
Though the Rhodophyta phylum has been probably the most studied when it comes to photoprotective exercise, probably the most considerable algae within the Caribbean Sea are these belonging to the Heterokontophyta phylum, whose abundance has notably elevated in response to coral reef degradation. Contemplating this excessive biomass availability and the variation of those organisms to excessive daylight publicity year-round, they possess photoprotection mechanisms, when it comes to metabolite manufacturing, that may be explored.
To take action, a strategy for the screening of promising photoprotective brown macroalgae extracts was carried out, enabling the analysis and monitoring of this exercise in every pattern set. For this goal, 17 algae samples belonging to the Ochrophyta phylum had been collected in Providencia Island in September 2021, by which the Dictyota, Canistrocarpus, Lobophora, Padina, Sargassum, Stypopodium, and Turbinaria genera are represented (Supplementary Desk S.1). The choice included samples from completely different genera, in addition to samples from the identical genera, with the purpose of figuring out variations in metabolic manufacturing and photoprotection exercise throughout the carried out screening methodology.
From the 17 samples and their organic triplicates (51 in whole), 204 extracts had been obtained: FO, FB, FM, and FW. The extraction yield ranged from 2 to 9% for the FO extracts, from 0.01 to 24% for the FB extracts, from 3 to fifteen% for the FM extracts, and from 0.5 to three% for the FW extracts.
Basic remarks on the UV–vis absorption spectra of crude extracts
The UV–Vis spectra of the crude extracts in Fig. 1 present the variations between the extracts. The FO extracts (Fig. 1a and e) introduced the best absorbance on the examined wavelengths, adopted by the FB (Fig. 1b and f), FW (Fig. 1d and h), and FM extracts (Fig. 1c and g).
UV–Vis absorption spectra (λ = 290–500 nm) of the extracts obtained from 17 chosen brown seaweed samples collected in Providencia. (a–d) spectra of extracts from Dictyota ( DI01,
DI02,
DI03,
DI04,
DI05,
DM,
DP01, and
DP02) and Canistrocarpus (
CCe and
CCr) genera: (a) FO extracts, (b) FB extracts, (c) FM extracts, and (d) FW extracts. (e–h) spectra of extracts from Stypopodium (
SS01,
SS02, and
SS03), Saragassum (
SP), Lobophora (
LV), Padina (
PG), and Turbinaria (
TT) genera: (e) FO extracts, (f) FB extracts, (g) FM extracts, and (h) FW extracts.
The samples from the Dictyota and Canistrocarpus genera (Fig. 1a–d) introduced absorbance maxima throughout the 350–480 nm area which corresponds to typical UVA absorbance bands of pigments equivalent to chlorophylls and carotenoids, that are the principle photoprotection mechanisms in photosynthetic organisms. Though there have been variations between the samples, the Canistrocarpus cervicornis (CCe)–FO extract had the best absorbance and the broadest absorption band (λmax = 420 nm and 440 nm) (Fig. 1a). An analogous development was noticed in samples belonging to the Stypopodium (SS), Turbinaria (TT), Padina (PG), Sargassum (SP), and Lobophora (LV) genera (Fig. 1e–h), by which there have been two absorbance maxima areas: between 290 and 295 nm and between 415 and 445 nm. Amongst these samples, SS03-FO confirmed the best absorbances within the studied ranges (λmax = 295 nm and 415 nm, respectively) (Fig. 1e).
The FB extracts (Fig. 1b and f) introduced distinctive traits, notably in samples from different genera than the Dictyota or Canistrocarpus genera. Amongst these extracts, the absorbance maxima had been distributed throughout the 290–500 nm vary, with maxima within the PG (λmax = 295, 312, 326, and 437 nm), LV (λmax = 450 nm) and SS (λmax = 297 nm) extracts, whereas the Dictyota (DI, DM, and DP) and Canistrocarpus (CCe and CCr) extracts introduced decrease absorbance maxima at roughly 430–450 nm, with no vital variations amongst them. Comparatively, there have been minimal variations between the FM and FW extracts (Fig. 1c, d, g, and h); nonetheless, they constantly yielded the bottom absorbances amongst all of the studied extracts.
In vitro photoprotection and antioxidant exercise assays
SPF, UVAr, and λc are parameters used to estimate the photoprotection capability of the extract in opposition to UVA and UVB radiation. Integration of those parameters into the early-stage seek for photoprotective compounds is proposed as a instrument to information the choice of promising extracts. The outcomes from the in vitro photoprotection and antioxidant exercise assays (Supplementary Desk S.2) had been concurrently analyzed by means of a heatmap, grouping samples on the idea of their similarities (Determine 2).
Heatmap illustration of the calculated in vitro solar safety issue (SPF at 50 ppm), UVA ratio (UVAr*), essential wavelength (λc), and antioxidant exercise (AOX) assays. FO/FB/FM/FW stands for extract sort. The numbers on the left differentiate the seven recognized clusters, and the numbers in purple point out the clusters with probably the most promising photoprotective exercise. * UVAr values correspond to the imply of three replicates at completely different concentrations, as no vital variations had been noticed between extracts on the evaluated concentrations.
The outcomes confirmed that extracts spontaneously grouped into seven clusters primarily based on their polarity. Clusters 5 to 7 grouped primarily FO extracts which displayed probably the most promising photoprotective exercise. Particularly, cluster 5 included the extract with the best recorded SPF worth (SS03-FO = 2915), whereas cluster 7 had the best UVAr worth (CCe-FO = 3623). All of the extracts inside clusters 5 and 6 demonstrated antioxidant exercise. Clusters 2 and three included FB and FW extracts with intermediate values for the studied photoprotection parameters, whereas cluster 1 included FM and FB extracts with no antioxidant exercise and the bottom SPF (DI01-FM = 0.146), UVAr (DI01-FM = 0.167), and λc (DI03-FM = 336) values. The λc parameter didn’t have a transparent most among the many evaluated extracts, however the larger values had been recognized in clusters 6 and seven, whereas the minimal values had been in cluster 1.
These outcomes mirror not solely the variations between extracts from completely different species but in addition the variations between extracts from the identical species obtained with a number of solvents. Whereas the primary one is an estimation of the chemical range of the evaluated samples, the second exhibits how the extraction process allowed extracts with completely different compositions to be obtained, a useful trait in additional purification steps. On this method, extracts positioned in clusters 5, 6, and seven had been prioritized with probably the most promising photoprotection exercise, yielding the choice of 17-FO and one FB extract.
The 18 preselected extracts and their triplicates (54 samples in whole) had been additional analyzed utilizing HPLC-DAD. Every chromatogram was baseline-corrected utilizing the Uneven Least Squares (AsLs) algorithm, fragmented into 28 retention-time variable fragments, and processed through MCR-ALS. With this system, chromatograms within the dataset had been concurrently corrected, aligned, and in contrast, acquiring pure spectra (UV–Vis spectra) and elution profile data for every detected part within the chromatographic dataset. This additionally affords retention time-shifts, coelution, and uneven peak phenomena, amongst different traits of HPLC-DAD chromatographic datasets39.
This evaluation resulted within the detection of 268 parts for the 28 segments within the 54 samples within the dataset. Particulars on the estimated variety of detected parts and figures of advantage for every fragment are introduced in Supplementary Desk S.3. The R2 (defined variance) and %LOF (% lack of match) values had been above 98% and fewer than 12% of the fragments, respectively. These values had been interpreted as acceptable for a lot of the fragments, contemplating the complexity and variations between samples and their replicates. The MCR-BANDS algorithm confirmed small variations between the utmost (fnmax) and minimal (fnmin) values of the relative contribution operate (fn) for every part, indicating that rotation ambiguities had been eliminated throughout the utilized constraints39,40.
After MCR-ALS evaluation, the outcomes had been retrieved in a relative focus matrix (RCM) comprising 18 samples (54 chromatograms) and 268 parts, every characterised by a UV-Vis spectrum between 200 and 800 nm. We utilized the rule proposed by Vinaixa and coworkers41 to tell apart between analytical and organic variations within the detected options, recognized as parts (N) with particular retention occasions (tr) and UV-Vis spectra. As proposed by the authors, options had been retained when the Coefficient of Variation Threshold (CVT) exceeded 20%, indicating that the variation was predominantly attributed to organic variations amongst samples. When this rule was utilized, no parts had been excluded from the dataset, and subsequent multivariate evaluation was carried out on the 54 X 268 RCM matrix.
Principal part evaluation (PCA), Determine 3, revealed that the primary two parts accounted for 60.4% of the variance, yielding an R2 worth of 0.853 and a Q2 worth of 0.502. The rating plot introduced in Determine 3a exhibits the clustering of the samples, with no vital variations amongst them. Nevertheless, inside this illustration, the three replicates of C. cervicornis (CCe-FO) didn’t cluster collectively, both due to modifications of their chemical composition or the quantity of the detected parts. In distinction, C. crispatus (CCr), a pattern belonging to the identical genus, had no such differentiation, grouping throughout the largest cluster with Dictyota, Sargassum, and Lobophora samples. Together with the talked about variations for C. cervicornis extracts, Hierarchical Cluster Evaluation (HCA) in Determine 3b revealed that every one S. zonale FO extracts (SS01-FO, SS02-FO, and SS03-FO) clustered collectively as a second differentiable group.
Principal Part Evaluation (PCA) of the relative focus matrix (RCM). (a) Rating plot, (b) Hierarchical cluster evaluation (HCA), (c) Loadings plot, and (d) UV–Vis spectra of chosen loadings. C: Canistrocarpus cervicornis (CCe) cluster; S: Stypopodium zonale (SS) cluster; O: different samples cluster. The pure spectra matrix (ST) was used to characterize the absorbance areas and shade the variables within the loadings plot: () (UV absorption; λ = 290–400 nm); (
) (seen absorption; λ = 400–800 nm); (
) (UV–Vis absorption; λ = 290–800). The black circumferences symbolize pattern clusters and essential variables for extract differentiation.
The pure spectra matrix (ST) was used to characterize the absorbance areas corresponding to every (N) part. This characterization was included into the loading plot, as depicted in Determine 3c. Variables exhibiting absorption within the UV area are represented in inexperienced, these absorbing within the seen area are denoted in blue, and variables absorbing within the UV and Vis areas are indicated in purple. The outcomes confirmed that variables which have a robust affect on the PCA parts and the situation of the observations within the Rating plot (e.g., 51, 159, 254, 220, 18) soak up primarily within the seen and UV-Vis areas, with absorption maxima at 270 nm and between 400 and 600 nm, as proven in Determine 3d. These findings recommend that these pigments are photosynthetic pigments with distinct distributions amongst samples.
Partial Least Squares (PLS) evaluation, Determine 4, integrates the chemical data obtained from HPLC-DAD metabolic profiling (RCM: 54 X 268) and the measured photoprotective exercise parameters (SPF, UVAr, λc, and antioxidant exercise) as outputs (Y-matrix: 54 X 4) within the evaluation. The Rating Plot (Determine 4a) exhibits three distinct teams: C. cervicornis (C), S. zonale (S), and different genera (D) extracts. As soon as once more, the C. cervicornis (Ce-FO) extract, on the one hand, and the S. zonale (SS-FO) extract, however, had been completely different from the opposite extracts, reflecting each larger photoprotection parameters and probably the most differentiable chemical profile. The VIP plot (Determine 4b) confirmed that 74 variables had been essential for this projection, every characterised by its retention time (tr) and UV–Vis spectrum. The principle essential variables for S. zonale extracts differentiation had been 81, with two absorption maxima within the UV area (tr = 19.0–19.9 min; λmax = 210 and 310 nm), and 107, with absorption maxima within the UV area (tr = 21.9-22.5 min; λmax = 204, 230, 250 and 296 nm) (Determine 4d). In C. cervicornis extracts, variables that affect their differentiation embrace 254 (tr = 40.0–43.0 min; λmax = 480, 524, and 648 nm), 220 (tr = 33.5–36.0 min; λmax = 472, 528, and 648 nm), and 18 (tr = 5.0–7.9 min; λmax = 476, 524, and 648 nm). To the identical extent, the loading plot (Determine 4c), confirmed that different essential variables in extract differentiation had been 127 (tr = 23.2–24.0 min; λmax = 432, 664 nm), 51 (tr = 15.5–17.3 min; λmax = 268, 452 nm), and 159 (tr = 24.6–25.3 min; λmax = 264, 340, 432, 624 and 660 nm).
Partial least sq. evaluation (PLS) of the relative focus matrix (RCM). The outputs (Y-matrix) corresponded to the measured in vitro photoprotection parameters (SPF at 50 ppm, UVAr*, λc, and antioxidant exercise). (a) Rating plot, (b) VIP plot for variables with VIP scores grater than 1 (essential variables), (c) Loadings plot, and (d) UV–Vis spectra of chosen variables (81, 107, and 127). C: Canistrocarpus cervicornis (CCe) cluster; S: Stypopodium zonale (SS) cluster; O: Different samples cluster. The pure spectra matrix (ST) was used to characterize the absorbance areas and shade the variables: ( ) (UV absorption; λ = 290–400 nm); (
) (seen absorption; λ = 400 -800 nm); and (
) (UV–VIS absorption; λ = 290–800). The black circumferences symbolize pattern clusters and essential variables for extract differentiation.* UVAr values correspond to the imply of three replicates at completely different concentrations, as no vital variations had been noticed between extracts on the evaluated concentrations.
Contemplating these variations, which grouped probably the most promising photoprotection exercise and probably the most differentiated chemical profile, C. cervicornis (CCe-FO) and S. zonale (SS01, SS03, and SS03 FO) extracts had been chosen for additional identification of the compounds accounting for the tracked photoprotection exercise.
Chemical compositions of the natural extracts of C. cervicornis (CCe) and S. zonale (SS)
Supplementary Determine S.1 exhibits the outcomes of the 1H-NMR evaluation of the C. cervicornis and S. zonale extracts. The spectra revealed that the extracts primarily consisted of fatty acids with attribute indicators at δH 1.27 for methylene protons in fatty acids, δH 2.1 for protons in allylic methylene, δH 2.3 for α methylene in carbonyl carbons, and δH 2.8 for protons of the methylene allylic to 2 double bonds. Downfield, there are much less intense indicators between δH 6 and seven and between δH 9 and 10.5 which can be related to pigments (chlorophylls42 and carotenoids43) within the extracts. As well as, indicators between δH 3 and 6 had been associated to the presence of terpenes44.
Fractionation of the CCe-FO extract by column chromatography allowed the restoration of 10 fractions. The photoprotection exercise assays for the fractions are introduced in Supplementary Determine S.2. The outcomes had been used to trace the exercise, indicating the best SPF values in fractions D09, D08, and D07 and the best UVAr worth noticed for fraction D08. The choice criterion didn’t embrace λc, as no vital variations had been noticed between the crude extract and its fractions.
The 1H- and 13C-NMR spectra of fractions D07 and D08 indicated the presence of pheophytin a45,46 (Supplementary Determine S.3 and Supplementary Determine S.4) as the main part, which was attributed to the noticed exercise in these fractions. Fraction D09 was subjected to preparative HPLC attributable to its abundance and vital worth in photoprotection exercise assays (Supplementary Determine S.2). This course of yielded 32 new fractions (D0901 to D0932) by which NMR evaluation revealed the presence of isolinearol (D0914; 1.7 mg; tr = 21.6–23.0 min; Determine 5a, Supplementary Determine S.5 and Supplementary Determine S.6)47,48, and fucoxanthin (D0924; 3.0 mg; tr = 40.5–41.8 min; Determine 5b, Supplementary Determine S.7 and Supplementary Determine S.8)49 with absorbance maxima at 210 nm and 470 nm, respectively.
Fucoxanthin was remoted as an orange strong, that was subjected to additional photoprotection exercise assays, as introduced in Fig. 6. These outcomes present that fucoxanthin is the compound that accounts for the tracked photoprotective exercise of the CCe-FO extract and demonstrates the potential for photoprotection throughout the UV area and safety in opposition to seen gentle, which isn’t measured with classical in vitro photoprotection exercise assays however is essential due to the unfavourable results of this radiation on pores and skin well being5,8.
Isolinearol was recognized by comparability of its 1H- and 13C-NMR spectra with these beforehand reported by Texeira and coworkers47. This secodolastane diterpenoid is taken into account a chemotaxonomic marker for the Dictyota/Canistrocarpus genera50 and particularly for Canistrocarpus cervicornis within the tropical Atlantic American area51.
Then again, the S. zonale (SS02-FO) extract was fractionated by column chromatography yielding 17 fractions (S01 to S17). The outcomes for the photoprotection exercise of those fractions are introduced in Supplementary Determine S.2. The info steered that the best photoprotection exercise was concentrated in fractions S06, S07, and S11, as proven by the SPF values, whereas the best photoprotective exercise in opposition to UVA was present in fraction S06. The λc values weren’t thought-about, as each the crude extract and the fractions introduced comparable values. 1H- and 13C-NMR spectral evaluation allowed the identification of pheophorbide a52 (Supplementary Determine S.9) and fucosterol53 in fraction S06.
Fraction S07 was separated by preparative HPLC, contemplating the upper values in photoprotection exercise assays (Supplementary Determine S.2). The outcomes yielded 6 new fractions (S0701 to S0706). NMR evaluation revealed the presence of compound 1 in fraction S0705 (10.1 mg; tr= 17.8-20 min; Determine 5c, Supplementary Determine S.10, Supplementary Determine S.11, Supplementary Determine S.12, Supplementary Determine S.13, and Supplementary Determine S.14) and stypodiol in fraction S0703 (2.7 mg; tr = 10–10.3 min; Determine 5d, Supplementary Determine S.15 and Supplementary Determine S.16)54, with absorption maxima at 207, 227, 287, and 320 nm, and 205 nm, respectively. Stypoldione was recognized in fractions S08 and S0955,56 (Determine 5e, Supplementary Determine S.17 and Supplementary Determine S.18), fucoxanthin was the principle compound in fraction S10, and fatty acids constituted fractions S11 and S12.
Compound 1 was remoted as an off-white strong, with a molecular system of C24H38O5 primarily based on its (+) HRESIMS information calculated for the protonated molecular ion [M+H]+ at m/z 407.2789 (calculated for C24H39O5+ at m/z 407.2792, Δ 0.74 ppm). The 13C-NMR spectrum confirmed the presence of 20 indicators, together with one quaternary carbonylic carbon (δC 197.16), three sp2 oxygenated quaternary carbons (δC 168.17, 165.0, and 162.17), one hemiacetal carbon (δC 103.62), one sp2 quaternary carbon (δC 103.09), two sp2 methines (δC 96.88, 96.68), one sp3 methylene (δC 46.00), one sp3 methyl (δC 14.46) and sp3 aliphatic methylenes attribute of an aliphatic chain (between δC 41.98 and δC 24.76). The 1H-NMR spectrum steered the presence of an aliphatic methyl proton (δH 0.88), three protons of aliphatic carbons (δH 1.27, 1.47, and 1.85), two protons of cyclic methylenes (δH 2.60, 2.91), two fragrant protons (δH 5.82, 6.00) and two fragrant hydroxyl protons (δH 10.22, 11.94). (+) HRESIMS information (Supplementary Determine S.19) was used to establish the C-15 facet chain size of compound 1. As well as, the HRESIMS/MS spectra of 407.2789 confirmed ions at 153.0185 (C7H5O4+, Δ 1.31 ppm) and 127.0395 (C6H7O3+, Δ 3.94 ppm), confirming the proposed construction. The primary ion corresponds to fragmentation between C2 and O1, C3 and C4, and the oxidation of the alcohol in C5, whereas the second ion corresponds to the fragmentation between C10 and C4, and between C2 and O1 (Supplementary Determine S.19).
These spectroscopic information are equivalent to these reported for two,5,7-trihydroxy-2-pentadecylchroman-4-one (spiralisone C), which was beforehand remoted from the brown alga Zonaria spiralis57, and the plant seeds of Syzygium cumini58. Chroman-4-ones are essential heterobicyclic secondary metabolites with numerous reported organic actions together with anticancer, antioxidant, antimicrobial, and antifungal actions [58,59]. That is the primary report of a chromanone within the Stypopodium genus, though the corresponding chromones have been beforehand recognized within the brown algae Zonaria tournefortii59, Stypopodium flabelliforme60, and Stypopodium zonale61 (Phaeophyceae). As mentioned by Kamboj and Singh (2020), chromones and chromanones are structurally associated, however the absence of the C2–C3 double bond in chromanones ends in completely different organic actions.
The outcomes of additional photoprotection exercise assays of pure compound 1, proven in Determine 6, revealed the best SPF values and the bottom UVAr values from all of the remoted compounds, in addition to a λc at 335.9 nm. These outcomes are comparable with these of BP-3, which was used as a constructive management, indicating its potential as a UVB filter. In distinction, the UVAr and λc values for compound 1 are decrease than these reported for pheophytin a, pheophorbide a, and fucoxanthin, which introduced higher UVA photoprotection and demanding wavelengths that classify them as broad-spectrum filters. These variations spotlight the potential of utilizing mixtures of the remoted compounds in sunscreen formulations to supply full UV photoprotection.
Outcomes of the in vitro photoprotection exercise assays for benzophenone-3 (BP-3), pheophytin a, fucoxanthin, 2,5,7-trihydroxy-2-pentadecylchroman-4-one (compound 1), and pheophorbide a recognized in C. cervicornis (CCe-FO) and S. zonale (SS02-FO) natural extracts. (a) SPF (Solar Safety Issue); (b) UVAr (UVA ratio); (c) λc (essential wavelength). The error bars symbolize the usual deviation (For SPF, n = 3, imply of three replicates at every focus. For UVAr, n = 3, imply of three replicates at completely different concentrations as no vital variations had been noticed between extracts on the evaluated concentrations).
UV-Vis spectra of the VIP loadings recognized in PCA and PLS evaluation in Determine 3 and 4, had been in contrast with these of the recognized compounds, and it was noticed that fucoxanthin corresponded to variable 51. The Relative Focus Matrix of the extracts (Supplementary Determine S.20) revealed that despite the fact that this variable was detected within the natural extracts of all of the species, the C. cervicornis (CCe) and S. polyceratium (SP) extracts introduced comparatively excessive relative concentrations of this compound. This explains the extract differentiation and the upper UVAr values present in these FO extracts. Equally, the UV-Vis spectra of variables 127 and 159 correspond to chlorophyll a derivatives equivalent to pheophytin a and pheophorbide a which had been recognized in extracts with the best UVAr values. These variables had been detected in a lot of the samples, with excessive concentrations in DP02, PG, and DM, and CCe, LV, and SP, respectively. For the S. zonale extracts, variable 81 corresponds to compound 1, and variable 33 corresponds to stypoldione, with absorption maxima within the UVC and UVB areas. These variables have a restricted distribution amongst samples, with S. zonale (SS01, SSS02, and SS03) having the best concentrations, accounting for this extract differentiation and the best SPF values discovered for these FO extracts.
Briefly, a photoprotective exercise screening methodology was carried out for the analysis of algae extracts. This strategy integrates in vitro photoprotection analysis, antioxidant TLC exercise assays, and HPLC-DAD metabolic profiling. Algal samples with promising photoprotective exercise and distinct metabolic profiles had been chosen, and photoprotective exercise was monitored till chemical characterization of the lively compounds. This work represents one of many first reported methodologies aiming to discover algae range as a supply of photoprotective compounds, utilizing extensively used and reasonably priced methodologies in pure product analysis laboratories. This technique could possibly be additional carried out for steady screening, monitoring, and comparability of photoprotective exercise in several algal species.