Phenolic Profile, Toxicity, Enzyme Inhibition, In Silico Studies, and Antioxidant Properties of Cakile maritima Scop. (Brassicaceae) from Southern Portugal

Cakile maritima Scop. (sea rocket) is an edible halophyte plant with several ethnomedicinal uses. This work reports the chemical profile and bioactivities of food grade extracts from sea rocket organs. Toxicity was determined on mammalian cells, and phenolic profiling and the quantitation of the main metabolites were made by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Enzymatic inhibition was determined towards acetyl- and butyrylcholinesterase (AChE, BuChE), α-glucosidase, α-amylase, and tyrosinase. Docking studies were performed to tyrosinase, on the major metabolites, and samples were tested for antioxidant properties. Extracts were not toxic, were constituted mainly by flavonoids, and some compounds (roseoside and oleuropein) are here described for the first time in the species. The aerial organs’ ethanol extract had relevant activity towards 2,2-diphenyl-1-picrylhydrazyl [DPPH, half maximal inhibitory concentration (IC50) = 0.59 mg/mL], and ferric-reducing activity power (FRAP, IC50 = 0.99 mg/mL). All samples were more active towards AChE than on BuChE. The ethanol fruits’ extract inhibited α-glucosidase [2.19 mmol of equivalent of acarbose (ACAE)/g]. Samples were active against tyrosinase, especially the aerial organs’ ethanol extracts [25.9 mg of equivalent of kojic acid (KAE)/g]. Quercetin and kaempferol glycosides fit well into the enzymatic pocket of tyrosinase. Our results suggest sea rocket as a candidate to be further explored as a source of bioactive products.


Introduction
Research on bioactive natural products has focused mainly in marine organisms, such as microand macro-algae and marine invertebrates, and in terrestrial glycophyte plants. Halophytes, which are naturally salt-tolerant plants, represent about 2% of terrestrial plant species and are present in about half the higher plant families. They exhibit a high diversity of plant forms and are currently Results are expressed as the mean of the % of viability relative to a control containing methanol (0.2%, v/v) ± standard error of mean (SEM), n = 6. For the same column, different letters are significantly different (Multiple Comparisons of Means: Tukey Contrast, 95% family-wise confidence level).

Chemical Composition of the Extracts: Qualitative Profiling
The phytochemical qualitative profile of sea rocket's extracts is depicted in Table 2. Compounds were numbered by their order of elution keeping the numbering constant in all extracts, and were identified using analytical standards and bibliographic information ( Table 2). The chromatogram obtained for the water extract of sea rocket's fruits is shown in Figure 1, as an example. The chemical composition of the extracts depended on the type of extract and organ (Table 2).
Twenty-five compounds were characterized in the analyzed extracts, most of them quercetin glycosides (compounds 10, 11, 12, 15, 18, and 21) and kaempferol glycosides (compounds 13, 14, 16, 24, and 25) ( Table 2, Figures 1 and 2). Both types of compounds were generally present in both types of organs, and for all of them, the aglycones were observed at m/z 301 for quercetin (fragment ions at m/z 271, 179, and 151) and m/z 285 for kaempferol (fragment ions at m/z 255 and 151). The neutral losses of 308, 162, and 146 Da were indicative of rutinoside, hexoside, and deoxyhexoside moieties, respectively.     Tables 2 and 3.  Tables 2 and 3.  Quercetin glycosides and kaempferol glycosides are flavonoids, a group of compounds associated with several health-promoting effects, like preventing cardiovascular complications and cancer, and are important ingredients in several products, including nutraceuticals, cosmetics, and medicinal commodoties [19]. Quercetin derivatives, such as glycosides, are present in different plant-derived food foodtuffs like tea, wine, onions, apples, and broccoli, and can prevent and treat non-infectious chronic diseases, such as diabetes, obesity, and hyperlipidemia [20,21]. Kaempferol glycosides also display interesting biological properties, including cytotoxicity toward human cancer cells, free radical scavenging activity, and inhibition of pancreatic lipase [22]. Quercetin and its derivatives, including quercetin-3-O-rhamnoside, were previously identified in aerial parts of sea rocket [3,19]. Likewise, kaempferol and kaempferol glycosides Quercetin glycosides and kaempferol glycosides are flavonoids, a group of compounds associated with several health-promoting effects, like preventing cardiovascular complications and cancer, and are important ingredients in several products, including nutraceuticals, cosmetics, and medicinal commodoties [19]. Quercetin derivatives, such as glycosides, are present in different plant-derived food foodtuffs like tea, wine, onions, apples, and broccoli, and can prevent and treat non-infectious chronic diseases, such as diabetes, obesity, and hyperlipidemia [20,21]. Kaempferol glycosides also display interesting biological properties, including cytotoxicity toward human cancer cells, free radical scavenging activity, and inhibition of pancreatic lipase [22]. Quercetin and its derivatives, including quercetin-3-O-rhamnoside, were previously identified in aerial parts of sea rocket [3,19]. Likewise, kaempferol and kaempferol glycosides were already detected in the same species, including kaempferol 7-O-rhamnoside, kaempférol 4 -O-glucoside, and kaempférol3-O-glucoside de 7-O-rhamnoside [23].
Compound 1 suffered the neutral loss of 36 Da (HCl) to yield a disaccharide (two hexoside moieties) at m/z 341. Its characterization was based in the neutral loss of 162 Da and the fragment ions characteristic of hexoside moieties. Compound 7 was also tentatively characterized as a hexoside derivative.
Two glucosinolates-isomers of dihydrogluconapin (compounds 2 and 5)-were characterized based on bibliographic information [24], and were present in both organs. Glucosinolates are naturally occurring S-linked glucosides characteristic in Brassicaceae species [25]. They originate different metabolites by enzymatic hydrolises, including isothiocyanates, which are likely linked to antimicrobial properties [25]. To our best knowledge, these compounds have not been previously reported in sea rocket.
Compound 8 was characterized as roseoside ( Figure 2; formate adduct), also known as vomifoliolglucoside or drovomifoliol-O-β-D-glucopyranoside, and was only detected in the extract from aerial organs. Roseoside is a megastigmane glycoside, is present in different plant species, such as Ludwigia stolonifera [26], and is first described here in sea rocket. This compound has several biological properties, including in vitro and in vivo anticarcinogenic and in vitro hypertensive [27].
Compound 20 was identified as oleuropein by comparison with an analytical standard. It was only detected in fruits and is firstly described here in the species. Oleuropein is a secoiridoid, one of the major compounds present in the olive fruit, and is endowed with several functional properties, such as anticancer, antibacterial, antifungal, antiparasitic, and antiplatelet aggregation [28].
Compound 22 suffered the neutral loss of 224 Da (591→367), which corresponds to sinapic acid. In addition, the MS 3 [591→223] fragmentation correlates with sinapic acid. We tentatively characterized this compound as disinapoyl-hexoside, which was only detected in the aerial organs. Similarly, compound 23 was characterized as trisinapoylgentiobioside, and was only present in fruits. Disinapoyl-hexoside and trisinapoylgentiobioside are sinapic acid derivatives, characteristic in the Brassicaceae family [29]. The most important characteristic property of sinapic acid derivatives is their antioxidant capacity, and thus, have potential use in in the food processing, cosmetics, and the pharmaceutical industry [30].
Compounds 27 and 28 were present in both organs and were characterized as the oxylipins oxo-dihydroxy-octadecenoic acid and trihydroxy-octadecenoic acid, respectively. Oxylipins are secondary metabolites resulting from the oxidative metabolism of polyunsaturated fatty acids (PUFAs) through the addition of oxygen. The production of these compounds increases in response to herbivory, pathogenesis, and wounding, and therefore, several oxylipins add for the regulation of innate defense-related and developmental processes in plants, having, for example, antimicrobial or insecticidal properties [31].

Chemical Composition: Quantitative Profiling of Phenolic Compounds
The phytochemical quantitative profile of sea rocket's extracts is presented in Table 3. Twelve phenolic compounds were quantified in the extracts of sea rocket: ten flavonoids, oleuropein, and disinapoyl-hexoside. Total individual phenolic content (TIPC) was defined as the sum of all the individual compounds that were quantified by HPLC with diode array detection (HPLC-DAD): flavonoids at 350 nm, oleuropein at 240 nm and disinapoyl at 320 nm. The aerial organs' extracts presented higher TIPC (acetone: 3.94 mg/g dried extract (DE), water: 7.92 mg/g DE and ethanol: 11.5 mg/g DE) than the fruits' extracts (acetone: 1.28 mg/g DE, ethanol: 4.42 mg/g DE and water: 5.48 mg/g DE). The total phenolic content of leaves of sea rocket was assayed elsewhere by a spectrophotometric assay [30], showing values of 5 to 7 mg gallic acid equivalent per g DE, which are similar to the ones obtained here by liquid chromatography. In all extracts, the majority of the content corresponded to flavonoids (mainly quercetin and kaempferol glycosides), which represented more than 90% of TIPC. This predominance of flavonoids is in agreement with a previous report on C. maritima [16]. Quercetin glycosides were present in similar amounts in both organs while kaempferol glycosides were generally higher in the aerial organs. For both compounds, the highest concentrations were found in ethanol and water, whereas acetone extracts presented the lowest concentrations in all cases.

Enzyme Inhibitory Activities
Molecules from natural sources have a high resemblance and binding potential with biological structures, increasing the odds of an interaction with different biological targets [32]. Those targets includes for example enzymes related with human diseases, such as AChE and BuChE for Alzheimer's disease (AD), α-glucosidase and α-amylase for Type 2 Diabetes Mellitus (T2DM), and tyrosinase, for hyperpigmentation disorders. Several drugs with enzymatic inhibitory properties towards such enzymes are currently in clinical use to treat the mentioned disorders (e.g., acarbose for T2DM, galantamine for AD, and kojic acid for hyperpigmentation). Nevertheless, their potential undesirable side effects (e.g., gastrointestinal disturbances and hepatotoxicity) promotes a growing interest by the pharmaceutical and cosmetic industries to identify innovative, more efficient, and less toxic natural enzymatic inhibitors. In this work, we evaluated the enzyme inhibitory effects of the sea rocket extracts towards the above-mentioned enzymes, and the results are depicted in Table 4. Table 4. Enzymatic inhibitory activity on acetyl-(AChE), butyrylcholinesterase (BuChE), α-glucosidase, α-amilase and tyrosinase, of different extracts prepared from aerial vegetative organs and fruits of sea rocket (Cakile maritima) *. Results are expressed as IC 50 values (mg/mL). Samples had similar capacities to inhibit AChE (aprox. 1.3 mg GALAE/g), except the water extracts from aerial vegetative organs, with reduced activity (0.53 mg GALAE/g), but were less active towards BuChE (from 0.26 to 0.92 mg GALAE/g). AD is one of the major causes of dementia. It is a multifactorial disease whose progression is linked to a considerable loss of the neurotransmitter acetylcholine (ACh), which is responsible for memory and cognition, to the accumulation of β-amyloid protein and to the formation of neurotoxic neurofibrillary tangles. The inhibition of AChE not only adds to the maintenance of normal ACh levels, but also decreases β-amyloid accumulation and neurotoxic fibrils formation, being considered as the main therapeutic approach to manage AD symptoms. To the best of our knowledge, this is the first report of the cholinesterase inhibitory properties of sea rocket.

Organs
The extracts had low inhibitory activity towards α-amylase (0.02 to 0.26 mmol ACAE/g extract). The same was observed for α-glucosidase, except for the ethanol extract from fruits that displayed a significant inhibition (2.19 mmol ACAE/g). A similar result towards the latter enzyme was observed for hydroethanolic stems extracts from the same species [10]. Our results suggests that the the highest α-glucosidase inhibitory activity displayed by the ethanol extract from fruits may be related with other compounds, rather than those identified in this work. While α-amylase hydrolyzes polysaccharides to oligosaccharides, α-glucosidase hydrolizes oligosaccharides to monosaccharides. Therefore, inhibition of these enzymes decreases the digestion of carbohydrates and the postprandial rise of blood glucose levels after a mixed carbohydrate meal, and is an important therapeutical instruments for the control of T2DM [33][34][35][36]. Moreover, hypoglycemic products with slight amylase inhibitory activity but significant glucosidase inhibitory properties are favored, as an excessive inhibition of amylase often results in gastrointestinal disorders [37]. Therefore, our results suggests ethanol extracts of sea rocket's fruits as candidates to further studies exploiting its possible antidiabetic assets.
Aditionally, all tested samples, except for water extract from fruits, showed a potent inhibition of tyrosinase. The best results were observed with extracts from the aerial vegetative organs, with values ranging from 19.9 mg KAE/g (water extracts), to 25.9 mg KAE/g (ethanol). Tyrosinase is a copper containing enzyme involved in melanogenesis and therefore, it is a key target in the search for depigmenting agents that can be used in the treatment of epidermal hyperpigmentation disorders, such as lentigo, melasma, age spots, inflammatory hypermelanosis, and trauma-induced hyperpigmentation [38][39][40]. Moreover, tyrosinase catalyzes the oxidation of phenolic compounds to quinones, which results in the enzymatic browning of plant-derived foods [41]. Quinones have an unpleasant flavour and color and react with proteins, reducing its digestibility and the bioavailability of essential amino acids, with consequent nutritional and economical losses during food storage [41,42]. Tyrosinase is also involved in melanogenesis and cuticle formation in insects [43]. Hence, the use of tyrosinase inhibitors as food preservatives, especially from natural sources, is therefore considered as an alternative approach not only to prevent the enzymatic browning of plant-derived foods but also in insect pest control [41]. The highest tyrosinase inhibition observed after treatment with the extracts from aerial organs of the studied species can be related with their higher content in phenolic compounds, since it is known that such molecules can significantly inhibit this enzyme [43].

Molecular Modeling
The inhibition assays performed on the extracts (Section 2.4) have shown a solid anti-tyrosinase activity. Among the substances identified by HPLC, and by comparison of the concentrations and their relative presence in the samples, we have selected four compounds, namely quercetin-di-Hex-dHex (11), quercetin-Hex-dHex (12), Kaempferol-Hex-Rut (13), and kaempferol-dHex-Hex (14), which have been subjected to molecular docking and prooved for their ability to interact with the tyrosinase enzymatic cavity (Figures 2 and 3). The in silico experiments have shown that these substances are able to fit well into the enzymatic pocket of tyrosinase and bind to several residues. The presence of the sugar moieties on the four selected molecules enhances the capacity to interact with the copper atoms present in the catalytic cavity of the tyrosinase. On the other hand, the size of the substance could create a steric hindrance which does not allow to obtain a perfect insertion of the substances in the deep of the enzymatic pocket, considering the fact that the tyrosinase should recognize phenol-like substances.
Compounds 12, 13, and 14 are all able to interact with the Cu atoms of tyrosinase, thus these molecules are potentially able to strongly inhibit tyrosinase, whereas compound 11 has the lowest gold score (ChemScore), and the lowest calculated ∆G, probably due to the steric hindrance of the di-glucoside group present in the structure. Compound 13 has the best score (15.44) and the best ∆G (-25.98) and it is able to bind to Cu401, and to establish an H bond to Arg268 and stacks to His61 and His85, which are key residues of the enzyme. Compounds 12 and 14 are also able to bind respectively to Cu401 and Cu400, and to establish several hydrogen bonds and stacks; however, their scores and ∆G are lower than compound 13 probably due to steric factors (for detailed interactions see Table 5).  Taking the scores into consideration, the ∆G energy values and the ability to bind to the Cu atoms of tyrosinase, it can be hypothesized that compunds 12-14 may play a role in the anti-tyrosinase activity of the extracts. Also, it must be taken into consideration that the ∆G binding energy of the reference compounds, kojic acid and tropolone, are significantly higher (respectively −30.11 and −28.62 Kcal/mol) than that of compound 12-14. Also, the chemscore values of the reference compounds are the higher of this series (Table 5). Moreover, considering that the correlation between the relative concentration of these substances and the anti-tyrosinase activity of the extracts is not linear, and that the docking experiments revealed medium-weak affinity for the tyrosinase enzyme, the total inhibitory activity found for the extracts might be influenced by the concomitant presence of other bioactive substances with synergistic or additive activity.

In Silico Absorption, Distribution, Metabolism, and Excretion (ADME) Evaluation
The ADME parameters of compounds 11-14 were predicted computationally, and results are depicted in Table 6. Acording to the obtained data, these molecules exhibit low oral absorption, no activity in the central nervous system (CNS), and, in general, unfavorably pharmacokinetic properties, as it could be expected from isolated flavonoids. In Section 2.4, it was observed that the studied extracts from sea rocket exhibited low to moderate activity towards enzymes related with the onset of neurodegenerative disorders, namely AChE and BuChE, and on T2DM, specifically α-glucosidase and α-amylase. This is supported by the ADME studies, that unravelled the poor pharmacokinetic properties of the main molecules present in such extracts, coupled with limited oral availability and nil activity in the CNS. Samples showed, however, strong inhibition towards tyrosinase, which is implicated in hyperpigmentation disorders, thus suggesting that they could be useful for external uses, such as for dermatological aplications. In fact, it is well known that phenolic glicosides, such as those identified in the sea rocket extracts, are well absorbed into skin dermis and have wide applications for cosmetic and dermatological purposes [44]. However, the final effect of orally administered extracts should also consider the biotransformations that may occur in the GI tract by enzymes and microbiota, thus, it is not completely possible to run out other possible systemic effects.  50 (concern below −5).

Antioxidant Activity
As a general rule, plants contain phenolic compounds with antioxidant properties, with potential applications as health promoters (e.g., to prevent oxidative stress-related diseases such as cardiovascular disorders and cancer), or as food additives (e.g., in food processing to improve the conservation processes and to extent shelf life). In this work, the extracts were appraised for in vitro antioxidant properties by five assays, and the results are summarized in Table 7. Table 7.
Radical scavenging on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals, ferric reducing antioxidant power (FRAP), copper chelating (CCA), and iron chelating activities (ICA) of different extracts prepared from aerial vegetative organs and fruits of sea rocket (Cakile maritima). Results are expressed as IC 50 values (mg/mL). Samples had, in general, nil to low antioxidant activity, and only the ethanol extract from the aerial organs had shown RSA towards the DPPH radical (IC 50 = 0.59 mg/mL), and ferric reducing capacity (IC 50 = 0.99 mg/mL). The higher activity detected in this extracts is likely due to its higher TIPC. Moreover, no relevant differences in antioxidant properties were observed between extracts from the different organs. The DPPH RSA of the aerial organs' ethanol extracts from sea rocket presented in this work is in accordance with the values reported for methanol extracts from leaves of cultivated sea rocket plants in Tunisia, where IC 50 values of at least 0.6 mg/mL were obtained, depending on the irrigation salinity [12]. However, a significant antiradical activity towards DPPH was detected in 80% methanol extracts from shoots of cultivated sea rocket plants, in Tunisia, in vegetative and flowering stages, with IC 50 values ranging from 240 to 650 µg/mL (depending on harvesting period and irrigation salinity [13]). A high capacity to scavenge the DPPH radical was also observed for ethanol extracts from leaves, stems, and flowers of the same species [3]. Additionally, a significant activity on 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) (IC 50 = 0.144 mg/mL) was observed in water-methanol extracts from sea rocket leaves, collected in Brittany (France) [14]. The differences between our results and other reports can be related with genetics and environmental factors (e.g., salinity), along with the tested extract, all factors known to significantly influence the chemical composition of plants, and therefore, its antioxidant potential.

Plant Material
Sea rocket plants (voucher number XBH42) were collected in Faro Beach, South of Portugal (coordinates: 37 • 0 0.163" N, −7 • 9 86.070" W), in July of 2018. Plants were divided into aerial vegetative organs (leaves and stems) and mature fruits. Samples were cleaned from external contaminations with tap water, dried in an oven for 3 days at 50 • C, powdered, and stored at −20 • C until analysis.

Preparation of the Extracts
Extracts were prepared by an ultrasound-assisted extraction method using food grade solvents. In brief, dried biomass was mixed with ethanol, acetone, and water (1:40, w/w), and extracted for 30 min in an ultrasonic water bath, at room temperature (RT, circa 20 • C). Organic extracts were filtered (Whatman paper no. 4) to remove solid debris, and the solvent was completely removed under reduced pressure at 40 • C, in a rotary evaporator. Water extracts were freeze-dried. The obtained dried extracts were weighed, dissolved in methanol at the concentration of 50 mg/mL, and stored at −20 • C until analysis.

Cell Culture
To determine the in vitro toxic effects of the extracts, the following mammalian cell lines were used: RAW 264.7 macrophages (murine, provided by the Faculty of Pharmacy and Centre for Neurosciences and Cell Biology, University of Coimbra, Portugal), HEK 293 (human embryonic kidney, provided by the Functional Biochemistry and Proteomics group, Centre of Marine Sciences, Portugal), and HepG2 (human hepatocellular carcinoma, Marine Molecular Bioengineering groups, Centre of Marine Sciences, Portugal). RAW cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 culture media, while HEK 293 and HepG2 cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) culture media, both supplemented with 10% heat-inactivated FBS, 1% L-glutamine (2 mM), and 1% penicillin (50 U/mL)/streptomycin (50 µg/mL), and were kept at 37 • C in a moistened atmosphere with 5% CO 2 .

Citotoxicity
Cells were seeded in 96-well microplates in different densities, according to the cell line, as follows: RAW: 1 × 10 4 cells/well, HEK and HepG2: 5 × 10 3 cells/well. Cells were left to adhere for 24 h and treated with the extracts at the concentration of 100 µg/mL for 72 h. Control cells were treated with methanol at the highest concentration used in the treatments (0.2%). Cellular viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colourimetric assay [45]. Results were expressed in terms of cellular viability (%).

Identification and Quantification of Phenolic Compounds by HPLC-MS
The phenolic fraction of the extracts was characterized by resorting to an HPLC Agilent 1100 Series with a G1315B diode array detector. A Luna Omega Polar C 18 analytical column of 150 × 3.0 mm and 5 µm particle size (Phenomenex), with a Polar C 18 Security Guard cartridge (Phenomenex) of 4 × 3.0 mm, was used. The HPLC system was connected to an ion trap mass spectrometer (Esquire 6000, Bruker Daltonics) equipped with an electrospray interface operating in negative mode. Detailed conditions are reported elsewhere [46]. Compounds identification was carried out based on analytical standards and mass spectra, whereasultra violet (UV) spectra were used for quantification purposes.

Cholinesterase Inhibition
The cholinesterase inhibition potential of the extracts (0.5, 1, and 5 mg/mL) was evaluated by the Ellman's method towards AChE and BuChE, as described in Zengin [47]. Galantamine (0.5-5 mg/mL) was used as the standard and results were expressed as the equivalent of galantamine (mg GALAE/g extract).

Tyrosinase Inhibition
The tyrosinase inhibition assay was performed as described by Zengin [47], on the samples at concentrations between 0.5 and 5 mg/mL. Kojic acid was used as standard inhibitor (at the same concentration as the samples) and results were presented as the equivalent of kojic acid (mg KAE/g).

Alpha-Amylase and α-Glucosidase Inhibition
The α-amylase and α-glucosidase inhibition capacity was evaluated according to the method reported by Uysal et al. [48]. Samples' concentration were between 0.5 and 5 mg/mL and acarbose was used as standard (at the same concentration as the samples). Results were expressed as the equivalent of acarbose (mmol ACAE/g).

Receptor Preparation
The extracts reported in this paper have shown a considerable anti-tyrosinase activity. To study the possible interaction between selected molecules identified in the extracts and the tyrosinase catalytic pocket, a computational study by molecular docking was conducted. The in silico part of this work was oriented through the metalloenzyme tyrosinase. The crystallographic structure of mushroom tyrosinase (pdb: 2Y9X) [49] has been downloaded from the Protein Data Bank (PDB). Tropolone is co-crystallized in this structure, thus it has been used as a guide to delimitate the enzymatic cavity. The enzyme was cleaned as previously reported. Non-catalytic water was also removed, not being involved in the catalytic mechanism, and the enzyme was neutralized at pH 7.4 by the module PropKa of Maestro. All the errors of the crystal were fixed automatically be the PrepWizard tool embedded in Maestro 10.2 suite [50].

Ligands Preparation
Considering the interesting anti-tyrosinase activity found for almost all the extracts, the effort was focused on four flavonoids, selected as the most representative polyphenols compounds, namely The chemical structures of compound 11-14 were drawn by ChemDraw 14 and then the two-dimensional (2D) structures were converted into three-dimensioanl (3D) by Maestro and the chirality was carefully checked. The module LigPrep was used to neutralize the molecules at pH 7.4 and the module ionizer was used to minimize the energy of the structure by applying the force field OPLS3 [51].

ADME Estimation
After the preparation of the structures of compound 11-14 by the Schrodinger 2015-2 package, ADME of chemicals were computed by QikProp [52], implemented in the Schrodinger package as previopusly performed by our group [53] 3.7.4. Self-Docking and Docking Method Validation Following a previously reported paper by Stefanucci et al., the docking software Gold was tested for validation [54]. At this stage, the software GOLD 5.5 was configured for self-docking involving the crystallographic tyrosinase inhibition-enzyme complex (2Y9X), as described above. All the scoring functions of GOLD (ASP, PLP, GOLDSCORE, and CHEMSCORE) were considered to conduct the validation tests, performing a self-docking of the crystallographic ligands, and by comparing the root-mean-square deviation (RMSD) of the best docked crystallographic pose with the original crystallographic pose. An area of 15 Å around the co-crystallized ligand was defined as the binding site. At the end of the docking calculations, the ChemScore scoring function returned with the lowest RMSD value. Both ASP and PLP methods were not able to find any interactions for tropolone with the copper atomes, thus have been discarded. On the other hand, both Goldscore and Chemscore were able to find a similar pose of tropolone superimposable to the crystallographic one with similar RMSD in the range of 2-3 angstorms. The best RMSD value self-docking of tropolone was obtained by using the Chemscore methods (RMSD = 2.33 Å), which was considerate good following the data reported in other works e.g., References [55,56]. The self docking also revealed that the best pose is similar to the crystallographic pose of tropolone cocrystallized to the tyrosinase-like 1 enzyme [57].
Moreover, further docking experiments were performed on the chemical standard, used in this work to calculate the inhibitory potency, namely kojic acid. Kojic acid is indeed considered a powerful inhibitor of tyrosinase. The docking was performed in a similar manner as for tropolone and the G-binding and chemscore values obtained were compared to those of the other selected substances and are reported in Table 5. A similar study has been already conducted by Nokinsee et al. [58]. In this study, a comprehensive in silico predictive study on the tyrosinase inhibitors by comparing the binding data and the IC 50 value for kojic acid and tropolone and other substances has been reported. It has been found that tropolone has a binding energy lower than that of kojic acid, also in agreement with the experimental IC 50 values of tropolone (IC50 = 0.4 µM) and of kojic acid (IC 50 = 7.4 µM).
Our results are reported in Table 5 and are in full agreement with previously reported data [58]. After this validation process, the docking experiments were carried out also on compounds 11-14 and for a better understanding of the results, were compared with the results of tropolone and kojic acid.

Molecular Docking
Dockings of the selected molecules were performed on the prepared tyrosinase structure by using the software Gold 5.5, developed by the Cambridge University [59]. ChemScore scoring function was employed for the docking calculations. The docking area was determined by centering the grid on the crystallographic inhibitor tropolone and extended in a radius of 15 Angstroms around the ligand center, similarly to the previous published works [60][61][62]. The best pose for each compound docked to tyrosinase was selected and depicted in Figures 2 and 3. The binding energy and the chemscore values are reported in Table 5.

In Vitro Antioxidant Properties
Extracts were evaluated for in vitro antioxidant properties, at six different concentrations, ranging from 10 to 2000 µg/mL. Assays were performed in 96-well flat bottom microtitration plates, and absorbances were read on a multi-plate reader (EZ read 400, Biochrom) (Cambridge, UK). Results were expressed as a percentage of inhibition relative to the negative control containing methanol, and as IC50 values (mg/mL) whenever possible.

Targeting Free Radicals: Radical Scavenging Activity (RSA) on DPPH and ABTS Radicals
The RSA on DPPH and ABTS radicals was performed as decribed previously [10]. The synthetic antioxidant compound BHT was used as positive control, in the same concentrations as the samples. CCA and ICA were determined as described elsewhere [63]. The synthetic metal chelator, ethylenediaminetetraacetic acid (EDTA), was used as the positive control, in the same concentrations as the extracts. Samples were also tested for FRAP [11], where an increase in samples' absorbance represents an increase in the reducing power of the extracts. Results were expressed as percentage of inhibition relative to the standard (BHT) at the concentration of 1 mg/mL, and as IC50 (µg/mL) whenever possible.

Statistical Analyses
Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) Statistics software v.22 (IBM SPSS Statistics for Windows, IBM Corp., Chicago, USA). Data of all analyses, in triplicate, are expressed as mean ± standard error of the mean (SEM). A one-way analysis of variance (ANOVA) with Tukey's honest significant difference (HSD) post-hoc test (p < 0.05) was used to look for statistically significant differences among results. Differences amongst samples were considered significant if p values

In Vitro Antioxidant Properties
Extracts were evaluated for in vitro antioxidant properties, at six different concentrations, ranging from 10 to 2000 µg/mL. Assays were performed in 96-well flat bottom microtitration plates, and absorbances were read on a multi-plate reader (EZ read 400, Biochrom) (Cambridge, UK). Results were expressed as a percentage of inhibition relative to the negative control containing methanol, and as IC 50 values (mg/mL) whenever possible.

Targeting Free Radicals: Radical Scavenging Activity (RSA) on DPPH and ABTS Radicals
The RSA on DPPH and ABTS radicals was performed as decribed previously [10]. The synthetic antioxidant compound BHT was used as positive control, in the same concentrations as the samples. CCA and ICA were determined as described elsewhere [63]. The synthetic metal chelator, ethylenediaminetetraacetic acid (EDTA), was used as the positive control, in the same concentrations as the extracts. Samples were also tested for FRAP [11], where an increase in samples' absorbance represents an increase in the reducing power of the extracts. Results were expressed as percentage of inhibition relative to the standard (BHT) at the concentration of 1 mg/mL, and as IC 50 (µg/mL) whenever possible.

Statistical Analyses
Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) Statistics software v.22 (IBM SPSS Statistics for Windows, IBM Corp., Chicago, USA). Data of all analyses, in triplicate, are expressed as mean ± standard error of the mean (SEM). A one-way analysis of variance (ANOVA) with Tukey's honest significant difference (HSD) post-hoc test (p < 0.05) was used to look for statistically significant differences among results. Differences amongst samples were considered significant if p values were equal or inferior to 0.05. Half-maximal inhibitory concentration (IC 50 ) values were determined through data sigmoidal fitting in the GraphPad Prism v. 5.0 software.

Conclusions
Ethanol, acetone, and water extracts from aerial organs and fruits of sea rocket were in general not toxic towards mammalian cell lines, and were mainly constituted by flavonoids, especially quercetin and kaempferol glycosides. The extracts displayed moderate and low capacity to inhibit AChE and BuChE, respectively. The extracts had reduced capacity to inhibit amylase and glucosidase, except for the ethanol fruits extracts, which could significantly inhibit the latter enzyme. ADME studies suggest that these molecules also exhibit low oral absorption. On the other hand, interestingly, except for the water extracts from fruits, tested samples had a strong inhibitory capacity towards tyrosinase containing quercetin and kaempferol glycosides that fit well into the enzymatic pocket of the latter enzyme. Samples had reduced antioxidant capacity. Our results suggest that the sea rocket can be further explored for its potential to provide pharmacological active natural products, especially with the capacity to inhibit tyrosinase, for cosmetic and dermatological purposes.