Pigments
The main pigments found in H. stipulacea belong into the two photoprotective cycles, violaxanthin, antheraxanthin and zeaxanthin (xanthophyll cycle), and the siphonein-siphonaxanthin cycle, together with other carotenoids neoxanthin, lutein, α- and β-carotenes and the chlorophylls a and b (chl. a and chl. b). Pigment composition was identical in old and young leaves, but their concentration significantly varied with leaf aging (Fig. 1A). Indeed, the concentrations of xanthophyll-cycling pigments (sum of violaxanthin, antheraxanthin and zeaxanthin), as well as the sum of siphonein and siphonaxanthin pigments, were significantly higher in old leaves than in the young ones (p<0.001). The sum of α- and β-carotenes was similar in the two types of leaf (Fig. 1A), while chl. a concentration was significantly higher in young than in old leaves (p<0.01).
The ratio between accessory pigments and chl.a was used as an index of change in photosystems’ structure in old vs young leaves (Fig. 1B). Except for chlorophyll.b and the α- and β-carotenes, all the accessory pigments vs chl. a ratios were significantly higher in old leaves compared to young ones (p<0.001). Among the xanthophyll-cycling pigments, the two photoprotective antheraxanthin and zeaxanthin displayed the greatest increase with leaf aging (Fig. 1B). In synthesis, carotenoids with known role in photoprotection and/or protection against reactive oxygen species enhanced with aging (Fig. 1 A, B).
Macromolecular composition
Protein, carbohydrate and lipid concentrations in young leaves were significantly higher than in old ones (p<0.05; Table 1). In particular, protein concentrations decreased from 21.3 ± 0.5 to 8.2 ± 0.6, carbohydrates from 19 ± 0.6 to 10.9 ± 1.1 and lipids from 5.8 ± 0.1 to 1.6 ± 0.3 mg. g-1 in young and old leaves, respectively (Table 1).
In young leaves, proteins and carbohydrates represented the main macromolecular components, contributing for 46.2 % and 41.2 % to the total organic matter pool, respectively, followed by lipids (12.6 %). Conversely, in old leaves carbohydrates represented the dominant component (accounting for 52.6% of the total organic matter pool) followed by proteins and lipids (39.6 % and 7.8 %, respectively).
Table 1 Macromolecular composition. Protein, carbohydrate and lipid concentrations (mg.g-1 dry leaf) of young and old leaves of Halophila stipulacea. Values are reported as mean ± S.D.
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Proteins
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Carbohydates
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Lipids
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Young leaves
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21.3±0.5
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19.0±0.6
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5.8±0.1
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Old leaves
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8.2±0.6
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10.9±1.1
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1.6±0.3
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Antioxidant activity
Old leaf extracts provided a significantly (p<0.001) much higher antioxidant power than young leaves (Fig. 2). Indeed, at the two low concentrations, young leaf extracts did not exert any antioxidant function, while old leaf extract was already bioactive.
Cytotoxicity, scavenging and repair effects of the leaf extracts
The old leaf extract did not induce any cytotoxicity on WI-38 cells for none of the three tested concentrations (Fig. 3A). Conversely, the young leaf extract displayed a slight cytotoxicity on WI-38 cells (Fig. 3A), with a cell viability decreasing from 83% (at 1 μg ml-1) to 67% (at 100 μg ml-1).
For the scavenging assay, positive control, corresponding to WI-38 cells injured by 10 mM of H2O2 for 48 hours, resulted in very low cell viability (38%). When WI-38 cells were pre-treated with young leaf extract before H2O2 injury, cell viability enhanced up to 64%, 71% and 81% at 1, 10 and 100 μg ml-1, respectively (Fig. 3B). Using old leaf extract as pre-treatment, a further enhancement of cell viability was noticed up to 82%, 96% and 113% at 1, 10 and 100 μg ml-1, respectively (Fig. 3B).
For the repair assay, the positive control represented by WI-38 cells injured with 10 mM of H2O2 for 1 hour, displayed 52% of viable cells. Recovery treatment using 1, 10 and 100 μg ml-1 of young leaf extract increased cell viability up to 75%, 79% and 86%, respectively (Fig. 3C). Using the old leaf extract, cell viability became 62%, 94% and 108%, respectively (Fig. 3C).
Oxidative stress gene expression in WI-38 cells treated with old leaf extracts
WI-38 cells treated with old leaf extract upregulated MBL2 and MPO (17.1 and 31.1 in fold regulation, respectively) and downregulated EPHX2 and GSR (-18.2 and -17.1 in fold regulation, respectively) (Tables S1, S2 and Fig. 4). Conversely, the WI-38 cells pre-treated with old leaf extract, before the prooxidant treatment with 10 mM of H2O2 induced an upregulation of all genes involved in antioxidant activity analysed (e.g., CCL5, GPX5, GSR, KRT1, LPO, MBL2, MPO, MT3, NOX5, TPO), with the exception of EPHX2 (-9.9 in fold regulation, Fig. 4, Tables S1, S2).
The prooxidant treatment alone of the WI-38 cell line with 10 mM of H2O2 induced an upregulation of the genes MBL2, MPO and SPINK1 (7.0, 8.8 and 33.7 in fold regulation, respectively) and the downregulation of the GSR gene (-2.7 in fold regulation) (Tables S1, S2 and Fig. 4).
Recovery treatment with old leaf extract after the injury with H2O2 revealed by the upregulation of all genes analysed (e.g., CCL5, EPHX2, GPX5, KRT1, LPO, MBL2, MT3, NOX5, TPO) with the exception of the GSR gene (Fig. 4 and Tables S1, S2).