 0.05) (B) Fe (III)-reducing power assay, measured by the potassium ferricyanide method. Data are expressed as a percentage of reducing power compared to the Fe reduction of ascorbic acid (AA, concentration 30 μg/mL) positive control. Symbols indicate significance in paired Tukey test (ANOVA p < 0.000001; Tukey of DMSO apex vs. DMSO thallus’ respective concentration, * p < 0.05; Tukey of EtOH apex vs. EtOH thallus’ respective concentration, $ p < 0.005). (C) OH radical scavenging activity by Mohr’s salt assay. Data are expressed as a percentage of inhibition of OH radical production with respect to the absorbance of the OH radical-producing solution alone (Control). Symbols indicate significance in paired Tukey (ANOVA p < 0.0001; Tukey of DMSO apex vs. DMSO thallus’ respective concentration, * p < 0.05; Tukey of EtOH apex vs. EtOH thallus’ respective concentration, $ p < 0.05). (D) NO radical scavenging activity was measured by the sodium nitroprusside method coupled with the Griess assay. Data are expressed as a percentage of inhibition of NO production with respect to the absorbance of the NO radical-producing solution alone (Control).

Figure 2

Cell viability evaluation. (A,B) L929 fibroblast cell growth evaluation by MTT test at 24 h in the presence of increasing concentrations of E. amentacea 50%-ethanol (A) and DMSO (B) extracts—gray bars, apices extracts; white bars, thallus extracts, respectively. Results are expressed as cell percentages with respect to the control. Asterisks indicate significance in paired Tukey test (ANOVA, p < 0.00005; Tukey of respective bar vs. C: * p < 0.01 and ** p < 0.005). (C,D) HaCaT keratinocyte cell growth evaluation by MTT test at 24 h in the presence of increasing concentrations of E. amentacea 50%-ethanol (A) and DMSO (B) extracts (ANOVA, p < 0.000001; Tukey of respective bar vs. C: * p < 0.05 and ** p < 0.005).

Figure 2

Cell viability evaluation. (A,B) L929 fibroblast cell growth evaluation by MTT test at 24 h in the presence of increasing concentrations of E. amentacea 50%-ethanol (A) and DMSO (B) extracts—gray bars, apices extracts; white bars, thallus extracts, respectively. Results are expressed as cell percentages with respect to the control. Asterisks indicate significance in paired Tukey test (ANOVA, p < 0.00005; Tukey of respective bar vs. C: * p < 0.01 and ** p < 0.005). (C,D) HaCaT keratinocyte cell growth evaluation by MTT test at 24 h in the presence of increasing concentrations of E. amentacea 50%-ethanol (A) and DMSO (B) extracts (ANOVA, p < 0.000001; Tukey of respective bar vs. C: * p < 0.05 and ** p < 0.005).

Figure 3

Cell death rescue evaluation after UV challenge. (A–D) Cell death rescue from 2 min UV (A,B) and 5 min UV (C,D) challenge of L929 fibroblasts in the presence of 50%-ethanol and DMSO extracts, evaluated by MTT test 24 h after the stimulus. Gray bars, apices extracts; white bars, thallus extracts, respectively. Results are expressed as a cell percentage with respect to control, untreated cells. Symbols indicate significance in paired Tukey test (ANOVA, p < 0.0005 in all panels; Tukey of UV2 or UV5 vs. C $ p < 0.005; Tukey of respective bar vs. UV2: * p < 0.05 and ** p < 0.001; Tukey of respective bar vs. UV5: * p < 0.05 and ** p < 0.0005). (E–H) Cell death rescue from 2 min UV (E,F) and 5 min UV (G,H) challenge of HaCaT keratinocytes in the presence of 50%-ethanol and DMSO extracts, evaluated by MTT test 24 h after the stimulus (ANOVA, p < 0.000001 in all panels; Tukey of UV2 or UV5 vs. C $ p < 0.0001; Tukey of respective bar vs. UV2: * p < 0.01 and ** p < 0.0005; Tukey of respective bar vs. UV5: * p < 0.05 and ** p < 0.005).

Figure 3

Cell death rescue evaluation after UV challenge. (A–D) Cell death rescue from 2 min UV (A,B) and 5 min UV (C,D) challenge of L929 fibroblasts in the presence of 50%-ethanol and DMSO extracts, evaluated by MTT test 24 h after the stimulus. Gray bars, apices extracts; white bars, thallus extracts, respectively. Results are expressed as a cell percentage with respect to control, untreated cells. Symbols indicate significance in paired Tukey test (ANOVA, p < 0.0005 in all panels; Tukey of UV2 or UV5 vs. C $ p < 0.005; Tukey of respective bar vs. UV2: * p < 0.05 and ** p < 0.001; Tukey of respective bar vs. UV5: * p < 0.05 and ** p < 0.0005). (E–H) Cell death rescue from 2 min UV (E,F) and 5 min UV (G,H) challenge of HaCaT keratinocytes in the presence of 50%-ethanol and DMSO extracts, evaluated by MTT test 24 h after the stimulus (ANOVA, p < 0.000001 in all panels; Tukey of UV2 or UV5 vs. C $ p < 0.0001; Tukey of respective bar vs. UV2: * p < 0.01 and ** p < 0.0005; Tukey of respective bar vs. UV5: * p < 0.05 and ** p < 0.005).

Figure 4

Cell death rescue evaluation after H₂O₂-challenge. (A,B) Cell death rescue from 200 μM H₂O₂-challenge of L929 fibroblasts in the presence of 50%-ethanol and DMSO extracts, evaluated by MTT test 24 h after the stimulus. Gray bars, apices extracts; white bars, thallus extracts, respectively. Results are expressed as cell percentages with respect to control, untreated cells. Symbols indicate significance in paired Tukey test (ANOVA, p < 0.000001; Tukey of H₂O₂ vs. C, $ p < 0.0001; Tukey of respective bar vs. H₂O₂: * p < 0.05 and ** p < 0.001). (C,D) Cell death rescue from 500 μM H₂O₂-challenge of HaCaT keratinocytes in the presence of 50%-ethanol and DMSO extracts, evaluated by MTT test 24 h after the stimulus (ANOVA, p < 0.0005; Tukey of H₂O₂ vs. C, $ p < 0.0005; Tukey of respective bar vs. H₂O₂: * p < 0.05 and ** p < 0.0005).

Figure 5

ROS scavenging activity in cellular assays. (A,B) Intracellular ROS production, measured by DCF fluorometric analysis in L929 fibroblasts incubated for 2 h in the presence or absence of 50 and 100 μg/mL of 50%-ethanol (A) or DMSO (B) extracts, respectively, after a 3 min UV challenge. Gray bars, apices extracts; white bars, thallus extracts, respectively. Results are expressed as a percentage of ROS production with respect to control, untreated cells. Asterisks indicate significance in the Tukey test (ANOVA p < 0.000001; Tukey of respective bar vs. UV3, * p < 0.05 and ** p < 0.005). (C,D) Intracellular ROS production in HaCaT keratinocytes incubated with 50 and 100 μg/mL of 50%-ethanol (C) or DMSO (D) extracts after a 3 min UV challenge (ANOVA p < 0.000001; Tukey of respective bar vs. UV3, * p < 0.05 and ** p < 0.005). (E,F) Intracellular ROS production in L929 fibroblasts incubated for 2 h with 200 μM H₂O₂ in the presence or absence of 50 and 100 μg/mL of 50%-ethanol (E) or DMSO (F) extracts (ANOVA p < 0.000001; Tukey of respective bar vs. H₂O₂, ** p < 0.0001). (G,H) Intracellular ROS production in HaCaT keratinocytes incubated for 2 h with 200 μM H₂O₂ with 50 and 100 μg/mL of 50%-ethanol (G) or DMSO (H) extracts (ANOVA p < 0.000001; Tukey of respective bar vs. H₂O₂, * p < 0.001 and ** p < 0.0001).

Figure 5

ROS scavenging activity in cellular assays. (A,B) Intracellular ROS production, measured by DCF fluorometric analysis in L929 fibroblasts incubated for 2 h in the presence or absence of 50 and 100 μg/mL of 50%-ethanol (A) or DMSO (B) extracts, respectively, after a 3 min UV challenge. Gray bars, apices extracts; white bars, thallus extracts, respectively. Results are expressed as a percentage of ROS production with respect to control, untreated cells. Asterisks indicate significance in the Tukey test (ANOVA p < 0.000001; Tukey of respective bar vs. UV3, * p < 0.05 and ** p < 0.005). (C,D) Intracellular ROS production in HaCaT keratinocytes incubated with 50 and 100 μg/mL of 50%-ethanol (C) or DMSO (D) extracts after a 3 min UV challenge (ANOVA p < 0.000001; Tukey of respective bar vs. UV3, * p < 0.05 and ** p < 0.005). (E,F) Intracellular ROS production in L929 fibroblasts incubated for 2 h with 200 μM H₂O₂ in the presence or absence of 50 and 100 μg/mL of 50%-ethanol (E) or DMSO (F) extracts (ANOVA p < 0.000001; Tukey of respective bar vs. H₂O₂, ** p < 0.0001). (G,H) Intracellular ROS production in HaCaT keratinocytes incubated for 2 h with 200 μM H₂O₂ with 50 and 100 μg/mL of 50%-ethanol (G) or DMSO (H) extracts (ANOVA p < 0.000001; Tukey of respective bar vs. H₂O₂, * p < 0.001 and ** p < 0.0001).

Figure 6

Counteraction of skin hydrolytic enzyme activity. (A) Inhibition of collagenase (from C. histolyticum) by enzymatic kinetic assay, measured as a reduced degradation of FALGPA, a synthetic peptide that mimics the collagen structure, in the presence of 50 μg/mL of algal extracts as compared to the activity of the control enzyme (in the presence of the respective vehicle solvents). EA and ET, apices and thallus 50%-ethanol extracts; DA and DT, apices and thallus DMSO extracts, respectively. Asterisks indicate significance in paired Tukey test (ANOVA p < 0.0005, Tukey of ET vs. EA, * p < 0.05). (B) Inhibition of hyaluronidase (from bovine testes) by enzyme kinetic assay measured as a reduced degradation of hyaluronic acid in the presence of 50 μg/mL algal extracts as compared to the control enzyme activity (in the presence of the respective vehicle solvents). Asterisks indicate significance in paired Tukey test (ANOVA p < 0.0005, Tukey of ET vs. EA, * p < 0.05). (C) Inhibition of mushroom tyrosinase by the relative enzyme kinetic assay, measured as a reduced conversion of L-Dopa to Dopachrome in the presence of 100 μg/mL of algal extracts as compared to the activity of the control enzyme (in the presence of the respective vehicle solvents). Asterisks indicate significance in paired Tukey test (ANOVA p < 0.0005, Tukey of ET vs. EA, * p < 0.05; Tukey of DT vs. DA, ** p < 0.01).

Figure 7

Inhibition of gene expression in UV-challenged HaCaT human keratinocytes. Gene expression was measured by qPCR analyses of IL-1β (A), IL-6 (B), and IL-8 (C) at 6 h, after a 3 min UV challenge and in the presence of 50 μg/mL algal extracts. EA and ET, apices and thallus 50%-ethanol extracts; DA and DT, apices and thallus DMSO extracts, respectively. Data are normalized on the HPRT-1 housekeeping gene and expressed as mRNA-fold increase compared to control cells. Asterisks indicate significance in Tukey test (IL-1β ANOVA p < 0.000001, Tukey of respective bar vs. UV, * p < 0.0005 and ** p < 0.0001, respectively; IL-6 ANOVA p < 0.000001, Tukey of respective bar vs. UV, ** p < 0.0005; IL-8 ANOVA p < 0.000001, Tukey of respective bar vs. UV, * p < 0.05 and ** p < 0.005).

Figure 8

Inhibition of gene expression in UV-challenged L929 murine fibroblasts. Gene expression was measured by qPCR analyses of IL-1β (A), IL-6 (B), and CXCL5 (C) at 6 h, after a 3 min UV challenge and in the presence of 50 μg/mL algal extracts. EA and ET, apices and thallus ethanol extracts; DA and DT, apices and thallus DMSO extracts, respectively. Data are normalized on the GAPDH housekeeping gene and expressed as mRNA-fold increase compared to control, untreated cells. Asterisks indicate significance in the Tukey test (IL-1β ANOVA p < 0.0005, Tukey of respective bar vs. UV, ** p < 0.005; IL-6 ANOVA p < 0.00005, Tukey of respective bar vs. UV, * p < 0.005 and ** p < 0.0005; CXCL5 ANOVA p < 0.000001, Tukey of respective bar vs. UV, * p < 0.05 and ** p < 0.001).

Figure 9

HPLC/MS analyses of the EtOH extracts. Total ion current (TIC) and extracted ion chromatograms (EIC) obtained by full-scan MS/MS analysis coupled to HPLC separation of an aliquot of the E. amentacea apices hydroalcoholic (A) and thalli (B) extracts at a starting dilution of 2.5 mg/mL (injection volume, 8 μL), acquiring the most abundant species under each peak. The red chromatogram in both panels represents the TIC of each extract, while the following blue, green, pink, black and gray chromatograms in both panels represent the EICs of the molecules indicated in the respective panels. The acquisition was performed on negative and positive ions in the 100–1000 mass range and analyzed using integrated software.

Figure 9

HPLC/MS analyses of the EtOH extracts. Total ion current (TIC) and extracted ion chromatograms (EIC) obtained by full-scan MS/MS analysis coupled to HPLC separation of an aliquot of the E. amentacea apices hydroalcoholic (A) and thalli (B) extracts at a starting dilution of 2.5 mg/mL (injection volume, 8 μL), acquiring the most abundant species under each peak. The red chromatogram in both panels represents the TIC of each extract, while the following blue, green, pink, black and gray chromatograms in both panels represent the EICs of the molecules indicated in the respective panels. The acquisition was performed on negative and positive ions in the 100–1000 mass range and analyzed using integrated software.

Figure 10

E. amentacea specimen. (A) Photograph from the site of collection in the Ligurian Sea, in the lower intertidal zone; (B) photograph of freshly collected seaweed thalli with a scale bar.