Author Contributions
Conceptualization, M.M. and L.P.; Data curation, C.B., M.M., M.R.C., C.O., R.O. and L.P.; Formal analysis, C.P., P.C., C.B., M.R.C., M.C., E.O., R.O. and M.R.; Funding acquisition, M.M., S.S., M.C.T. and L.P.; Investigation, C.P., M.M., T.B., C.O., C.B., S.S. and L.P.; Methodology, C.P., M.M., C.B., T.B., C.O. and L.P.; Resources, M.M., T.B., C.O., S.S., C.B. and L.P.; Supervision, C.P., M.M., M.R., M.C.T. and L.P.; Validation, P.C., C.B., M.R.C., M.C., E.O. and R.O.; Visualization, C.P., M.M., T.B., C.O., S.S. and L.P.; Writing—original draft, C.P., M.M., T.B., C.O., M.R., S.S. and L.P.; Writing—review and editing, C.P., M.M., M.R.C., C.O., R.O., M.C.T. and L.P. All authors have read and agreed to the published version of the manuscript.
Figure 1.
(A) Total phenol content (TPC) of the extracts obtained by the four different extraction conditions. Data are expressed as mg of GA/mg of freeze-dried extract and represent the mean of six samples, each measured in triplicate; * p ≤ 0.05 (one-way ANOVA test). (B) Total reducing capacity (TRC) of the extracts obtained by the four different extraction conditions. Data are expressed as mg of GA/mg of freeze-dried extract and represent the mean of six samples, each measured in triplicate. * p ≤ 0.05. (C) Radical scavenging capacity (RSC) of the extracts obtained by the four different extraction conditions. Data are expressed as mg of GA/mg of freeze-dried extract and represent the mean of six samples, each measured in triplicate; * p ≤ 0.05 (one-way ANOVA test).
Figure 1.
(A) Total phenol content (TPC) of the extracts obtained by the four different extraction conditions. Data are expressed as mg of GA/mg of freeze-dried extract and represent the mean of six samples, each measured in triplicate; * p ≤ 0.05 (one-way ANOVA test). (B) Total reducing capacity (TRC) of the extracts obtained by the four different extraction conditions. Data are expressed as mg of GA/mg of freeze-dried extract and represent the mean of six samples, each measured in triplicate. * p ≤ 0.05. (C) Radical scavenging capacity (RSC) of the extracts obtained by the four different extraction conditions. Data are expressed as mg of GA/mg of freeze-dried extract and represent the mean of six samples, each measured in triplicate; * p ≤ 0.05 (one-way ANOVA test).
Figure 2.
(A) Chromatographic separation of OLE constituents monitored in full mass ion positive mode and (B) chromatographic separation of OLE constituents monitored in full mass ion negative mode.
Figure 2.
(A) Chromatographic separation of OLE constituents monitored in full mass ion positive mode and (B) chromatographic separation of OLE constituents monitored in full mass ion negative mode.
Figure 3.
(A) LPS-activated cells were treated for 24 h with OLE at the indicated concentrations. Cells were stained using the PerCP-Annexin V and FVD 780 and analyzed by flow cytometry. Annexin V/FVD—double negative cells (lower left quadrant) represented live cells, annexin V/FVD—double positive cells (upper right quadrant) represented apoptotic cells and annexin V-negative/FVD-positive cells (lower right quadrant) indicated dead cells. A representative dot plot is shown. The percentage of viable, apoptotic and dead cells was reported in (B) for each OLE concentration. Data are the mean percentage of two different experiments. Evaluation of OLE cytotoxicity and safety on HaCaT cell line by (C) Trypan Blue exclusion and (D) MTT assays. Dotted lines indicate the 50% and 75% of cell viability. ns, not significant OLE-treated versus untreated group (one-way ANOVA test).
Figure 3.
(A) LPS-activated cells were treated for 24 h with OLE at the indicated concentrations. Cells were stained using the PerCP-Annexin V and FVD 780 and analyzed by flow cytometry. Annexin V/FVD—double negative cells (lower left quadrant) represented live cells, annexin V/FVD—double positive cells (upper right quadrant) represented apoptotic cells and annexin V-negative/FVD-positive cells (lower right quadrant) indicated dead cells. A representative dot plot is shown. The percentage of viable, apoptotic and dead cells was reported in (B) for each OLE concentration. Data are the mean percentage of two different experiments. Evaluation of OLE cytotoxicity and safety on HaCaT cell line by (C) Trypan Blue exclusion and (D) MTT assays. Dotted lines indicate the 50% and 75% of cell viability. ns, not significant OLE-treated versus untreated group (one-way ANOVA test).
Figure 4.
(A) LPS-activated RAW 264.7 cells were in vitro stimulated using different OLE concentrations for 24 h. NO release in the supernatant culture was quantified by using Griess reagent. Results are reported as mean ± SD of three independent experiments, each conducted in triplicate. (B) *** p < 0.0001, OLE–treated versus LPS-treated group (one-way ANOVA test). Concentration-response curve was obtained for the determination of the IC50. Results are reported as mean of two independent experiments, each conducted in triplicate. LPS-activated RAW 264.7 cells were in vitro stimulated with different concentrations of OLE for 24 h. Supernatants were collected and the concentrations of IL-6 and IL-1β were determined by ELISA test (C,D). ** p < 0.001, *** p < 0.0001, OLE-treated versus LPS-treated group (one-way ANOVA test). Concentration-response curves were obtained for the determination of the IC50. For each curve results are reported as mean of two independent experiments, each conducted in triplicate (E,F).
Figure 4.
(A) LPS-activated RAW 264.7 cells were in vitro stimulated using different OLE concentrations for 24 h. NO release in the supernatant culture was quantified by using Griess reagent. Results are reported as mean ± SD of three independent experiments, each conducted in triplicate. (B) *** p < 0.0001, OLE–treated versus LPS-treated group (one-way ANOVA test). Concentration-response curve was obtained for the determination of the IC50. Results are reported as mean of two independent experiments, each conducted in triplicate. LPS-activated RAW 264.7 cells were in vitro stimulated with different concentrations of OLE for 24 h. Supernatants were collected and the concentrations of IL-6 and IL-1β were determined by ELISA test (C,D). ** p < 0.001, *** p < 0.0001, OLE-treated versus LPS-treated group (one-way ANOVA test). Concentration-response curves were obtained for the determination of the IC50. For each curve results are reported as mean of two independent experiments, each conducted in triplicate (E,F).

Figure 5.
Skin used for the assay (A) and simulation of an open wound (B).
Figure 5.
Skin used for the assay (A) and simulation of an open wound (B).
Figure 6.
(A) OLE In vitro release profile of from the hydrogel film B2 represented as mg/mL vs. time and (B) mg/cm2 vs. time.
Figure 6.
(A) OLE In vitro release profile of from the hydrogel film B2 represented as mg/mL vs. time and (B) mg/cm2 vs. time.
Figure 7.
Evaluation of hydrogel film B2 cytotoxicity and safety on HaCaT cell line by an MTT assay. Dotted lines indicate the 50% and 75% of cell viability. ns, not significant OLE-treated versus untreated group (one-way ANOVA test).
Figure 7.
Evaluation of hydrogel film B2 cytotoxicity and safety on HaCaT cell line by an MTT assay. Dotted lines indicate the 50% and 75% of cell viability. ns, not significant OLE-treated versus untreated group (one-way ANOVA test).
Table 1.
Recovery extract yield.
Table 1.
Recovery extract yield.
Extraction Conditions: Solvent, Temperature | Recovery Yield (%) a |
---|
abs EtOH, RT b | 6.4 |
70% EtOH, RT b | 8.7 |
abs EtOH, 60 °C | 7.9 |
70% EtOH, 60 °C | 9.7 |
Table 2.
Minimum inhibitory (MIC) and minimum bactericidal (MBC) values of OLE and the reference antibiotic ampicillin expressed as mg/mL ± SD (n = 3).
Table 2.
Minimum inhibitory (MIC) and minimum bactericidal (MBC) values of OLE and the reference antibiotic ampicillin expressed as mg/mL ± SD (n = 3).
| S. epidermidis | S. aureus | L. innocua | E. faecalis |
---|
OLE MIC | 0.47 ± 0.00 | 0.94 ± 0.00 | 3.75 ± 0.00 | 3.75 ± 0.00 |
OLE MBC | 0.94 ± 0.00 | 1.88 ± 0.00 | 7.50 ± 0.00 | 7.50 ± 0.00 |
Ampicillin MIC | 0.13 ± 0.00 | 0.13 ± 0.00 | 0.50 ± 0.00 | 0.50 ± 0.00 |
Ampicillin MBC | 0.50 ± 0.00 | 0.25 ± 0.00 | 1.00 ± 0.00 | 4.00 ± 0.00 |
Table 3.
Inhibition halos measured for the hydrogel films B1–B3. Results are expressed as mm ± SD (n = 3, n.i. = no inhibition).
Table 3.
Inhibition halos measured for the hydrogel films B1–B3. Results are expressed as mm ± SD (n = 3, n.i. = no inhibition).
Hydrogel Film (OLE mg) | S. epidermidis | S. aureus | L. innocua | E. faecalis |
---|
B1 (3.64) | 23.00 ± 0.00 | 16.33 ± 0.58 | n.i. | n.i. |
B2 (10.92) | 28.67 ± 0.58 | 20.33 ± 0.58 | 21.67 ± 0.58 | 21.00 ± 0.00 |
B3 (18.21) | 25.67 ± 0.58 | 21.00 ± 0.00 | 21.67 ± 0.58 | 21.33 ± 0.58 |